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Giulio RASTELLI
Professore Ordinario
Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103
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Pubblicazioni
2023
- Design, synthesis, biological evaluation and crystal structure determination of dual modulators of carbonic anhydrases and estrogen receptors
[Articolo su rivista]
Tinivella, Annachiara; Nwachukwu, J. C.; Angeli, A.; Foschi, F.; Benatti, Annalaura; Pinzi, Luca; Izard, T.; Ferraroni, M.; Erumbi, R.; Christodoulou, M. S.; Passarella, D.; Supuran, C.; Nettles, K. W.; Rastelli, Giulio
abstract
2023
- Discovery of potent pyrrolo-pyrimidine and purine HDAC inhibitors for the treatment of advanced prostate cancer
[Articolo su rivista]
Moi, Davide; Bonanni, Davide; Belluti, Silvia; Linciano, Pasquale; Citarella, Andrea; Franchini, Silvia; Sorbi, Claudia; Imbriano, Carol; Pinzi, Luca; Rastelli, Giulio
abstract
2023
- Identification of Promising Drug Candidates against Prostate Cancer through Computationally-Driven Drug Repurposing
[Articolo su rivista]
Bernal, Leonardo; Pinzi, Luca; Rastelli, Giulio
abstract
2023
- Insights into the Structural Conformations of the Tau Protein in Different Aggregation Status
[Articolo su rivista]
Pinzi, L.; Bisi, N.; Sorbi, C.; Franchini, S.; Tonali, N.; Rastelli, G.
abstract
Tau is a protein characterized by large structural portions displaying extended conformational changes. Unfortunately, the accumulation of this protein into toxic aggregates in neuronal cells leads to a number of severe pathologies, collectively named tauopathies. In the last decade, significant research advancements were achieved, including a better understanding of Tau structures and their implication in different tauopathies. Interestingly, Tau is characterized by a high structural variability depending on the type of disease, the crystallization conditions, and the formation of pathologic aggregates obtained from in vitro versus ex vivo samples. In this review, we reported an up-to-date and comprehensive overview of Tau structures reported in the Protein Data Bank, with a special focus on discussing the connections between structural features, different tauopathies, different crystallization conditions, and the use of in vitro or ex vivo samples. The information reported in this article highlights very interesting links between all these aspects, which we believe may be of particular relevance for a more informed structure-based design of compounds able to modulate Tau aggregation.
2023
- Structure-activity exploration of a small-molecule allosteric inhibitor of T790M/L858R double mutant EGFR
[Articolo su rivista]
Foschi, FRANCESCA MADDALENA; Tinivella, Annachiara; Crippa, Valeria; Pinzi, Luca; Mologni, Luca; Passarella, Daniele; Rastelli, Giulio
abstract
2022
- Development of machine learning classifers to predict compound activity on prostate cancer cell lines.
[Articolo su rivista]
Bonanni, Davide; Pinzi, Luca; Rastelli, Giulio
abstract
2022
- Dual Targeting Strategies On Histone Deacetylase 6 (HDAC6) And Heat Shock Protein 90 (Hsp90)
[Articolo su rivista]
Bonanni, Davide; Citarella, Andrea; Moi, Davide; Pinzi, Luca; Bergamini, Elisa; Rastelli, Giulio
abstract
The design of multi-target drugs acting simultaneously on multiple signaling pathways is a growing field in medicinal chemistry, especially for the treatment of complex diseases, such as cancer. Histone deacetylase 6 (HDAC6) is an established anticancer drug target involved in tumor cells transformation. Being an epigenetic enzyme at the interplay of many biological processes, HDAC6 has become an attractive target for polypharmacology studies aimed at improving the therapeutic efficacy of anticancer drugs. For example, the molecular chaperone Heat shock protein 90 (Hsp90) is a substrate of HDAC6 deacetylation, and several lines of evidence demonstrate that simultaneous inhibition of HDAC6 and Hsp90 promotes synergistic antitumor effects on different cancer cell lines, highlighting the potential benefits of developing a single molecule endowed with multi-target activity. This review will summarize the complex interplay between HDAC6 and Hsp90, providing also useful hints for multi-target drug design and discovery approaches in this field. To this end, crystallographic structures of HDAC6 and Hsp90 complexes will be extensively reviewed in light of discussing binding pockets features and pharmacophore requirements and providing useful guidelines for the design of dual inhibitors. The few examples of multi-target inhibitors obtained so far, mostly based on chimeric approaches, will be summarized and put into context. Finally, the main features of HDAC6 and Hsp90 inhibitors will be compared, and ligand- and structure-based strategies potentially useful for the development of small molecular weight dual inhibitors will be proposed and discussed.
2022
- Identification of potential biological targets of oxindole scaffolds via in silico repositioning strategies
[Articolo su rivista]
Tinivella, Annachiara; Pinzi, Luca; Gambacorta, Guido; Baxendale, Ian; Rastelli, Giulio
abstract
2022
- Synthesis of potent and selective HDAC6 inhibitors led to unexpected opening of a quinazoline ring
[Articolo su rivista]
Moi, D.; Citarella, A.; Bonanni, D.; Pinzi, L.; Passarella, D.; Silvani, A.; Giannini, C.; Rastelli, G.
abstract
Histone deacetylase (HDAC) inhibitors are highly involved in the regulation of many pharmacological responses, which results in anti-inflammatory and anti-cancer effects. In the present work, chemoinformatic analyses were performed to obtain two potent and selective aminotriazoloquinazoline-based HDAC6 inhibitors. We unexpectedly obtained an aminotriazole from a water-driven ring opening of the triazoloquinazoline scaffold. Both compounds were evaluated as HDAC6 inhibitors, resulting in subnanomolar inhibitory activity and high selectivity with respect to class I HDAC1 and HDAC8. Importantly, the compounds were about 3- and 15-fold more potent compared to the reference compound trichostatin A. Additionally, the predicted binding modes were investigated with docking. Considering that the aminotriazole scaffold has never been embedded into the chemical structure of HDAC6 inhibitors, the present study suggests that both the aminotriazoloquinazoline and aminotriazole classes of compounds could be excellent starting points for further optimization of potential anticancer compounds, introducing such novel groups into a relevant and new area of investigation.
2021
- Antifungal Activity and DNA Topoisomerase Inhibition of Hydrolysable Tannins from Punica granatum L.
[Articolo su rivista]
Brighenti, V.; Iseppi, R.; Pinzi, L.; Mincuzzi, A.; Ippolito, A.; Messi, P.; Sanzani, S. M.; Rastelli, G.; Pellati, F.
abstract
Punica granatum L. (pomegranate) fruit is known to be an important source of bioactive phenolic compounds belonging to hydrolysable tannins. Pomegranate extracts have shown antifungal activity, but the compounds responsible for this activity and their mechanism/s of action have not been completely elucidated up to now. The aim of the present study was the investigation of the inhibition ability of a selection of pomegranate phenolic compounds (i.e., punicalagin, punicalin, ellagic acid, gallic acid) on both plant and human fungal pathogens. In addition, the biological target of punicalagin was identified here for the first time. The antifungal activity of pomegranate phenolics was evaluated by means of Agar Disk Diffusion Assay and minimum inhibitory concentration (MIC) evaluation. A chemoinformatic analysis predicted for the first time topoisomerases I and II as potential biological targets of punicalagin, and this prediction was confirmed by in vitro inhibition assays. Concerning phytopathogens, all the tested compounds were effective, often similarly to the fungicide imazalil at the label dose. Particularly, punicalagin showed the lowest MIC for Alternaria alternata and Botrytis cinerea, whereas punicalin was the most active compound in terms of growth control extent. As for human pathogens, punicalagin was the most active compound among the tested ones against Candida albicans reference strains, as well as against the clinically isolates. UHPLC coupled with HRMS indicated that C. albicans, similarly to the phytopathogen Coniella granati, is able to hydrolyze both punicalagin and punicalin as a response to the fungal attack. Punicalagin showed a strong inhibitory activity, with IC50 values of 9.0 and 4.6 µM against C. albicans topoisomerases I and II, respectively. Altogether, the results provide evidence that punicalagin is a valuable candidate to be further exploited as an antifungal agent in particular against human fungal infections.
2021
- Chemoinformatics Analyses of Tau Ligands Reveal Key Molecular Requirements for the Identification of Potential Drug Candidates against Tauopathies
[Articolo su rivista]
Pinzi, Luca; Tinivella, Annachiara; Rastelli, Giulio
abstract
Tau is a highly soluble protein mainly localized at a cytoplasmic level in the neuronal cells, which plays a crucial role in the regulation of microtubule dynamic stability. Recent studies have demonstrated that several factors, such as hyperphosphorylation or alterations of Tau metabolism, may contribute to the pathological accumulation of protein aggregates, which can result in neuronal death and the onset of a number of neurological disorders called Tauopathies. At present, there are no available therapeutic remedies able to reduce Tau aggregation, nor are there any structural clues or guidelines for the rational identification of compounds preventing the accumulation of protein aggregates. To help identify the structural properties required for anti-Tau aggregation activity, we performed extensive chemoinformatics analyses on a dataset of Tau ligands reported in ChEMBL. The performed analyses allowed us to identify a set of molecular properties that are in common between known active ligands. Moreover, extensive analyses of the fragment composition of reported ligands led to the identification of chemical moieties and fragment combinations prevalent in the more active compounds. Interestingly, many of these fragments were arranged in recurring frameworks, some of which were clearly present in compounds currently under clinical investigation. This work represents the first in-depth chemoinformatics study of the molecular properties, constituting fragments and similarity profiles, of known Tau aggregation inhibitors. The datasets of compounds employed for the analyses, the identified molecular fragments and their combinations are made publicly available as supplementary material.
2021
- Design and Synthesis of Hsp90 Inhibitors with B-Raf and PDHK1 Multi-Target Activity
[Articolo su rivista]
Pinzi, L.; Foschi, F.; Christodoulou, M. S.; Passarella, D.; Rastelli, G.
abstract
The design of multi-target ligands has become an innovative approach for the identification of effective therapeutic treatments against complex diseases, such as cancer. Recent studies have demonstrated that the combined inhibition of Hsp90 and B-Raf provides synergistic effects against several types of cancers. Moreover, it has been reported that PDHK1, which presents an ATP-binding pocket similar to that of Hsp90, plays an important role in tumor initiation, maintenance and progression, participating also to the senescence process induced by B-Raf oncogenic proteins. Based on these premises, the simultaneous inhibition of these targets may provide several benefits for the treatment of cancer. In this work, we set up a design strategy including the assembly and integration of molecular fragments known to be important for binding to the Hsp90, PDHK1 and B-Raf targets, aided by molecular docking for the selection of a set of compounds potentially able to exert Hsp90-B-Raf-PDHK1 multi-target activities. The designed compounds were synthesized and experimentally validated in vitro. According to the in vitro assays, compounds 4 a, 4 d and 4 e potently inhibited Hsp90 and moderately inhibited the PDHK1 kinase. Finally, molecular dynamics simulations were performed to provide further insights into the structural basis of their multi-target activity.
2021
- Development and application of LigAdvisor, a user-friendly web-platform for polypharmacology and drug repurposing
[Abstract in Atti di Convegno]
Pinzi, Luca; Tinivella, Annachiara; Rastelli, Giulio
abstract
2021
- Drug Repurposing and Polypharmacology to Fight SARS-CoV-2 Through Inhibition of the Main Protease
[Articolo su rivista]
Pinzi, Luca; Tinivella, Annachiara; Caporuscio, Fabiana; Rastelli, Giulio
abstract
The outbreak of a new coronavirus (SARS-CoV-2), which is responsible for the COVID-19 disease and is spreading rapidly around the world, urgently requires effective therapeutic treatments. In this context, drug repurposing represents a valuable strategy, as it enables accelerating the identification of drug candidates with already known safety profiles, possibly aiding in the late stages of clinical evaluation. Moreover, therapeutic treatments based on drugs with beneficial multi-target activities (polypharmacology) may show an increased antiviral activity or help to counteract severe complications concurrently affecting COVID-19 patients. In this study, we present the results of a computational drug repurposing campaign that aimed at identifying potential inhibitors of the main protease (Mpro) of the SARS-CoV-2. The performed in silico screening allowed the identification of 22 candidates with putative SARS-CoV-2 Mpro inhibitory activity. Interestingly, some of the identified compounds have recently entered clinical trials for COVID-19 treatment, albeit not being assayed for their SARS-CoV-2 antiviral activity. Some candidates present a polypharmacology profile that may be beneficial for COVID-19 treatment and, to the best of our knowledge, have never been considered in clinical trials. For each repurposed compound, its therapeutic relevance and potential beneficial polypharmacological effects that may arise due to its original therapeutic indication are thoroughly discussed.
2021
- Hydroxamic Acid Derivatives: From Synthetic Strategies to Medicinal Chemistry Applications
[Articolo su rivista]
Citarella, A.; Moi, D.; Pinzi, L.; Bonanni, D.; Rastelli, G.
abstract
Since the approval of three hydroxamic acid-based HDAC inhibitors as anticancer drugs, such functional groups acquired even more notoriety in synthetic medicinal chemistry. The ability of hydroxamic acids (HAs) to chelate metal ions makes this moiety an attractive metal binding group - in particular, Fe(III) and Zn(II) - so that HA derivatives find wide applications as metalloenzymes inhibitors. In this minireview, we will discuss the most relevant features concerning hydroxamic acid derivatives. In a first instance, the physicochemical characteristics of HAs will be summarized; then, an exhaustive description of the most relevant methods for the introduction of such moiety into organic substrates and an overview of their uses in medicinal chemistry will be presented.
2021
- Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group
[Articolo su rivista]
Linciano, P.; Pinzi, L.; Belluti, S.; Chianese, U.; Benedetti, R.; Moi, D.; Altucci, L.; Franchini, S.; Imbriano, C.; Sorbi, C.; Rastelli, G.
abstract
Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC50 of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.
2021
- Investigation of the effect of different linker chemotypes on the inhibition of histone deacetylases (HDACs)
[Articolo su rivista]
Linciano, P.; Benedetti, R.; Pinzi, L.; Russo, F.; Chianese, U.; Sorbi, C.; Altucci, L.; Rastelli, G.; Brasili, L.; Franchini, S.
abstract
Histone Deacetylases (HDACs) are among the most attractive and interesting targets in anticancer drug discovery. The clinical relevance of HDAC inhibitors (HDACIs) is testified by four FDA-approved drugs for cancer treatment. However, one of the main drawbacks of these drugs resides in the lack of selectivity against the different HDAC isoforms, resulting in severe side effects. Thus, the identification of selective HDACIs represents an exciting challenge for medicinal chemists. HDACIs are composed of a cap group, a linker region, and a metal-binding group interacting with the catalytic zinc ion. While the cap group has been extensively investigated, less information is available about the effect of the linker on isoform selectivity. To this aim, in this work, we explored novel linker chemotypes to direct isoform selectivity. A small library of 25 hydroxamic acids with hitherto unexplored linker chemotypes was prepared. In vitro tests demonstrated that, depending on the linker type, some candidates selectively inhibit HDAC1 over HDAC6 isoform or vice versa. Docking calculations were performed to rationalize the effect of the novel linker chemotypes on biologic activity. Moreover, four compounds were able to increase the levels of acetylation of histone H3 or tubulin. These compounds were also assayed in breast cancer MCF7 cells to test their antiproliferative effect. Three compounds showed a significant reduction of cancer proliferation, representing valuable starting points for further optimization.
2021
- LigAdvisor: A versatile and user-friendly web-platform for drug design
[Articolo su rivista]
Pinzi, L.; Tinivella, A.; Gagliardelli, L.; Beneventano, D.; Rastelli, G.
abstract
Although several tools facilitating in silico drug design are available, their results are usually difficult to integrate with publicly available information or require further processing to be fully exploited. The rational design of multi-target ligands (polypharmacology) and the repositioning of known drugs towards unmet therapeutic needs (drug repurposing) have raised increasing attention in drug discovery, although they usually require careful planning of tailored drug design strategies. Computational tools and data-driven approaches can help to reveal novel valuable opportunities in these contexts, as they enable to efficiently mine publicly available chemical, biological, clinical, and disease-related data. Based on these premises, we developed LigAdvisor, a data-driven webserver which integrates information reported in DrugBank, Protein Data Bank, UniProt, Clinical Trials and Therapeutic Target Database into an intuitive platform, to facilitate drug discovery tasks as drug repurposing, polypharmacology, target fishing and profiling. As designed, LigAdvisor enables easy integration of similarity estimation results with clinical data, thereby allowing a more efficient exploitation of information in different drug discovery contexts. Users can also develop customizable drug design tasks on their own molecules, by means of ligand- and target-based search modes, and download their results. LigAdvisor is publicly available at https://ligadvisor.unimore.it/.
2021
- LigAdvisor: a web platform designed for charting novel polypharmacology and drug repurposing routes from crystallographic ligands and known drugs
[Poster]
Pinzi, Luca; Tinivella, Annachiara; Rastelli, Giulio
abstract
2021
- LigAdvisor: a web server to perform in silico explorations on crystallographic ligands and known drugs for polypharmacology and drug repurposing.
[Abstract in Atti di Convegno]
Pinzi, Luca; Tinivella, Annachiara; Rastelli, Giulio
abstract
2021
- Prediction of activity and selectivity profiles of human Carbonic Anhydrase inhibitors using machine learning classification models
[Articolo su rivista]
Tinivella, Annachiara; Pinzi, Luca; Rastelli, Giulio
abstract
The development of selective inhibitors of the clinically relevant human Carbonic Anhydrase (hCA) isoforms IX and XII has become a major topic in drug research, due to their deregulation in several types of cancer. Indeed, the selective inhibition of these two isoforms, especially with respect to the homeostatic isoform II, holds great promise to develop anticancer drugs with limited side effects. Therefore, the development of in silico models able to predict the activity and selectivity against the desired isoform(s) is of central interest. In this work, we have developed a series of machine learning classification models, trained on high confidence data extracted from ChEMBL, able to predict the activity and selectivity profiles of ligands for human Carbonic Anhydrase isoforms II, IX and XII. The training datasets were built with a procedure that made use of flexible bioactivity thresholds to obtain well-balanced active and inactive classes. We used multiple algorithms and sampling sizes to finally select activity models able to classify active or inactive molecules with excellent performances. Remarkably, the results herein reported turned out to be better than those obtained by models built with the classic approach of selecting an a priori activity threshold. The sequential application of such validated models enables virtual screening to be performed in a fast and more reliable way to predict the activity and selectivity profiles against the investigated isoforms.
2021
- Promising Non-cytotoxic Monosubstituted Chalcones to Target Monoamine Oxidase-B
[Articolo su rivista]
Iacovino, L. G.; Pinzi, L.; Facchetti, G.; Bortolini, B.; Christodoulou, M. S.; Binda, C.; Rastelli, G.; Rimoldi, I.; Passarella, D.; Di Paolo, M. L.; Dalla Via, L.
abstract
A library of monosubstituted chalcones (1-17) bearing electron-donating and electron-withdrawing groups on both aromatic rings were selected. The cell viability on human tumor cell lines was evaluated first. The compounds unable to induce detectable cytotoxicity (1, 13, and 14) were tested using the monoamine oxidase (MAO) activity assay. Interestingly, they inhibit MAO-B, acting as competitive inhibitors, with 13 and 14 showing the best profiles. In particular, 13 exhibited a potency higher than that of safinamide, taken as a reference. Docking studies and crystallographic analysis showed that in human MAO-B 13 binds with the halogen-substituted aromatic ring in the entrance cavity, similar to safinamide, whereas 14 is accommodated in the opposite way. The main conclusion of this cell biology, biochemistry, and structural study is to highlights 13 as a chalcone derivative that is worth consideration for the development of novel MAO-B-selective inhibitors for the treatment of neurodegenerative diseases.
2021
- Tackling polypharmacology and drug repurposing with the LigAdvisor webserver.
[Poster]
Pinzi, Luca; Tinivella, Annachiara; Rastelli, Giulio
abstract
2021
- Targeting the allosteric sites of the B-Raf protein kinase through an in silico approach
[Poster]
Pinzi, Luca; Rastelli, Giulio
abstract
2020
- Breakthroughs in medicinal chemistry: New targets and mechanisms, new drugs, new hopes-6
[Articolo su rivista]
Vanden Eynde, J. J.; Mangoni, A. A.; Rautio, J.; Leprince, J.; Azuma, Y. -T.; Garcia-Sosa, A. T.; Hulme, C.; Jampilek, J.; Karaman, R.; Li, W.; Gomes, P. A. C.; Hadjipavlou-Litina, D.; Capasso, R.; Geronikaki, A.; Cerchia, L.; Sabatier, J. -M.; Ragno, R.; Tuccinardi, T.; Trabocchi, A.; Winum, J. -Y.; Luque, F. J.; Prokai-Tatrai, K.; Spetea, M.; Gutschow, M.; Kosalec, I.; Guillou, C.; Vasconcelos, M. H.; Kokotos, G.; Rastelli, G.; De Sousa, M. E.; Manera, C.; Tosi, Gemma; Mangani, S.; Siciliano, C.; Galdiero, S.; Liu, H.; Scott, P. J. H.; De Los Rios, C.; Agrofoglio, L. A.; Collina, S.; Guedes, R. C.; Munoz-Torrero, D.
abstract
Breakthroughs in Medicinal Chemistry: New Targets and Mechanisms, New Drugs, New Hopes is a series of Editorials that is published on a biannual basis by the Editorial Board of the Medicinal Chemistry section of the journal Molecules [...].
2020
- Breakthroughs in medicinal chemistry: New targets and mechanisms, new drugs, new hopes-7
[Articolo su rivista]
Gutschow, M.; Eynde, J. J. V.; Jampilek, J.; Kang, C.; Mangoni, A. A.; Fossa, P.; Karaman, R.; Trabocchi, A.; Scott, P. J. H.; Reynisson, J.; Rapposelli, S.; Galdier, S.; Winum, J. -Y.; Brullo, C.; Prokai-Tatrai, K.; Sharma, A. K.; Schapira, M.; Azuma, Y. -T.; Cerchia, L.; Spete, M.; Torri, G.; Collina, S.; Geronikaki, A.; Garcia-Sosa, A. T.; Helena Vasconcelos, M.; Sousa, M. E.; Kosalec, I.; Tuccinardi, T.; Duarte, I. F.; Salvador, J. A. R.; Bertinaria, M.; Pellecchia, M.; Amato, J.; Rastelli, G.; Gomes, P. A. C.; Guedes, R. C.; Sabatier, J. -M.; Estevez-Braun, A.; Pagano, B.; Mangani, S.; Ragno, R.; Kokotos, G.; Brindisi, M.; Gonzalez, F. V.; Borges, F.; Miloso, M.; Rautio, J.; Munoz-Torrero, D.
abstract
Breakthroughs in Medicinal Chemistry [...].
2020
- Design of dual inhibitors of histone deacetylase 6 and heat shock protein 90
[Articolo su rivista]
Pinzi, L.; Benedetti, R.; Altucci, L.; Rastelli, G.
abstract
Histone deacetylase 6 (HDAC6) and heat shock protein 90 (Hsp90) are widely investigated anticancer drug targets. Importantly, several lines of evidence indicate that their regulation and activity are intimately linked, and that their combined inhibition may lead to impressive therapeutic benefits. In this study, we developed and applied an integrated computational strategy to design dual inhibitors of HDAC6 and Hsp90. Although the two targets share very little homology, an integrated ligand-based and structure-based virtual screening approach indicated a subset of compounds possessing the key structural requirements for binding at both targets. In vitro tests demonstrated that some of the selected candidates are able to selectively inhibit HDAC6 over HDAC1, to increase the acetylation levels of tubulin on cell assays and to reduce cell proliferation. The discovered compounds represent valuable starting points for further hit optimization.
2020
- Identification of Target Associations for Polypharmacology from Analysis of Crystallographic Ligands of the Protein Data Bank
[Articolo su rivista]
Pinzi, L.; Rastelli, G.
abstract
The design of a chemical entity that potently and selectively binds to a biological target of therapeutic relevance has dominated the scene of drug discovery so far. However, recent findings suggest that multitarget ligands may be endowed with superior efficacy and be less prone to drug resistance. The Protein Data Bank (PDB) provides experimentally validated structural information about targets and bound ligands. Therefore, it represents a valuable source of information to help identifying active sites, understanding pharmacophore requirements, designing novel ligands, and inferring structure-activity relationships. In this study, we performed a large-scale analysis of the PDB by integrating different ligand-based and structure-based approaches, with the aim of identifying promising target associations for polypharmacology based on reported crystal structure information. First, the 2D and 3D similarity profiles of the crystallographic ligands were evaluated using different ligand-based methods. Then, activity data of pairs of similar ligands binding to different targets were inspected by comparing structural information with bioactivity annotations reported in the ChEMBL, BindingDB, BindingMOAD, and PDBbind databases. Afterward, extensive docking screenings of ligands in the identified cross-targets were made in order to validate and refine the ligand-based results. Finally, the therapeutic relevance of the identified target combinations for polypharmacology was evaluated from comparison with information on therapeutic targets reported in the Therapeutic Target Database (TTD). The results led to the identification of several target associations with high therapeutic potential for polypharmacology.
2020
- Repositioning natural products in drug discovery
[Articolo su rivista]
Rastelli, G.; Pellati, F.; Pinzi, L.; Gamberini, M. C.
abstract
No abstract available
2019
- 2-Phenyloxazole-4-carboxamide as a Scaffold for Selective Inhibition of Human Monoamine Oxidase B
[Articolo su rivista]
Di Paolo, M. L.; Christodoulou, M. S.; Calogero, A. M.; Pinzi, L.; Rastelli, G.; Passarella, D.; Cappelletti, G.; Dalla Via, L.
abstract
A series of 2-phenyloxazoles bearing an amide group at position 4 were designed and synthesized for evaluation as potential inhibitors of human recombinant monoamine oxidases (hrMAOs). Results of kinetics experiments demonstrated that all compounds behave as competitive MAO inhibitors, with good selectivity toward the MAO-B isoform. The most potent and selective derivatives are characterized by inhibition constant (Ki) values in the sub-micromolar range and a good selectivity index (Ki MAO-A/Ki MAO-B>50). Some derivatives were also found to be able to inhibit MAO activity in nerve growth factor (NGF)-differentiated PC12 cells, taken as a model of neuronal cells. In particular, 2-(2-hydroxyphenyl)-N-phenyloxazole-4-carboxamide (compound 4 a) may be a promising new scaffold, exerting the highest selectivity and inhibitory effect toward MAOs in NGF-differentiated PC12 cell lysates, without compromising cell viability. Molecular docking analysis allowed a rationalization of the experimentally observed binding affinity and selectivity.
2019
- Evaluation of Amides, Carbamates, Sulfonamides, and Ureas of 4-Prop-2-ynylidenecycloalkylamine as Potent, Selective, and Bioavailable Negative Allosteric Modulators of Metabotropic Glutamate Receptor 5
[Articolo su rivista]
Graziani, Davide; Caligari, Silvia; Callegari, Elisa; De Toma, Carlo; Longhi, Matteo; Frigerio, Fabio; Dilernia, Roberto; Menegon, Sergio; Pinzi, Luca; Pirona, Lorenza; Tazzari, Valerio; Valsecchi, Anna Elisa; Vistoli, Giulio; Rastelli, Giulio; Ruga Riva, Carlo
abstract
Negative allosteric modulators (NAMs) of the metabotropic glutamate receptor 5 (mGlu 5 ) hold great promise for the treatment of a variety of central nervous system disorders. We have recently reported that prop-2-ynylidenecycloalkylamine derivatives are potent and selective NAMs of the mGlu 5 receptor. In this work, we explored the amide, carbamate, sulfonamide, and urea derivatives of prop-2-ynylidenecycloalkylamine compounds with the aim of improving solubility and metabolic stability. In silico and experimental analyses were performed on the synthesized series of compounds to investigate structure-activity relationships. Compounds 12, 32, and 49 of the carbamate, urea, and amide classes, respectively, showed the most suitable cytochrome inhibition and metabolic stability profiles. Among them, compound 12 showed excellent selectivity, solubility, and stability profiles as well as suitable in vitro and in vivo pharmacokinetic properties. It was highly absorbed in rats and dogs and was active in anxiety, neuropathic pain, and lower urinary tract models.
2019
- In silico repositioning of cannabigerol as a novel inhibitor of the enoyl acyl carrier protein (ACP) reductase (INHA)
[Articolo su rivista]
Pinzi, L.; Lherbet, C.; Baltas, M.; Pellati, F.; Rastelli, G.
abstract
Cannabigerol (CBG) and cannabichromene (CBC) are non-psychoactive cannabinoids that have raised increasing interest in recent years. These compounds exhibit good tolerability and low toxicity, representing promising candidates for drug repositioning. To identify novel potential therapeutic targets for CBG and CBC, an integrated ligand-based and structure-based study was performed. The results of the analysis led to the identification of CBG as a low micromolar inhibitor of the Enoyl acyl carrier protein (ACP) reductase (InhA) enzyme.
2019
- Molecular docking: Shifting paradigms in drug discovery
[Articolo su rivista]
Pinzi, L.; Rastelli, G.
abstract
Molecular docking is an established in silico structure-based method widely used in drug discovery. Docking enables the identification of novel compounds of therapeutic interest, predicting ligand-target interactions at a molecular level, or delineating structure-activity relationships (SAR), without knowing a priori the chemical structure of other target modulators. Although it was originally developed to help understanding the mechanisms of molecular recognition between small and large molecules, uses and applications of docking in drug discovery have heavily changed over the last years. In this review, we describe how molecular docking was firstly applied to assist in drug discovery tasks. Then, we illustrate newer and emergent uses and applications of docking, including prediction of adverse effects, polypharmacology, drug repurposing, and target fishing and profiling, discussing also future applications and further potential of this technique when combined with emergent techniques, such as artificial intelligence.
2019
- Refinement and rescoring of virtual screening results
[Articolo su rivista]
Rastelli, G.; Pinzi, L.
abstract
High-throughput docking is an established computational screening approach in drug design. This methodology enables a rapid identification of biologically active hit compounds, providing an efficient and cost-effective complement or alternative to experimental high-throughput screenings. However, limitations inherent to the methodology make docking results inevitably approximate. Two major Achille’s heels include the use of approximated scoring functions and the limited sampling of the ligand-target complexes. Therefore, docking results require careful evaluation and further post-docking analyses. In this article, we will overview our approach to post-docking analysis in virtual screenings. BEAR (Binding Estimation After Refinement) was developed as a post-docking processing tool that refines docking poses by means of molecular dynamics (MD) and then rescores the ligands based on more accurate scoring functions (MM-PB(GB)SA). The tool has been validated and used prospectively in drug discovery applications. Future directions regarding refinement and rescoring in virtual screening are discussed.
2019
- Unveiling target associations for polypharmacology from analysis of crystallographic ligands in the Protein Data Bank
[Abstract in Atti di Convegno]
Pinzi, Luca; Rastelli, Giulio
abstract
2019
- Virtual screening for dual Hsp90/B-Raf inhibitors
[Capitolo/Saggio]
Anighoro, A.; Pinzi, L.; Rastelli, G.; Bajorath, J.
abstract
In this chapter, we describe a computational strategy leading to the identification of the first dual inhibitors of Heat Shock Protein 90 (Hsp90) and protein kinase B-Raf. Both proteins are validated targets for anti-cancer drug discovery. There is strong evidence that the simultaneous inhibition of Hsp90 and B-Raf provides therapeutic benefits compared to exclusive engagement of one or the other target. Hence, we have been interested in searching for dual Hsp90/B-Raf inhibitors. Virtual compound screening led to the identification of two compounds with micromolar activity against both targets. The computational approach faced a number of challenges that needed to be overcome, as described herein.
2018
- Exploration and Comparison of the Geometrical and Physicochemical Properties of an αc Allosteric Pocket in the Structural Kinome
[Articolo su rivista]
Sturm, Noe Joseph; Tinivella, Annachiara; Rastelli, Giulio
abstract
In this work, a comprehensive analysis of the local geometrical and physicochemical properties of a type III allosteric pocket located between the regulatory αC helix and the activation loop of protein kinases was made by comparing available crystal structures in the structural kinome. We first explored the structural kinome to outline the possible conformations of this site. Subsequently we characterized the positions of cocrystallized ligands of the structural kinome with respect to the structural variability of the allosteric site. Then, we searched for kinase structures with similar allosteric site conformation. The search returned 26 kinases with a DFG-in/αC-out conformation potentially prone to bind allosteric inhibitors, as well as different scaffolds that can be useful starting points for the design of new inhibitors. These promising allosteric pockets were probed by performing molecular docking of known active compounds taken from ChEMBL. Interestingly, none of the active compounds reported in ChEMBL had a purely allosteric binding mode, and none of the ATP-competitive ligands had chemical moieties extending into the allosteric pocket in more than two-thirds of the investigated kinases, indicating that the allosteric pocket is accessible but still largely unexplored by available inhibitors. Finally, we compared the physicochemical properties of the allosteric site in the structural kinome and discussed the peculiar and conserved features. These analyses may help the design of allosteric ligands tailored toward the intended kinase(s).
2018
- Identification of 4-aryl-1H-pyrrole[2,3-b]pyridine derivatives for the development of new B-Raf inhibitors
[Articolo su rivista]
Pinzi, Luca; Anighoro, Andrew; Bajorath, Jürgen; Rastelli, Giulio
abstract
During the last years, a significant interest in the identification of new classes of B-Raf inhibitors has emerged. In this study, which was conceived within an effort that culminated in the recent report of the first dual inhibitors of B-Raf and Hsp90, we describe the identification of four compounds based on 4-aryl-1H-pyrrole[2,3-b]pyridine scaffold as interesting starting points for the development of new B-Raf inhibitors. Structure-activity relationships and predicted binding modes are discussed. Moreover, the novelty of the newly identified structures with respect to currently known B-Raf inhibitors was assessed through a ligand-based dissimilarity assessment. Finally, structural modifications with the potential ability to improve the activity toward B-Raf are put forward. This article is protected by copyright. All rights reserved.
2018
- Identification of small-molecule EGFR allosteric inhibitors by high-Throughput docking
[Articolo su rivista]
Caporuscio, Fabiana; Tinivella, Annachiara; Restelli, Valentina; Semrau, Marta S; Pinzi, Luca; Storici, Paola; Broggini, Massimo; Rastelli, Giulio
abstract
Aim: The EGFR inhibitors represent the first-line treatment of non-small-cell lung cancer. However, the emergence of resistance urgently requires the development of new inhibitors targeting drug-resistant mutants. Methodology: A recently released structure of an EGFR kinase domain in complex with an allosteric inhibitor and a mutant protein model derived from it were used to set up a low-cost high-Throughput docking protocol for the fast identification of EGFR allosteric inhibitors. Conclusion: The virtual screening of commercially available compounds led to the identification of interesting new hit compounds. The most promising hit was confirmed to be a new allosteric inhibitor of wild-Type and T790M/L858R double mutant EGFR which was able to inhibit the growth of non-small-cell lung cancer cell lines.
2018
- Investigating the Selectivity of Allosteric Inhibitors for Mutant T790M EGFR over Wild Type Using Molecular Dynamics and Binding Free Energy Calculations
[Articolo su rivista]
Tinivella, Annachiara; Rastelli, Giulio
abstract
The recent discovery of the fourth generation EAI045 allosteric inhibitor, which potently and selectively inhibits mutant EGFR, represents an important step forward for the treatment of non-small cell lung cancer. However, the structural determinants of EAI045 selectivity with respect to the wild type (wt) protein have not been fully investigated. To this aim, we performed a comparative analysis of long-scale molecular dynamics simulations and binding free energy calculations on wt and T790M EGFR in complexes with the EAI001 and EAI045 allosteric ligands. Unexpectedly, we found that the observed selectivity for T790M EGFR over wt is not due to more favorable interactions of the two ligands with the mutated gatekeeper residue, as previously suggested. Rather, the allosteric ligands were engaged in a direct hydrogen bond with the Asp855 residue of the DFG motif in mutant T790M but not in wt, in which the hydrogen bond was found to be water-mediated. Per-residue decomposition of binding free energies suggests that the loss of a direct interaction with Asp855 is the main cause of inhibitor selectivity. Moreover, the possibility that the allosteric ligands and adenosine triphosphate may have synergistic binding effects, as previously observed in MEK allosteric inhibitors, was investigated. Altogether, the results suggest that ligand selectivity arises from direct hydrogen bonds with the Asp855 side chain, and that the design of mutant-selective inhibitors should be focused on ligands that form direct hydrogen bonds with Asp855 in T790M EGFR but not in wt EGFR. These results may provide useful hints for future structural design of mutant-selective allosteric inhibitors that spare wt EGFR, which is a highly desirable goal.
2018
- Selection of protein conformations for structure-based polypharmacology studies
[Articolo su rivista]
Pinzi, Luca; Caporuscio, Fabiana; Rastelli, Giulio
abstract
Several drugs exert their therapeutic effect through the modulation of multiple targets. Structure-based approaches hold great promise for identifying compounds with the desired polypharmacological profiles. These methods use knowledge of the protein binding sites to identify stereoelectronically complementary ligands. The selection of the most suitable protein conformations to be used in the design process is vital, especially for multitarget drug design in which the same ligand has to be accommodated in multiple binding pockets. Herein, we focus on currently available techniques for the selection of the most suitable protein conformations for multitarget drug design, compare the potential advantages and limitations of each method, and comment on how their combination could help in polypharmacology drug design.
2018
- Structure–Activity Relationships of Hexahydrocyclopenta[c]quinoline Derivatives as Allosteric Inhibitors of CDK2 and EGFR
[Articolo su rivista]
Carlino, Luca; Christodoulou, Michael S.; Restelli, Valentina; Caporuscio, Fabiana; Foschi, FRANCESCA MADDALENA; Semrau, MARTA STEFANIA; Costanzi, Elisa; Tinivella, Annachiara; Pinzi, Luca; Lo Presti, Leonardo; Battistutta, Roberto; Storici, Paola; Broggini, Massimo; Passarella, Daniele; Rastelli, Giulio
abstract
Following the discovery of a type III allosteric modulator of cyclin-dependent kinase 2 (CDK2) characterized by a hexahydrocyclopenta[c]quinolone scaffold, three different series of its derivatives were synthesized and biologically evaluated. Docking of the synthesized compounds into the allosteric pocket of CDK2 allowed the elucidation of structure–activity relationships (SARs). Moreover, the compounds were tested on the wild-type epidermal growth factor receptor (EGFR) kinase domain (KD) and its clinically relevant T790M/L858R mutant form. Herein we describe the first SAR investigation of allosteric ligands that bind to the type III inhibitor pocket of CDK2 and EGFR-KD. Although the activity of the synthesized inhibitors needs to be improved, the obtained results provide clear-cut indications about pharmacophore requirements and selectivity determinants. Remarkably, this study led to the identification of a selective T790M/L858R EGFR allosteric inhibitor that is inactive toward both wild-type EGFR and CDK2. Finally, docking into the T790M/L858R EGFR-KD led us to hypothesize that the compounds bind to the double-mutant EGFR-KD by adopting a binding mode different from that in CDK2, thus rationalizing the observed selectivity profile.
2017
- Heat shock protein 90 and serine/threonine kinase B-Raf inhibitors have overlapping chemical space
[Articolo su rivista]
Anighoro, A.; Pinzi, Luca; Marverti, Gaetano; Bajorath, J; Rastelli, Giulio
abstract
Heat shock protein 90 (Hsp90) and B-Raf are validated targets for anticancer drug discovery. Although there is strong evidence that concomitant inhibition of Hsp90 and B-Raf may provide significant therapeutic benefits, molecules endowed with dual activity against the two targets have not been reported. For the first time, we show that Hsp90 and B-Raf inhibitors have overlapping chemical space and we disclose the first-in-class dual inhibitors. The compounds were identified through a computational strategy especially devised for detecting ligands with dual-target activity. Although the two targets had only remote binding site similarity, we were able to identify dual inhibitors with well-balanced in vitro potencies and relatively low molecular weight. Remarkably, they also inhibited the V600E mutant form of B-Raf with similar potency. This study provides the first direct proof that designing dual ligands of Hsp90 and a kinase is possible, thus opening the way to new interesting possibilities in drug discovery.
2017
- On the integration of in silico drug design methods for drug repurposing
[Articolo su rivista]
MARCH VILA, Eric; Pinzi, Luca; Sturm, Noã; Tinivella, Annachiara; Engkvist, Ola; Chen, Hongming; Rastelli, Giulio
abstract
Drug repurposing has become an important branch of drug discovery. Several computational approaches that help to uncover new repurposing opportunities and aid the discovery process have been put forward, or adapted from previous applications. A number of successful examples are now available. Overall, future developments will greatly benefit from integration of different methods, approaches and disciplines. Steps forward in this direction are expected to help to clarify, and therefore to rationally predict, new drug-target, target-disease, and ultimately drug-disease associations.
2017
- Probing an allosteric pocket of CDK2 with small-molecules
[Articolo su rivista]
Christodoulou, Michael S; Caporuscio, Fabiana; Restelli, Valentina; Carlino, Luca; Cannazza, Giuseppe; Costanzi, Elisa; Citti, Cinzia; Lo Presti, Leonardo; Pisani, Pasquale; Battistutta, Roberto; Broggini, Massimo; Passarella, Daniele; Rastelli, Giulio
abstract
The availability of well characterized allosteric modulators is of crucial importance for investigating allosteric regulation of protein function. In a recently identified inactive conformation of CDK2 an open allosteric pocket has been detected and proposed as a site to accommodate allosteric inhibitors. Previous structure-based approaches allowed the identification of a hit compound expected to bind to this pocket. Here, we report the characterization of this compound by X-ray crystallography, which surprisingly provided a chemical structure different from the one previously reported. Therefore, the compound was synthesized and completely characterized. X-ray structures of the synthesized and purchased compounds were superimposable. A reaction mechanism was proposed to explain the formation of the structure indicated by X-ray. Moreover, a stereoselective synthesis was developed to evaluate the biological activity of the pure stereoisomers. Modeling studies were performed to unveil the details of the interaction with CDK2. Then, the activity of the obtained compounds was evaluated with different biological assays. Mutagenesis experiments confirmed binding to the allosteric pocket. Finally, the allosteric ligands were shown to inhibit the growth of A549 and SKOV3 cancer cell lines. Therefore, this paper presents a thorough chemical and biological characterization of the first small-molecule ligands to be used as probes to study the allosteric modulation of CDK2 activity.
2017
- Promiscuity of inhibitors of human protein kinases at varying data confidence levels and test frequencies
[Articolo su rivista]
Stumpfe, Dagmar; Tinivella, Annachiara; Rastelli, Giulio; Bajorath, Jürgen
abstract
More than 141 000 inhibitors of human kinases and their activity data were assembled to perform an in-depth analysis of inhibitor promiscuity (single- versus multi-kinase activity) at varying activity data confidence levels. For â¼20% of these inhibitors, it was also possible to consider test frequency and inactivity information. Only small subsets of highly promiscuous inhibitors were identified. Nearly 95% of more than 45 000 inhibitors with high-confidence data were only active against one or at most two kinases. At decreasing data confidence levels, more than 92 000 kinase inhibitors were on average active against two kinases. When taking all activity information without any restrictions into account, the mean promiscuity degree of kinase inhibitors was less than four and notably biased by small numbers of highly promiscuous inhibitors. Even under these conditions, more than 70% of all inhibitors were active against a single kinase. There was only small-scale progression of inhibitor promiscuity when data confidence criteria were iteratively removed during the analysis. Furthermore, the majority of inhibitors that were tested against 10 to 20 different kinases were only active against a single kinase. The results of our activity data-driven analysis indicate that promiscuity of kinase inhibitors cannot generally be assumed. Many inhibitors retain single-kinase activity at decreasing data confidence criteria or increasing test frequency. Hence, on the basis of currently available data, many kinase inhibitors are selective, which is an important aspect for drug development.
2017
- Synthesis and Biological Evaluation of Migrastatin Macrotriazoles
[Articolo su rivista]
Passarella, Daniele; Rastelli, Giulio; Murphy, Paul V; Robakiewicz, Stefania; Taciak, Bortlomej; Ulewicz, Katarzyna; Broggini, Gianluigi; Krol, Magdalena; Gabba, Adele
abstract
The synthesis of three macrotriazoles that are analogues
of migrastatin is reported. The synthesis is based on copper(I)- and
ruthenium catalyzed azide alkyne cycloaddition. The preparation of
the enantiopure terminal alkyne derivative is based on Trost
desymmetrisation, Brown alkoxyallylation and the efficient Colvin
reaction. Biological evaluation of the obtained compounds evidenced
a promising efficacy in reducing the ability of MDA-MB-361 cell line
to migrate.
2017
- The 1,2,3-triazole ring as a bioisostere in medicinal chemistry
[Articolo su rivista]
Bonandi, Elisa; Christodoulou, Michael S.; Fumagalli, Gaia; Perdicchia, Dario; Rastelli, Giulio; Passarella, Daniele
abstract
1,2,3-Triazole is a well-known scaffold that has a widespread occurrence in different compounds characterized by several bioactivities, such as antimicrobial, antiviral, and antitumor effects. Moreover, the structural features of 1,2,3-triazole enable it to mimic different functional groups, justifying its wide use as a bioisostere for the synthesis of new active molecules. Here, we provide an overview of the 1,2,3-triazole ring as a bioisostere for the design of drug analogs, highlighting relevant recent examples.
2016
- An unexpected reversal in the pharmacological stereoselectivity of benzothiadiazine AMPA positive allosteric modulators
[Articolo su rivista]
Battisti, UMBERTO MARIA; Citti, Cinzia; Rastelli, Giulio; Pinzi, Luca; Puja, Giulia; Ravazzini, Federica; Ciccarella, Giuseppe; Braghiroli, Daniela; Cannazza, Giuseppe
abstract
Benzothiadiazine type compounds (BTDs) have gained great attention for their potential therapeutic activity as nootropic and neuroprotective agents. BTDs, acting as AMPA positive allosteric modulators, potentiate the glutamatergic neurotransmission without the side effects typically associated with direct agonists. Studies regarding the binding mode of racemic BTDs into the receptor binding pocket demonstrated that one enantiomer establishes a more favourable interaction and possesses a higher biological activity with respect to the other one. The S enantiomer was proved to be the eutomer for both IDRA21 and S18986, two of the most studied BTD AMPA positive allosteric modulators. However, recent data highlighted an opposite stereoselectivity for some substituted BTDs (7-chloro-9-(furan-3-yl)-2,3,3a,4-tetrahydro-1H-benzo[e]pyrrolo[2,1-c][1,2,4]thiadiazine 5,5-dioxide and 7-chloro-2,3,4-trimethyl-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide) showing unexpected structure-activity relationships. In this work, the synthesis and configuration assignment of the stereoisomers of 7-chloro-5-(3-furanyl)-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide, one of the most active BTDs, are reported. Electrophysiological tests demonstrated that the R form is the eutomer. Docking and molecular dynamics simulations on the AMPA GluA2 binding site revealed new insights into the stereodiscrimination process. Lastly, metabolic studies disclosed a stereoselective hepatic metabolization of this chiral BTD.
2016
- Dual Kinase-Bromodomain Inhibitors in Anticancer Drug Discovery: A Structural and Pharmacological Perspective
[Articolo su rivista]
Carlino, Luca; Rastelli, Giulio
abstract
Protein kinases play crucial roles in several cell transformation processes and are validated drug targets for many human diseases, including cancer. Nevertheless, most tumors have eluded the effects of inhibition of a single kinase by activating resistance mechanisms and/or alternative pathways and escape mechanisms. In recent years, multitarget approaches directed toward inhibition of kinases and targets of different families have received increasing attention. In particular, co-targeting kinases and bromodomain epigenetic reader proteins has rapidly emerged as a promising approach to cancer drug development. In this manuscript, we will review the recent discoveries that led to the identification and optimization of dual kinase/bromodomain inhibitors. We will analyze and compare the structural features required for dual inhibition and comment on the potential of this approach in anticancer drug discovery. Moreover, we will introduce computational approaches useful for the identification of dual kinase/bromodomain inhibitors and generate ad hoc pharmacophore and docking models.
2016
- Exploiting computationally derived out-of-the-box protein conformations for drug design
[Articolo su rivista]
Caporuscio, Fabiana; Rastelli, Giulio
abstract
Structural plasticity is an intrinsic property of proteins that allows each gene product to accomplish its tasks in a strictly regulated manner at a precise time and cellular location. Moreover, protein motions allow protein-ligand and protein-protein recognition. The knowledge of the conformational ensemble that a drug target populates may be crucial for the design of small molecules that can differently modulate its function. X-ray crystallography and NMR have endlessly provided snapshots of protein states. However, experimental structure determination is not always straightforward. Therefore, attempts have been made to depict protein conformational landscapes through molecular dynamics and enhanced sampling methods. Here, we review how accounting for protein dynamics through in silico generated out-of-the-box protein conformations has started to impact on drug discovery.
2016
- G48A, a new KRAS mutation found in lung adenocarcinoma
[Articolo su rivista]
Marabese, Mirko; Caiola, Elisa; Garassino, Marina C.; Rastelli, Giulio; Settanni, Giulio; Brugnara, Sonia; Broggini, Massimo; Ganzinelli, Monica
abstract
A new somatic mutation in the coding region of Kirsten rat sarcoma viral oncogene homolog gene (KRAS), G48A, has been identified in a patient with non-small cell lung cancer (NSCLC). No other mutations were found by screening several genes known to be mutated in NSCLC. The patient responded to first-line therapy and is still under maintenance treatment 18 months after diagnosis. Normal and cancer cells were engineered to express the KRAS(G48A) mutation. KRAS(G48A) overexpression did not change the growth or the response to treatment compared with KRAS(wild type)-expressing cells. Analysis of the structure of the KRAS(G48A) mutant predicted altered interactions with other proteins. Analysis of KRAS binding to B-Raf proto-oncogene, serine/threonine kinase showed that the KRAS(G48A) mutant behaves more like a wild-type than a classical KRAS(G12) mutant. In conclusion, this new mutation in the coding region of KRAS, found in NSCLC, does not induce phenotypic changes similar to those induced by G12 mutants but presumably affects KRAS binding to proteins other than B-Raf proto-oncogene, serine/threonine kinase.
2016
- Molecular dynamics simulations and classical multidimensional scaling unveil new metastable states in the conformational landscape of CDK2
[Articolo su rivista]
Pisani, Pasquale; Caporuscio, Fabiana; Carlino, Luca; Rastelli, Giulio
abstract
Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS). The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the aC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms throughout the human kinome.
2016
- Novel and less explored chemotypes of natural origin for the inhibition of Hsp90
[Articolo su rivista]
Pellati, Federica; Rastelli, Giulio
abstract
The Hsp90 chaperone is a promising target for the treatment of cancer. Well known inhibitors, e.g.
geldanamycin, radicicol, novobiocin and their derivatives, have been extensively investigated and optimized
as inhibitors of Hsp90. More recently, new chemotypes of natural origin were reported to modulate Hsp90
function via various and often “non-conventional” mechanisms. This review focuses on these novel classes
of natural products whose structures have not yet been thoroughly explored for medicinal chemistry purposes.
These novel chemotypes may constitute interesting starting points for future drug discovery
campaigns.
2016
- Polypharmacology predictions in the Protein Data Bank
[Poster]
Pinzi, Luca; Rastelli, Giulio
abstract
2016
- Predicting drug polypharmacology using structural databases
[Abstract in Atti di Convegno]
Pinzi, Luca; Rastelli, Giulio
abstract
2015
- Analytical and Simulation-Based Models for Drug Release and Gel-Degradation in a Tetra-PEG Hydrogel Drug-Delivery System
[Articolo su rivista]
Reid, Ralph; Sgobba, Miriam; Raveh, Barak; Rastelli, Giulio; Sali, Andrej; Santi, Daniel V.
abstract
We have recently reported drug-releasing, degradable Tetra-PEG hydrogels as a new drug delivery system. The gels contain two self-cleaving β-eliminative linkers: one that covalently tethers the drug to the gel and releases it at a predictable rate, and another with slower cleavage that is installed in each cross-link of the polymer to control gel degradation. By balancing the two cleavage rates, the system can be designed to discharge most or all of the drug before the gel undergoes significant degradation. If polymer degradation is too rapid, undesirable gel-fragments covalently bound to the drug are released; if too slow, the gel remains in the body as an inert substance for prolonged periods. Here, we describe an analytical theory as well as a Monte Carlo simulation of concurrent drug release from and degradation of Tetra-PEG polymers. Considerations are made for an ideal network as well as networks containing missing bonds and double link defects. The analytical and simulation approaches are in perfect agreement with each other and with experimental data in the regime of interest. Using these models, we are able to (a) compute the time courses of drug release and gel degradation as well as the amount of fragment-drug conjugate present at any time and (b) estimate the rate constants of drug release and gel degradation necessary to control each of the above. We can also account for the size-dependent elimination of gel fragments from a localized semipermeable compartment and hence estimate fragment mass vs time curves in such in vivo compartments. The models described allow design of an optimal Tetra-PEG drug delivery vehicle for a particular use.
2015
- Computational polypharmacology analysis of the heat shock protein 90 interactome
[Articolo su rivista]
Anighoro, Andrew; Stumpfe, Dagmar; Heikamp, Kathrin; Beebe, Kristin; Neckers, Leonard M.; Bajorath, Jürgen; Rastelli, Giulio
abstract
The design of a single drug molecule that is able to simultaneously and specifically interact with multiple biological targets is gaining major consideration in drug discovery. However, the rational design of drugs with a desired polypharmacology profile is still a challenging task, especially when these targets are distantly related or unrelated. In this work, we present a computational approach aimed at the identification of suitable target combinations for multitarget drug design within an ensemble of biologically relevant proteins. The target selection relies on the analysis of activity annotations present in molecular databases and on ligand-based virtual screening. A few target combinations were also inspected with structure-based methods to demonstrate that the identified dual-activity compounds are able to bind target combinations characterized by remote binding site similarities. Our approach was applied to the heat shock protein 90 (Hsp90) interactome, which contains several targets of key importance in cancer. Promising target combinations were identified, providing a basis for the computational design of compounds with dual activity. The approach may be used on any ensemble of proteins of interest for which known inhibitors are available.
2015
- Computational polypharmacology comes of age
[Articolo su rivista]
Rastelli, Giulio; Pinzi, Luca
abstract
No abstract
2015
- Corrigendum to "Theoretical analysis of the addition of hydroxylamine to uracil and 5-fluorouracil as a model for the thymidylate synthase reaction" [J. Mol. Struct. (Theochem) 343 (1995) 1-9]
[Articolo su rivista]
Rastelli, G.; Costi, M. P.
abstract
2015
- Discovery of Multitarget Antivirals Acting on Both the Dengue Virus NS5-NS3 Interaction and the Host Src/Fyn Kinases
[Articolo su rivista]
Vincetti, Paolo; Caporuscio, Fabiana; Kaptein, Suzanne; Gioiello, Antimo; Mancino, Valentina; Suzuki, Youichi; Yamamoto, Naoki; Crespan, Emmanuele; Lossani, Andrea; Maga, Giovanni; Rastelli, Giulio; Castagnolo, Daniele; Neyts, Johan; Leyssen, Pieter; Costantino, Gabriele; Radi, Marco
abstract
This study describes the discovery of novel dengue virus inhibitors targeting both a crucial viral protein-protein interaction and an essential host cell factor as a strategy to reduce the emergence of drug resistance. Starting from known c-Src inhibitors, a virtual screening was performed to identify molecules able to interact with a recently discovered allosteric pocket on the dengue virus NS5 polymerase. The selection of cheap-to-produce scaffolds and the exploration of the biologically relevant chemical space around them suggested promising candidates for chemical synthesis. A series of purines emerged as the most interesting candidates able to inhibit virus replication at low micromolar concentrations with no significant toxicity to the host cell. Among the identified antivirals, compound 16i proved to be 10 times more potent than ribavirin, showed a better selectivity index and represents the first-in-class DENV-NS5 allosteric inhibitor able to target both the virus NS5-NS3 interaction and the host kinases c-Src/Fyn.
2015
- Histone deacetylases: Structural determinants of inhibitor selectivity
[Articolo su rivista]
Micelli, Carmina; Rastelli, Giulio
abstract
Histone deacetylases (HDACs) are epigenetic targets with an important role in cancer, neurodegeneration, inflammation, and metabolic disorders. Although clinically effective HDAC inhibitors have been developed, the design of inhibitors with the desired isoform(s) selectivity remains a challenge. Selective inhibitors could help clarify the function of each isoform, and provide therapeutic agents having potentially fewer adverse effects. Crystal structures of several HDACs have been reported, enabling structure-based drug design and providing important information to understand enzyme function. Here, we provide a comprehensive review of the structural information available on HDACs, discussing both conserved and isoform-specific structural and mechanistic features. We focus on distinctive aspects that help rationalize inhibitor selectivity, and provide structure-based recommendations for achieving the desired selectivity.
2015
- Insights into the interaction of negative allosteric modulators with the metabotropic glutamate receptor 5: Discovery and computational modeling of a new series of ligands with nanomolar affinity
[Articolo su rivista]
Anighoro, Andrew; Graziani, Davide; Bettinelli, Ilaria; Cilia, Antonio; De Toma, Carlo; Longhi, Matteo; Mangiarotti, Fabio; Menegon, Sergio; Pirona, Lorenza; Poggesi, Elena; Riva, Carlo; Rastelli, Giulio
abstract
Metabotropic glutamate receptor 5 (mGlu5) is a biological target implicated in major neurological and psychiatric disorders. In the present study, we have investigated structural determinants of the interaction of negative allosteric modulators (NAMs) with the seven-transmembrane (7TM) domain of mGlu5. A homology model of the 7TM receptor domain built on the crystal structure of the mGlu1 template was obtained, and the binding modes of known NAMs, namely MPEP and fenobam, were investigated by docking and molecular dynamics simulations. The results were validated by comparison with mutagenesis data available in the literature for these two ligands, and subsequently corroborated by the recently described mGlu5 crystal structure. Moreover, a new series of NAMs was synthesized and tested, providing compounds with nanomolar affinity. Several structural modifications were sequentially introduced with the aim of identifying structural features important for receptor binding. The synthesized NAMs were docked in the validated homology model and binding modes were used to interpret and discuss structure-activity relationships within this new series of compounds. Finally, the models of the interaction of NAMs with mGlu5 were extended to include important non-aryl alkyne mGlu5 NAMs taken from the literature. Overall, the results provide useful insights into the molecular interaction of negative allosteric modulators with mGlu5 and may facilitate the design of new modulators for this class of receptors.
2015
- Rational design of dual inhibitors of Hsp90 and B-Raf as a novel pharmacological approach against melanomas
[Poster]
Pinzi, Luca; Rastelli, Giulio
abstract
2015
- Rational design of dual inhibitors of Hsp90 and Braf as a novel pharmacological approach against melanomas
[Abstract in Atti di Convegno]
Pinzi, Luca; Rastelli, Giulio
abstract
2015
- Role of bifidobacteria in the hydrolysis of chlorogenic acid
[Articolo su rivista]
Raimondi, Stefano; Anighoro, Andrew; Quartieri, Andrea; Amaretti, Alberto; Tomás Barberán, Francisco A.; Rastelli, Giulio; Rossi, Maddalena
abstract
This study aimed to explore the capability of potentially probiotic bifidobacteria to hydrolyze chlorogenic acid into caffeic acid (CA), and to recognize the enzymes involved in this reaction. Bifidobacterium strains belonging to eight species occurring in the human gut were screened. The hydrolysis seemed peculiar of Bifidobacterium animalis, whereas the other species failed to release CA. Intracellular feruloyl esterase activity capable of hydrolyzing chlorogenic acid was detected only in B. animalis. In silico research among bifidobacteria esterases identified Balat_0669 as the cytosolic enzyme likely responsible of CA release in B. animalis. Comparative modeling of Balat_0669 and molecular docking studies support its role in chlorogenic acid hydrolysis. Expression, purification, and functional characterization of Balat_0669 in Escherichia coli were obtained as further validation. A possible role of B. animalis in the activation of hydroxycinnamic acids was demonstrated and new perspectives were opened in the development of new probiotics, specifically selected for the enhanced bioconversion of phytochemicals into bioactive compounds.
2014
- Dimerization hot spots in the structure of human Hsp90
[Articolo su rivista]
Rastelli, Giulio
abstract
Heat shock protein 90 (Hsp90) is an ubiquitous molecular chaperone responsible for the assembly and regulation of many signal transduction and regulatory client proteins. Since Hsp90 refolds, stabilizes and regulates the trafficking of many proteins responsible for uncontrolled proliferation and apoptotic resistance, including multiple protein kinases, steroid hormone receptors, mutated p53, survivin and others, this chaperone is an emerging target for the development of anticancer drugs.1,2
Hsp90 is a large and conformationally dynamic protein that undergoes dramatic conformational changes upon ATP binding and hydrolysis. Crystallography, small-angle X-ray scattering and electron microscopy techniques have revealed an underlying conformational complexity of Hsp90, which is composed by three highly dynamic domains, the N-terminal (NTD), middle (MD) and C-terminal (CTD) domains.3 Dimerization is an essential step of the Hsp90 cycle. Recently, the crystal structure of yeast Hsp90 in complex with an ATP analogue and the co-chaperone p23/Sba1 revealed the architecture of the closed and compact homodimer, providing a view of Hsp90 in the ATP-bound state, which represents an obliged conformation along the ATPase cycle.4
This work reports the results of a molecular dynamics and dimerization free energy analysis performed on the structure of a human Hsp90 homology model in the closed conformation. Decomposition of free energies on a residue basis led to the prediction of five clusters of dimerization hot spots, three of which are located in the NTD while the other two in the CTD. These residues represent valuable candidates for future mutagenesis studies and may provide new sites for the design of allosteric inhibitors.
2014
- Polypharmacology: challenges and opportunities in drug discovery
[Articolo su rivista]
Anighoro, Andrew; Bajorath, Jürgen; Rastelli, Giulio
abstract
At present, the legendary magic bullet, i.e., a drug with high potency and selectivity toward a specific biological target, shares the spotlight with an emerging and alternative polypharmacology approach. Polypharmacology suggests that more effective drugs can be developed by specifically modulating multiple targets. It is generally thought that complex diseases such as cancer and central nervous system diseases may require complex therapeutic approaches. In this respect, a drug that "hits" multiple sensitive nodes belonging to a network of interacting targets offers the potential for higher efficacy and may limit drawbacks generally arising from the use of a single-target drug or a combination of multiple drugs. In this review, we will compare advantages and disadvantages of multitarget versus combination therapies, discuss potential drug promiscuity arising from off-target effects, comment on drug repurposing, and introduce approaches to the computational design of multitarget drugs.
2014
- Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2
[Articolo su rivista]
Rastelli, Giulio; Anighoro, Andrew; Chripkova, Martina; Carrassa, Laura; Broggini, Massimo
abstract
Allosteric targeting of protein kinases via displacement of the structural αC helix with type III allosteric inhibitors is currently gaining a foothold in drug discovery. Recently, the first crystal structure of CDK2 with an open allosteric pocket adjacent to the αC helix has been described, prospecting new opportunities to design more selective inhibitors, but the structure has not yet been exploited for the structure-based design of type III allosteric inhibitors. In this work we report the results of a virtual screening campaign that resulted in the discovery of the first-in-class type III allosteric ligands of CDK2. Using a combination of docking and post-docking analyses made with our tool BEAR, 7 allosteric ligands (hit rate of 20%) with micromolar affinity for CDK2 were identified, some of them inhibiting the growth of breast cancer cell lines in the micromolar range. Competition experiments performed in the presence of the ATP-competitive inhibitor staurosporine confirmed that the 7 ligands are truly allosteric, in agreement with their design. Of these, compound 2 bound CDK2 with an EC50 value of 3 μM and inhibited the proliferation of MDA-MB231 and ZR-75-1 breast cancer cells with IC50 values of approximately 20 μM, while compound 4 had an EC50 value of 71 μM and IC50 values around 4 μM. Remarkably, the most potent compound 4 was able to selectively inhibit CDK2-mediated Retinoblastoma phosphorylation, confirming that its mechanism of action is fully compatible with a selective inhibition of CDK2 phosphorylation in cells. Finally, hit expansion through analog search of the most potent inhibitor 4 revealed an additional ligand 4g with similar in vitro potency on breast cancer cells.
2013
- BEAR, a Molecular Docking Refinement and Rescoring Method
[Articolo su rivista]
Anighoro, Andrew; Rastelli, Giulio
abstract
BEAR (Binding Estimation After Refinement) is a computational method for structure-based virtual screening. It was set up as a post-docking processing tool for the refinement of ligand binding modes predicted by molecular docking programs and the accurate evaluation of free energies of binding. BEAR has been validated in a number of computational drug discovery applications. It performed well in discriminating active ligands with respect to molecular decoys of biological targets belonging to different protein families as well as in discovering biologically active hits. Recently, it has been validated also in the emerging field of G-protein coupled receptors structure based virtual screening.
2013
- Emerging Topics in Structure-Based Virtual Screening
[Articolo su rivista]
Rastelli, Giulio
abstract
Molecular dynamics simulations and the generation of ad hoc chemical libraries are playing an increasingly important and recognized role in structure-based virtual screening. These approaches are important for treating target flexibility and improving the drug discovery pipeline. In this article I will comment on these two topics and put them into perspective.
2013
- Enrichment Factor Analyses on G-Protein Coupled Receptors with Known Crystal Structure
[Articolo su rivista]
Anighoro, Andrew; Rastelli, Giulio
abstract
G-protein coupled receptors (GPCRs) are highly relevant drug targets. Four GPCRs with known crystal structure were analyzed with docking (AutoDock4) and postdocking (MM-PBSA) in order to evaluate the ability to recognize known antagonists from a larger database of molecular decoys and to predict correct binding modes. Moreover, implications on multitarget drug screening are put forward. The results suggest that these methods may be of interest to the growing field of GPCR structure-based virtual screening.
2013
- Targeting the Hsp90 interactome using in silico polypharmacology approaches
[Articolo su rivista]
Anighoro, Andrew; Stumpfe, D.; Heikamp, K.; Bajorath, J.; Rastelli, Giulio
abstract
In recent years, polypharmacology has gained popularity in drug discovery. [1] Especially for complex diseases such as cancer, the ability of a drug to bind to and interfere with multiple targets provides new opportunities for therapeutic intervention In this article, we focus on Hsp90 and its interactome, whose pivotal role in survival and proliferation of cancer cells renders this array of targets particularly attractive polypharmacological drug design strategies.
The primary goal of our work is the identification and selection of suitable target proteins from the interactome that might be combined with Hsp90 to explore and exploit a multi-target inhibition approach. This task is accomplished by applying computational methods to mine the structural and biological information associated with potential ligands in public databases and assess the degree of structural similarity between known inhibitors of different targets. Therefore, we propose an integrated ligand- and structure-based approach to select small molecules from databases suitable for consideration as multi-target inhibitors .
2013
- αC helix displacement as a general approach for allosteric modulation of protein kinases
[Articolo su rivista]
Palmieri, Lorenzo; Rastelli, Giulio
abstract
Owing to their crucial role in the modulation of cell pathways, protein kinases are important targets for several human diseases, including but not limited to cancer. The classic approach of targeting the ATP active site has recently come up against selectivity issues, which can be considerably reduced by following an allosteric modulation approach. Being closely related to protein kinase inactivation, allosteric targeting via displacement of the conserved structural αC helix enables a direct and specific modulation mechanism. A structure-based survey of the allosteric regulation of αC helix conformation in various kinase families is provided, highlighting key allosteric pockets and modulation mechanisms that appear to be more broadly conserved than was previously thought.
2012
- Advances and applications of binding affinity prediction methods in drug discovery
[Articolo su rivista]
M. D., Parenti; Rastelli, Giulio
abstract
Nowadays, the improvement of R&D productivity is the primary commitment in pharmaceutical research, both in big pharma and smaller biotech companies. To reduce costs, to speed up the discovery process and to increase the chance of success, advanced methods of rational drug design are very helpful, as demonstrated by several successful applications. Among these, computational methods able to predict the binding affinity of small molecules to specific biological targets are of special interest because they can accelerate the discovery of new hit compounds. Here we provide an overview of the most widely used methods in the field of binding affinity prediction, as well as of our own work in developing BEAR, an innovative methodology specifically devised to overtake some limitations in existing approaches. The BEAR method was successfully validated against different biological targets, and proved its efficacy in retrieving active compounds from virtual screening campaigns. The results obtained so far indicate that BEAR may become a leading tool in the drug discovery pipeline. We primarily discuss advantages and drawbacks of each technique and show relevant examples and applications in drug discovery.
2012
- Application of a post-docking procedure based on MM-PBSA and MM-GBSA on single and multiple protein conformations
[Articolo su rivista]
Sgobba, Miriam; Caporuscio, Fabiana; Anighoro, Andrew; Portioli, Corinne; Rastelli, Giulio
abstract
In the last decades, molecular docking has emerged as an increasingly useful tool in the modern drug discovery process, but it still needs to overcome many hurdles and limitations such as how to account for protein flexibility and poor scoring function performance. For this reason, it has been recognized that in many cases docking results need to be post-processed to achieve a significant agreement with experimental activities. In this study, we have evaluated the performance of MM-PBSA and MM-GBSA scoring functions, implemented in our post-docking procedure BEAR, in rescoring docking solutions. For the first time, the performance of this post-docking procedure has been evaluated on six different biological targets (namely estrogen receptor, thymidine kinase, factor Xa, adenosine deaminase, aldose reductase, and enoyl ACP reductase) by using i) both a single and a multiple protein conformation approach, and ii) two different software, namely AutoDock and LibDock. The assessment has been based on two of the most important criteria for the evaluation of docking methods, i.e., the ability of known ligands to enrich the top positions of a ranked database with respect to molecular decoys, and the consistency of the docking poses with crystallographic binding modes. We found that, in many cases, MM-PBSA and MM-GBSA are able to yield higher enrichment factors compared to those obtained with the docking scoring functions alone. However, for only a minority of the cases, the enrichment factors obtained by using multiple protein conformations were higher than those obtained by using only one protein conformation.
2011
- Assessing protein kinase selectivity with molecular dynamics and MM-PBSA binding free energy calculations.
[Articolo su rivista]
E., Muzzioli; A., Del Rio; Rastelli, Giulio
abstract
An application of molecular dynamics and molecular mechanics Poisson-Boltzmann surface area techniques to the prediction of protein kinase inhibitor selectivity is presented. A highly active and selective ERK2 inhibitor was placed in equivalent orientations in five different protein kinases (SRC, LCK, GSK3, JNK3 and Aurora-A). Binding free energies were then computed with the molecular mechanics Poisson-Boltzmann surface area approach using 15 nanosecond fully solvated molecular dynamics trajectories of the corresponding protein-ligand complexes. The results show correlation with experimentally determined selectivities and provide useful insights into the underlying structural determinants for selectivity.
2011
- BEAR, a novel virtual screening methodology for drug discovery
[Articolo su rivista]
Degliesposti, Gianluca; C., Portioli; Parenti, Marco Daniele; Rastelli, Giulio
abstract
BEAR (binding estimation after refinement) is a new virtual screening technology based on the conformational refinement of docking poses through molecular dynamics and prediction of binding free energies using accurate scoring functions. Here, the authors report the results of an extensive benchmark of the BEAR performance in identifying a smaller subset of known inhibitors seeded in a large (1.5 million) database of compounds. BEAR performance proved strikingly better if compared with standard docking screening methods. The validations performed so far showed that BEAR is a reliable tool for drug discovery. It is fast, modular, and automated, and it can be applied to virtual screenings against any biological target with known structure and any database of compounds.
2011
- Structure-Based Design of Potent Aromatase Inhibitors by High-Throughput Docking
[Articolo su rivista]
Caporuscio, Fabiana; Rastelli, Giulio; Imbriano, Carol; Del Rio, Alberto
abstract
Cytochrome P450 aromatase catalyzes the conversion of androgen substrates into estrogens. Aromatase inhibitors (AIs) have been used as first-line drugs in the treatment of estrogen-dependent breast cancer in postmenopausal women. However, the search for new, more potent, and selective AIs still remains necessary to avoid the risk of possible resistances and reduce toxicity and side effects of current available drugs. The publication of a high resolution X-ray structure of human aromatase has opened the way to structure-based virtual screening to identify new small-molecule inhibitors with structural motifs different from all known AIs. In this context, a high-throughput docking protocol was set up and led to the identification of nanomolar AIs with new core structures.
2010
- A computational workflow for the design of irreversible inhibitors of protein kinases.
[Articolo su rivista]
DEL RIO, Alberto; Sgobba, Miriam; Parenti, Marco Daniele; Degliesposti, Gianluca; Forestiero, R; Percivalle, C; Conte, Pf; Freccero, M; Rastelli, Giulio
abstract
Design of irreversible inhibitors is an emerging and relatively less explored strategy for the design of protein kinase inhibitors. In this paper, we present a computational workflow that was specifically conceived to assist such design. The workflow takes the form of a multi-step procedure that includes: the creation of a database of already known reversible inhibitors of protein kinases, the selection of the most promising scaffolds that bind one or more desired kinase templates, the modification of the scaffolds by introduction of chemically reactive groups (suitable cysteine traps) and the final evaluation of the reversible and irreversible protein-ligand complexes with molecular dynamics simulations and binding free energy predictions. Most of these steps were automated. In order to prove that this is viable, the workflow was tested on a database of known inhibitors of ERK2, a protein kinase possessing a cysteine in the ATP site. The modeled ERK2-ligand complexes and the values of the estimated binding free energies of the putative ligands provide useful indicators of their aptitude to bind reversibly and irreversibly to the protein kinase. Moreover, the computational data are used to rank the ligands according to their computed binding free energies and their ability to bind specific protein residues in the reversible and irreversible complexes, thereby providing a useful decision-making tool for each step of the design. In this work we present the overall procedure and the first proof of concept results.
2010
- BEAR: una piattaforma di screening virtuale al servizio delle aziende farmaceutiche e biotecnologiche
[Articolo su rivista]
Rastelli, Giulio; G., Degliesposti; M., Parenti; C., Portioli
abstract
BEAR è un sistema computazionale, automatizzato e innovativo di screening virtuale per l’identificazione di potenziali farmaci ideato
e messo a punto all’Università di Modena e Reggio Emilia grazie a lunghi anni di ricerca farmaceutica.
2010
- Binding estimation after refinement: BEARing out an innovative virtual screening methodology
[Abstract in Rivista]
M. D., Parenti; G., Degliesposti; C., Portioli; Rastelli, Giulio
abstract
Binding estimation after refinement: BEARing out an innovative virtual screening methodology
2010
- Exploring the binding site of C-terminal Hsp90 inhibitors
[Articolo su rivista]
Sgobba, Miriam; Forestiero, Rosetta; Degliesposti, Gianluca; Rastelli, Giulio
abstract
The 90 kDa heat shock protein (Hsp90) is a prominent target for anticancer drug discovery. While its N-terminal domain has been widely exploited, several lines of evidence are emerging in favor of targeting its C-terminal domain to conceive innovative drugs based on perturbation of the dimer interface. Here, we describe the application of several computational approaches useful to predict the location of the C-terminal binding site.
2010
- Fast and accurate predictions of relative binding free energies using MM-PBSA and MM-GBSA
[Articolo su rivista]
Rastelli, Giulio; A., Del Rio; G., Degliesposti; M., Sgobba
abstract
In the drug discovery process, accurate methods of computing the affinity of small molecules with a biological target are strongly needed. This is particularly true for molecular docking and virtual screening methods, which use approximated scoring functions and struggle in estimating binding energies in correlation with experimental values. Among the various methods, MM-PBSA and MM-GBSA are emerging as useful and effective approaches. Although these methods are typically applied to large collections of equilibrated structures of protein-ligand complexes sampled during molecular dynamics in water, the possibility to reliably estimate ligand affinity using a single energy-minimized structure and implicit solvation models has not been explored in sufficient detail. Herein, we thoroughly investigate this hypothesis by comparing different methods for the generation of protein-ligand complexes and diverse methods for free energy prediction for their ability to correlate with experimental values. The methods were tested on a series of structurally diverse inhibitors of Plasmodium falciparum DHFR with known binding mode and measured affinities. The results showed that correlations between MM-PBSA or MM-GBSA binding free energies with experimental affinities were in most cases excellent. Importantly, we found that correlations obtained with the use of a single protein-ligand minimized structure and with implicit solvation models were similar to those obtained after averaging over multiple MD snapshots with explicit water molecules, with consequent save of computing time without loss of accuracy. When applied to a virtual screening experiment, such an approach proved to discriminate between true binders and decoy molecules and yielded significantly better enrichment curves
2010
- Improving enrichment and hit rate in virtual screening
[Abstract in Rivista]
Rastelli, Giulio; Degliesposti, Gianluca; Parenti, Marco Daniele; C., Portioli
abstract
In the drug discovery process, accurate methods of computing the affinity of small molecules with a desired biological target are strongly needed. Even if, in the last years, the accuracy and efficiency of the available virtual screening algorithms have been improved, many drawbacks and limitations still exist. For example, docking methods lack a reliable simulation of both ligand and receptor flexibilities, as well as good scoring functions able to estimate ligand binding energies in reasonable agreement with experimental data. These limitations often lead to a high level of false positives or false negatives in the hit list. For that reason, it is generally agreed that docking results need to be post-processed with more accurate tools.To this end, we developed Binding Estimation After Refinement (BEAR), a new and automated post-docking procedure for the conformational refinement of docking poses through molecular dynamics (MD) followed by accurate prediction of binding free energies using MM-PBSA and MM-GBSA1 (Figure1). The BEAR performance in virtual screening was evaluated on several macromolecular targets and related sets of known ligands, determining the enrichment factors and assessing the correlation between predicted and experimental binding affinities. These analyses suggested critical improvements with respect to standard docking softwares2,3. Moreover, when applied in virtual screening campaigns, BEAR was able to discover novel and potent inhibitors of Plasmodium falciparum plasmepsin II with an impressive hit rate 4, and has been successful in identifying promising scaffolds for the design of irreversible protein kinase inhibitors5. Therefore, taken as a whole, the results obtained so far prospect that BEAR may become a prominent tool in the drug discovery pipeline.The BEAR virtual screening procedure is reliable and strongly automated, and can be tailored to the needs of the end-user in terms of computational time and the desired accuracy of the results. BEAR is under constant development and validation on additional biological targets in order to further improve accuracy, automation and calculation speed.
2009
- Activity prediction and structural insights of Extracellular Signal-Regulated Kinase 2 inhibitors with molecular dynamics simulations
[Articolo su rivista]
A., Del Rio; B. F., Baldi; Rastelli, Giulio
abstract
A computational application to predict, probe and interpret the activities of a series of congeneric compounds inhibiting extracellular signal-regulated kinase 2 protein kinase is presented. The study shows that molecular dynamics coupled with molecular mechanics Poisson-Boltzmann solvent accessible surface area free energy estimation is a suitable tool for investigating the experimental binding activities of ligands to protein kinases. Computed and experimental binding activities were found to be significantly correlated. Moreover, the interpretation of the X-ray co-crystal structure in conjunction with computational results shows that the hinge region of the protein insure the principal binding site via multiple hydrogen bonding interactions, whereas fine-modulation of biological activities along the series is accomplished through the combination of weak and strong interactions that compete with water. These are located in the substituent moieties of the ligands interfacing with the DFG motif, the sugar region and the hydrophobic pocket of extracellular signal-regulated kinase 2. The study suggests that a wider interaction framework that is well beyond the hinge region is required to predict and rationalize at molecular level the experimental biological activities of congeneric compound series.
2009
- Binding estimation after refinement, a new automated procedure for the refinement and rescoring of docked ligands in virtual screening
[Articolo su rivista]
Rastelli, Giulio; Degliesposti, Gianluca; A., Del Rio; Sgobba, Miriam
abstract
Binding estimation after refinement (BEAR) is a novel automated computational procedure suitable for correcting and overcoming limitations of docking procedures such as poor scoring function and the generation of unreasonable ligand conformations. BEAR makes use of molecular dynamics simulation followed by MM-PBSA and MM-GBSA binding free energy estimates as tools to refine and rescore the structures obtained from docking virtual screenings. As binding estimation after refinement relies on molecular dynamics, the entire procedure can be tailored to the needs of the end-user in terms of computational time and the desired accuracy of the results. In a validation test, binding estimation after refinement and rescoring resulted in a significant enrichment of known ligands among top scoring compounds compared with the original docking results. Binding estimation after refinement has direct and straightforward application in virtual screening for correcting both false-positive and false-negative hits, and should facilitate more reliable selection of biologically active molecules from compound databases.
2009
- Design and discovery of plasmepsin inhibitors using an automated workflow on large scale grids
[Articolo su rivista]
Degliesposti, Gianluca; V., Kasam; A., Da Costa; H. K., Kang; N., Kim; D. W., Kim; V., Breton; D., Kim; Rastelli, Giulio
abstract
Novel and potent inhibitors of Plasmodium falciparum plasmepsin II were identified by post-processing the results of a docking screening with BEAR, a recently reported procedure for the refinement and rescoring of docked ligands in virtual screening. FRET substrate degradation assays performed on the 30 most promising compounds resulted in 26 inhibitors with IC(50) values ranging from 4.3 nM to 1.8 microM.Herein we report the discovery of novel and potent inhibitors of Plasmodium falciparum plasmepsin II using GRID computing infrastructures. These compounds were identified by post-processing the results of a large docking screen of commercially available compounds using an automated procedure based on molecular dynamics refinement and binding free-energy estimation using MM-PBSA and MM-GBSA. Among the best-scored compounds, four highly populated and promising chemical classes were identified: N-alkoxyamidines, guanidines, amides, and ureas and thioureas. Thirty hit compounds representative of each class were selected on the basis of their favourable binding free energies and molecular interactions with key active site residues. These were experimentally validated using an inhibition assay based on FRET substrate degradation. Remarkably, 26 of the 30 tested compounds proved to be active as plasmepsin II inhibitors, with IC(50) values ranging from 4.3 nM to 1.8 microM.
2009
- Disegnare i farmaci al computer
[Articolo su rivista]
Rastelli, Giulio
abstract
Non solo chimica e biologia: anche l’informatica è
diventata fondamentale per progettare e sviluppare
nuovi farmaci. Un progettista molecolare ci spiega
come, partendo da informazioni genetiche e
strutturali, il computer semplifichi e velocizzi la
ricerca di sostanze terapeutiche.
2009
- Fragment-Based Drug Discovery: a Practical Approach
[Recensione in Rivista]
Rastelli, Giulio
abstract
This book presents a company-based view of the recent progresses in fragment-based drug discovery (FBDD). The book is highly focussed on methodologies.
However, theory is only briefly introduced leaving ample space for the discussion of their implementation in drug discovery campaigns, thereby giving the reader a
clear sense of their applicability and usefulness. Most of the examples come from pharmaceutical drug discovery programs led by the authors themselves,
most of which are from the industry side. The opening chapter provides an overview of the advantages of using chemical fragments in drug discovery.
These include an analysis of the potential advantages over HTS screening, a popular methodology that proved to be problematic in the development of druglike
compounds. Compared with other approaches, fragment-based drug discovery has the potential of finding novel molecules that can be optimized into patentable compounds more easily, have greater molecular diversity, higher hit rates, and are ideally suited for “undruggable” and novel targets.
2009
- Membreo dell'Editorial Board dell'International Journal of Medicinal Chemistry
[Direzione o Responsabilità Riviste]
Mai Abd El, Hamid; Rastelli, Giulio
abstract
2009
- Pharmaceutical composition for preventing and treating malaria containing compounds that inhibit Plasmepsin II activity, and method of treating malaria using the same
[Brevetto]
Doman, Kim; Hee Kyoung, Kang; Do Won, Kim; Rastelli, Giulio; Ana Lucia Da, Costa; Vinod, Kasam; Vincent, Breton
abstract
A pharmaceutical composition containing a compound that binds to active sites of plasmepsin II to inhibit activity, and a method of preventing and treating malaria, including administering an effective dose of the pharmaceutical composition to a mammal. The pharmaceutical composition of the invention contains at least one compound selected from the group consisting of an N- alkoxyamidine derivative, a guanidine derivative, an amide derivative, a urea or thiourea derivative, and N- (2-{[3-(l,3 -benzodioxol- 5 -yl) - 3 -oxo- 1 -propene- 1 -yl] amino } phenyl)-4-nitrobenzene sul¬ fonamide. The compound contained in the pharmaceutical composition is used to prevent and treat malaria since it binds to active sites of plasmepsin II to inhibit activity. The compound is effective to malaria that is resistant to existing anti-malarial drugs.
2009
- Structure-based and in silico design of Hsp90 inhibitors
[Articolo su rivista]
M., Sgobba; Rastelli, Giulio
abstract
The molecular chaperone Hsp90 is responsible for activation and stabilization of several oncoproteins in cancer cells, and has emerged as an important target in cancer treatment because of this pivotal role. In recent years, interests have arisen around structure-based design of small molecules aimed at inhibiting the chaperone activity of Hsp90. In this review, we illustrate the recent advances in structure-based and in silico strategies aimed at discovering and optimizing Hsp90 inhibitors.
2009
- WISDOM, a grid enabled drug discovery initiative against malaria
[Capitolo/Saggio]
V., Breton; D., Kim; Rastelli, Giulio
abstract
The goal of this chapter is to present the WISDOM initiative, which is one of
the main accomplishments in the use of grids for biomedical sciences
achieved on grid infrastructures in Europe. Researchers in life sciences are
among the most active scientifi c communities on the EGEE infrastructure.
As a consequence, the biomedical virtual organization stands fourth in
terms of resources consumed in 2007, with an average of 7000 jobs submitted
every day to the grid and more than 4 million hours of CPU consumed in
the last 12 months. Only three experiments on the CERN Large Hadron
Collider have used more resources. Compared to particle physics, the use of
resources is much less centralized as about 40 different scientifi c applications
are now currently deployed on EGEE. Each of them requires an amount
of CPU which ranges from a few to a few hundred CPU years. Thanks to the
20,000 processors available to the users of the biomedical virtual organization,
crunching factors in the hundreds are witnessed routinely. Such
performances were already achieved on supercomputers but at the cost of
reservation and long delays in the access to resources. On the contrary, grid
infrastructures are constantly open to the user communities.
Such changes in the scale of the computing resources made continuously
available to the researchers in biomedical sciences open opportunities for
exploring new fi elds or changing the approach to existing challenges. In
this chapter, we would like to show the potential impact of grids in the fi eld
of drug discovery through the example of the WISDOM initiative.
2009
- WISDOM-II: Screening against multiple targets implicated in malaria using computational grid infrastructures
[Articolo su rivista]
V., Kasam; J., Salzeman; M., Botha; A., Dacosta; G., Degliesposti; R., Isea; D., Kim; A., Maass; C., Kenyon; Rastelli, Giulio; M., Hofmann Apitius; V., Breton
abstract
Background: Despite continuous efforts of the international community to reduce the impact of malaria on developing countries, no significant progress has been made in the recent years and the discovery of new drugs is more than ever needed. Out of the many proteins involved in the metabolic activities of the Plasmodium parasite, some are promising targets to carry out rational drug discovery. MotivationRecent years have witnessed the emergence of grids, which are highly distributed computing infrastructures particularly well fitted for embarrassingly parallel computations like docking. In 2005, a first attempt at using grids for large-scale virtual screening focused on plasmepsins and ended up in the identification of previously unknown scaffolds, which were confirmed in vitro to be active plasmepsin inhibitors. Following this success, a second deployment took place in the fall of 2006 focussing on one well known target, dihydrofolate reductase (DHFR), and on a new promising one, glutathione-S-transferase.MethodsIn silico drug design, especially vHTS is a widely and well-accepted technology in lead identification and lead optimization. This approach, therefore builds, upon the progress made in computational chemistry to achieve more accurate in silico docking and in information technology to design and operate large scale grid infrastructures.ResultsOn the computational side, a sustained infrastructure has been developed: docking at large scale, using different strategies in result analysis, storing of the results on the fly into MySQL databases and application of molecular dynamics refinement are MM-PBSA and MM-GBSA rescoring. The modeling results obtained are very promising. Based on the modeling results, In vitro results are underway for all the targets against which screening is performed.ConclusionThe current paper describes the rational drug discovery activity at large scale, especially molecular docking using FlexX software on computational grids in finding hits against three different targets (PfGST, PfDHFR, PvDHFR (wild type and mutant forms) implicated in malaria. Grid-enabled virtual screening approach is proposed to produce focus compound libraries for other biological targets relevant to fight the infectious diseases of the developing world.
2008
- In vitro effects of Plasmodium falciparum dihydrofolate reductase inhibitors on normal and cancer cell proliferation
[Articolo su rivista]
Rossi, Tiziana; Coppi, A; Bruni, E; Sgobba, Miriam; Degliesposti, Gianluca; Rastelli, Giulio
abstract
Toxicological evaluations were performed on two novel Plasmodium falciparum dihydrofolate reductase inhibitors and other known antimalarial drugs. Cytotoxicity tests were performed on Vero and MCF-7 cells and apoptotic and/or proliferative markers p21 and p53 and A, B1, D1, and D2 cyclines. The results are discussed and show that this molecule can be considered an interesting new candidate for further development.
2008
- Membro dell'Editorial Board di Annals of Tropical Medicine and Public Health
[Direzione o Responsabilità Riviste]
Abubakar, Yaro; Rastelli, Giulio
abstract
2008
- Membro dell'Editorial Board di ScienceAsia
[Direzione o Responsabilità Riviste]
Worachart, Sirawaraporn; Rastelli, Giulio
abstract
2008
- Molecular modeling and crystal structure of ERK2-Hypothemycin complexes
[Articolo su rivista]
Rastelli, Giulio; R., Rosenfeld; R., Reid; D. V., Santi
abstract
Resorcylic acid lactones containing a cis-enone-such as hypothemycin-are susceptible to Michael addition reactions and are potent and specific inhibitors of about 45 of the known Ser/Thr/Tyr protein kinases. These inhibitors bind reversibly, and then form a covalent adduct with a completely conserved cysteine in the ATP binding site of their target kinases. As a paradigm for the structures of the cis-enone resorcylic acid lactone complexes with this subset of kinases, we have modeled the structure of ERK2-hypothemycin reversible and covalent complexes using docking and extended molecular dynamics simulations. Subsequently, we determined the 2.5A resolution crystal structure of the complex that was in excellent accord with the modeled structure. The results were used to discuss structure-activity relationships, and provide a structural template for the development of irreversible inhibitors that complement the ATP binding site of kinases.
2008
- Structural models and binding site prediction of the C-terminal domain of human Hsp90: a new target for anticancer drugs
[Articolo su rivista]
Sgobba, Miriam; Degliesposti, Gianluca; A. M., Ferrari; Rastelli, Giulio
abstract
Heat shock protein 90 is a valuable target for anticancer drugs because of its role in the activation and stabilization of multiple oncogenic signalling proteins. While several compounds inhibit heat shock protein 90 by binding the N-terminal domain, recent studies have proved that the C-terminal domain is important for dimerization of the chaperone and contains an additional binding site for inhibitors. Heat shock protein 90 inhibition achieved with molecules binding to the C-terminal domain provides an additional and novel opportunity to design and develop drugs. Therefore, for the first time, we have investigated the structure and the dynamic behaviour of the C-terminal domain of human heat shock protein 90 with and without the small-middle domain, using homology modelling and molecular dynamics simulations. In addition, secondary structure predictions and peptide folding simulations proved useful to investigate a putative additional alpha-helix located between H18 and beta20 of the C-terminal domain. Finally, we used the structural information to infer the location of the binding site located in the C-terminal domain by using a number of computational tools. The predicted pocket is formed by two grooves located between helix H18, the loop downstream of H18 and the loop connecting helices H20 and H21 of each monomer of the C-terminal domain, with only two amino acids contributing from each middle domain.
2007
- Grid-enabled high throughput virtual screening
[Relazione in Atti di Convegno]
Jacq, N; Breton, V; CHEN H., Y; HO L., Y; Hofmann, M; LEE H., C; Legré, Y; LIN S., C; Maas, A; Medernach, E; Merelli, I; Milanesi, L; Rastelli, Giulio; Reichstadt, M; Salzemann, J; Schwichtenger, H; Sridhar, M; Kasam, V; WU Y., T; Zimmermann, M.
abstract
Large scale grids for in silico drug discovery open opportunities of
particular interest to neglected and emerging diseases. In 2005 and 2006, we
have been able to deploy large scale virtual docking within the framework of
the WISDOM initiative against malaria and avian influenza requiring about 100
years of CPU on the EGEE, Auvergrid and TWGrid infrastructures. These
achievements demonstrated the relevance of large scale grids for the virtual
screening by molecular docking. This also allowed evaluating the performances
of the grid infrastructures and to identify specific issues raised by large scale
deployment.
2007
- Relationship between quantum-chemical descriptors of proton dissociation and experimental acidity contants of variously hydroxylated coumarins. Identification of the biologically active species for xanthine oxidase inhibition
[Articolo su rivista]
Ferrari, Anna Maria; Sgobba, Miriam; Gamberini, Maria Cristina; Rastelli, Giulio
abstract
Quantum-chemical descriptors related to proton dissociation constants of a set of coumarins hydroxylated in various positions have been computed and related to the experimental pKa values. An excellent correlation was found between the computed deprotonation energies of hydroxycoumarins in water and their experimental pKa values, and the results were used to predict the pKa of other hydroxycoumarins. Then, predicted and experimental pKa values were used as a basis for interpreting and discussing the variation of xanthine oxidase inhibitory activities within a subset of coumarins, with the aim of identifying the molecular species most relevant for enzyme inhibition.
2007
- Validation of an automated procedure for the prediction of relative free energies of binding on a set of aldose reductase inhibitors
[Articolo su rivista]
A. M., Ferrari; Degliesposti, Gianluca; Sgobba, Miriam; Rastelli, Giulio
abstract
Among the available methods for predicting free energies of binding of ligands to a protein, the molecular mechanicsPoisson–Boltzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) approacheshave been validated for a relatively limited number of targets and compounds in the training set. Here, we report the results ofan extensive study on a series of 28 inhibitors of aldose reductase with experimentally determined crystal structures and inhibitoryactivities, in which we evaluate the ability of MM-PBSA and MM-GBSA methods in predicting binding free energies using a numberof different simulation conditions. While none of the methods proved able to predict absolute free energies of binding in quantitativeagreement with the experimental values, calculated and experimental free energies of binding were significantly correlated.Comparing the predicted and experimental DG of binding, MM-PBSA proved to perform better than MM-GBSA, and within theMM-PBSA methods, the PBSA of Amber performed similarly to Delphi. In particular, significant relationships between experimentaland computed free energies of binding were obtained using Amber PBSA and structures minimized with a distance-dependentdielectric function. Importantly, while free energy predictions are usually made on large collections of equilibrated structures sampledduring molecular dynamics in water, we have found that a single minimized structure is a reasonable approximation if relativefree energies of binding are to be calculated. This finding is particularly relevant, considering that the generation of equilibrated MDensembles and the subsequent free energy analysis on multiple snapshots is computationally intensive, while the generation andanalysis of a single minimized structure of a protein–ligand complex is relatively fast, and therefore suited for high-throughput virtualscreening studies. At this aim, we have developed an automated workflow that integrates all the necessary steps required togenerate structures and calculate free energies of binding. The procedure is relatively fast and able to screen automatically and iterativelymolecules contained in databases and libraries of compounds. Taken altogether, our results suggest that the workflow can bea valuable tool for ligand identification and optimization, being able to automatically and efficiently refine docking poses, whichsometimes may not be accurate, and rank the compounds based on more accurate scoring functions.
2007
- Virtual screening on large scale grids
[Articolo su rivista]
N., Jacq; V., Breton; H. Y., Chen; L. Y., Ho; M., Hofmann; V., Kasam; H. C., Lee; Y., Legre´; S. C., Lin; A., Maaß; E., Medernach; I., Merelli; L., Milanesi; Rastelli, Giulio; M., Reichstadt; J., Salzemann
abstract
Large scale grids for in silico drug discovery open opportunities of particular interest to neglected and emerging diseases.
In 2005 and 2006, we have been able to deploy large scale virtual docking within the framework of the WISDOM
initiative against malaria and avian influenza requiring about 100 years of CPU on the EGEE, Auvergrid and TWGrid
infrastructures. These achievements demonstrated the relevance of large scale grids for the virtual screening by molecular
docking. This also allowed evaluating the performances of the grid infrastructures and to identify specific issues raised by
large scale deployment.
2006
- Valutazione tossicologica di due nuovi inibitori della DHFR del P. falciparum e del P. vivax
[Relazione in Atti di Convegno]
Rastelli, Giulio; Baggio, Giosuè Gabriele; Ulivi, P.; Ruberto, Ippazio Antonio; Rossi, Tiziana
abstract
Secondo una segnalazione del 2004 dell'UNiCEF "La malaria uccide più di un milionedi persone all'anno". Gli antifolato sono una classe di antimalarici cheagiscono sulla via biosintetica del folato dei vari Plasmodi inibendo gli enzimi in essacoinvolti. Sono farmaci estremamente sfruttati e, per questo motivo spesso inefficaci acausa dell'insorgenza di ceppi di Plasmodium divenuti resistenti. Oltre a ciò, nostri studirecenti in vitro hanno evidenziato la capacità di alcuni antimalarici di interferire con lareplicazione di cellule sane e cellule tumorali. Per lapresenza di gravi effetti collaterali e di ceppi resistenti, diviene sempre piiì urgente lanecessità di disporre di nuovi farmaci antimalarici. Nel presente studio sono state utilizzatedue molecole di nuova sintesi non analoghe del folato, ma in grado di inibire la DHFR(diidrofolato reduttasi) del Plasmodium falciparum e del Plasmodium vivax con una IC50compresa nel range micromolare e sub-micromolare. I composti, siglati come 1B e 6G sonorispettivamente una molecola N-idrossiamidina simile e un derivato della tiourea e sonostati saggiati in modo comparativo con l'antifolato pirimetamina nei confronti di celluleVERO e cellule MCF-7 (tumore mammario). Le cellule sono state coltivate sterilmente inpiastre con terreno EMEM arricchito. Al raggiungimento della semiconfluenza, sono stateaggiunte alle colture concentrazioni crescenti di 1B, 6G e pirimetamina (1,56-3,125-6,25-12,5-25-50 mg/l). Dopo 48 ore di incubazione sono stati eseguiti test: MTT e WesternBlotting. I risultati preliminari indicano che il composto 1B non interferisce in alcun modosulla crescita delle cellule sane o tumorali, mentre il derivato tioureico (6G) esercita unsignificativo stimolo sulla crescita delle cellule VERO (+ 60%), ma non sulle MCF-7. Ilfarmaco di riferimento pirimetamina stimola in modo dose dipendente la replicazione deileMCF-7. L'espressione delle proteine p53 e p21 immodificata nelle colture di MCF-7trattate con le due nuove molecole ne conferma l'assenza di tossicità. Questi risultatipreliminari, associati alla conferma che le due molecole sono attive specialmente verso iceppi di Plasmodium multiresistenti sono incoraggianti per il proseguimento dello studio dinuovi strumenti terapeutici caratterizzati anche da scarsa citotossicità.
2005
- Second Joint Italian-Swiss Meeting on Medicinal Chemistry , Italy, Modena 19-20 February 2005.
[Relazione in Atti di Convegno]
Brasili, Livio; Benvenuti, Stefania; Costantino, Luca; Costi, Maria Paola; Philipp, Floersheim; Franchini, Silvia; Gamberini, Gianfranco; Parenti, Carlo; Rastelli, Giulio; Rustichelli, Cecilia; Tait, Annalisa; Tondi, Donatella
abstract
The Second Joint Italian-Swiss Meeting on Medicinal Chemistry (ITCHMC2005) was held in Modena, (Italy) from September 12 to 16, 2005, under the auspices of the European Federation of Medicinal Chemistry (EFMC).This year, the annual meeting of the Division of Medicinal Chemistry of the Italian Chemical Society was co-organized with the Division for Medicinal Chemistry of the Swiss Chemical Society and it followed the first, successful one held in Torino in September 1997. This important event in the field of medicinal chemistry brought together scientists from both academia and industry to discuss different aspects of modern medicinal chemistry. Top-ranking scientists from medicinal chemistry and clinical development had the opportunity to meet and discuss the following topics: Carbohydrate Chemistry in Drug Design, Nuclear Receptors, Progress in Design and Development of Protease Inhibitors, Progress in Oncology Research and finally, Pain and Neurodegenerative Diseases.IntroductionThe conference was attended by 300 scientists from 13 countries, with most of the participants from Italy and Switzerland. The meeting allowed an extensive exchange of information and widespread networking. Of the 134 posters on display, 19 were selected for short oral presentations and two were selected for the Farminidustria awards. The meeeting was organized in six sessions with 6 Plenary (PL), 16 Main Lectures (ML) and 19 short communications (SC).The scientific sessions were held at the Forum Guido Monzani, a modern complex with multifunctional facilities; the opening ceremony took place at Accadenia Militare di Modena, housed in the seventeenth century Palazzo Ducale.Modena, city of art, culture and prosperous economy, offered an exciting background for stimulating scientific interactions. Participants were mainly from academia and other research centers together with 46 pharmaceutical companies; among them, six presented their work, namely Novartis Basel , Roche Basel, Novartis East Hannover USA, IRBM Pomezia Italy, Santhera Pharmaceutical AG, Heidelberg, GlaxoSmithKline (GSK) Verona, S-IN Soluzioni Informatiche, Vicenza. The areas covered by the meeting were advanced medicinal chemistry including computational chemistry, established drug targets, libraries and screens, inhibitor design and clinical advances. There were two poster sessions, with presentations given mainly by young scientists.
2005
- Structure-based design of 7-carbamate analogs of geldanamycin
[Articolo su rivista]
Rastelli, Giulio; Zq, Tian; Z., Wang; D., Myles; Y., Liu
abstract
The 7-carbamate groups of geldanamycin and its 17-(2-dimethylaminoethyl)amino-17-demethoxy derivative (17-DMAG) bind the N-terminal domain of Hsp90 by establishing a network of hydrogen bonds which involve four buried water molecules. In this study, a structure-based approach was used to investigate the effects of displacing some of these waters by modification of the 7-carbamate. A general loss of binding to human Hsp90 was observed, except for replacement of the carbamate with a hydroxamate group which gave an analog with weak activity. Modeling of Hsp90-ligand interactions suggested that the hydroxamate was not able to displace the buried water molecules, while bulkier substituents able to do so proved inactive.
2004
- Medicinal chemistry in parasitology
[Esposizione]
Costi, Maria Paola; Ferrari, Stefania; Rastelli, Giulio
abstract
"Medicinal Chemistry in Parasitology", January 23, 2004. Modena(Italy).The congress has been organized to give an overview of the medicinal chemistry in the area of parasitic/tropical diseases. The aim of the workshop is to stimulate coordination, research initiatives and collaborations among different groups working in the area.
2004
- Synthesis and biological activities of novel 17-aminogeldanamycin derivatives
[Articolo su rivista]
Z. Q., Tian; Y., Liu; D., Zhang; Z., Wang; S. D., Dong; C. W., Carreras; Y., Zhou; Rastelli, Giulio; D. V., Santi; D. C., Myles
abstract
Geldanamycin interferes with the action of heat shock protein 90 (Hsp90) by binding to the N-terminal ATP binding site and inhibiting an essential ATPase activity. In a program directed toward finding potent, water soluble inhibitors of Hsp90, we prepared a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogs, and compared their affinity for Hsp90, ability to inhibit growth of SKBr3 mammalian cells, and in selected cases, water solubility. Over 20 analogs showed cell growth inhibition potencies similar to that of 17-allylamino-17-demethoxygeldanamycin (17-AAG), the front-runner geldanamycin analog that is currently in multiple clinical trials. Many of these analogs showed water solubility properties that were desirable for formulation. One of the most potent and water-soluble analogs in the series was 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG), which was independently prepared by the NCI and will soon enter clinical trials. Importantly, the binding affinity of these analogs to the molecular target Hsp90 does not correlate well with their cytotoxicity in SKBr3 cells.
2004
- Three-dimensional quantitative structure-activity relationship analysis of a set of Plasmodium falciparum dihydrofolate reductase inhibitors using a pharmacophore generation approach
[Articolo su rivista]
M. D., Parenti; S., Pacchioni; A. M., Ferrari; Rastelli, Giulio
abstract
A 3D pharmacophore model able to quantitatively predict inhibition constants was derived for a series of inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), a validated target for antimalarial therapy. The data set included 52 inhibitors, with 23 of these comprising the training set and 29 an external test set. The activity range, expressed as Ki, of the training set molecules was from 0.3 to 11 300 nM. The 3D pharmacophore, generated with the HypoGen module of Catalyst 4.7, consisted of two hydrogen bond donors, one positive ionizable feature, one hydrophobic aliphatic feature, and one hydrophobic aromatic feature and provided a 3D-QSAR model with a correlation coefficient of 0.954. Importantly, the type and spatial location of the chemical features encoded in the pharmacophore were in full agreement with the key binding interactions of PfDHFR inhibitors as previously established by molecular modeling and crystallography of enzyme-inhibitor complexes. The model was validated using several techniques, namely, Fisher's randomization test using CatScramble, leave-one-out test to ensure that the QSAR model is not strictly dependent on one particular compound of the training set, and activity prediction in an external test set of compounds. In addition, the pharmacophore was able to correctly classify as active and inactive the dihydrofolate reductase and aldose reductase inhibitors extracted from the MDDR database, respectively. This test was performed in order to challenge the predictive ability of the pharmacophore with two classes of inhibitors that target very different binding sites. Molecular diversity of the data sets was finally estimated by means of the Tanimoto approach. The results obtained provide confidence for the utility of the pharmacophore in the virtual screening of libraries and databases of compounds to discover novel PfDHFR inhibitors.
2003
- Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90
[Articolo su rivista]
Jm, Jez; Jch, Chen; Rastelli, Giulio; Rm, Stroud; Dv, Santi
abstract
Hsp90 is an attractive chemotherapeutic target because it chaperones the folding of proteins found in multiple signal transduction pathways. We describe the 1.75 A resolution crystal structure of human Hsp90alpha (residues 9-236) complexed with 17-desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG). The structure revealed an altered set of interactions between the 17-substituent and the protein compared to geldanamycin and the 17-dimethylaminoethyl moiety pointing into solvent, but otherwise was similar to that reported for the complex with geldanamycin. Targeted molecular dynamics simulations and energetic analysis indicate that geldanamycin undergoes two major conformational changes when it binds Hsp90, with the key step of the conversion being the trans to cis conformational change of the macrocycle amide bond. We speculate that 17-DMAG analogs constrained to a cis-amide in the ground state could provide a significant increase in affinity for Hsp90.
2003
- Docking and database screening reveal new classes of Plasmodium falciparum dihydrofolate reductase inhibitors
[Articolo su rivista]
Rastelli, Giulio; S., Pacchioni; W., Sirawaraporn; R., Sirawaraporn; Md, Parenti; Am, Ferrari
abstract
Plasmodium falciparum dihydrofolate reductase (PfDHFR) is an important target for antimalarial chemotherapy. Unfortunately, the emergence of resistant parasites has significantly reduced the efficiency of classical antifolate drugs such as cycloguanil and pyrimethamine. In this study, an approach toward molecular docking of the structures contained in the Available Chemicals Directory (ACD) database to search for novel inhibitors of PfDHFR is described. Instead of docking the whole ACD database, specific 3D pharmacophores were used to reduce the number of molecules in the database by excluding a priori molecules lacking essential requisites for the interaction with the enzyme and potentially unable to bind to resistant mutant PfDHFRs. The molecules in the resulting focused database were then evaluated with regard to their fit into the PfDHFR active site. Twelve new compounds whose structures are completely unrelated to known antifolates were identified and found to inhibit, at the micromolar level, the wild-type and resistant mutant PfDHFRs harboring A16V, S108T, A16V + S108T, C59R + S108N + I164L, and N51I + C59R + S108N + 1164L mutations. Depending on the functional groups interacting with key active site residues of the enzyme, these inhibitors were classified as N-hydroxyamidine, hydrazine, urea, and thiourea derivatives. The structures of the complexes of the most active inhibitors, as refined by molecular mechanics and molecular dynamics, provided insight into how these inhibitors bind to the enzyme and suggested prospects for these novel derivatives as potential leads for antimalarial development.
2003
- Ortho-halogen naphthaleins as specific inhibitors of Lactobacillus casei thymidylate synthase. Conformational properties and biological activity
[Articolo su rivista]
S., Ghelli; M., Rinaldi; D., Barlocco; A., Gelain; P., Pecorari; Tondi, Donatella; Rastelli, Giulio; Costi, Maria Paola
abstract
Thymidylate synthase (TS) (EC 2.1.1.45), an enzyme involved in the DNA synthesis of both prokaryotic and eukaryotic cells, is a potential target for the development of anticancer and antinfective agents. Recently, we described a series of phthalein and naphthalein derivatives as TS inhibitors. These compounds have structures unrelated to the folate (Non-Analogue Antifolate Inhibitors, NAAIs) and were selective for the bacterial versus the human TS (hTS). In particular, halogen-substituted molecules were the most interesting. In the present paper the halogen derivatives of variously substituted 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[2,3-c]furan-1-one (1-5) and 3,3-bis(4-hydroxyphenyl)-1H,3H-naphtho[1,8-c,d]pyran-1-one (6-14) were synthesized to investigate the biological effect of halogen substitution on the inhibition and selectivity for the TS enzymes. Conformational properties of the naphthalein series were explored in order to highlight possible differences between molecules that show species-specific biological profile with respect to non species-specific ones. With this aim, the conformational properties of the synthesized compounds were investigated by NMR, in various solvents and at different temperatures, and by computational analysis. The apparent inhibition constants (K-i) for Lactobacillus casei TS (LcTS) were found to range from 0.7 to 7.0 muM, with the exception of the weakly active iodo-derivatives (4, 10, 13); all] the compounds were poorly active against hTS. The di-halogenated compounds 7, 8, 14 showed the highest specificity towards LcTS, their specificity index (SI) ranging between 40 and > 558. The di-halogenated 1,8-naphthalein derivatives (7-10) exhibited different conformational properties with respect to the tetra-haloderivatives. Though a clear explanation for the observed specificity by means of conformational analysis is difficult to find, some interesting conformational effects are discussed in the context of selective recognition of the compounds investigated by the LcTS enzyme.
2003
- Structure of Plasmodium vivax dihydrofolate reductase determined by homology modeling and molecular dynamics refinement
[Articolo su rivista]
Rastelli, Giulio; S., Pacchioni; M. D., Parenti
abstract
The structure of Plasmodium vivax dihydrofolate reductase (PvDHFR), a potentially important target for antimalarial chemotherapy, was determined by means of homology modeling and molecular dynamics refinement. The structure proved to be consistent with DHFRs of known crystal structure. The comparison of the complexes of the antifolate inhibitor pyrimethamine bound at the active sites of PvDHFR and PfDHFR, the related enzyme from Plasmodium falciparum, prospected the possibility of using structure-based drug design to develop inhibitors that are effective against both malarial enzymes. This study constitutes a first step toward understanding of the antifolate-PvDHFR molecular interactions and possible rationalization of resistance in vivax malaria.
2002
- Binding of 1-benzopyran-4-one derivatives to aldose reductase: A free energy perturbation study
[Articolo su rivista]
Rastelli, Giulio; Costantino, Luca; Gamberini, Maria Cristina; A., Del Corso; U., Mura; Jm, Petrash; Am, Ferrari; S., Pacchioni
abstract
The relative binding affinities to human aldose reductase (ALR2) of three new 7-hydroxy-2-benzyl-4H-1-benzopyran-4-one inhibitors were predicted by free energy perturbation (FEP) simulations. Molecular substitutions were specifically designed to investigate the role of hydrogen bonding at the active site of ALR2. Starting from the lead inhibitor 7-hydroxy-2-(4'-hydroxy-benzyl)-4H-1-benzopyran-4-one, the 4'-hydroxyl was mutated to methyl and to trifluoromethyl, and an hydroxyl at position 8 was additionally introduced. Once synthesized and tested as inhibitors of ALR2, the compounds displayed variations of K-i that were in qualitative to quantitative agreement with the calculated relative free energies of binding. The results, discussed in terms of balance between free energies of solvation and free energies of binding to ALR2, elucidate the importance of hydrogen bonding with Thr113 and with Trp111 and cofactor, and provide a rationale to the observed differences in binding affinities. (C) 2002 Elsevier Science Ltd. All rights reserved.
2002
- Discovery of new inhibitors of aldose reductase from molecular docking and database screening
[Articolo su rivista]
Rastelli, Giulio; Am, Ferrari; Costantino, Luca; Gamberini, Maria Cristina
abstract
Aldose reductase (ALR2) is a target enzyme for the treatment of diabetic complications. Owing to the limited number of currently available drugs for the treatment of diabetic complications, the discovery of new inhibitors of ALR2 that can potentially be optimized as drugs appears highly desirable. In this study, a molecular docking analysis of the structures of more than 127,006 organic compounds contained in the National Cancer Institute database was performed to find and score molecules that are complementary to ALR2. Besides retrieving several carboxylic acid derivatives, which are known to generally inhibit aldose reductase, docking proposed other families of putative inhibitors such as sulfonic acids, nitro-derivatives, sulfonamides and carbonyl derivatives. Twenty-five compounds, chosen as the highest-scoring representatives of each of these families, were tested as aldose reductase inhibitors. Five of them were found to inhibit aldose reductase in the micromolar range. For these active compounds, selectivity with respect to the closely-related aldehyde reductase was determined by measuring the corresponding inhibitory activities. The structures of the complexes between the new lead inhibitors and aldose reductase, here refined with molecular mechanics and molecular dynamics calculations. suggest that new pharmacophoric groups can bind aldose reductase very efficiently. In the case of the family of the nitro-derivative-inhibitors. a class of particularly interesting compounds, a round of optimizations was performed with the synthesis and biological evaluation of a series of derivatives aimed at testing the proposed binding mode and at improving interaction with active site residues. Starting from a hit compound having an IC50 of 42 muM, the most potent compound synthesized showed a 10-fold increase in inhibitory activity and 10-fold selectivity with respect to ALR1, and structure-activity relationships of the designed compounds were in agreement with the proposed mode of binding at the active site.
2002
- Mutational analysis of Plasmodium falciparum dihydrofolate reductase: the role of aspartate 54 and phenylalanine 223 on catalytic activity and antifolate binding
[Articolo su rivista]
W., Sirawaraporn; R., Sirawaraporn; S., Yongkiettrakul; A., Amornpol; Rastelli, Giulio; S., Kamchonwongpaisan; Y., Yuthavong
abstract
The catalytic activity and ability to confer resistance to antifolates of Plasmodium falciparum dihydrofolate reductase (pfDHFR) through single and double mutations at Asp-54 and Phe-223 were investigated. A single Asp54Glu (D54E) mutation in the pfDHFR domain greatly decreased the catalytic activity of the enzyme and affected both the K, values for the substrate dihydrofolate and the K-i values for pyrimethamine, cycloguanil and WR99210. The Phe223Ser (F223S) single mutant had unperturbed kinetics but had very poor affinity with the first two antifolates. The ability to confer high resistance to the antifolates of F223S enzyme was, however, abolished in the D54E + F223S double mutant enzyme. When D54E mutation was present together with the A16V + S108T double mutation, the effects on the Km values for the substrate dihydrofolate and the binding affinity of antifolates were much more pronounced. The severely impaired kinetics and poor activity observed in A16V+S108T+D54E enzyme could, however, be restored when F223S was introduced, while the binding affinities to the antifolates remained poor. The experimental findings can be explained with a model for substrate and inhibitor binding. Our data not only indicate the importance of Asp-54 of pfDHFR in catalysis and inhibitor binding, but also provide evidence that infer the potentially crucial function of the C-terminal portion of pfDHFR domain. (C) 2002 Elsevier Science B.V. All rights reserved.
2002
- Nitrophenyl derivatives as Aldose Reductase inhibitors
[Articolo su rivista]
Costantino, Luca; A. M., Ferrari; Gamberini, Maria Cristina; Rastelli, Giulio
abstract
Nitrophenyl derivatives were recently discovered as a new class of ALR2 inhibitors by means of docking and database screening of the National Cancer Institute database of organic molecules. The nitro group was predicted to bind to the Tyr48 and His110 active site residues of the enzyme, the site where acidic ALR2 inhibitors such as carboxylic acids bind in their anionic form. Given the novelty of these compounds, we decided to expand their structure-activity relationships by synthesizing and testing a series of derivatives and the corresponding compounds having a carboxylic group instead of the nitro moiety; the results obtained were rationalized by means of docking and molecular dynamics simulations. On the whole there is an agreement between inhibitory data and the results of molecular modeling experiments, supporting the hypothesized binding mode of these compounds.
2002
- Oxidative Modification of Aldose Reductase Induced By Copper Ion. Definition Of The Metal-Protein Interaction Mechanism
[Articolo su rivista]
Cecconi, I.; Scaloni, A.; Rastelli, Giulio; Moroni, M.; Vilardo, P. G; Costantino, Luca; Cappiello, M.; Garl, D.; Carper, D.; Petrash, J. M.; Del Corso, A.; Mura, U.
abstract
Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys(303) appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys(298)-Cys(303). However, a Cys(80)-Cys(303) disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys(80)-Cys(303) disulfide to the more stable product Cys(298)-Cys(303) is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys(80)-Cys(303) disulfide. However, no evidence of conditions favoring the formation of the Cys(298)-Cys(303) disulfide was observed. Our proposal is that the generation of the Cys(298)-Cys(303) disulfide, either directly or by rearrangement of the Cys(80)-Cys(303) disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.
2001
- 7-Hydroxy-2-substituted-4-H-1-benzopyran-4-one derivatives as aldose reductase inhibitors: a SAR study
[Articolo su rivista]
Costantino, Luca; A., DEL CORSO; Rastelli, Giulio; Jm, Petrash; U., Mura
abstract
On the basis of the results of molecular modelling studies performed on the aldose reductase (ALR2) inhibitor 7-hydroxy-2-(4'-hydroxybenzyl)-4H-1-benzopyran-4-one (compound A) bound at the active site of the enzyme, we synthesised and tested on bovine and human ALR2 several derivatives modified at position 2 of the benzopyran moiety, in order to confirm the hypothesised binding mode of this compound. The substitution of the methylene bridge with the isosteric sulphur substituent gives an active derivative, while substitution with a polar NH causes a decrease in inhibitory activity; this is in accordance to the previously reported structure in which the methylene linker was found to be adjacent to a hydrophobic aminoacid (Leu300). Among the substituents at 4' position examined, the most favourable for inhibitory activity are those able to act as hydrogen bond donors, supporting the hypothesis of the importance of the interaction with Thr113 for the inhibition of the enzyme.
2001
- Synthesis and cytotoxicity of bis(benzo[g]indole-3-carboxamides) and related compounds
[Articolo su rivista]
G. A., Pinna; M. A., Pirisi; G. E., Grella; L., Gherardini; J. M., Mussinu; G., Paglietti; A. M., Ferrari; Rastelli, Giulio
abstract
A series of bis(benzo[g]indoles) bridged by CX-(CH2)nN(Me)(CH2)n-CX (X = O, S, H2; n = 2,3) was synthesized as bifunctional antitumor agents and evaluated for cytotoxic activity against diverse human cancer cell lines by the National Cancer Institute. The parent compounds 2a,b exhibited a good level of activity and derivates 2c-g,i,k demonstrated significant inhibitory effects, all with IC50 values in the low micromolar range. The thioamide analogue 2j showed less potency. It is interesting to note that introduction of substituents on the benzene ring of the benzo[g]indole portion of 2a,b did not affect activity, with the only exception of the 7,8-dichloro derivative 2h which became less potent. One member of this series, 2i, was then tested in the hollow fiber cell assay to evaluate, in a preliminary fashion, its in vivo antineoplastic activity. Molecular modelling studies were performed on amide 2a and thioamide 2j to explain the loss of activity of 2j as to 2a. Finally, compound 2a behaved as a typical DNA intercalating agent, as judged from viscosity measurements with Poly(dA-dT)...poly(dA-dT).
2000
- A series of diarylsubstituted oximes as potential substrates for new aldose reductase inhibitors
[Articolo su rivista]
D., Rakowitz; G., Heinisch; P., Lukavsky; S., Kiendler; C., Trenkwalder; D., Barlocco; Rastelli, Giulio; Costantino, Luca
abstract
In the course of our research aimed at the discovery of new compounds acting as aldose reductase inhibitors, we tested a series of some (E)- and (Z)-ω-[[(aryldiazinylmethylene)amino]oxy]alkanoic acids, which were found to have moderate in vitro inhibitory activity. On this basis we have now prepared several new derivatives modified both at the length of the chain and at its terminal carboxylic group, together with compounds carrying various substituents at the phenyl ring. This paper describes their synthesis and biological properties.
2000
- Aldose reductase does catalyse the reduction of glyceraldehyde through a stoichiometric oxidation of NADPH.
[Articolo su rivista]
A., Del Corso; Costantino, Luca; Rastelli, Giulio; F., Buono; U., Mura
abstract
In order to define the ability of bovine lens aldose reductase (ALR2) to generate polyols from aldoses, the quantitative determination of glycerol in the presence of glyceraldehyde was performed by gas chromatography after derivatization with trifluoroacetic anhydride. The proposed method appears to be useful in quantifying low amounts of glycerol in the presence of relatively high concentrations of glyceraldehyde and in following glycerol formation in enzyme assay conditions. The generation of one equivalent of glycerol in the presence of ALR2, is paralleled by the oxidation of one equivalent of NADPH. A similar result was obtained when S-glutathionyl-modified ALR2 was used, instead of the native enzyme, as a catalyst of glyceraldehyde reduction. Sorbinil, a classical ALR2 inhibitor, present in the enzyme assay mixture, inhibits to the same extent both NADPH oxidation and glycerol formation. The demonstration of the stoichiometric ratio of 1:1 occurring in the presence of bovine lens ALR2 between the synthesis of glycerol from D, L -glyceraldehyde and the oxidation of NADPH, rules out doubts concerning the ability of the enzyme to catalyse the reduction of aldoses to the corresponding polyalcohols. Possible autooxidation processes of glyceraldehyde, in the enzyme assay conditions, appear to be irrelevant with respect to the enzyme-catalysed reduction of the aldose. This would indicate that the spectrophotometric monitoring of NADPH oxidation at 340 nm, in the presence of ALR2, is a reliable method to assay the enzyme activity.
2000
- Interaction of pyrimethamine, cycloguanil, WR99210 and their analogues with Plasmodium falciparum dihydrofolate reductase: Structural basis of antifolate resistance
[Articolo su rivista]
Rastelli, Giulio; W., Sirawaraporn; P., Sompornpisut; T., Vilaivan; S., Kamchonwongpaisan; R., Quarrell; G., Lowe; Y., Thebtaranonth; Y., Yuthavong
abstract
The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (K-i). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The K-i values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance. (C) 2000 Elsevier Science Ltd. All rights reserved.
2000
- Pharmacological approaches to the treatment of diabetic complications
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; Gamberini, Maria Cristina; D., Barlocco
abstract
Diabetes is often accompanied by several long-term complications such as neuropathy, nephropathy, retinopathy, cataract and angiopathy; their occurrence has been linked to the modification of the physiological levels of glycaemia. Several interrelated metabolic pathways have been implicated in the toxic effects of glucose; the polyol pathways was one of the first considered. However, while in diabetic animal models the inhibitors of aldose reductase (ALR2, the first enzyme of this pathway) seem to be active, 16 years of clinical trials, based mainly on neuropathy, have been inconclusive; only one drug currently being marketed. Newer potent and selective aldose reductase inhibitors have been discovered in the last few years, but the lack of commercial success has probably led to the very rapid decrease in the number of patents relating to newer aldose reductase inhibitors. Inhibition of the second enzyme of this pathway, sorbitol dehydrogenase (SDH), has been shown to be detrimental. Other approaches for the prevention and the delay of progression of diabetic complications seem to be more promising, namely, the inhibition of the formation of advanced glycated end products (AGEs) or protein kinase C (PKC) β2 inhibition; compounds acting on these two pathways have proved effective in retarding the development of diabetic complications in animal models and some products are in clinical trials at the moment. Renewed attention has been paid to vascular involvement in the pathogenesis of diabetic neuropathy; the biological activity of C-peptide and the role of endothelin-1 (ET-1) in diabetic vascular disease are emerging as a new research area for the treatment of diabetic complications.
2000
- Preparation of thieno[3,2-h]cinnolinones as matrix metalloproteinase inhibitors
[Articolo su rivista]
Ga, Pinna; Mm, Curzu; G., Murineddu; G., Chelucci; G., Cignarella; E., Menta; Hw, Krell; Rastelli, Giulio; Am, Ferrari
abstract
A new series of thieno[3,2-h]cinnolinone analogues was synthesized which is structurally related to 2,3,4,4a,5,6-hexahydro thieno[3,2-h]cinnolin-3-one 1, a weak inhibitor of the matrix metalloproteinase MMP-8 (human neutrophil collagenase). Preliminary SAR studies have shown that while C-4a-methyl, C-7-acetylamino, C-7 and C-8-nitro substitution, and C-4-C-4a olefination provided no increase in activity relative to 1, Cs-acetylamino substitution as in 5 and 8 was favourable. Moreover, to predict how the thieno[3,3-h]cinnolinone inhibitors might bind to MMP-8, the unsubstituted compound 9 was docked into the MMP-8 crystal structure. These studies revealed that inhibitor 9 does not seem to be able to coordinate the catalytically-active zinc ion but preferably interact with the peptide-binding region of the active site.
2000
- Structural bases for the inhibition of aldose reductase by phenolic compounds
[Articolo su rivista]
Rastelli, Giulio; L., Antolini; Benvenuti, Stefania; Costantino, Luca
abstract
Aldose reductase (ALR2) is an enzyme involved in the development of long-term diabetic complications. In the search for aldose reductase inhibitors less acidic than carboxylic acids, phenolic compounds related to benzopyran-4-one and chalcone are particularly interesting because they possess good inhibitory properties. In order to investigate the similarities between these two classes of compounds and to provide a structural basis for their inhibition of ALR2, the existing structure-activity relationships were reconsidered. To this end, the acidity constants of a set of chalcones were measured and compared with those of benzopyran-4-one derivatives. Then, having established the relevant protonation state of these phenolics at physiological pH, a conformational analysis was performed on the most active benzopyran-4-one and chalcone derivatives' and the results were compared with the crystal structures of some analogues. Finally, molecular docking of the most active chalcone into the ALR2 binding site was performed, and the structure of the enzyme-inhibitor complex was compared with that of the complex formed between ALR2 and a previously-obtained benzopyran-4-one derivative. (C) 2000 Elsevier Science Ltd. All rights reserved.
2000
- Synthesis and aldose reductase inhibitory activity of a new series of benzo[h]cinnolinone derivatives
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; G., Cignarella; D., Barlocco
abstract
Following our previous studies on pyridazinone carboxylic acids as potent and selective aldose reductase (ALR2) inhibitors, a new series of benzo[h]cinnolinone carboxylic acids, variously substituted at the positions 4, 7-10 and differently modified both at the central ring and at the acidic side chain, were synthesized and tested as inhibitors of ALR2. Comparison with previously synthesized compounds allows us to define more precisely structure-activity relationships for this class of compounds. In fact, in addition to the importance of the acidic side chain, their properties are highly influenced by the substituents present on the benzo[h]cinnolinone nucleous, with potency ranging from that of Sorbinil to very weakly active compounds.
1999
- 1-Benzopyran-4-one antioxidants as aldose reductase inhibitors
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; Gamberini, Maria Cristina; Ja, Vinson; P., Bose; Iannone, Anna; M., Staffieri; L., Antolini; A., DEL CORSO; U., Mura; A., Albasini
abstract
Starting from the inhibitory activity of the flavonoid Quercetin, a series of 4H-1-benzopyran-4-one derivatives was synthesized and tested for inhibition of aldose reductase, an enzyme involved in the appearance of diabetic complications. Some of the compounds obtained display inhibitory activity similar to that of Sorbinil but are more selective than Quercetin and Sorbinil with respect to the closely related enzyme, aldehyde reductase, and also possess antioxidant activity. Remarkably, these compounds possess higher pK(a) values than carboxylic acids, a characteristic which could make the pharmacokinetics of these compounds very interesting. Molecular modeling investigations on the structures of inhibitors bound at the active site of aldose reductase were performed in order to suggest how these new inhibitors might bind to the enzyme and also to interpret structure-activity relationships.
1999
- Diabetes complications and their potential prevention: Aldose reductase inhibition and other approaches
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; P., Vianello; G., Cignarella; D., Barlocco
abstract
Despite recent advances both in the chemistry and molecular pharmacology of antidiabetic drugs, diabetes still remains a life-threatening disease, which tends to spread all over the world. The clinical profile of diabetic subjects is often worsened by the presence of several long-term complications, namely neuropathy, nephropathy, retinopathy, and cataract. Several attempts have been made to prevent or at least to delay them. The most relevant are reported in this review, including the development of compounds acting as aldose reductase inhibitors, anti-advanced glycation end- product drugs, free radical scavengers, vasoactive agents, essential fatty acid supplementation, and neurotropic growth factors.
1999
- Isoxazolo-[3,4-d]-pyridazin-7-(6H)-one as a potential substrate for new aldose reductase inhibitors
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; Gamberini, Maria Cristina; Mp, Giovannoni; V., DAL PIAZ; P., Vianello; D., Barlocco
abstract
The isoxazolo-[3,4-d]-pyridazin-7-(6H)-one (2) and its corresponding open derivatives 5-acetyl-4-amino-(4-nitro)-6-substituted-3(2H)pyridazinones (3, 4) were used as simplified substrates for the synthesis of new aldose reductase inhibitors with respect to the previously reported 5, 6-dihydrobenzo[h]cinnolin-3(2H)one-2 acetic acids (1). Moreover, a few derivatives lacking the 5-acetyl group were prepared. Several compounds derived from 2 displayed inhibitory properties comparable to those of Sorbinil. In this class the presence at position 6 of a phenyl carrying an electron-withdrawing substituent proved to be beneficial, independently from its position on the ring (5g,j-l). Acetic acid derivatives were more effective than propionic and butyric analogues. On the contrary, all the monocyclic compounds (6-8) were either inactive or only weakly active. The 3-methyl-4-(p-chlorophenyl)isoxazolo-[3,4-d]-pyridazin-7-(6H )-one acetic acid (5g), which proved to be the most potent derivative, was also investigated in molecular modeling studies, to assess possible similarities in its interaction with the enzyme, with respect to the model 1
1998
- Free energy perturbation studies on binding of the inhibitor 5,6-dihydrobenzo[h]cinnolin-3(2H)one-2-acetic acid and its methoxylated analogs to aldose reductase
[Articolo su rivista]
Rastelli, Giulio; Costantino, Luca; P., Vianello; D., Barlocco
abstract
Free energy perturbation simulations have been employed to rationalize the binding differences between a benzocinnolinone carboxylic acid inhibitor of aldose reductase;Ind its methoxylated analogs in four selected substitution sites. The calculated free energy differences are in qualitative agreement with the experimental results. The balance between the cost for desolvation and the gain in enzyme binding correctly predicts and rationalizes the different inhibitory activities of each methoxylated compound. The implications for perturbations occurring at the interface between protein residues and water molecules present at the active site are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.
1998
- Fruits of ribes, rubus, vaccinium and prunus genus. Metal contents and genome
[Articolo su rivista]
Plessi, Maria; Bertelli, Davide; Rastelli, Giulio; Albasini, Albano; Monzani, Agar
abstract
The production of fruits of genus Ribes, Rubus, Vaccinium and Prunus is particularly important in mountain communities. The contents of macro- and microelements in fruits from different cultivars of blueberry (Vaccinium corimbosus L.), red currant (Ribes rubrum L.), raspberry (Rubus idaeus L.) and cherry (Prunus avium L.) were determined. The anthocyanin and total polyphenol contents of the fruits were also determined. The results were analyzed with statistical methods. By using the one-way analysis of variance (ANOVA) the various genera of the fruits were found to be differentiable on the basis of their metal contents. Multivariate statistical analysis, performed using principal component analysis (PCA), confirms that the different fruits can also be well discriminated by their contents of metals, total anthocyanins, and polyphenols.
1998
- Molecular dynamics simulations of the structure of aldose reductase complexed with the inhibitor Tolrestat
[Articolo su rivista]
Rastelli, Giulio; Costantino, Luca
abstract
This study reports a molecular dynamics (MD) investigation on the structure of aldose reductase (ALR2) complexed with the potent inhibitor tolrestat. The simulations predict four different orientations of tolrestat into the ALR2 binding site; these orientations have in common a strong interaction of the anionic carboxylate with Tyr48, His110, Trp111 and NADP+, but completely differ for the orientation of the aromatic portion of the inhibitor. Interestingly, the orientation in which tolrestat gives the most attractive interaction energy with the enzyme is in full accord with the x-ray crystal structure of the complex that has been reported in the literature after this work was completed. In addition, the suggestion of more than one orientation of tolrestat during MD is in agreement with recent electrospray mass spectrometry experiments on the ALR2-tolrestat complex.
1998
- Oxidative modification of aldose reductase induced by copper ion. Factors and conditions affecting the process
[Articolo su rivista]
I., Cecconi; M., Moroni; P. G., Vilardo; M., Dal Monte; Borella, Paola; Rastelli, Giulio; Costantino, Luca; D., Garland; D., Carper; J. M., Petrash; A., Del Corso; U., Mura
abstract
Bovine lens aldose reductase (ALR2) is inactivated by copper ion [Cu(II)] through an oxygen-independent oxidative modification process. A stoichiometry of 2 equiv of Cu(II)/enzyme mol is required to induce inactivation. While metal chelators such as EDTA or o-phenantroline prevent but do not reverse the ALR2 inactivation, DTT allows the enzyme activity to be rescued by inducing the recovery of the native enzyme form. The inactive enzyme form is characterized by the presence of 2 equiv of bound copper, at least one of which present as Cu(I), and by the presence of two lesser equivalents, with respect to the native enzyme, of reduced thiol residues. Data are presented which indicate that the Cu-induced protein modification responsible for the inactivation of ALR2 is the generation on the enzyme of an intramolecular disulfide bond. GSH significantly interferes with the Cu-dependent inactivation of ALR2 and induces, through its oxidation to GSSG, the generation of an enzyme form linked to a glutathionyl residue by a disulfide bond.
1997
- 1H-NMR conformational studies of some phtalein derivatives acting as thymidylate synthase inhibitors
[Articolo su rivista]
S., Ghelli; Costi, Maria Paola; D., Barlocco; M., Rinaldi; Tondi, Donatella; P., Pecorari; Rastelli, Giulio
abstract
1997
- A model of the interaction of substrates and inhibitors with xanthine oxidase
[Articolo su rivista]
Rastelli, Giulio; Costantino, Luca; A., Albasini
abstract
A model of the interaction of substrates and inhibitors with xanthine oxidase (XO) based on similarity concepts and molecular modeling is introduced and discussed, and previous literature is reexamined in the light of recent insights into the mechanism and structure of XO. Use is made of quantum-chemical calculations with the inclusion of solvent effects, molecular superimposition with least-squares fitting algorithms, and molecular electrostatic potentials. First, the relative stabilities of the tautomeric forms of the physiological substrates, xanthine and hypoxanthine, are calculated both in vacuo and in water in order to select the most abundant form(s) at physiological pH: the two substrates prove to be stable in their lactam forms, with a dominance of the N-7-H tautomer for xanthine and of N-9-H for hypoxanthine. The structures of xanthine and hypoxanthine are then superimposed, and their relative orientation with respect to the molybdenum center of XO is suggested. The criteria used for superimposition reflect the importance of functional groups of xanthine and hypoxanthine, as inferred from experimental work. In particular, the carbonyl oxygen common to the two substrates is given special consideration on account of its determinant role. The results show that the most important functional groups of the two substrates can be successfully superimposed by means of a rotation that exchanges the five-membered with the six-membered rings of xanthine and hypoxanthine with respect to molybdenum. The close similarity of the electrostatic potentials of the two superimposed molecules adds weight to the proposed orientation of the substrates in the binding site. The model of interaction is then tested and further developed using a series of previously-synthesized dimensional analogs of xanthine and hypoxanthine. The results confirm that the correct positioning of the carbonyl group is essential if a productive interaction with XO is to be achieved and allow us to map the dimensions of the active site starting from the superimposition of the physiological substrates. Two hypotheses regarding the amino acid residues interacting with the important carbonyl oxygen of the substrates are then put forward on the basis of spectroscopic and biochemical evidence: they are postulated to be one lysine or one protonated glutamic acid residue. In an attempt to unify the binding of substrates and inhibitors, the model is extended to the inhibitors of XO by superimposing the most interesting inhibitors developed by Robins on xanthine and hypoxanthine. This allows us to define the most suitable location of the phenyl rings of these inhibitors with respect to the superimposition of the substrates. Intriguingly, the superimpositions of the most active inhibitors are consistent with a unique location of their phenyl rings, even though they are in different positions on the purine ring. Finally, the flavone, which is a potent inhibitor of XO and is currently under investigation by the authors, is accounted for by these findings and successfully included in the model. This model incorporates many important insights into XO and can be of general interest. Moreover, it represents a clear-cut alternative to a previous model developed by Robins on the basis of the coordination of substrates and inhibitors to molybdenum.
1997
- Fruit of ribes, rubus, vaccinium and prunus genus, metals content and genome
[Abstract in Atti di Convegno]
Plessi, Maria; Bertelli, Davide; Rastelli, Giulio; Albasini, Albano; Monzani, Agar
abstract
The metal content of fruits of genus rubus, ribes, vaccinium and prunus has been determined by indictively coupled plasma emission spectrometry
1997
- New aldose reductase inhibitors as potential agents for the prevention of long-term diabetic complications
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; G., Cignarella; P., Vianello; D., Barlocco
abstract
The results of recent clinical trials emphasise the importance of an improved glycaemic control in diabetic patients in order to prevent or at least to delay long-term complications. The difficulty in obtaining normalisation of blood glucose values has underlined the importance of the search for new and effective aldose reductase inhibitors (ARIs) to control the consequences of elevated glucose levels, therefore delaying the onset and retarding the progression of diabetic complications such as neuropathy, nephropathy, retinopathy and cataract. Although the physiological role of aldose reductase (ALR2) has not been clearly elucidated yet, it has been shown that this enzyme, the first of the polyol pathway, is responsible for the production of sorbitol from glucose. There are several pieces of evidence which link this process to the occurrence of diabetic complications. Orally active aldose reductase inhibitors can be grouped into two chemical classes: cyclic imide and carboxylic acid derivatives. This review describes the recent insights into these two classes of inhibitors, and the further development of ARIs provided with antidiabetic and antihyperlipidaemic properties. The most recent developments in understanding of the structure, catalytic mechanism and biochemical behaviour of ALR2 are also reported.
1997
- Structure-based design of an inhibitor modeled at the substrate active site of aldose reductase
[Articolo su rivista]
Rastelli, Giulio; P., Vianello; D., Barlocco; Costantino, Luca; G., Cignarella; A., DEL CORSO; U., Mura
abstract
This study presents the first successful example of structure-based drug design on aldose reductase in the extant literature. Starting from the structure of the modeled complex of aldose reductase with a pyridazinone acetic acid inhibitor that we previously disclosed, using the tools of molecular modeling for structure manipulation and molecular mechanics for energy minimization, we were able to design and synthesize a new analog that showed remarkably improved activity. We hope that a proper account of the most important enzyme-inhibitor interactions revealed by this study mill allow, in the future, the design of new lead compounds having structures unrelated to carboxylic acids.
1996
- A rational approach to the design of flavones as xanthine oxidase inhibitors
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; A., Albasini
abstract
In the light of previous QSAR studies on flavones as inhibitors of xanthine oxidase, we synthesized and tested a new series of 7-hydroxyflavones carrying a wide and balanced variety of substituents (pi, sigma(p)) at the 4' position in order to explore the effect of substituents at this position on the xanthine oxidase inhibitory activity. The results of pK(a) determinations show that the electronic effects of the substituents are not transferred to the hydroxyl at C7, previously found to be fundamental for activity. An excellent correlation is found between molar refractivity of the substituents and the inhibitory activity. These results, applied to the more active 5,7-dihydroxyflavones, allowed the design and synthesis of a very active inhibitor, with an IC50 in the nanomolar range. On interpretative grounds, C4' substituents of flavones are involved in dispersion interactions with the enzyme. The calculation of quantum chemical polarizabilities and solvent accessible surface areas suggests the existence of pi-pi stacking interactions with an aromatic aminoacidic residue of the enzyme.
1996
- Conformational analysis of phthalein derivatives acting as thymidylate synthase inhibitors by means of 1H NMR and quantum chemical calculations
[Articolo su rivista]
S., Ghelli; Rastelli, Giulio; D., Barlocco; M., Rinaldi; Tondi, Donatella; P., Pecorari; Costi, Maria Paola
abstract
The conformations of a set of phthalein derivatives with bacterial thymidylate synthase (TS) inibitory activity were investigated by H-1 NMR spectra, performed at both room and low temperature, and by quantum chemical calculations. Since the crystal structure of the binary complex of phenolphthalein with the enzyme is known, we set out to study the conformation of various of its analogues in solution in order to observe the effects of the substituents on the phenolic rings, of the alpha-naphthol derivative and of the rigid analogue, fluorescein, and compare the results with the X-ray crystal structure studies. A relationship between the chemical shift of the proton on C4 (H4) of the phthalidic ring and the averaged angle formed by the phthalidic and the aromatic ring planes was found in which the most perpendicular conformations have the lowest H4 chemical shift values. At room temperature, the rotational freedom of all the studied compounds was similar, while at lower temperature the naphthol derivative assumed a partially blocked conformation. Finally, a qualitative relationship between the inhibitory properties of the compounds and their conformations is discussed. Copyright
1996
- Model chemical studies of thymidylate synthase. Nucleophilic addition of hydroxylamine to uracil and 5-fluorouracil
[Articolo su rivista]
Costi, Maria Paola; Rastelli, Giulio; M., Rinaldi; M., Cevolani; P., Pecorari
abstract
To explain the reaction of 5-fluoro-2'-deoxyuridine-5'-monophosphate towards Thymidylate Synthase (TS), a chemical model formed by hydroxylamine and 5-fluorouracil (FU) or uracil (U) was suggested. The reaction mechanism would involve a nucleophilic attack of the nitrogen of the free base to C-6 of the uracil ring; the reaction is catalyzed by the cationic form of the base. The kinetic constants, the activation energies, the entropies of activation for the forward and the reverse reactions and the equilibrium constants for the reactions of FU and U with hydroxylamine were measured. The value of the activation energy for the forward reaction of FU with hydroxylamine is 1.95 Kcal/mol lower than with U. The value of the equilibrium constant for the reaction of FU with hydroxylamine is 23 times higher than that of U at 25 degrees C. Therefore the product of the first reaction is the most stable one. The reactivity of FU towards hydroxylamine is, in its complex, higher with respect to the reactivity of U in the same conditions, because the reaction proceeds largely towards higher Keg values; the activation energy of the forward reaction is lower for FU and the entropy of activation too. Thus indicating that the activated complex is more ordered than that of U. The results indicate that the increased reactivity and stability of the complex with FU in the model reaction may account for the competition observed in the biological reaction.
1996
- Natural polyhydroxylated compounds as inhibitors of xanthine oxidase
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; A., Albasini
abstract
1996
- Synthesis, activity, and molecular modeling of a new series of tricyclic pyridazinones as selective aldose reductase inhibitors
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; K., Vescovini; G., Cignarella; P., Vianello; A., DEL CORSO; M., Cappiello; U., Mura; D., Barlocco
abstract
Three new series of tricyclic pyridazinones have been synthesized and tested in vitro in order to assess (i) their ability to inhibit aldose reductase enzyme (ALR2) and (ii) their specificity toward the target enzyme with respect to other related oxidoreductases, such as aldehyde reductase, sorbitol dehydrogenase, and glutathione reductase. The inhibitory capability of the most effective compounds (IC50 values ranging from 6.44 to 12.6 mu M) appears to be associated with a rather significant specificity for ALR2. Molecular mechanics and molecular dynamic calculations performed on the ALR2-inhibitor complex give indications of specific interaction sites responsible for the binding, thus providing information for the design of new inhibitors with improved affinity for the enzyme.
1995
- ANTHOCYANIDINES AS INHIBITORS OF XANTHINE-OXIDASE
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; A., Albasini
abstract
Anthocyanidines as inhibitors of xanthine oxidase
1995
- Insight into the specificity of Thymidylate synthase from molecular dynamics and free-energy perturbation calculations
[Articolo su rivista]
Rastelli, Giulio; B., Thomas; P. A., Kollman; D. V., Santi
abstract
Molecular dynamics and free energy pertubation calculations have been used to calculate the relative free energies of binding of 2′-deoxyuridine 5′-monophosphate (dUMP) and 2′-deoxycytidine 5′-monophosphate (dCMP) to thymidylate synthase (TS) and two asparagine 229 mutants. Calculations qualitatively reproduce experimentally observed dissociation constants of the protein-nucleotide complexes. Furthermore, they provide insight into structural aspects of binding and catalysis of these nucleotides to the protein. The simulations of the wild-type TS complexes with dUMP and dCMP support the key role asparagine 229 in causing tighter binding of dUMP than dCMP; repulsion between the base of dCMP support the key role of asparagine 229 side chain reduces the ΔG of binding to the protein from that found in aqueous solution and causes the displacement of this nucleotide into a position unsuitable for reaction. The free energy calculation of the aspartate 229 mutant of TS interacting with either dUMP or dCMP suggest a synergism between the aspartate 229 side chain and the vicinal histidine 199 in binding. The best agreement between the calculated and the experimental ΔΔG of binding has been obtained when the aspartate side chain is anionic and the histidine 199 is either protonated or in its δH tautomer. Under these conditons, dUMP and dCMP are both properly positioned for nucleophilic attack. In contrast, calculations with a neutral aspartic acid side chain suggest a strong discriminating power of the neutral 229 side chain in binding the two nucleotides, the preferred one depending on which of the two oxygens of the aspartate is protonated. We speculate that protonation of the aspartate 229 side chain can be the key to rationalizing why the aspartate 229 mutant selectively methylates dCMP. Finally, calculations of the valine 229 mutant demonstrate that substitution of the polar asparagine side chain with a hydrophobic residue does not result in a significant change in the location of the two nucleotides in the active site, except that dUMP seems to be better positioned for nuclephilic attack than dCMP.
1995
- Quantitative measurement of proton dissociation and tautomeric constants of apigeninidin
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; Mc, Rossi; A., Albasini
abstract
Depending on the pH of the medium, the anthocyanidin apigeninidin [5,7-dihydroxy-2-(4'-hydroxyphenyl) benzopyrilium chloride] may deprotonate, leading to the formation of three tautomeric neutral and anionic forms. In order to determine the relative percentages of the various forms which are present in solution at a given pH, the three possible derivatives of apigeninidin in which two hydroxys are replaced by methoxy groups and the three in which only one hydroxy is replaced by a methoxy group were synthesized. From the first three derivatives only one neutral form and from the second three only one anionic form can be generated by proton dissociation; the study of these compounds made possible the measurement of the proton dissociation constants of each tautomer of apigeninidin and allowed us to calculate the tautomeric ratio of its three neutral and anionic forms. Moreover, our results give evidence of the existence of a dianionic form present at pH close to neutrality (pK'(a) = 8.06). From the results obtained it is possible to calculate the composition of the mixture of the different species which can originate from the cationic form of apigeninidin at any pH by proton dissociation and tautomeric equilibria.
1995
- Theoretical analysis of the addition of hydroxylamine to uracil and 5-fluorouracil as a model for the Thymidylate synthase reaction
[Articolo su rivista]
Rastelli, Giulio; Costi, Maria Paola
abstract
In the present paper we report a quantum chemical (PM3) investigation of reagents, transition structures, intermediates and final products of the nucleophilic addition of hydroxylamine to uracil (U) and 5-fluorouracil (FU). This reaction serves as a model for the more complex enzymatic methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) by thymidylate synthase. From the analysis of the frontier orbitals of the isolated and complexed species, as well as from the calculation of activation barriers, we propose that nucleophilic attack usually proceeds after formation of an initial complex between U (or FU) and one neutral and one protonated molecule of hydroxylamine. Our results give some insight into the mechanism of these reactions and account for the higher rate of addition of hydroxylamine to FU, compared to U. The main connection between the chemical simulation and the biological scheme is that in both reactions hydrogen bonding residues are found to be necessary to assist catalysis.
1995
- Theoretical and experimental study of flavones as inhibitors of xanthine oxidase
[Articolo su rivista]
Rastelli, Giulio; Costantino, Luca; A., Albasini
abstract
Inhibitory activities of 19 polyhydroxylated and polymethoxylated flavones towards xanthine oxidase have been obtained by accurate measurements of the dissociation constants of enzyme-inhibitor complexes (K-EI). A topological description of the congeneric series has been adopted to derive quantitative structure-activity relationships (QSAR) with the use of multiple linear regression analysis. For interpretative purposes, molecular orbital calculations have also been performed. Inhibition appears to involve flavones as donors and the anion at the C-7 hydroxyl as the most active form in solution. Substituents in the 4H-1-benzopyran-4-one moiety can directly affect the availability of the C-7 anion in solution; substituents in the 2-phenyl moiety are probably involved in secondary local interactions with the enzyme.
1994
- Composition, Superoxide Radicals Scavenging and Antilipoperoxidant Activity of some Edible Fruits
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; T., Rossi; M., Bertoldi; A., Albasini
abstract
The scavenging activities against chemically-generated superoxide radicals and the antilipoperoxidant activities of the methanolic crude extracts of fruits of Ribes Rubus, Vaccinium etc. genera have been investigated. These extracts possess interesting properties, which appear to be related to their total polyphenol content.
1994
- Solvent effects on the tautomerism of apigeninidin
[Articolo su rivista]
Rastelli, Giulio; Costantino, Luca; A., Albasini
abstract
Solvent effects are found to be responsible for the predominance in water solution of a highly unstable tautomer of apigeninidin in vacuo. We present free energy perturbation in molecular dynamics simulations and self-consistent reaction field calculations of the relative solvation of the anionic tautomers of apigeninidin.
1993
- Activité Antilipoperoxydante d'Extraits Polyphenoliques de Ribes Nigrum L.
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; T., Rossi; M., Bertoldi; A., Albasini
abstract
The methanolic crude extracts of fruits, leaves and buds of Ribes nigrum L. were tested to inhibit lipid peroxdation in relation to their polyphenolic content. The polyphenols present in buds show the highest activity and therefore the buds seem to be a promising source of polyphenolic antioxidants.
1993
- Crystal and molecular structures of 5-Fluoro-3-methyl, 5-chloro-3-methyl and 5-1,3-dimethyl -uracil hydrogen bond patterns through X-ray and molecular orbital analysis
[Articolo su rivista]
G., Vampa; L., Antolini; Rastelli, Giulio; Costi, Maria Paola; P., Pecorari; M., Rinaldi
abstract
CRYSTAL AND MOLECULAR-STRUCTURES OF 5-FLUORO-3-METHYL-URACIL, 5-CHLORO-3-METHYL-URACIL AND 5-CHLORO-1,3-DIMETHYL-URACIL - HYDROGEN-BOND PATTERNS THROUGH X-RAY AND MOLECULAR-ORBITAL ANALYSIS
1993
- Physico-chemical properties of anthocyanidins. Part 1. Theoretical evaluation of the stability of the neutral and anionic tautomeric forms
[Articolo su rivista]
Rastelli, Giulio; Costantino, Luca; A., Albasini
abstract
Quantum chemical (AM1) and solvation model calculations have been applied to the study of tautomeric stabilities in three anthocyanidins, so as to single out the dominant tautomeric forms (neutral and anionic) at physiological pH. The theoretical information concerning the tautomeric stabilities and electronic structures of anthocyanidins are essential for the development of quantitative structure-activity relationships. The present results allow us to discriminate between the wide variety of tautomeric forms, and to infer that, depending on the pH, only two neutral tautomers and two anionic tautomers should be present in solution. On interpretative grounds, the main factors determining stability are chiefly ascribable to the extent of pi-electron delocalization and to the possibility of classical resonance structures. Several theoretical descriptors have been calculated for use in quantitative structure-activity relationships.
1992
- Activity of polyphenolic crude extracts as scavengers of superoxide radicals and inhibitors of xanthine oxidase
[Articolo su rivista]
Costantino, Luca; A., Albasini; Rastelli, Giulio; Benvenuti, Stefania
abstract
In view of the pharmacological interest in phenolic substances, we have determined the total amount of anthocyanins and polyphenols present in the berries of several cultivars of Ribes, Rubus, and Vaccinium genera. The in vitro antiradical activity of the crude extracts on chemically-generated superoxide radicals as well as the inhibitory activity towards the enzyme xanthine oxidase were studied. All the crude extracts examined showed a remarkably high activity towards chemically-generated superoxide radicals. The activities were greater than those expected on the basis of the quantities of anthocyanins and polyphenols present in the samples. Furthermore, the extracts showed a certain inhibitory activity towards xanthine oxidase. Ribes nigrum extracts exhibit the highest activity, being the richest in both anthocyanins and polyphenols. On the other hand, Ribes rubrum extracts seem to contain more active substances than the other crude extracts.
1992
- Inhibitory activity of flavonols toward the xanthine oxidase enzyme
[Articolo su rivista]
Costantino, Luca; Rastelli, Giulio; A., Albasini
abstract
The spectrophotometric methods of analysis adopted for the study of the inhibition of the enzyme, xanthine oxidase, are very different as regards the time required for the assay. With a view to studying the inhibitory effect of flavonoids, we examined the stability and the inhibitory activity of quercetin and myricetin in relation to environment and time. The results show that, in a buffer solution of pH 7.6 at 20-degrees-C, these flavonols undergo transformation in accordance with the environment and that their transformation products have a lower inhibitory capacity towards xanthine oxidase. The method that assesses the initial rate of formation of uric acid is therefore that which affords the most reliable results regarding the activity of flavonols; accordingly, this was the method used to determine the kinetic parameters of the enzymatic inhibition exerted by quercetin and myricetin.
1991
- Conformational analysis, molecular modeling and quantitative structure-activity relationships studies of 2,4-diamino-6,7-dimethoxy-2-substituted quinazoline α1-adrenergic antagonists
[Articolo su rivista]
Rastelli, Giulio; Fanelli, Francesca; Menziani, Maria Cristina; Cocchi, Marina; DE BENEDETTI, Pier Giuseppe
abstract
Conformational analysis (AM1), modeling of the molecular shape (QUANTA 3.0) and quantitative structure-activity relationship analysis were done on a set of 16 2,4-diamino-6,7-dimethoxy-2-substituted quinazoline alpha-1-adrenoceptor antagonists (prazosin analogs). The results obtained show that the 2-substituents of the analogs considered are quite flexible. Furthermore, they suggest that, once the electronic requirements of the common quinazoline moiety are satisfied, the binding affinities are modulated by the molecular shape of the quinazoline 2-substituent, through the optimization of both dispersive and steric interactions and the hydrophobic contribution.
1991
- MOLECULAR-ORBITAL STUDY OF THE NITROGEN BASICITY OF PRAZOSIN ANALOGS IN RELATION TO THEIR ALPHA-1-ADRENOCEPTOR BINDING-AFFINITY
[Articolo su rivista]
DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Rastelli, Giulio; Cocchi, Marina
abstract
AM1 theoretical molecular descriptors were computed for prazosin analogues (2-substituted 4-amino-6,7-dimethoxy derivatives of quinazoline, quinoline and isoquinoline) and and correlated with both their experimental acidity constants and alpha-1-adrenoceptor binding affinity data values. The results confirm the crucial role of the N1 protonated form of these derivatives for a selective and productive binding interaction with the alpha-1 adrenergic receptor.
1991
- QSAR Analysis Using Theoretical Molecular Descriptors in 2,4-Diamino-6,7-Dimethoxy Quinazoline 1-Adrenoceptor Antagonists.
[Abstract in Atti di Convegno]
DE BENEDETTI, Pier Giuseppe; Menziani, Maria Cristina; Rastelli, Giulio; Cocchi, Marina
abstract
QSAR Analysis,Theoretical Molecular Descriptors,Quinazoline,a1-Adrenoceptor Antagonists.
1990
- QSAR analysis in 2,4-diamino-6,7-dimethoxy quinoline derivatives - α1-adrenoceptor antagonists - using the partial least squares (PLS) method and theoretical molecular descriptors
[Articolo su rivista]
Cocchi, Marina; Menziani, Maria Cristina; Rastelli, Giulio; DE BENEDETTI, Pier Giuseppe
abstract
QSAR analysis in 2,4-diamino-6,7-dimethoxy quinoline derivatives - α1-adrenoceptor antagonists - using the partial least squares (PLS) method and theoretical molecular descriptors