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Filippo GENOVESE

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Pubblicazioni

2024 - Proteomics Analysis of Formalin-Fixed Paraffine-Embedded Tissue Reveals Key Proteins Related to Lung Dysfunction in Idiopathic Pulmonary Fibrosis. [Articolo su rivista]
Samarelli, ANNA VALERIA; Tonelli, Roberto; Raineri, Giulia; Bruzzi, Giulia; Andrisani, Dario; Gozzi, Filippo; Marchioni, Alessandro; Costantini, Matteo; Fabbiani, Luca; Genovese, Filippo; Pinetti, Diego; Manicardi, Linda; Castaniere, Ivana; Masciale, Valentina; Aramini, Beatrice; Tabbi', Luca; Rizzato, Simone; Bettelli, Stefania; Manfredini, Samantha; Dominici, Massimo; Clini, Enrico; Cerri, Stefania
abstract

Idiopathic pulmonary fibrosis (IPF) severely affects the lung leading to aberrant deposition of extracellular matrix and parenchymal stiffness with progressive functional derangement. The limited availability of fresh tissues represents one of the major limitations to study the molecular profiling of IPF lung tissue. The primary aim of this study was to explore the proteomic profiling yield of archived formalin-fixed paraffin-embedded (FFPE) specimens of IPF lung tissues. We further determined the protein expression according to respiratory functional decline at the time of biopsy. The total proteins isolated from 11 FFPE samples of IPF patients compared to 3 FFPE samples from a non-fibrotic lung defined as controls, were subjected to label-free quantitative proteomic analysis by liquid chromatography-mass spectrometry (LC-MS/MS) and resulted in the detection of about 400 proteins. After the pairwise comparison between controls and IPF, functional enrichment analysis identified differentially expressed proteins that were involved in extracellular matrix signaling pathways, focal adhesion and transforming growth factor β (TGF‐β) signaling pathways strongly associated with IPF onset and progression. Five proteins were significantly over-expressed in the lung of IPF patients with either advanced disease stage (Stage II) or impaired pulmonary function (FVC<75, DLco<55) compared to controls; these were lymphocyte cytosolic protein 1 (LCP1), peroxiredoxin-2 (PRDX2), transgelin 2 (TAGLN2), lumican (LUM) and mimecan (OGN) that might play a key role in the fibrogenic processes. Our work showed that the analysis of FFPE samples was able to identify key proteins that might be crucial for the IPF pathogenesis. These proteins are correlated with lung carcinogenesis or involved in the immune landscape of lung cancer, thus making possible common mechanisms between lung carcinogenesis and fibrosis progression, two pathological conditions at risk for each other in the real life.

2023 - A dual-Omics approach to identify the modulated proteins/genes in THP-1 cells infected with different drug resistant L. infantum clinical isolates. [Abstract in Atti di Convegno]
Tagliazucchi, Lorenzo; Perea-Martinez, Ana; Fiorini, Greta; Ignacio Manzano, José; García-Hernández, Raquel; Genovese, Filippo; Pinetti, Diego; Gamarro, Francisco; Costi, Maria Paola
abstract

2023 - Corrigendum to “Quantitative comparison of the protein corona of nanoparticles with different matrices” [Int J Pharm X 2022 Oct 21;4: 100136] (International Journal of Pharmaceutics: X (2022) 4, (S2590156722000251), (10.1016/j.ijpx.2022.100136)) [Articolo su rivista]
Ottonelli, I.; Duskey, J. T.; Genovese, F.; Pederzoli, F.; Caraffi, R.; Valenza, M.; Tosi, G.; Vandelli, M. A.; Ruozi, B.
abstract

: [This corrects the article DOI: 10.1016/j.ijpx.2022.100136.].

2023 - Insights into host-target interaction from the proteome modulation analysis through untargeted LC-MS/MS Proteomics of drug resistant L infantum-THP1 infected cells [Relazione in Atti di Convegno]
Tagliazucchi, Lorenzo; Malpezzi, Giulia; Perea Martinez, Ana; Ignacio Manzano, José; Garcia-Hernandez, Raquel; Pinetti, Diego; Genovese, Filippo; Thorè, Eli; Brooks, Bryan W.; Bertram, Michael G.; Gamarro, Francisco; Costi, Maria Paola
abstract

2023 - LC-MS/MS proteomics for the rapid and selective screening of drug resistances in Leishmania infantum clinical isolates [Abstract in Atti di Convegno]
Tagliazucchi, Lorenzo; Malpezzi, Giulia; Perea Martinez, Ana; Ignacio Manzano, José; Garcia-Hernandez, Raquel; Pinetti, Diego; Genovese, Filippo; Costi, Maria Paola
abstract

2023 - Label-Free Mass Spectrometry Proteomics Reveals Different Pathways Modulated in THP-1 Cells Infected with Therapeutic Failure and Drug Resistance Leishmania infantum Clinical Isolates [Articolo su rivista]
Tagliazucchi, Lorenzo; Perea-Martinez, Ana; Fiorini, Greta; Ignacio Manzano, José; Genovese, Filippo; García-Hernández, Raquel; Pinetti, Diego; GAMARRO CONDE, Francisco; Costi, Maria Paola
abstract

2022 - Destabilizers of the thymidylate synthase homodimer accelerate its proteasomal degradation and inhibit cancer growth [Articolo su rivista]
Costantino, Luca; Ferrari, Stefania; Santucci, Matteo; MH Salo-Ahen, Outi; Carosati, Emanuele; Franchini, Silvia; Lauriola, Angela; Pozzi, Cecilia; Trande, Matteo; Gozzi, Gaia; Saxena, Puneet; Cannazza, Giuseppe; Losi, Lorena; Cardinale, Daniela; Venturelli, Alberto; Quotadamo, Antonio; Linciano, Pasquale; Tagliazucchi, Lorenzo; Moschella, MARIA GAETANA; Guerrini, Remo; Pacifico, Salvatore; Luciani, Rosaria; Genovese, Filippo; Henrich, Stefan; Alboni, Silvia; Santarem, Nuno; CORDEIRO DA SILVA, Anabela; Giovannetti, Elisa; J Peters, Godefridus; Pinton, Paolo; Rimessi, Alessandro; Cruciani, Gabriele; M Stroud, Robert; C Wade, Rebecca; Mangani, Stefano; Marverti, Gaetano; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola
abstract

2022 - LC-MS/MS Helps Characterizing THP-1 Cells Proteome After Infection with Drug Resistant Leishmania Strains [Abstract in Atti di Convegno]
Tagliazucchi, Lorenzo; Perea-Martinez, Ana; Fiorini, Greta; Ignacio Manzano, José; García-Hernández, Raquel; Genovese, Filippo; Pinetti, Diego; Gamarro, Francisco; Costi, Maria Paola
abstract

2022 - Quantitative comparison of the protein corona of nanoparticles with different matrices [Articolo su rivista]
Ottonelli, Ilaria; Duskey, Jason Thomas; Genovese, Filippo; Pederzoli, Francesca; Caraffi, Riccardo; Valenza, Marta; Tosi, Giovanni; Vandelli, Maria Angela; Ruozi, Barbara
abstract

: Nanoparticles (NPs) are paving the way for improved treatments for difficult to treat diseases diseases; however, much is unknown about their fate in the body. One important factor is the interaction between NPs and blood proteins leading to the formation known as the "protein corona" (PC). The PC, consisting of the Hard (HC) and Soft Corona (SC), varies greatly based on the NP composition, size, and surface properties. This highlights the need for specific studies to differentiate the PC formation for each individual NP system. This work focused on comparing the HC and SC of three NPs with different matrix compositions: a) polymeric NPs based on poly(lactic-co-glycolic) acid (PLGA), b) hybrid NPs consisting of PLGA and Cholesterol, and c) lipidic NPs made only of Cholesterol. NPs were formulated and characterized for their physico-chemical characteristics and composition, and then were incubated in human plasma. In-depth purification, identification, and statistical analysis were then performed to identify the HC and SC components. Finally, similar investigations demonstrated whether the presence of a targeting ligand on the NP surface would affect the PC makeup. These results highlighted the different PC fingerprints of these NPs, which will be critical to better understand the biological influences of the PC and improve future NP designs.

2021 - Development and validation of a new storage procedure to extend the in-use stability of azacitidine in pharmaceutical formulations [Articolo su rivista]
Iudicello, A.; Genovese, F.; Strusi, V.; Dominici, M.; Ruozi, B.
abstract

Stability studies performed by the pharmaceutical industry are principally designed to fulfill licensing requirements. Thus, post-dilution or post-reconstitution stability data are frequently limited to 24 h only for bacteriological reasons, regardless of the true physicochemical stability which could, in many cases, be longer. In practice, the pharmacy-based centralized preparation may require preparation in advance for administration, for example, on weekends, holidays, or in general when pharmacies may be closed. We report an innovative strategy for storing resuspended solutions of azacitidine, a well-known chemotherapic agent, for which the manufacturer lists maximum stability of 22 h. By placing the syringe with the azacitidine reconstituted suspension between two refrigerant gel packs and storing it at 4 °C, we found that the concentration of azacitidine remained above 98% of the initial concentration for 48 h, and no change in color nor the physicochemical properties of the suspension were observed throughout the study period. The physicochemical and microbiological properties were evaluated by HPLC–UV and UHPLC-HRMS analysis, FTIR spectroscopy, pH determination, visual and subvisual examination, and sterility assay. The HPLC-UV method used for evaluating the chemical stability of azacitidine was validated according to ICH. Precise control of storage temperature was obtained by a digital data logger. Our study indicates that by changing the storage procedure of azacitidine reconstituted suspension, the usage window of the drug can be significantly extended to a time frame that better copes with its use in the clinical environment.

2021 - Discovery and preliminary characterization of a novel inhibitor of the SOS response in Pseudomonas aeruginosa [Abstract in Atti di Convegno]
Vascon, Filippo; Chinellato, Monica; Genovese, Filippo; Romanyuk, Zhanna; De Felice, Sofia; Tondi, Donatella; Kavaliauskas, Povilas; Cendron, Laura
abstract

Bacterial antibiotic resistance (AR) is becoming one of the biggest threats to human health, progressively disarming the current arsenal of antimicrobial drugs. Besides efforts to develop new antimicrobial agents, strategies to avoid the onset of novel resistance mechanisms are strongly needed. The bacterial SOS response to DNA damage (a common outcome of antibiotic treatment), mainly orchestrated by LexA and RecA proteins, is one of the crucial pathways involved in AR acquisition. In previous studies, the SOS response suppression has proved to be an efficient strategy to delay the appearance of drug resistance, but currently known inhibitors of the RecA-LexA axis are limited to few compounds. From a Fluorescence Polarization (FP)-based high-throughput screening of a small molecule library, a novel hit compound (hereafter “A12”) acting as inhibitor of the Pseudomonas aeruginosa SOS system was discovered. In-vitro dose-response characterization of A12 revealed an inhibitory potency in the high micromolar range, while biophysical assays including differential scanning fluorimetry (DSF) and isothermal titration calorimetry (ITC) assessed RecA as the main target. We are currently producing and screening a sub-library based on the structure of compound A12 in order to select more potent derivatives to be tested on P. aeruginosa cultures and determine their effectiveness as antibiotic adjuvants to synergize with bactericidal treatment and delay the onset of resistance.

2019 - Enamel peptides reveal the sex of the Late Antique ‘Lovers of Modena’ [Articolo su rivista]
Lugli, F.; Di Rocco, G.; Vazzana, A.; Genovese, F.; Pinetti, D.; Cilli, E.; Carile, M. C.; Silvestrini, S.; Gabanini, G.; Arrighi, S.; Buti, L.; Bortolini, E.; Cipriani, A.; Figus, C.; Marciani, G.; Oxilia, G.; Romandini, M.; Sorrentino, R.; Sola, M.; Benazzi, S.
abstract

Recent work has disclosed the critical role played by enamel peptides in sex classification of old skeletal remains. In particular, protein AMELY (amelogenin isoform Y) is present in the enamel dental tissue of male individuals only, while AMELX (isoform X) can be found in both sexes. AMELY can be easily detected by LC-MS/MS in the ion extracted chromatograms of the SM(ox)IRPPY peptide (monoisotopic [M + 2 H]+2 mass = 440.2233 m/z). In this paper, we exploited the dimorphic features of the amelogenin protein to determine the sex of the so-called ‘Lovers of Modena’, two Late Antique individuals whose skeletons were intentionally buried hand-in-hand. Upon discovery, mass media had immediately assumed they were a male-female couple, even if bad preservation of the bones did not allow an effective sex classification. We were able to extract proteins from the dental enamel of both individuals (~1600 years old) and to confidently classify them as males. Results were compared to 14 modern and archaeological control samples, confirming the reliability of the ion chromatogram method for sex determination. Although we currently have no information on the actual relationship between the ‘Lovers of Modena’ (affective? Kin-based?), the discovery of two adult males intentionally buried hand-in-hand may have profound implications for our understanding of funerary practices in Late Antique Italy.

2018 - An autoregulatory loop controls the expression of the transcription factor NF-Y [Articolo su rivista]
Belluti, Silvia; Semeghini, Valentina; Basile, Valentina; Rigillo, Giovanna; Salsi, Valentina; Genovese, Filippo; Dolfini, Diletta; Imbriano, Carol
abstract

The heterotrimeric NF-Y complex is a pioneer factor that binds to CCAAT-genes and regulates their transcription. NF-Y cooperates with multiple transcription factors and co-regulators in order to positively or negatively influence gene transcription. The recruitment of NF-Y to CCAAT box is significantly enriched in cancer-associated gene promoters loci and positively correlates with malignancy. NF-Y subunits, in particular the DNA-binding subunit NF-YA and the histone-fold subunit NF-YC, appear overexpressed in specific types of cancer. Here we demonstrate that NF-Y subunits expression is finely regulated through transcriptional and post-translational mechanisms thus allowing control over basal expression levels. NF-Y negatively regulates the transcription of the genes encoding for its subunits. DNA pull-down/affinity purification assay coupled with Mass Spectrometry identified putative co-regulators, such as Lamin A, involved in NF-YA gene transcription level. We also evidentiate how the stability of the complex is severely affected by the absence of one subunit. Our results identified for the first time one of the mechanisms responsible for NF-Y expression, which may be involved in the aberrant expression and activity observed in tumor cells and other pathological conditions.

2018 - Oxytetracycline-Protein Complex: The Dark Side of Pet Food [Articolo su rivista]
DI CERBO, Alessandro; Scarano, Antonio; Pezzuto, Federica; Guidetti, Gianandrea; Canello, Sergio; Pinetti, Diego; Genovese, Filippo; Corsi, Lorenzo
abstract

Background: Worldwide antibiotic abuse represents a huge burden, which can have a deep impact on pet and human health through nutrition and medicalization representing another way of antibiotic resistance transmission. Objective: We aimed our research to determine a possible complex formation between biological bone substrates, such as proteins, and Oxytetracycline (OTC), an approved antibiotic for use in zootechny, which might determine a toxic effect on K562 cells. Method: Cell viability and HPLC-ESI/QqToF assays were used to assess potential toxicity of bone extract derived from OTC-treated chickens according to standard withdrawal times and from untreated chickens at 24, 48 and 72h of incubation. Results: Cell culture medium with ground bone from chickens reared in the presence of OTC (OTC-CCM) resulted significantly cytotoxic at every incubation time regardless of the bone concentration while cell culture medium with ground bone from chickens reared without OTC (BIO-CCM) resulted significantly cytotoxic only after 72h of incubation. HPLC-ESI/QqToF assay ruled out the possible presence of OTC main derivatives possibly released by bone within culture medium until 1 μg/mL. Conclusion: The presence of a protein complex with OTC is able to exert a cytotoxic effect once released in the medium after 24-48h of incubation.

2018 - Proteomic and bioinformatic studies for the characterization of response to pemetrexed in platinum drug resistant ovarian cancer [Articolo su rivista]
Severi, Leda; Losi, Lorena; Fonda, Sergio; Taddia, Laura; Gozzi, Gaia; Marverti, Gaetano; Magni, Fulvio; Chinello, Clizia; Stella, Martina; Sheouli, Jalid; Braicu, Elena I.; Genovese, Filippo; Lauriola, Angela; Marraccini, Chiara; Gualandi, Alessandra; D'Arca, Domenico; Ferrari, Stefania; Costi, Maria P.
abstract

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

2018 - Qualitative and semiquantitative analysis of the protein coronas associated to different functionalized nanoparticles [Articolo su rivista]
Pederzoli, Francesca; Tosi, Giovanni; Genovese, Filippo; Belletti, Daniela; Vandelli, Maria Angela; Ballestrazzi, Antonio; Forni, Flavio; Ruozi, Barbara
abstract

The investigation on protein coronas (PCs) adsorbed onto nanoparticle (NP) surface is representing an open issue due to difficulties in detection and clear isolation of the adsorbed proteins. In this study, we investigated protocols able to isolate the compositions of PCs of three polymeric NPs.

2017 - Conveying a newly designed hydrophilic anti-human thymidylate synthase peptide to cisplatin resistant cancer cells: are pH-sensitive liposomes more effective than conventional ones? [Articolo su rivista]
Sacchetti, Francesca; D'Arca, Domenico; Genovese, Filippo; Pacifico, Salvatore; Maretti, Eleonora; Hanuskova, Miriam; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Context: LR-peptide, a novel hydrophilic peptide synthetized and characterized in previous work, is able to reduce the multi-drug resistance response in cisplatin (cDPP) resistant cancer cells by inhibiting human thymidylate synthase overexpressed in several tumors, including ovarian and colon-rectal cancers, but it is unable to enter the cells spontaneously. Objective: The aim of this work was to design and characterize liposomal vesicles as drug delivery systems for the LR peptide, evaluating the possible benefits of the pH-responsive feature in improving intracellular delivery. Materials and methods For this purpose, conventional and pH-sensitive liposomes were formulated, compared regarding their physical-chemical properties (size, PDI, morphology, in vitro stability and drug release) and studied for in vitro cytotoxicity against a cDDP-resistant cancer cells. Results and discussion Results indicated that LR peptide was successfully encapsulated in both liposomal formulations but at short incubation time only LR loaded pH-sensitive liposomes showed cell inhibition activity while for long incubation time the two kinds of liposomes demonstrated the same efficacy. Conclusions Data provide evidence that acidic pH-triggered liposomal delivery is able to significantly reduce the time required by the systems to deliver the drug to the cells without inducing an enhancement of the efficacy of the drug.

2017 - Design, synthesis and biological evaluation of non-covalent AmpC Beta-Lactamases inhibitors [Articolo su rivista]
Genovese, Filippo; Lazzari, Sandra; Venturi, Ettore; Costantino, Luca; Blazquez, Jesus; Ibacache Quiroga, · Claudia; Costi, Maria Paola; Tondi, Donatella
abstract

Bacterial resistance represents a worldwide emergency threatening the efficacy of all available antibiotics. Among the several resistance mechanisms developed by bacteria, beta-lactamases enzymes (BLs), able to inactivate most beta-lactam core antibiotics, represent a key target to block prolonging antibiotics half-life. Several approaches aimed at inhibiting BLs have been so far undertaken, mainly involving beta-lactam like or covalent inhibitors. Applying a structure based de novo design approach, we recently discovered a novel, non-covalent and competitive inhibitor of AmpC beta lactamases: it has a Ki of 1 µM, a ligand efficiency of 0.38 kcal ∙ mol–1 and lead-like physical properties. Moreover, it reverts resistance to ceftazidime in bacterial pathogens expressing AmpC and does not up-regulate beta-lactamase expression in cell culture. Its features make it a good candidate for chemical optimization: starting from its crystallographic complex with AmpC, eleven analogues were designed to complement additional AmpC sites, then synthesized and tested against clinical resistant pathogens. While the new inhibitors maintain similar in vitro activity as the starting lead, some of them exert a higher potency in in vivo tests, showing improved synergic potency with ceftazidime in resistant clinically isolated strains.

2015 - pH sensitive PEGylated Liposomes delivering active hydrophilic peptide with anticancer activity: in vitro study on cDDP-resistant ovarian cell line [Abstract in Atti di Convegno]
Sacchetti, Francesca; Marraccini, Chiara; D'Arca, Domenico; Pinetti, Diego; Genovese, Filippo; Maretti, Eleonora; Iannuccelli, Valentina; Costi, Maria Paola; Leo, Eliana Grazia
abstract

Thymidylate synthase (TS) can be considered a very interesting molecular target for the therapy of the ovarian cancer.. Recently, specific octapeptides able to reduce the growth of platinum-resistant cells by inhibiting the enzyme human thymidylate synthase (hTS), have been identified. Similarly to the majority of peptides, they cannot cross the cell membrane and require a delivery system for transport into the cells and pH sensitive liposomes, destabilizing at mildly acidic pH, are considered efficient tools for delivering water-soluble drugs into the cell cytoplasm. In the present study in order to attain the peptide triggering in the cells promoting endosomal escape, stealth pH-sensitive liposomes were developed and characterized. Results suggested that pH sensitive liposomes seemed suitable carriers for the encapsulation of small hydrophilic molecules like peptides. The appreciable difference in cytotoxicity between loaded and unloaded liposomes demonstrated that the peptide, whose activity is held in the cytoplasm, was triggered in the proper biological site.

2014 - EVALUATION OF LR STABILITY BY LC CHIP Q-TOF AND QUANTITATIVE DETERMINATION OF LR PEPTIDE CELL PENETRATION BY LC-MS/MS [Articolo su rivista]
Cazzato, ADDOLORATA STEFANIA; Cannazza, Giuseppe; Ponterini, Glauco; Marraccini, Chiara; Pirondi, Silvia; Genovese, Filippo; Costi, Maria Paola
abstract

In the present work the degradation profile of an anticancer peptide in different biological matrixes like DMEM (Dulbecco’s Modified Eagle Medium) and cell lysates by LC Chip Q-TOF was shown. Subsequently, an LC-MS/MS method for the quantitative analysis of LR in cell lysates was developed and fully validated.

2014 - Mass Spectrometric/Bioinformatic Identification of a Protein Subset That Characterizes the Cellular Activity of Anticancer Peptides [Articolo su rivista]
Genovese, Filippo; A., Gualandi; L., Taddia; Marverti, Gaetano; S., Pirondi; C., Marraccini; P., Perco; M., Pelà; R., Guerrini; M. R., Amoroso; F., Esposito; A., Martello; Ponterini, Glauco; D'Arca, Domenico; Costi, Maria Paola
abstract

The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [D-Gln4]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides’ cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.

2014 - Optimization of Peptides That Target Human Thymidylate Synthase to Inhibit Ovarian Cancer Cell Growth [Articolo su rivista]
M., Pelà; Saxena, Puneet; Luciani, Rosaria; Santucci, Matteo; Ferrari, Stefania; Marverti, Gaetano; Marraccini, Chiara; Martello, Andrea; Pirondi, Silvia; Genovese, Filippo; S., Salvadori; D'Arca, Domenico; Ponterini, Glauco; Costi, Maria Paola; R., Guerrini
abstract

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The D-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.

2013 - A proteomic approach to investigate the mechanism of action of anticancer peptides [Relazione in Atti di Convegno]
Genovese, Filippo; Gualandi, Alessandra; Taddia, Laura; Caselli, Monica; Ponterini, Glauco; Ferrari, Stefania; Marverti, Gaetano; R., Guerrini; M., Pela'; Pavesi, Giorgia; C., Trapella; Costi, Maria Paola
abstract

Many efforts to improve survival of patients affected by Ovarian Cancer (OC) have focused on more effective systemic therapies and on the search for new therapeutic targets. One of the molecular targets for OC is human Thymidylate Synthase (hTS), a homodimeric enzyme essential for DNA biosynthesis. The main goal of our research is to identify compounds able to inhibit hTS by interfering with its dimerization, without causing its over-expression and the onset of cellular drug resistance against the traditional hTStargeted compounds. We have recently discovered some peptides which specifically target the hTS dimer interface and inhibit the enzyme by stabilizing its di-inactive form [1]. These molecules have been recently investigated for their SAR profile. LR, our lead compound, inhibits the intracellular enzyme in both cisplatin (cDDP)-sensitive and -resistant ovarian cancer cells without causing protein overexpression, thus showing a potential for overcoming the limits of OC chemotherapy. This work aims at setting up a proteomic approach able to provide information on the changes in the protein expression profile induced in OC cells by treatment with LR with respect to a well-known folate antimetabolite, Pemetrexed (PTX) and identify key proteins that are involved in its mechanism of action.

2013 - Chairperson EUTROC- Mayo Conference-Berlin - Preliminary characterization of pharmacodynamic biomarkers for LR-peptide growth inhibition of ovarian cancer (OC) cell models. [Altro]
Costi, Maria Paola; Amoroso, MARIA ROSARIA; Marverti, Gaetano; Genovese, Filippo
abstract

Preliminary characterization of pharmacodynamic biomarkers for LR-peptide growth inhibition of ovarian cancer (OC) cell models. M.R.Amorosoa,b, G.Marvertib, F.Genovese, DS. Matassab, J.HEllemanc, E.Bernsc, F.Espositob*, MP Costib*. aDept. Biomedical Sciences and cDept. Life Science, Via Campi 287-183, University of Modena and Reggio Emilia, 41125, Modena, Italy; Dept. of Medical Oncology, Erasmus University Medical Center - Cancer Center, PO Box 2040, 3000 CA, Rotterdam, Netherlands. bDepartment of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, Naples 80131, Italy. LR-peptide is an inhibitor of human Thymidylate synthase (TS) through the stabilization of its inactive form as shown in drug-protein interaction experiments (1). Our previous findings demonstrated an inhibition of ovarian cancer cell growth at 2-4 μM in four cis-platin sensitive and resistant ovarian cancer cell-lines, i.e. A2780 and A2780/CP, 2008 and C13. The level of inhibition caused by LR-peptide is similar to that of paclitaxel, a first line drug in OC therapy. Mass spectrometry differential proteomic studies were performed on A2780 OC cell models in which untreated cells were examined in comparison with LR-peptide treated cells (2). Bioinformatic analysis of the results led to the identification of at least 10 proteins that were modulated upon LR-peptide treatment. Among them TRAP1 an antiapoptotic mitochondrial HSP75 protein was observed. This result was the starting point for a deeper understanding of the role of the combined TRAP1/DHFR/hTS modulation in ovarian cancer cells. We first studied the role of TRAP1 alone in OC cell models. We treated a panel of ovarian cancer cells (including IGROV1 and COV504) with paclitaxel, whose cytotoxic activity involves the activation of ER (endoplasmic reticulum) stress pathways. TRAP1 shows a protective role from ER stress, as reported previously (3). We observed that upon 1-hour exposure to a sub-lethal concentration of paclitaxel, cells expressing higher TRAP1 levels (IGROV1) showed a weak activation of ER stress sensors. such as phospho PERK, phospho eIf2alpha and Grp78/BiP. COV504 cell line express lower level of TRAP1, are more sensitive to paclitaxel treatment, showing an hyperactivation of ER stress markers. Higher concentrations of paclitaxel for longer times led to apoptotic processes as confirmed by stronger activation of the ER-stress induced caspase 12, in COV cells than in IGROV cell line. These data confirm the protective role of TRAP1 against paclitaxel induced ER stress, offering a possible mechanism of drug resistance in ovarian cancer. 1.Cardinale D. et al., PNAS 2011. 2. Proceedings EPS32 Athen 2013 p_466 3. Amoroso MR, et al.Cell Death Differ. 2012 Acknowledgement This work has been supported by AIRC-DROC 10474 project to MPC.

2013 - Fragment Based Discovery of Thymidylate Synthase Dimeric Interface Inhibitors Through Mass Spectrometry. Invited lecture to the Fragment Based Lead Discovery track. [Relazione in Atti di Convegno]
Costi, Maria Paola; Ponterini, Glauco; Ferrari, Stefania; Costantino, Luca; Franchini, Silvia; Venturelli, Alberto; Genovese, Filippo
abstract

Fragment-based drug design has been applied to Thymidylate synthase.The strategy applied is Mass Spectrometry related. identification of small molecule library that can bind to the protein represents the starting point for further drug design of a novel class of TS inhibitors with high potential as anticancer agents.

2013 - Identification and Characterization of New Proteins in Podocyte Dysfunction of Membranous Nephropathy by Proteomic Analysis of Renal Biopsy [Articolo su rivista]
Ligabue, Giulia; Magistroni, Riccardo; Marco, Cantu'; Lupo, Valentina; Genovese, Filippo; Cavazzini, Fabrizio; Luciana, Furci; Cappelli, Gianni
abstract

Interstitial fluid, obtained by gentle centrifugation of the renal biopsy specimen, is highly enriched in elements directly secreted by the kidney tissue and is suitable for proteomic analysis. Here we describe the first clinical application of renal interstitial fluid analysis in a subset of samples obtained from patients affected by idiopathic membranous nephropathy. We included in the study fifty-one patients with different pathologic diagnoses. We identified the proteomic pattern of idiopathic membranous nephropathy with mass spectrometry analysis by comparing these samples with two controls: normal kidney and IgA nephropathy. Proteomic results were validated by immunofluorescence analysis of renal tissues and Western blot of serum, urines and podocyte cell cultures. We observed an increased expression of PDZ and LIM domain protein 5 (PDLI5) and LIM domain binding protein 3 (LDB3) providing first evidence of the differential expression of these LIM domain-related proteins in kidney and urines of patients with idiopathic membranous nephropathy. Interstitial fluid can be considered a valuable biological fluid in the discovery phase of biomarkers. In order to validate its clinical use, it is pivotal to assess the availability of the biomarkers in ‘usual’ samples: blood and/or urine. PDLI5 and LDB3 share a common LIM domain suggesting a possible role in the cytoskeleton organization and they appear upregulated in glomeruli of patients affected by idiopathic membranous nephropathy. Furthermore the two proteins become highly abundant in the urine of patients affected by idiopathic membranous nephropathy. In conclusion, our approach may be considered a novel method for identifying candidate biomarkers for patients suffering from membranous nephropathy and other glomerulonephrites

2013 - Invited lecture to 18th World Congress on Advances in Oncology and 16th International Symposium on Molecular Medicine 10-12 October, 2013, Creta Maris, Hersonissos, Crete, Greece [Relazione in Atti di Convegno]
Genovese, Filippo; Gualandi, Alessandra; Marverti, Gaetano; Taddia, Laura; Glauco, Ponterini; Pirondi, Silvia; Pela', Michela; Pavesi, Giorgia; Costi, Maria Paola
abstract

Proteomic approach to the identification of early phase biomarker for anticancer peptides targeting thefolate pathway. F.Genovesea, A.Gualandia,b L.Taddiaa, G.Ponterinia, G.Marvertia, S.Pirondia, R.Guerrinic, M.Pelàa,c G.Pavesia, C.Trapellac, M.P.Costia aUniversity of Modena and Reggio Emilia, via Campi 183, 41125 Modena, Italy, bCRBA, S. Orsola University Hospital, Bologna, Italy, cDepartment of Pharmaceutical Science, University of Ferrara, Italy Many efforts to improve survival of patients affected by Ovarian Cancer (OC) have focused on more effective systemic therapies and on the search for new therapeutic targets. One of the molecular targets for OC is human Thymidylate Synthase (hTS), a homodimeric enzyme essential for DNA biosynthesis. In order to investigate the effects of hTS-interface-mimicking peptides at a cellular level, we started a study in which the cellular behavior of the peptides was investigated in combination with the proteomic differential analysis of the cytoplasmatic proteins of treated vs. untreated OC cells. The same experiment was performed with pemetrexed (PTX), a well known antifolate, for control purposes. The bioinformatic analysis of the effects of our peptide drug candidate indicates that deregulations can be mainly assigned to modulation of translational initiation, termination of RNA Pol-II transcription, transport, and protein catabolic events. Although apparently folate pathway members are not directly altered at a protein level, as the selection of ions to be sequenced is stochastic and biased towards abundant peptides, the bioinformatic analysis of peptide-modulated proteins suggested cellular investigations on the proteins of the folate-associated genes showing the largest number of dependencies to the species of the core set, which is required for the phosphorylation of several deoxyribonucleosides and nucleoside analogues. Comparison with the PTX-modulated proteins shows that some proteins of the proteasome complex and ribonucleoproteins are involved in both cases. These differences suggest that the two compounds may show a different mechanism of action which is in agreement with the hypothesized pharmacological model. Detailed cellular proteins profile based on the inferred roles of the identified proteins will further clarify the biological effects. 1.A proteomic approach to investigate the mechanism of action of anticancer peptides. F. Genovese, A. Gualandi, L. Taddia, M. Caselli, G. Ponterini, S.Ferrari, G. Marverti, R. Guerrini, M. Pela, G. Pavesi, C. Trapella, M.P. Costi, Proceeding 32EPS, p.466, ISBN 978-960-466-121-3). This work is supported by AIRC-DROC IG10474. www.unimore.airc-droc.it

2012 - Proteomic Approach to the Detection of the Mechanism of Action of Anticancer Peptides [Abstract in Rivista]
Genovese, Filippo; Gualandi, Alessandra; Taddia, Laura; Caselli, Monica; Ponterini, Glauco; Marverti, Gaetano; Pirondi, Silvia; R., Guerrini; M., Pela'; Pavesi, Giorgia; C., Trapella; Costi, Maria Paola
abstract

A label-free quantitative proteomic approach has been undertaken to study the effects of the peptide on the proteins involved in the modulated metabolic pathways, in particular those involved in the folate metabolism. Structure-activity relationships (SAR) have been performed to improve the lead peptide pharmacodynamics. All the compounds have been assayed and a protein profile set was studied to mark and validate their behavior as inhibitor of OC cell growth.

2012 - Unusual Targeting of the human Thymidylate synthase interface: a tethering approach with mass-spectrometric detection. [Relazione in Atti di Convegno]
Costi, Maria Paola; Franchini, Silvia; Ferrari, Stefania; R., Wade; S., Henrich; O., Salo; Genovese, Filippo; S., Mangani; Pozzi, Cristina; M., Santucci; Costantino, Luca; Sammak, Susan; G., Cruciani; E., Carosati; Ponterini, Glauco
abstract

Ovarian cancer is the fifth most common cause of death from cancer in women. The standard first-line treatment is a combination of paclitaxel and carboplatin (DDP) or carboplatin alone. In the case of progressive disease or drug resistance treatment with platinum, either alone or in combination, investigational compounds should be used (1). In the human ovarian carcinoma cell DDP-resistance was associated with cross-resistance to the thymidylate synthase (TS) inhibitor 5-fluorouracil (prodrug of 5FdUMP, fluorodeoxyuridine monophosphate) and methotrexate (2). We aim at restoring the sensibility to Platinum-based drugs through direct inhibition of Thymidylate synthase (TS) changing the mechanism of action from active site binders (classical TS inhibitors) to inhibitors of the regulatory function of monomeric TS through small molecule cellular perturbation. To this aim we applied a multidisciplinary approach to identify new molecules that could bind to specific pocket at the protein interface. We applied the tethering approach (3) that leads to the selection of disulfide compounds to anchor at the cystein on the monomeric interface (gray coloured balls on the left in the picture). Mass Spectrometry (MS) identification of the covalent TS-thiol complexes, and medicinal chemistry development of the identified hits (coloured structure on the right in the picture). We included cystein mutants design, site directed mutagenesis, disulfide library selection, tethering of thiol ligands at the protein interface through Mass spectrometry, X-ray crystallography, structure-based drug design and synthetic chemistry. The validation of the TS-interface inhibitor binders was accomplished trough FRET (Fluorescence resonance energy transfer) and enzyme kinetic assay. The strategies adopted and the midpoint/final results of the discovery processes will be presented. 1. Ozols RF et al. Lancet 2002;. 2. D.Cardinale et al, CMC, 2010, D.Cardinale et al, PNAS, 2011 ; 3. Stroud R et al PNAS 2000 and www.light-eu.org. The project is supported by FP6 european grant (LIGHTS, www.light-eu.org), LSH 038752 and AIRC-DROC-2012(http://www.droc-airc.unimore.it/site/home.html).

2011 - Interstitial fluid obtained from kidney biopsy as new source of renal biomarkers [Articolo su rivista]
Magistroni, Riccardo; Cantu, M.; Ligabue, Giulia; Masellis, M.; Spisni, E.; Furci, L.; Lupo, Valentina; Genovese, F.; Cavazzini, Fabrizio; Albertazzi, Alberto
abstract

Introduction: Development of renal biomarkers is required to improve on diagnostic accuracy, prognosis and prediction of response to therapy in renal disease. We describe a new method of obtaining from renal specimens a biologic fluid potentially enriched in secreted proteins. Methods: A renal biopsy specimen was centrifuged, and the interstitial fluid (IF) obtained was evaluated by SELDI-ToF, 1D and 2D gel electrophoresis. Twelve spots were extracted from the 2D gel and characterized by MALDI-TOF-MS. Results: The SELDI diagrams demonstrated abundant peptide peaks. One-dimensional gel electrophoresis demonstrated the presence of many bands indicating a diversity of proteins in the sample. Comparison of serum to IF demonstrated a number of bands that were not shared, suggesting that the IF is not a simple "replica" of plasma fluid. Employing 2D-PAGE, 418 spots were identified in the IF sample; 12 spots were selected and analyzed by mass spectrometry. Conclusions: We have described a novel technique to obtain a biologic fluid that contains a significant quantity and diversity of proteins from renal tissue. The procedure to obtain the fluid is simple and easily applicable to standard renal biopsy procedures. This fluid has the potential to identify informative proteins that are more concentrated than in any other renal biologic fluid previously analyzed and strictly related to renal pathophysiology. Future work includes the development of a clinical protocol to identify and validate informative biomarkers that have diagnostic and prognostic value.

2010 - Allosteric Inhibition of human Thymidylate Synthase. [Abstract in Atti di Convegno]
S., Henrich; O., Salo Ahen; D., Garg; Cardinale, Daniela; Ferrari, Stefania; Franchini, Silvia; Genovese, Filippo; Guaitoli, Giambattista; Lazzari, Sandra; Venturelli, Alberto; S., Mangani; Costi, Maria Paola; R. C., Wade
abstract

Thymidylate synthase (TS) takes part in the folate pathway and is a dimeric enzyme that catalyzes the conversion from dUMP to dTMP. This reaction step is essential for all cells, but especially for fast growing cancer cells, making TS an important target for cancer treatment. Most of the clinical drugs for inhibiting TS are substrate or cofactor analogues that give rise to resistance after a certain time. The aim of the LIGHTS EU project is to overcome such resistance to inhibitors by interfering with TS dimerizationusing low molecular weight compounds and peptides binding to allosteric sites.

2010 - Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescenceresonance energy transfer. [Articolo su rivista]
Genovese, Filippo; Ferrari, Stefania; Guaitoli, Giambattista; Caselli, Monica; Costi, Maria Paola; Ponterini, Glauco
abstract

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43' with an excitation energy donor/acceptor pair. The dimer-monomer equilibrium of the enzyme is then characterized through steady-state fluorescence determination of the inter-subunit resonance energy transfer efficiency.

2010 - Tethering low affinity ligands to the dimeric interface of human thymidylate synthase. [Relazione in Atti di Convegno]
Costi, Maria Paola; Genovese, Filippo; Franchini, Silvia; Venturelli, Alberto; Lazzari, Sandra; Farina, Davide Salvatore Francesco; Pirondi, Silvia; R. C., Wade; S., Mangani; C., Pozzi; S., Henrich; Ferrari, Stefania; Ponterini, Glauco; Guaitoli, Giambattista; G., Cruciani
abstract

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2009 - MALDI-TOF MS as a tool for rapid Identification of Low Protein Affinity Drug Fragments. [Abstract in Atti di Convegno]
Genovese, Filippo; Lazzari, Sandra; G., Cruciani; Franchini, Silvia; S., Henrich; R. C., Wade; Venturelli, Alberto; Costi, Maria Paola
abstract

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2008 - FRET-based assay for sensing a cancer key protein functional state and inhibition by oligopeptides: Sitespecific Protein Chemical Modifications [Abstract in Rivista]
Genovese, Filippo; M., Vargiu; M. A., Ariza Mateos; Guaitoli, Giambattista; Ferrari, Stefania; Ponterini, Glauco; Costi, Maria Paola
abstract

FRET-based assay for sensing a cancer key protein functional state and inhibition by oligopeptides: Sitespecific Protein Chemical Modifications

2008 - Merging disulphide bonds for drug design Interfering with a cancer key protein [Abstract in Atti di Convegno]
Lazzari, Sandra; Venturelli, Alberto; G., Cruciani; Franchini, Silvia; S., Henrich; Genovese, Filippo; R. C., Wade; Costi, Maria Paola
abstract

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2008 - Metodo per la funzionalizzazione sito specifica di molecole proteiche [Brevetto]
Genovese, Filippo; Ferrari, Stefania; Costi, Maria Paola; Ponterini, Glauco
abstract

Viene qui fornito un metodo in soluzione per le modifiche chimiche sito-specifiche di proteine, sfruttando l’affinità di cavità strutturate di proteine (come le cavità catalitiche degli enzimi) verso miscele di inibitori (ir)reversibili/(co)substrati o ligandi per mascherare i residui coinvolti nella formazione del complesso, permettendo la funzionalizzazione chemoselettiva di residui opportunamente selezionati al di fuori della tasca. Pertanto, grazie al mascheramento di residui di cavità strutturate, è possibile studiare ed esplorare siti allosterici a bassa affinità sia chimicamente, come porzioni funzionalizzabili, che funzionalmente. Alcune delle applicazioni in campo diagnostico, analitico e terapeutico dell’enzima ingegnerizzato risultante vengono qui discusse. Questa piattaforma di coniugazione potrebbe portare alla progettazione di un kit di coniugazione per eseguire modifiche chimiche sito-specifiche di proteine per applicazioni di ricerca, diagnostiche e terapeutiche.