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Emilia CASELLI
Professore Associato
Dipartimento Scienze della Vita sede ex Scienze Farmaceutiche Via Campi 103
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Pubblicazioni
- Acetamidomethaneboronic acids bearing the R1 side chains of beta-lactams as inhibitors of beta-lactamase enzymes
[Brevetto]
L. C., Blaszczak; Caselli, Emilia; Prati, Fabio; B. K., Shoichet
abstract
The invention provides a method of treating a .beta.-lactam-antibiotic-resistant bacterial infection with a combination of a .beta.-lactamanalog (acylaminomethaneboronic acid deriv.). The invention also provides a method of inhibiting a .beta.-lactamase comprising contacting the .beta.-lactamase with a .beta.-lactamanalog. Finally, the invention provides compds. which are .beta.-lactamanalogs and compns. contg. .beta.-lactam analogs.
- Boronic acid inhibitors of beta-lactamases as therapeutic agents in treatment of antibiotic-resistant infection diseases
[Brevetto]
Prati, Fabio; Caselli, Emilia
abstract
he invention relates to novel boronic acid compounds, a method for the preparation of such compounds, intermediate compounds for the preparation of such compounds, intermediate compounds for the use in a method for preparation of such compounds, a pharmaceutical composition, the use of one or more compounds discussed above or of a pharmaceutical composition in the manufacture of a medicament for the treatment of a bacterial infection, and a screening method.
- Sulfonamido-substituted boronic acids as β-lactamase inhibitors for treatment of antibiotic-resistant bacterial infections
[Brevetto]
Shoichet Brian, K; Prati, Fabio; Caselli, Emilia; Romagnoli, Chiara; Eidam, Oliv
abstract
Disclosed herein inter alia are Boron containing compounds and methods for treating infections related to antibiotic resistant microorganisms.
2023
- Boronic Acid Transition State Inhibitors as Potent Inactivators of KPC and CTX-M β-Lactamases: Biochemical and Structural Analyses
[Articolo su rivista]
Alsenani, Tahani A; Rodríguez, María Margarita; Ghiglione, Barbara; Taracila, Magdalena A; Mojica, Maria F; Rojas, Laura J; Hujer, Andrea M; Gutkind, Gabriel; Bethel, Christopher R; Rather, Philip N; Introvigne, Maria Luisa; Prati, Fabio; Caselli, Emilia; Power, Pablo; van den Akker, Focco; Bonomo, Robert A
abstract
Design of novel beta-lactamase inhibitors (BLIs) is one of the currently accepted strategies to combat the threat of cephalosporin and carbapenem resistance in Gram-negative bacteria. Boronic acid transition state inhibitors (BATSIs) are competitive, reversible BLIs that offer promise as novel therapeutic agents. In this study, the activities of two alpha-amido-beta-triazolylethaneboronic acid transition state inhibitors (S02030 and MB_076) targeting representative KPC (KPC-2) and CTX-M (CTX-M-96, a CTX-M-15-type extended-spectrum beta-lactamase [ESBL]) beta-lactamases were evaluated. The 50% inhibitory concentrations (IC(50)s) for both inhibitors were measured in the nanomolar range (2 to 135 nM). For S02030, the k(2)/K for CTX-M-96 (24,000 M-1 s(-1)) was twice the reported value for KPC-2 (12,000 M-1 s(-1)); for MB_076, the k(2)/K values ranged from 1,200 M-1 s(-1) (KPC-2) to 3,900 M-1 s(-1) (CTX-M-96). Crystal structures of KPC-2 with MB_076 (1.38-& ANGS; resolution) and S02030 and the in silico models of CTX-M-96 with these two BATSIs show that interaction in the CTX-M-96-S02030 and CTX-M-96-MB_076 complexes were overall equivalent to that observed for the crystallographic structure of KPC-2-S02030 and KPC-2-MB_076. The tetrahedral interaction surrounding the boron atom from S02030 and MB_076 creates a favorable hydrogen bonding network with S70, S130, N132, N170, and S237. However, the changes from W105 in KPC-2 to Y105 in CTX-M-96 and the missing residue R220 in CTX-M-96 alter the arrangement of the inhibitors in the active site of CTX-M-96, partially explaining the difference in kinetic parameters. The novel BATSI scaffolds studied here advance our understanding of structure-activity relationships (SARs) and illustrate the importance of new approaches to beta-lactamase inhibitor design.
2023
- Defining the minimum inhibitory concentration of 22 rifamycins in iron limited, physiologic medium against Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae clinical isolates
[Articolo su rivista]
Lu, Peggy; Alletto, Francesco; Lee, Bosul; Burk, Elizabeth; Martinez, Jasmine; Prati, Fabio; Caselli, Emilia; Spellberg, Brad; Luna, Brian
abstract
: Recently, we reported rifabutin hyper-activity against Acinetobacter baumannii. We sought to characterize if any additional rifamycins (n = 22) would also display hyper-activity when tested in iron-limited media against A. baumannii, K. pneumoniae, and E. coli. MICs were determined against representative clinical isolates using the iron-limited media RPMI-1640. Only rifabutin was hyperactive against A. baumannii.
2023
- Natural protein engineering in the Ω-loop: the role of Y221 in ceftazidime and ceftolozane resistance in Pseudomonas-derived cephalosporinase
[Articolo su rivista]
Mack, A. R.; Kumar, V.; Taracila, M. A.; Mojica, M. F.; O'Shea, M.; Schinabeck, W.; Silver, G.; Hujer, A. M.; Papp-Wallace, K. M.; Chen, S.; Haider, S.; Caselli, E.; Prati, F.; van den Akker, F.; Bonomo, R. A.
abstract
: A wide variety of clinically observed single amino acid substitutions in the Ω-loop region have been associated with increased minimum inhibitory concentrations and resistance to ceftazidime (CAZ) and ceftolozane (TOL) in Pseudomonas-derived cephalosporinase and other class C β-lactamases. Herein, we demonstrate the naturally occurring tyrosine to histidine substitution of amino acid 221 (Y221H) in Pseudomonas-derived cephalosporinase (PDC) enables CAZ and TOL hydrolysis, leading to similar kinetic profiles (k cat = 2.3 ± 0.2 µM and 2.6 ± 0.1 µM, respectively). Mass spectrometry of PDC-3 establishes the formation of stable adducts consistent with the formation of an acyl enzyme complex, while spectra of E219K (a well-characterized, CAZ- and TOL-resistant comparator) and Y221H are consistent with more rapid turnover. Thermal denaturation experiments reveal decreased stability of the variants. Importantly, PDC-3, E219K, and Y221H are all inhibited by avibactam and the boronic acid transition state inhibitors (BATSIs) LP06 and S02030 with nanomolar IC50 values and the BATSIs stabilize all three enzymes. Crystal structures of PDC-3 and Y221H as apo enzymes and complexed with LP06 and S02030 (1.35-2.10 Å resolution) demonstrate ligand-induced conformational changes, including a significant shift in the position of the sidechain of residue 221 in Y221H (as predicted by enhanced sampling well-tempered metadynamics simulations) and extensive hydrogen bonding between the enzymes and BATSIs. The shift of residue 221 leads to the expansion of the active site pocket, and molecular docking suggests substrates orientate differently and make different intermolecular interactions in the enlarged active site compared to the wild-type enzyme.
2023
- Sulfonamidoboronic Acids as “Cross-Class” Inhibitors of an Expanded-Spectrum Class C Cephalosporinase, ADC-33, and a Class D Carbapenemase, OXA-24/40: Strategic Compound Design to Combat Resistance in Acinetobacter baumannii
[Articolo su rivista]
Introvigne, Maria Luisa; Beardsley, Trevor J.; Fernando, Micah C.; Leonard, David A.; Wallar, Bradley J.; Rudin, Susan D.; Taracila, Magdalena A.; Rather, Philip N.; Colquhoun, Jennifer M.; Song, Shaina; Fini, Francesco; Hujer, Kristine M.; Hujer, Andrea M.; Prati, Fabio; Powers, Rachel A.; Bonomo, Robert A.; Caselli, Emilia
abstract
2023
- Synthesis of a Novel Boronic Acid Transition State Inhibitor, MB076: A Heterocyclic Triazole Effectively Inhibits Acinetobacter-Derived Cephalosporinase Variants with an Expanded-Substrate Spectrum
[Articolo su rivista]
Powers, R. A.; June, C. M.; Fernando, M. C.; Fish, E. R.; Maurer, O. L.; Baumann, R. M.; Beardsley, T. J.; Taracila, M. A.; Rudin, S. D.; Hujer, K. M.; Hujer, A. M.; Santi, N.; Villamil, V.; Introvigne, M. L.; Prati, F.; Caselli, E.; Bonomo, R. A.; Wallar, B. J.
abstract
Class C Acinetobacter-derived cephalosporinases(ADCs) represent an important target for inhibition in the multidrug-resistantpathogen Acinetobacter baumannii. ManyADC variants have emerged, and characterization of their structuraland functional differences is essential. Equally as important is thedevelopment of compounds that inhibit all prevalent ADCs despite thesedifferences. The boronic acid transition state inhibitor, MB076, a novel heterocyclic triazole with improved plasma stability, wassynthesized and inhibits seven different ADC & beta;-lactamase variantswith K (i) values MB076 acted synergistically in combination with multiple cephalosporinsto restore susceptibility. ADC variants containing an alanine duplicationin the & omega;-loop, specifically ADC-33, exhibited increased activityfor larger cephalosporins, such as ceftazidime, cefiderocol, and ceftolozane.X-ray crystal structures of ADC variants in this study provide a structuralcontext for substrate profile differences and show that the inhibitoradopts a similar conformation in all ADC variants, despite small changesnear their active sites.
2021
- A comprehensive and contemporary “snapshot” of β-lactamases in carbapenem resistant Acinetobacter baumannii
[Articolo su rivista]
Hujer, A. M.; Hujer, K. M.; Leonard, D. A.; Powers, R. A.; Wallar, B. J.; Mack, A. R.; Taracila, M. A.; Rather, P. N.; Higgins, P. G.; Prati, F.; Caselli, E.; Marshall, S. H.; Clarke, T.; Greco, C.; Venepally, P.; Brinkac, L.; Kreiswirth, B. N.; Fouts, D. E.; Bonomo, R. A.
abstract
Successful treatment of Acinetobacter baumannii infections require early and appropriate antimicrobial therapy. One of the first steps in this process is understanding which β-lactamase (bla) alleles are present and in what combinations. Thus, we performed WGS on 98 carbapenem-resistant A. baumannii (CR Ab). In most isolates, an acquired blaOXA carbapenemase was found in addition to the intrinsic blaOXA allele. The most commonly found allele was blaOXA-23 (n = 78/98). In some isolates, blaOXA-23 was found in addition to other carbapenemase alleles: blaOXA-82 (n = 12/78), blaOXA-72 (n = 2/78) and blaOXA-24/40 (n = 1/78). Surprisingly, 20% of isolates carried carbapenemases not routinely assayed for by rapid molecular diagnostic platforms, i.e., blaOXA-82 and blaOXA-172; all had ISAba1 elements. In 8 CR Ab, blaOXA-82 or blaOXA-172 was the only carbapenemase. Both blaOXA-24/40 and its variant blaOXA-72 were each found in 6/98 isolates. The most prevalent ADC variants were blaADC-30 (21%), blaADC-162 (21%), and blaADC-212 (26%). Complete combinations are reported.
2021
- Insights Into the Inhibition of MOX-1 β-Lactamase by S02030, a Boronic Acid Transition State Inhibitor
[Articolo su rivista]
Ishikawa, T.; Furukawa, N.; Caselli, E.; Prati, F.; Taracila, M. A.; Bethel, C. R.; Ishii, Y.; Shimizu-Ibuka, A.; Bonomo, R. A.
abstract
The rise of multidrug resistant (MDR) Gram-negative bacteria has accelerated the development of novel inhibitors of class A and C β-lactamases. Presently, the search for novel compounds with new mechanisms of action is a clinical and scientific priority. To this end, we determined the 2.13-Å resolution crystal structure of S02030, a boronic acid transition state inhibitor (BATSI), bound to MOX-1 β-lactamase, a plasmid-borne, expanded-spectrum AmpC β-lactamase (ESAC) and compared this to the previously reported aztreonam (ATM)-bound MOX-1 structure. Superposition of these two complexes shows that S02030 binds in the active-site cavity more deeply than ATM. In contrast, the SO3 interactions and the positional change of the β-strand amino acids from Lys315 to Asn320 were more prominent in the ATM-bound structure. MICs were performed using a fixed concentration of S02030 (4 μg/ml) as a proof of principle. Microbiological evaluation against a laboratory strain of Escherichia coli expressing MOX-1 revealed that MICs against ceftazidime are reduced from 2.0 to 0.12 μg/ml when S02030 is added at a concentration of 4 μg/ml. The IC50 and Ki of S02030 vs. MOX-1 were 1.25 ± 0.34 and 0.56 ± 0.03 μM, respectively. Monobactams such as ATM can serve as informative templates for design of mechanism-based inhibitors such as S02030 against ESAC β-lactamases.
2021
- Straightforward synthesis of chiral non-racemic α-boryl isocyanides
[Articolo su rivista]
Fini, Francesco; Zanni, Alessandro; Introvigne, Maria Luisa; Stucchi, Mattia; Caselli, Emilia; Prati, Fabio
abstract
A straightforward concise synthesis of chiral non-racemic aliphatic alpha-boryl isocyanides, relay intermediates for boron-based bioactive molecules in multicomponent reactions, is presented. The short synthetic sequence comprises as key steps copper-catalysed asymmetric borylation of imines, simultaneous nitrogen formylation/boron-protecting group interconversion and the final formamide dehydration reaction.
2020
- 1,2,3-Triazolylmethaneboronate: A Structure Activity Relationship Study of a Class of β-Lactamase Inhibitors against Acinetobacter baumannii Cephalosporinase
[Articolo su rivista]
Caselli, E.; Fini, F.; Introvigne, M. L.; Stucchi, M.; Taracila, M. A.; Fish, E. R.; Smolen, K. A.; Rather, P. N.; Powers, R. A.; Wallar, B. J.; Bonomo, R. A.; Prati, F.
abstract
Boronic acid transition state inhibitors (BATSIs) are known reversible covalent inhibitors of serine β-lactamases. The selectivity and high potency of specific BATSIs bearing an amide side chain mimicking the β-lactam's amide side chain are an established and recognized synthetic strategy. Herein, we describe a new class of BATSIs where the amide group is replaced by a bioisostere triazole; these compounds were designed as molecular probes. To this end, a library of 26 α-triazolylmethaneboronic acids was synthesized and tested against the clinically concerning Acinetobacter-derived cephalosporinase, ADC-7. In steady state analyses, these compounds demonstrated Ki values ranging from 90 nM to 38 μM (±10%). Five compounds were crystallized in complex with ADC-7 β-lactamase, and all the crystal structures reveal the triazole is in the putative amide binding site, thus confirming the triazole-amide bioisosterism. The easy synthetic access of these new inhibitors as prototype scaffolds allows the insertion of a wide range of chemical groups able to explore the enzyme binding site and provides insights on the importance of specific residues in recognition and catalysis. The best inhibitor identified, compound 6q (Ki 90 nM), places a tolyl group near Arg340, making favorable cation-π interactions. Notably, the structure of 6q does not resemble the natural substrate of the β-lactamase yet displays a pronounced inhibition activity, in addition to lowering the minimum inhibitory concentration (MIC) of ceftazidime against three bacterial strains expressing class C β-lactamases. In summary, these observations validate the α-triazolylboronic acids as a promising template for further inhibitor design.
2020
- Structural Insights into Inhibition of the Acinetobacter-Derived Cephalosporinase ADC-7 by Ceftazidime and Its Boronic Acid Transition State Analog
[Articolo su rivista]
Curtis, B. N.; Smolen, K. A.; Barlow, S. J.; Caselli, E.; Prati, F.; Taracila, M. A.; Bonomo, R. A.; Wallar, B. J.; Powers, R. A.
abstract
Extended-spectrum class C β-lactamases have evolved to rapidly inactivate expanded-spectrum cephalosporins, a class of antibiotics designed to be resistant to hydrolysis by β-lactamase enzymes. To better understand the mechanism by which Acinetobacter-derived cephalosporinase-7 (ADC-7), a chromosomal AmpC enzyme, hydrolyzes these molecules, we determined the X-ray crystal structure of ADC-7 in an acyl-enzyme complex with the cephalosporin ceftazidime (2.40 Å) as well as in complex with a boronic acid transition state analog inhibitor that contains the R1 side chain of ceftazidime (1.67 Å). In the acyl-enzyme complex, the carbonyl oxygen is situated in the oxyanion hole where it makes key stabilizing interactions with the main chain nitrogens of Ser64 and Ser315. The boronic acid O1 hydroxyl group is similarly positioned in this area. Conserved residues Gln120 and Asn152 form hydrogen bonds with the amide group of the R1 side chain in both complexes. These complexes represent two steps in the hydrolysis of expanded-spectrum cephalosporins by ADC-7 and offer insight into the inhibition of ADC-7 by ceftazidime through displacement of the deacylating water molecule as well as blocking its trajectory to the acyl carbonyl carbon. In addition, the transition state analog inhibitor, LP06, was shown to bind with high affinity to ADC-7 (Ki , 50 nM) and was able to restore ceftazidime susceptibility, offering the potential for optimization efforts of this type of inhibitor.
2020
- Structures of FOX-4 cephamycinase in complex with transition-state analog inhibitors
[Articolo su rivista]
Lefurgy, S. T.; Caselli, E.; Taracila, M. A.; Malashkevich, V. N.; Biju, B.; Papp-Wallace, K. M.; Bonanno, J. B.; Prati, F.; Almo, S. C.; Bonomo, R. A.
abstract
Boronic acid transition-state analog inhibitors (BATSIs) are partners with β-lactam antibiotics for the treatment of complex bacterial infections. Herein, microbiological, biochemical, and structural findings on four BATSIs with the FOX-4 cephamycinase, a class C β-lactamase that rapidly hydrolyzes cefoxitin, are revealed. FOX-4 is an extended-spectrum class C cephalosporinase that demonstrates conformational flexibility when complexed with certain ligands. Like other β-lactamases of this class, studies on FOX-4 reveal important insights into structure–activity relationships. We show that SM23, a BATSI, shows both remarkable flexibility and affinity, binding similarly to other β-lactamases, yet retaining an IC50 value < 0.1 μM. Our analyses open up new opportunities for the design of novel transition-state analogs of class C enzymes.
2020
- The β-Lactamase Inhibitor Boronic Acid Derivative SM23 as a New Anti-Pseudomonas aeruginosa Biofilm
[Articolo su rivista]
Peppoloni, S.; Pericolini, E.; Colombari, B.; Pinetti, D.; Cermelli, C.; Fini, F.; Prati, F.; Caselli, E.; Blasi, Elisabetta
abstract
Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great capacity to form biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), a cell-to-cell communication mechanism used by many bacteria. Selective inhibition of QS-controlled pathogenicity without affecting bacterial growth may represent a novel promising strategy to overcome the well-known and widespread drug resistance of P. aeruginosa. In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and virulence factors production by P. aeruginosa. Our results indicated that SM23: (1) inhibited biofilm development and production of several virulence factors, such as pyoverdine, elastase, and pyocyanin, without affecting bacterial growth; (2) decreased the levels of 3-oxo-C12-HSL and C4-HSL, two QS-related autoinducer molecules, in line with a dampened lasR/lasI system; (3) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; and (4) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model closely mimicking clinical settings. Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as powerful inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.
2020
- The β-lactamase Inhibitor Boronic Acid SM23 Inhibits Pseudomonas aeruginosa Biofilm Formation and Virulence Factor Production
[Poster]
Peppoloni, Samuele; Pericolini, Eva; Colombari, Bruna; Pinetti, Diego; Cermelli, Claudio; Fini, Francesco; Prati, Fabio; Caselli, Emilia; Blasi, Elisabetta
abstract
Introduction: Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often responsible of severe device-related infections, given its great ability to produce biofilm. P. aeruginosa finely regulates the expression of different virulence factors, including biofilm production, by Quorum Sensing (QS), an intercellular communication mechanism used by many bacteria. Biofilm formation
enhances bacterial resistance to antimicrobial agents due to a decreased penetration of antibiotics and a reduced rate of growth of embedded bacteria. Thus, novel agents capable of selective inhibiting biofilm formation may represent a promising strategy to overcome the well-known and widespread drug-resistance of P. aeruginosa.
Material and Methods: by using the bioluminescent P. aeruginosa strain P1242, we investigated the effects of SM23, a boronic acid derivative specifically designed as beta-lactamase inhibitor, on biofilm formation and virulence factor production by P. aeruginosa.
Results: we found that SM23: a) inhibited both biofilm formation and production of the virulence factors, pyoverdine, elastase and pyocyanin, without affecting bacterial growth; b) decreased the levels of QS-related autoinducers molecules, namely 3-oxo-C12-HSL and C4-HSL, by reducing lasR/lasI system gene expression in the biofilm; c) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; d) reduced significantly P. aeruginosa biofilm and pyoverdine production on endotracheal tubes, an in vitro condition closely mimicking clinical settings.
Discussion and Conclusions: taken together, our results indicate that, besides inhibiting beta-lactamase, the boronic acid SM23, can also act as potent inhibitor of P. aeruginosa virulence, by profoundly affecting biofilm and QS-related signals. These findings highlight potential application of this compound in the prevention and treatment of biofilm-associated P. aeruginosa infections.
2020
- The β-lactamase Inhibitor Boronic Acid SM23 as a new anti-Pseudomonas aeruginosa Biofilm Compound
[Poster]
Peppoloni, Samuele; Pericolini, Eva; Colombari, Bruna; Pinetti, Diego; Cermelli, Claudio; Fini, Francesco; Prati, Fabio; Blasi, Elisabetta; Caselli, Emilia
abstract
BACKGROUND: Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great ability to produce biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), an intercellular communication mechanism used by many bacteria. Biofilm formation can enhance bacterial resistance to antimicrobial agents due to a decreased penetration of the antibiotic and a reduced rate of bacterial cells in biofilm. Selective inhibition of biofilm formation may thus represent a novel promising strategy to overcome the well-known and widespread drug-resistance of P. aeruginosa.
METHODS: In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and production of virulence factors, using the P. aeruginosa bioluminescent strain P1242.
RESULTS: Our results indicated that SM23: a) inhibited the development of biofilm and the production of the virulence factors pyoverdine, elastase and pyocyanin, without affecting bacterial growth; b) decreased the levels of P. aeruginosa QS-related Autoinducers molecules 3-oxo-C12-HSL and C4-HSL by dampened lasR/lasI system gene expression in the biofilm; c) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; d) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model, closely mimicking the clinical settings.
CONCLUSION: Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as potent inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.
2020
- α-Triazolylboronic Acids: A Promising Scaffold for Effective Inhibitors of KPCs
[Articolo su rivista]
Introvigne, M. L.; Taracila, M. A.; Prati, F.; Caselli, E.; Bonomo, R. A.
abstract
Boronic acids are known reversible covalent inhibitors of serine β-lactamases. The selectivity and high potency of specific boronates bearing an amide side chain that mimics the β-lactam's amide side chain have been advanced in several studies. Herein, we describe a new class of boronic acids in which the amide group is replaced by a bioisostere triazole. The boronic acids were obtained in a two-step synthesis that relies on the solid and versatile copper-catalyzed azide–alkyne cycloaddition (CuAAC) followed by boronate deprotection. All of the compounds show very good inhibition of the Klebsiella pneumoniae carbapenemase KPC-2, with Ki values ranging from 1 nM to 1 μM, and most of them are able to restore cefepime activity against K. pneumoniae harboring blaKPC-2. In particular, compound 1 e, bearing a sulfonamide substituted by a thiophene ring, proved to be an excellent KPC-2 inhibitor (Ki=30 nM); it restored cefepime susceptibility in KPC-Kpn cells (MIC=0.5 μg/mL) with values similar to that of vaborbactam (Ki=20 nM, MIC in KPC-Kpn 0.5 μg/mL). Our findings suggest that α-triazolylboronates might represent an effective scaffold for the treatment of KPC-mediated infections.
2019
- Inhibition of Quorum Sensing-dependent biofilm formation and virulence factors in Pseudomonas aeruginosa by the boronic acid SM23
[Poster]
Peppoloni, S.; Pericolini, E.; Colombari, B.; Pinetti, D.; Fini, F.; Prati, F.; Blasi, E.; Caselli, E.
abstract
Introduction: Quorum sensing (QS) regulates the expression of virulence factors in P. aeruginosa. Inhibiting QS-controlled virulence factors without affecting the growth of P. aeruginosa may represent a promising strategy for overcoming its widespread and constantly increasing drug-resistance. In this study, we investigated the effects of SM23, a boronic acid, which was specifically designed as beta-lactamase inhibitor, on biofilm formation and virulence factor production by P. aeruginosa
Material and Methods: the bioluminescent P. aeruginosa strain P1242 was employed. The effect of the boronic acid SM23 on P. aeruginosa were assessed by evaluating a) the biofilm formation and its morphology by crystal violet staining/bioluminescence and confocal microscopy and b) the production in cell supernatant of the virulence factors, pyoverdines and elastase. The pyoverdine release was assessed by measuring the fluorescence emission with a multi-well fluorescence plate reader and mass spectrometry, while the elastase activity was determined by the Ohman’s method, using the Elastin-Congo red as substrate. Finally a qRT-PCR was employed to study the SM23-induced changes in the expression of the QS genes lasI and lasR.
Results: the SM23 significantly inhibited the development of biofilm and the production of virulence factors, as pyoverdines and elastase, without affecting bacterial growth. Preincubation of bacteria with P. aeruginosa-conditioned (24 h) medium completely prevented the binding of SM23 to the cells. By investigating the transcriptional changes related to QS, we found that Pseudomonas exposure to SM23 caused a notable decrease in the levels of lasI and lasR gene expression. Finally, the SM23 significantly reduced P. aeruginosa biofilm and pyoverdine production on endotracheal tubes, an in vitro condition closely mimicking clinical settings.
Discussion and Conclusions: taken together, our results indicate that boronic acid SM23, besides inhibiting beta-lactamase, can also act as potent inhibitor of QS in P. aeruginosa, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.
2018
- Deciphering the evolution of cephalosporin resistance to ceftolozane-tazobactam in pseudomonas aeruginosa
[Articolo su rivista]
Barnes, Melissa D.; Taracila, Magdalena A.; Rutter, Joseph D.; Bethel, Christopher R.; Galdadas, Ioannis; Hujer, Andrea M.; Caselli, Emilia; Prati, Fabio; Dekker, John P.; Papp-Wallace, Krisztina M.; Haider, Shozeb; Bonomo, Robert A.
abstract
Pseudomonas aeruginosa produces a class C β-lactamase (e.g., PDC-3) that robustly hydrolyzes early generation cephalosporins often at the diffusion limit; therefore, bacteria possessing these β-lactamases are resistant to many β-lactam antibiotics. In response to this significant clinical threat, ceftolozane, a 3' aminopyrazo-lium cephalosporin, was developed. Combined with tazobactam, ceftolozane promised to be effective against multidrug-resistant P. aeruginosa. Alarmingly, Ω-loop variants of the PDC β-lactamase (V213A, G216R, E221K, E221G, and Y223H) were identified in ceftolozane/tazobactam-resistant P. aeruginosa clinical isolates. Herein, we demonstrate that the Escherichia coli strain expressing the E221K variant of PDC-3 had the highest minimum inhibitory concentrations (MICs) against a panel of β-lactam antibiotics, including ceftolozane and ceftazidime, a cephalosporin that differs in structure largely in the R2 side chain. The kcat values of the E221K variant for both substrates were equivalent, whereas the Km for ceftolozane (341 ± 64 +M) was higher than that for ceftazidime (174 ± 20 +M). Timed mass spectrometry, thermal stability, and equilibrium unfolding studies revealed key mechanistic insights. Enhanced sampling molecular dynamics simulations identified conformational changes in the E221K variant Ω-loop, where a hidden pocket adjacent to the catalytic site opens and stabilizes ceftolozane for efficient hydrolysis. Encouragingly, the diazabi-cyclooctane β-lactamase inhibitor avibactam restored susceptibility to ceftolozane and ceftazidime in cells producing the E221K variant. In addition, a boronic acid transition state inhibitor, LP-06, lowered the ceftolozane and ceftazidime MICs by 8-fold for the E221K-expressing strain. Understanding these structural changes in evolutionarily selected variants is critical toward designing effective β-lactam/ β-lactamase inhibitor therapies for P. aeruginosa infections. IMPORTANCE The presence of β-lactamases (e.g., PDC-3) that have naturally evolved and acquired the ability to break down β-lactam antibiotics (e.g., ceftazidime and ceftolozane) leads to highly resistant and potentially lethal Pseudomonas aeruginosa infections. We show that wild-type PDC-3 β-lactamase forms an acyl enzyme complex with ceftazidime, but it cannot accommodate the structurally similar ceftolozane that has a longer R2 side chain with increased basicity. A single amino acid substitution from a glutamate to a lysine at position 221 in PDC-3 (E221K) causes the tyrosine residue at 223 to adopt a new position poised for efficient hydrolysis of both cephalosporins. The importance of the mechanism of action of the E221K variant, in particular, is underscored by its evolutionary recurrences in multiple bacterial species. Understanding the biochemical and molecular basis for resistance is key to designing effective therapies and developing new β-lactam/β-lactamase inhibitor combinations.
2018
- Inhibition of Acinetobacter -Derived Cephalosporinase: Exploring the Carboxylate Recognition Site Using Novel β-Lactamase Inhibitors
[Articolo su rivista]
Caselli, Emilia; Romagnoli, Chiara; Powers, Rachel A.; Taracila, Magdalena A.; Bouza, Alexandra A.; Swanson, Hollister C.; Smolen, Kali A.; Fini, Francesco; Wallar, Bradley J.; Bonomo, Robert A.; Prati, Fabio
abstract
Boronic acids are attracting a lot of attention as β-lactamase inhibitors, and in particular, compound S02030 (Ki= 44 nM) proved to be a good lead compound against ADC-7 (Acinetobacter-derived cephalosporinase), one of the most significant resistance determinants in A. baumannii. The atomic structure of the ADC-7/S02030 complex highlighted the importance of critical structural determinants for recognition of the boronic acids. Herein, to elucidate the role in recognition of the R2-carboxylate, which mimics the C3/C4found in β-lactams, we designed, synthesized, and characterized six derivatives of S02030 (3a). Out of the six compounds, the best inhibitors proved to be those with an explicit negative charge (compounds 3a-c, 3h, and 3j, Ki= 44-115 nM), which is in contrast to the derivatives where the negative charge is omitted, such as the amide derivative 3d (Ki= 224 nM) and the hydroxyamide derivative 3e (Ki= 155 nM). To develop a structural characterization of inhibitor binding in the active site, the X-ray crystal structures of ADC-7 in a complex with compounds 3c, SM23, and EC04 were determined. All three compounds share the same structural features as in S02030 but only differ in the carboxy-R2 side chain, thereby providing the opportunity of exploring the distinct binding mode of the negatively charged R2 side chain. This cephalosporinase demonstrates a high degree of versatility in recognition, employing different residues to directly interact with the carboxylate, thus suggesting the existence of a "carboxylate binding region" rather than a binding site in ADC enzymes. Furthermore, this class of compounds was tested against resistant clinical strains of A. baumannii and are effective at inhibiting bacterial growth in conjunction with a β-lactam antibiotic.
2018
- Structure-Based Analysis of Boronic Acids as Inhibitors of Acinetobacter-Derived Cephalosporinase-7, a Unique Class C β-Lactamase
[Articolo su rivista]
Bouza, Alexandra A; Swanson, Hollister C; Smolen, Kali A; Vandine, Alison L; Taracila, Magdalena A; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; Bonomo, Robert A; Powers, Rachel A; Wallar, Bradley J
abstract
Acinetobacter baumannii is a multidrug resistant pathogen that infects more than 12 000 patients each year in the US. Much of the resistance to β-lactam antibiotics in Acinetobacter spp. is mediated by class C β-lactamases known as Acinetobacter-derived cephalosporinases (ADCs). ADCs are unaffected by clinically used β-lactam-based β-lactamase inhibitors. In this study, five boronic acid transition state analog inhibitors (BATSIs) were evaluated for inhibition of the class C cephalosporinase ADC-7. Our goal was to explore the properties of BATSIs designed to probe the R1 binding site. Kivalues ranged from low micromolar to subnanomolar, and circular dichroism (CD) demonstrated that each inhibitor stabilizes the β-lactamase-inhibitor complexes. Additionally, X-ray crystal structures of ADC-7 in complex with five inhibitors were determined (resolutions from 1.80 to 2.09 Å). In the ADC-7/CR192 complex, the BATSI with the lowest Ki(0.45 nM) and greatest ΔTm(+9 °C), a trifluoromethyl substituent, interacts with Arg340. Arg340 is unique to ADCs and may play an important role in the inhibition of ADC-7. The ADC-7/BATSI complexes determined in this study shed light into the unique recognition sites in ADC enzymes and also offer insight into further structure-based optimization of these inhibitors.
2016
- A method of treating a bacterial infection in a subject in need thereof includes administering to the subject therapeutically effective amts. of at least one β-lactam antibiotic and at least one triazolylmethyl boronic acid β-lactamase inhibitor.
[Brevetto]
Bonomo, Robert; Prati, Fabio; Caselli, Emilia; Romagnoli, Chiara
abstract
A method of treating a bacterial infection in a subject in need thereof includes administering to the subject therapeutically effective amts. of at least one β-lactam antibiotic and at least one triazolylmethyl boronic acid β-lactamase inhibitor.
2016
- Boronic acid transition state inhibitors active against KPC and other class a β-lactamases: Structure-activity relationships as a guide to inhibitor design
[Articolo su rivista]
Rojas, Laura J.; Taracila, Magdalena A.; Papp Wallace, Krisztina M.; Bethel, Christopher R.; Caselli, Emilia; Romagnoli, Chiara; Winkler, Marisa L.; Spellberg, Brad; Prati, Fabio; Bonomo, Robert A.
abstract
Boronic acid transition state inhibitors (BATSIs) are competitive, reversible β-lactamase inhibitors (BLIs). In this study, a series of BATSIs with selectively modified regions (R1, R2, and amide group) were strategically designed and tested against representative class A β-lactamases of Klebsiella pneumoniae, KPC-2 and SHV-1. Firstly, the R1 group of compounds 1a to 1c and 2a to 2e mimicked the side chain of cephalothin, whereas for compounds 3a to 3c, 4a, and 4b, the thiophene ring was replaced by a phenyl, typical of benzylpenicillin. Secondly, variations in the R2 groups which included substituted aryl side chains (compounds 1a, 1b, 1c, 3a, 3b, and 3c) and triazole groups (compounds 2a to 2e) were chosen to mimic the thiazolidine and dihydrothiazine ring of penicillins and cephalosporins, respectively. Thirdly, the amide backbone of the BATSI, which corresponds to the amide at C-6 or C-7 of β-lactams, was also changed to the following bioisosteric groups: urea (compound 3b), thiourea (compound 3c), and sulfonamide (compounds 4a and 4b). Among the compounds that inhibited KPC-2 and SHV-1 β-lactamases, nine possessed 50% inhibitory concentrations (IC50s) of ≤600 nM. The most active compounds contained the thiopheneacetyl group at R1 and for the chiral BATSIs, a carboxy- or hydroxy-substituted aryl group at R2. The most active sulfonamido derivative, compound 4b, lacked an R2 group. Compound 2b (S02030) was the most active, with acylation rates (k2/K) of 1.2±0.2×104M-1 s-1 for KPC-2 and 4.7±0.6×103M-1 s-1 for SHV-1, and demonstrated antimicrobial activity against Escherichia coli DH10B carrying blaSHV variants and blaKPC-2 or blaKPC-3 and against clinical strains of Klebsiella pneumoniae and E. coli producing different class A β-lactamase genes. At most, MICs decreased from 16 to 0.5 mg/liter.
2016
- Crystal structures of KPC-2 and SHV-1 β-lactamases in complex with the boronic acid transition state analog S02030
[Articolo su rivista]
Nguyen, Nhu Q.; Krishnan, Nikhil P.; Rojas, Laura J.; Prati, Fabio; Caselli, Emilia; Romagnoli, Chiara; Bonomo, Robert A.; Van Den Akker, Focco
abstract
Resistance to expanded-spectrum cephalosporins and carbapenems has rendered certain strains of Klebsiella pneumoniae the most problematic pathogens infecting patients in the hospital and community. This broad-spectrum resistance to β-lactamases emerges in part via the expression of KPC-2 and SHV-1 β-lactamases and variants thereof. KPC-2 carbapenemase is particularly worrisome, as the genetic determinant encoding this β-lactamase is rapidly spread via plasmids. Moreover, KPC-2, a class A enzyme, is difficult to inhibit with mechanism-based inactivators (e.g., clavulanate). In order to develop new β-lactamase inhibitors (BLIs) to add to the limited available armamentarium that can inhibit KPC-2, we have structurally probed the boronic acid transition state analog S02030 for its inhibition of KPC-2 and SHV-1. S02030 contains a boronic acid, a thiophene, and a carboxyl triazole moiety. We present here the 1.54- and 1.87-Å resolution crystal structures of S02030 bound to SHV-1 and KPC-2 β-lactamases, respectively, as well as a comparative analysis of the S02030 binding modes, including a previously determined S02030 class C ADC-7 β-lactamase complex. S02030 is able to inhibit vastly different serine β-lactamases by interacting with the conserved features of these active sites, which includes (i) forming the bond with catalytic serine via the boron atom, (ii) positioning one of the boronic acid oxygens in the oxyanion hole, and (iii) utilizing its amide moiety to make conserved interactions across the width of the active site. In addition, S02030 is able to overcome more distantly located structural differences between the β-lactamases. This unique feature is achieved by repositioning the more polar carboxyl-triazole moiety, generated by click chemistry, to create polar interactions as well as reorient the more hydrophobic thiophene moiety. The former is aided by the unusual polar nature of the triazole ring, allowing it to potentially form a unique COH...O 2.9-Å hydrogen bond with S130 in KPC-2.
2015
- Click Chemistry in Lead Optimization of Boronic Acids as β-Lactamase Inhibitors
[Articolo su rivista]
Caselli, Emilia; Romagnoli, Chiara; Vahabi, Roza; Taracila, Magdalena A; Bonomo, Robert A; Prati, Fabio
abstract
Boronic acid transition-state inhibitors (BATSIs) represent one of the most promising classes of β-lactamase inhibitors. Here we describe a new class of BATSIs, namely, 1-amido-2-triazolylethaneboronic acids, which were synthesized by combining the asymmetric homologation of boronates with copper-catalyzed azide-alkyne cycloaddition for the stereoselective insertion of the amido group and the regioselective formation of the 1,4-disubstituted triazole, respectively. This synthetic pathway, which avoids intermediate purifications, proved to be flexible and efficient, affording in good yields a panel of 14 BATSIs bearing three different R1 amide side chains (acetamido, benzylamido, and 2-thienylacetamido) and several R substituents on the triazole. This small library was tested against two clinically relevant class C β-lactamases from Enterobacter spp. and Pseudomonas aeruginosa. The Ki value of the best compound (13a) was as low as 4 nM with significant reduction of bacterial resistance to the combination of cefotaxime/13a.
2015
- Inhibiting the β-Lactamase of Mycobacterium tuberculosis (Mtb) with Novel Boronic Acid Transition-State Inhibitors (BATSIs)
[Articolo su rivista]
Kurz, Sebastian G.; Hazra, Saugata; Bethel, Christopher R.; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; Blanchard, John S.; Bonomo, Robert A.
abstract
BlaC, the single chromosomally-encoded β-lactamase of Mycobacterium tuberculosis, has been identified as a promising target for novel therapies that rely upon β-lactamase inhibition. Boronic acid transition state inhibitors (BATSIs) are a class of β-lactamase inhibitors which permit rational inhibitor design by combinations of various R1 and R2 side chains. To explore the structural determinants of effective inhibition, we screened a panel of 25 BATSIs synthesized to explore key structure-function relationships. We identified a cefoperazone analogue, EC19, which displayed slow, tight-binding inhibition against BlaC. To further characterize the molecular basis of inhibition, we solved the three-dimensional structure of the EC19-BlaC complex and expanded our analysis to variant enzymes. The results of this structure-function analysis encourage the design of a novel class of β-lactamase inhibitors, BATSIs, to be used against Mycobacterium tuberculosis.
2015
- Negative Epistasis and Evolvability in TEM-1 β-Lactamase—The Thin Line between an Enzyme's Conformational Freedom and Disorder
[Articolo su rivista]
Dellus Gur, Eynat; Elias, Mikael; Tawfik, Dan S; Caselli, Emilia; Prati, Fabio; Salverda, Merijn L. M; De Visser, J. Arjan G. M; Fraser, James S.
abstract
Epistasis is a key factor in evolution since it determines which combinations of mutations provide adaptive solutions and which mutational pathways toward these solutions are accessible by natural selection. There is growing evidence for the pervasiveness of sign epistasis—a complete reversion of mutational effects, particularly in protein evolution—yet its molecular basis remains poorly understood. We describe the structural basis of sign epistasis between G238S and R164S, two adaptive mutations in TEM-1 β-lactamase— an enzyme that endows antibiotics resistance. Separated by 10 Å, these mutations initiate two separate trajectories toward increased hydrolysis rates and resistance toward second and third-generation cephalosporins antibiotics. Both mutations allow the enzyme's active site to adopt alternative conformations and accommodate the new antibiotics. By solving the corresponding set of crystal structures, we found that R164S causes local disorder whereas G238S induces discrete conformations. When combined, the mutations in 238 and 164 induce local disorder whereby nonproductive conformations that perturb the enzyme's catalytic preorganization dominate. Specifically, Asn170 that coordinates the deacylating water molecule is misaligned, in both the free form and the inhibitor-bound double mutant. This local disorder is not restored by stabilizing global suppressor mutations and thus leads to an evolutionary cul-de-sac. Conformational dynamism therefore underlines the reshaping potential of protein's structures and functions but also limits protein evolvability because of the fragility of the interactions networks that maintain protein structures.
2015
- Synthesis of [(1,2,3-Triazol-1-yl)methyl]boronic Acids Through Click Chemistry: Easy Access to a Potential Scaffold for Protease Inhibitors
[Articolo su rivista]
Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio
abstract
Stereoselective synthesis of previously unreported [(1,2,3-triazol-1-yl)methyl]boronic acids has been achieved from azidomethylboronates by copper-catalyzed azide–alkyne cycloaddition reaction. The proximity of the cycloaddition reaction center to the boronic group is not detrimental to the stability of the sp3 C–B bond or to the stereoisomeric composition, which further expands the field of application of click chemistry to new boronate substrates and offers a new potential scaffold for protease inhibitors.
2014
- Biochemical and structural analysis of inhibitors targeting the ADC-7 cephalosporinase of Acinetobacter baumannii
[Articolo su rivista]
R. A., Powers; H. C., Swanson; M. A., Taracila; N. W., Florek; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; R. A., Bonomo; B. J., Wallar
abstract
Beta-lactam resistance in Acinetobacter baumannii presents one of the greatest challenges to contemporary antimicrobial chemotherapy. Much of this resistance to cephalosporins derives from the expression of the class C β-lactamase enzymes, known as Acinetobacter-derived cephalosporinases (ADCs).
Currently, beta-lactamase inhibitors are structurally similar to beta-lactam substrates and are not effective inactivators of this class C cephalosporinase. Herein, two boronic acid transition state inhibitors
(BATSIs S02030 and SM23) that are chemically distinct from beta-lactams were designed and tested for inhibition of ADC enzymes. BATSIs SM23 and S02030 bind with high affinity to ADC-7, a chromosomal cephalosporinase from Acinetobacter baumannii (Ki = 21.1 ± 1.9 nM and 44.5 ± 2.2 nM, respectively). The X-ray crystal structures of ADC-7 were determined in both the apo form (1.73 Å resolution) and in complex with S02030 (2.0 Å resolution). In the complex, S02030 makes several canonical interactions: the O1 oxygen of S02030 is bound in the oxyanion hole, and the R1 amide group makes key interactions with conserved residues Asn152 and Gln120. In addition, the carboxylate group of the inhibitor is meant to mimic the C3/C4 carboxylate found in beta-lactams. The C3/C4 carboxylate recognition site in class C enzymes is comprised of Asn346 and Arg349 (AmpCnumbering), and these residues are conserved in ADC-7. Interestingly, in the ADC-7/S02030 complex, the inhibitor carboxylate group is observed to interact with Arg340, a residue that distinguishes ADC-7 from the related class C enzyme AmpC. A thermodynamic analysis suggests that DH driven compounds
may be optimized to generate new lead agents. The ADC-7/BATSI complex provides insight into recognition of non-beta-lactam inhibitors by ADC enzymes and offers a starting point for the structure based
optimization of this class of novel beta-lactamase inhibitors against a key resistance target.
2013
- Design and exploration of novel boronic acid Inhibitors reveals important Interactions with a clavulanic acid-resistant sulfhydryl-
variable (SHV) β‑ lactamase
[Articolo su rivista]
Marisa L., Winkler; Elizabeth A., Rodkey; Magdalena A., Taracila; Sarah M., Drawz; Christopher R., Bethel; Krisztina M., Papp Wallace; Kerri M., Smith; Yan, Xu; Jeffrey R., Dwulit Smith; Romagnoli, Chiara; Caselli, Emilia; Prati, Fabio; Focco van den, Akker; Robert A., Bonomo
abstract
Inhibitor resistant (IR) class A β -lactamases pose a signi fi cant threat to
many current antibiotic combinations. The K234R substitution in the SHV β -lactamase,
from Klebsiella pneumoniae, results in resistance to ampicillin/clavulanate. After site-
saturation mutagenesis of Lys-234 in SHV, microbiological and biochemical character-
ization of the resulting β -lactamases revealed that only − Arg conferred resistance to
ampicillin/clavulanate. X-ray crystallography revealed two conformations of Arg-234 and
Ser-130 in SHV K234R. The movement of Ser-130 is the principal cause of the observed
clavulanate resistance. A panel of boronic acid inhibitors was designed and tested against
SHV-1 and SHV K234R. A chiral ampicillin analogue was discovered to have a 2.4 ± 0.2
nM K i for SHV K234R; the chiral ampicillin analogue formed a more complex hydrogen-
bonding network in SHV K234R vs SHV-1. Consideration of the spatial position of Ser-
130 and Lys-234 and this hydrogen-bonding network will be important in the design of
novel antibiotics targeting IR β -lactamases.
2013
- Interactions of Oxyimino-Substituted Boronic Acids and β-Lactams with the CMY-2-Derived Extended-Spectrum Cephalosporinases CMY-30 and CMY-42
[Articolo su rivista]
S., Kotsakis; Caselli, Emilia; L., Tzouvelekis; E., Petinaki; Prati, Fabio; V., Miriagou
abstract
CMY-30 and CMY-42 are extended-spectrum (ES) derivatives of CMY-2. ES characteristics are due to substitutions of Gly (CMY-
30) and Ser (CMY-42) for Val211 in the -loop. To characterize the effects of 211 substitutions, we studied the interactions of
CMY-2, -30, and -42 with boronic acid transition state inhibitors (BATSIs) resembling ceftazidime and cefotaxime, assessed
thermal stability of the enzymes in their free forms and in complexes with BATSIs and oximino- -lactams, and simulated, using
molecular dynamics (MD), the CMY-42 apoenzyme and the CMY-42 complexes with ceftazidime and the ceftazidime-like
BATSI. Inhibition constants showed that affinities between CMY-30 and CMY-42 and the R1 groups of BATSIs were lower than
those of CMY-2. ES variants also exhibited decreased thermal stability either as apoenzymes or in covalent complexes with oxi-
mino compounds. MD simulations further supported destabilization of the ES variants. Val211Ser increased thermal factors of
the -loop backbone atoms, as previously observed for CMY-30. The similar effects of the two substitutions seemed to be due to
a less-constrained Tyr221 likely inducing concerted movement of elements at the edges of the active site ( -loop–Q120 loop–R2
loop/H10 helix). This inner-protein movement, along with the wider R1 binding cleft, enabled intense vibrations of the cova-
lently bound ceftazidime and ceftazidime-like BATSIs. Increased flexibility of the ES enzymes may assist the productive adapta-
tion of the active site to the various geometries of the oximino substrates during the reaction (higher frequency of near-attack
conformations).
2012
- Fragment-guided Design of Subnanomolar β-Lactamase Inhibitors Active in vivo
[Articolo su rivista]
O., Eidam; Romagnoli, Chiara; G., Dalmasso; S., Barelier; Caselli, Emilia; R., Bonnet; B. K., Shoichet; Prati, Fabio
abstract
Fragment-based design was used to guide derivatization of a lead series of β-lactamase inhibitors that had heretofore resisted optimization for in vivo activity. X-ray structures of fragments overlaid with the lead suggested new, unanticipated functionality and points of attachment. Synthesis of three derivatives improved affinity over 20-fold and improved efficacy in cell culture. Crystal structures were consistent with the fragment-based design, enabling further optimization to a Ki of 50 pM, a 500-fold improvement that required the synthesis of only six derivatives. One of these, compound 5, was tested in mice. Whereas cefotaxime alone failed to cure mice infected with β-lactamase-expressing E. coli, 65% were cleared of infection when treated with a cefotaxime:5 combination. Fragment complexes offer a path around design hurdles, even for advanced molecules; the series described here may provide leads to overcome β-lactamase-based resistance, a key clinical challenge.
2011
- Exploring sequence requirements for C3/C4 carboxylate recognition in the Pseudomonas aeruginosa cephalosporinase: Insights into plasticity of the AmpC β-lactamase
[Articolo su rivista]
Sarah M., Drawz; Magdalena, Taracila; Caselli, Emilia; Prati, Fabio; Robert A., Bonomo
abstract
Class C β-lactamases (AmpC cephalosporinases) contribute significantly to β-lactam treatment failure of infections caused by the clinically relevant Gram-negative pathogen Pseudomonas aeruginosa. Despite years of study of these important β-lactamases, the structure-function relationships of this enzyme are not well understood. An understanding of structure-function relationships is crucial to the design of both substrates and enzyme inhibitors to achieve high affinity but escape hydrolysis. To this end, we examined a molecular model of a P. aeruginosa AmpC β-lactamase, PDC-3, in complex with a boronate inhibitor and generated alanine variants at three putative C3/C4 β-lactam carboxylate binding amino acids. A panel of nine penicillin and cephalosporin analog boronates was synthesized to serve as active site probes of the PDC-3 enzyme and the Arg349Ala variant. In conjunction with substrate susceptibility testing, these boronates reveal important features about the specificity of the P. aeruginosa AmpC enzyme. Namely, this β-lactamase maintains a high level of activity despite the substitution of C3/C4 β-lactam carboxylate recognition residues. Unlike class A serine β-lactamases, our work indicates that PDC-3 possesses a “binding region” rather than specific binding amino acids. The multiplicity of regions interacting with the C3/C4 carboxylate has implications for the evolution and resistance profile of this enzyme.
2010
- Design, synthesis, crystal structures and antimicrobial activity of sulfonamide boronic acids as beta-lactamase inhibitors
[Articolo su rivista]
Oliv, Eidam; Romagnoli, Chiara; Caselli, Emilia; Joel, Karpiak; Kerim, Babaoglu; Denise Teotico, Pohlhaus; Richard, Bonnet; Brian K., Shoichet; Prati, Fabio
abstract
β-Lactamase-mediated resistance to β-lactam antibiotics threatens our antibiotic armamentarium. We investigated a new series of sulfonamide boronic acids against AmpC, a class C beta-lactamase that confers extended spectrum resistance to beta-lactams worldwide. Previously, we had explored transition-state analog carboxamide boronic acids and another, unrelated series of non-covalent inhibitors bearing sulfonamide side chains. Merging these two series resulted in boronic acids that differed in the replacement of the canonical carboxamide, which was inserted to mimic the amide in C6/C7 of peniccilins and cephalosporins, with a sulfonamide. Surprisingly, these sulfonamides had a highly distinct SAR from the carboxamides, with Ki values 23 times better for small analogs and Ki values 10 to 20 times worse than the carboxamide congener for larger molecules. X-ray crystal structures of three of the new sulfonamides explain this dramatically changed SAR. The most potent of these beta-lactamase inhibitors were tested in cell culture and showed good ability to reverse beta-lactamase mediated resistance to third generation cephalosporins.
2010
- Exploring novel inhibitors of FOX-4 Class C β-lactamase
[Abstract in Atti di Convegno]
S., Mallo; C., Bethel; F., Perez llarena; Prati, Fabio; Caselli, Emilia; M., Page; G., Bou; R., Bonomo
abstract
-------
2010
- Inhibition of the Class C β-Lactamase from Acinetobacter spp.: Insights into Effective Inhibitor Design
[Articolo su rivista]
S. M., Drawz; M., Babic; C. R., Bethel; M., Taracila; A. M., Distler; Ori, Claudia; Caselli, Emilia; Prati, Fabio; R. A., Bonomo
abstract
The need to develop β-lactamase inhibitors against class C cephalosporinases of Gram-negative pathogens represents an urgent clinical priority. To respond to this challenge, five boronic acid derivatives, including a new cefoperazone analogue, were synthesized and tested against the class C cephalosporinase ofAcinetobacter baumannii [Acinetobacter-derived cephalosporinase (ADC)]. The commercially availablecarbapenem antibiotics were also assayed. In the boronic acid series, a chiral cephalothin analogue with ameta-carboxyphenyl moiety corresponding to the C3/C4 carboxylate of β-lactams showed the lowest Ki (11 (1 nM). In antimicrobial susceptibility tests, this cephalothin analogue lowered the ceftazidime and cefotaximeminimum inhibitory concentrations (MICs) of Escherichia coli DH10B cells carrying blaADC from 16 to 4 μg/mL and from 8 to 1 μg/mL, respectively. On the other hand, each carbapenem exhibited a Ki of <20 μM, andtimed electrospray ionization mass spectrometry (ESI-MS) demonstrated the formation of adducts correspondingto acyl-enzyme intermediates with both intact carbapenem and carbapenem lacking the C6hydroxyethyl group. To improve our understanding of the interactions between the β-lactamase and theinhibitors, we constructed models of ADC as an acyl-enzyme intermediate with (i) the meta-carboxyphenylcephalothin analogue and (ii) the carbapenems, imipenem and meropenem. Our first model suggests that thischiral cephalothin analogue adopts a novel conformation in the β-lactamase active site. Further, the additionof the substituent mimicking the cephalosporin dihydrothiazine ring may significantly improve affinity for theADCβ-lactamase. In contrast, theADC-carbapenem models offer a novel role for theR2 side group and alsosuggest that elimination of the C6 hydroxyethyl group by retroaldolic reaction leads to a significantconformational change in the acyl-enzyme intermediate. Lessons from the diverse mechanisms andstructures of the boronic acid derivatives and carbapenems provide insights for the development of newβ-lactamase inhibitors against these critical drug resistance targets.
2010
- One-Pot Synthesis of Imidazole-4-Carboxylates by Microwave-Assisted1,5-Electrocyclization of Azavinyl Azomethine Ylides
[Articolo su rivista]
Preti, Lisa; Orazio A., Attanasi; Caselli, Emilia; Gianfranco, Favi; Ori, Claudia; Davoli, Paolo; Fulvia, Felluga; Prati, Fabio
abstract
Diversely functionalized imidazole-4-carboxylates were synthesized by microwave-assisted 1,5-eletrocyclization of 1,2-diaza-1,3-diene-derived azavinyl azomethine ylides. 1,2-Diaza-1,3-dienes were treated with primary aliphatic or aromatic amines and subjected to microwave irradiation in the presence of aldehydes. 3-Alkyl- and 3-arylimidazole-4-carboxylates were prepared in good yields through a one-pot multicomponent procedure. Modulation of the substituents at C-2, N-3 and C-5 was possible, and 2-unsubstituted imidazoles were obtained when paraformaldehyde was used.
2009
- Structural Studies of Inhibition of β-Lactamases by Boronic Acid Transition State Analogs and a Penam Sulfone
[Abstract in Atti di Convegno]
Ke, W.; Caselli, Emilia; Bethel, C.; Bunjak, J.; Prati, Fabio; Bonomo, R. A.
abstract
2009
- The role of a second-shell residue in modifying substrate and inhibitor interactions in the SHV β-lactamase: A study of Ambler position Asn276
[Articolo su rivista]
S. M., Drawz; C. R., Bethel; K. H., Hujer; K. N., Hurless; A. M., Distler; Caselli, Emilia; Prati, Fabio; R. A., Bonomo
abstract
Inhibitor-resistant class A β-lactamases of the TEM and SHV families that arise by single aminoacid substitutions are a significant threat to the efficacy of β-lactam/β-lactamase inhibitor combinations. Tobetter understand the basis of the inhibitor-resistant phenotype in SHV, we performed mutagenesis toexamine the role of a second-shell residue, Asn276. Of the 19 variants expressed in Escherichia coli, only theAsn276Asp enzyme demonstrated reduced susceptibility to ampicillin/clavulanate (MIC increased from 50/2f 50/8 μg/mL) while maintaining high-level resistance to ampicillin (MIC = 8192 μg/mL). Steady-statekinetic analyses of Asn276Asp revealed slightly diminished kcat/Km for all substrates tested. In contrast, weobserved a 5-fold increase in Ki for clavulanate (7.4(0.9 μMfor Asn276Asp vs 1.4(0.2 μMfor SHV-1) and a40% reduction in kinact/KI (0.013 ( 0.002 μM-1 s-1 for Asn276Asp vs 0.021 ( 0.004 μM-1 s-1 for SHV-1).Timed electrospray ionization mass spectrometry of clavulanate-inhibited SHV-1 and SHV Asn276Aspshowed nearly identical mass adducts, arguing for a similar pathway of inactivation. Molecular modelingshows that novel electrostatic interactions are formed between Arg244Nη2 and both 276AspOδ1 and Oδ2;these new forces restrict the spatial position of Arg244, a residue important in the recognition of the C3/C4carboxylate of β-lactam substrates and inhibitors. Testing the functional consequences of this interaction, wenoted considerable free energy costs (+ΔΔG) for substrates and inhibitors. A rigid carbapenem (meropenem)was most affected by the Asn276Asp substitution (46-fold increase in Ki vs SHV-1). We conclude that residue276 is an important second-shell residue in class A β-lactamase-mediated resistance to substrates andinhibitors, and only Asn is able to precisely modulate the conformational flexibility of Arg244 required forsuccessful evolution in nature.
2009
- α-Aminoester-Derived Imidazoles by 1,5-Electrocyclization of Azavinyl Azomethine Ylides
[Articolo su rivista]
O. A., Attanasi; Caselli, Emilia; Davoli, Paolo; G., Favi; F., Mantellini; Ori, Claudia; Prati, Fabio
abstract
An efficient and practical method for the preparation of α-imidazol-1-yl esters from 1,2-diaza-1,3-dienes (DDs), α-amino esters and aldehydes is described. The overall sequence features a Michael-type conjugate addition between the α-amino ester and the DD, followed by iminium ion formation via condensation with the aldehyde and 1,5-electrocyclization of the resulting thermally-generated azavinyl azomethine ylide to afford eventually α-imidazol-1-yl esters. Such a protocol allows the access to enantiomerically pure imidazoles from optically pure α-amino esters.
2008
- Structure-based optimization of cephalotin analogue boronic acids as beta-lactamase inhibitors
[Articolo su rivista]
Morandi, Stefania; Morandi, Federica; Caselli, Emilia; B. K., Shoichet; Prati, Fabio
abstract
Boronic acids have proved to be promising selective inhibitors of beta-lactamases, acting as transition state analogues. Starting from a previously described nanomolar inhibitor of AmpC beta-lactamase, three new inhibitors were designed to gain interactions with highly conserved residues, such as Asn343, and to bind more tightly to the enzyme. Among these, one was obtained by stereoselective synthesis and succeeded in placing its anionic group into the carboxylate binding site of the enzyme, as revealed by X-ray crystallography of the complex inhibitor/AmpC. Nevertheless, it failed at improving affinity, when compared to the lead from which it was derived. The origins of this structural and energetic discrepancy are discussed.
2007
- Using Boronic Acid Transition State Inhibitors to understand the extended spectrum (ES) and carbapenemase phenotypes of class A beta-lactamases in Klebsiella pneumoniae
[Abstract in Atti di Convegno]
BETHEL C., R; Prati, Fabio; Caselli, Emilia; Bonomo, R. A.
abstract
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2005
- Enantiomeric excess of 1,2-diols by formation of cyclic boronates: an improved method
[Articolo su rivista]
Morandi, Stefania; Caselli, Emilia; Forni, Arrigo; Bucciarelli, Maria; Torre, Giovanni; Prati, Fabio
abstract
A reliable method for determining the enantiomeric composition of 1,2-diols by the formation of diastereomeric cyclic
esters with boronic acid is described. Starting from a previously reported structure of boronic chiral derivatizing agent (CDA), seven
structurally related racemic CDAs were synthesized and their discriminating ability towards diols measured. The most promising
amongst these was synthesized in its enantiomerically pure form according to Mattesons protocol for the stereoselective homologation
of pinanediol boronates; this CDA quantitatively and rapidly reacts with 1,2-diols in very mild conditions affording a
couple of diastereoisomers, whose composition can be determined via 1H NMR analysis. In particular, an attractive feature is that
the resonance used for the analysis originated from the CDA as a couple of baseline-separated singlets (Dd up to 0.3 ppm) is useful
for integration.
2004
- Biocatalytic asymmetric synthesis of (S)- and (R)-Timolol
[Articolo su rivista]
Tosi, Giovanni; F., Zironi; Caselli, Emilia; Forni, Arrigo; Prati, Fabio
abstract
A new biocatalytic route for the synthesis of both enantiomers of Timolol (1) is described. Starting from 3,4-dichloro-1,2,5-thiadiazole (2), (R)- and (S)-Timolol (87% ee) were obtained in 35% and 30% overall yield, respectively. Asymmetric reduction of the intermediate haloketone 5 with baker's yeast afforded the corresponding halohydrin 6 in the optically active form (87% ee), which gave the R enantiomer (distomer) of Timolol. The S enantiomer (eutomer) was obtained via inversion of configuration of the halohydrin following the Mitsunobu procedure.
2003
- (S)-(+)-N-acetylphenylglycineboronic acid: A chiral derivatizing agent for Ee determination of 1,2-diols
[Articolo su rivista]
Caselli, Emilia; Danieli, Chiara; Morandi, Stefania; B., Bonfiglio; Forni, Arrigo; Prati, Fabio
abstract
A new chiral derivatizing agent for ee determination of 1,2-diols via H-1 NMR is described. (S)-(+)-N-Acetylphenylglycineboronic acid (1) is synthesized in enantiomerically pure form; its reaction with chiral diols quantitatively yields cyclic boronic esters 5a-g. The latter show a remarkably high diastereodifferentiation of proton NMR signals useful for de determination.
2003
- Acido alfa-Acetammido-alfa-Fenilmetanboronico come Agente Derivatizzante Chirale per la Determinazione dell’Eccesso Enantiomerico di 1,2-Dioli
[Abstract in Atti di Convegno]
B., Bonfiglio; Caselli, Emilia; Forni, Arrigo; Danieli, Chiara; Prati, Fabio
abstract
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2003
- Chemo-enzymatic synthesis of levodropropizine
[Articolo su rivista]
Caselli, Emilia; Tosi, Giovanni; Forni, Arrigo; Bucciarelli, Maria; Prati, Fabio
abstract
Levodropropizine, an antitussive drug, was prepared in high enantiomeric excess in three steps, starting from dichloroacetone (2).Monosubstitution of 2 with sodium benzoate and subsequent baker’s yeast reduction stereoselectively afforded the correspondingchlorohydrin in 73% ee, which was converted to levodropropizine and enantiomerically enriched up to 95% ee by fractionalcrystallisation
2003
- Nanomolar inhibitors of AmpC beta-lactamase
[Articolo su rivista]
Morandi, Federica; Caselli, Emilia; Morandi, Stefania; P. J., Focia; J., Blazquez; B. K., Shoichet; Prati, Fabio
abstract
beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and the cephalosporins. In an effort to combat these enzymes, a combination of stereoselective organic synthesis, enzymology, microbiology, and X-ray crystallography was used to design and evaluate new carboxyphenyl-glycylboronic acid transition-state analogue inhibitors of the class C beta-lactamase AmpC. The new compounds improve inhibition by over 2 orders of magnitude compared to analogous glycylboronic acids, with K-1 values as low as 1 nM. On the basis of the differential binding of different analogues, the introduced carboxylate alone contributes about 2.1 kcal/mol in affinity. This carboxylate corresponds to the ubiquitous C3(4)'carboxylate of beta-lactams, and this energy represents the first thermodynamic measurement of the importance of this group in molecular recognition by class C beta-lactamases. The structures of AmpC in complex with two of these inhibitors were determined by X-ray crystallography at 1.72 and 1.83 Angstrom resolution. These structures suggest a structural basis for the high affinity of the new compounds and provide templates for further design. The highest affinity inhibitor was 5 orders of magnitude more selective for AmpC than for characteristic serine proteases, such as chymotrypsin. This inhibitor reversed the resistance of clinical pathogens to the third generation cephalosporin ceftazidime; it may serve as a lead compound for drug discovery to combat bacterial resistance to beta-lactam antibiotics.
2003
- Recognition and resistance in TEM beta-lactamase
[Articolo su rivista]
Xj, Wang; G., Minasov; J., Blazquez; Caselli, Emilia; Prati, Fabio; Bk, Shoichet
abstract
Developing antimicrobials that are less likely to engender resistance has become an important design criterion as more and more drugs fall victim to resistance mutations. One hypothesis is that the more closely an inhibitor resembles a substrate, the more difficult it will be to develop resistant mutations that can at once disfavor the inhibitor and still recognize the substrate. To investigate this hypothesis, 10 transition-state analogues, of greater or lesser similarity to substrates, were tested for inhibition of TEM-1 beta-lactamase, the most widespread resistance enzyme to penicillin antibiotics. The inhibitors were also tested against four characteristic mutant enzymes: TEM-30, TEM-32, TEM-52, and TEM-64. The inhibitor most similar to the substrate, compound 10, was the most potent inhibitor of the WT enzyme, with a K-i value of 64 nM. Conversely, compound 10 was the most susceptible to the TEM-30 (R244S) mutant, for which inhibition dropped by over 100-fold. The other inhibitors were relatively impervious to the TEM30 mutant enzyme. To understand recognition and resistance to these transition-state analogues, the structures of four of these inhibitors in complex with TEM-1 were determined by X-ray crystallography. These structures suggest a structural basis for distinguishing inhibitors that mimic the acylation transition state and those that mimic the deacylation transition state; they also suggest how TEM-30 reduces the affinity of compound 10. In cell culture, this inhibitor reversed the resistance of bacteria to ampicillin, reducing minimum inhibitory concentrations of this penicillin by between 4- and 64-fold, depending on the strain of bacteria. Notwithstanding this activity, the resistance of TEM-30, which is already extant in the clinic, suggests that there can be resistance liabilities with substrate-based design.
2003
- Sintesi Stereoselettiva di Acidi Boronici Inibitori di beta-Lattamasi
[Abstract in Atti di Convegno]
Morandi, Stefania; Caselli, Emilia; S., Baldini; Prati, Fabio
abstract
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2002
- A Common Mechanism Underlying Promiscuous Inhibitors from Virtual and High-Throughput Screening
[Articolo su rivista]
Susan L., Mcgovern; Caselli, Emilia; Nikolaus, Grigorieff; Brian K., Shoichet
abstract
High-throughput and virtual screening are widely used to discover novel leads for drug design.
On examination,many screening hits appear non-drug-like: they act noncompetitively,show
little relationship between structure and activity,and have poor selectivity. Attempts to develop
these peculiar molecules into viable leads are often futile,and much time can be wasted on
the characterization of these “phony” hits. Despite their common occurrence,the mechanism
of action of these promiscuous molecules remains unknown. To investigate this problem,45
diverse screening hits were studied. Fifteen of these were previously reported as inhibitors of
various receptors,including â-lactamase,malarial protease,dihydrofolate reductase,HIV Tar
RNA,thymidylate synthase,kinesin,insulin receptor,tyrosine kinases,farnesyltransferase,gyrase,prions,triosephosphate isomerase,nitric oxide synthase,phosphoinositide 3-kinase,and integrase; 30 were from an in-house screening library of a major pharmaceutical company.
In addition to their original targets,35 of these 45 compounds were shown to inhibit several
unrelated model enzymes. These 35 screening hits included compounds,such as fullerenes,dyes,and quercetin,that have repeatedly shown activity against diverse targets. When tested
against the model enzymes,the compounds showed time-dependent but reversible inhibition
that was dramatically attenuated by albumin,guanidinium,or urea. Surprisingly,increasing
the concentration of the model enzymes 10-fold largely eliminated inhibition,despite a 1000-
fold excess of inhibitor; a well-behaved competitive inhibitor did not show this behavior. One
model to explain these observations was that the active form of the promiscuous inhibitors
was an aggregate of many individual molecules. To test this hypothesis,light scattering and
electron microscopy experiments were performed. The nonspecific inhibitors were observed to
form particles of 30-400 nm diameter by both techniques. In control experiments,a wellbehaved
competitive inhibitor and an inactive dye-like molecule were not observed to form
aggregates. Consistent with the hypothesis that the aggregates are the inhibitory species,the
particle size and IC50 values of the promiscuous inhibitors varied monotonically with ionic
strength; a competitive inhibitor was unaffected by changes in ionic strength. Unexpectedly,aggregate formation appears to explain the activity of many nonspecific inhibitors and may
account for the activity of many promiscuous screening hits. Molecules acting via this
mechanism may be widespread in drug discovery screening databases. Recognition of these
compounds may improve screening results in many areas of pharmaceutical interest.
2002
- Highly enantioselective reduction of ethyl 4-chloro-3-oxobutanoate to L-and D-3-hydroxyesters with baker`s yeast
[Articolo su rivista]
Forni, Arrigo; Caselli, Emilia; Prati, Fabio; Bucciarelli, Maria; Torre, Giovanni
abstract
Reduction with baker's yeast of ethyl or methyl 4-chloro-3-oxobutanoate in the presence of allyl bromide or allyl alcohol as additive afforded the corresponding L- and D-3-hydroxyesters. Several reduction conditions were tested, involving: different concentrations of additive; variations in the yeast/substrate ratio; the presence and absence of glucose; different pre-incubation periods; a range of temperatures. The best conditions gave a complete conversion of the substrate within 1-2 h and a very high enantioselectivity, 90-97% e.e., for the two enantiomers.
2002
- Lipase-Catalyzed Resolution and Desymmetrization of 2-Hydroxymethylaziridines
[Articolo su rivista]
Davoli, Paolo; Caselli, Emilia; Bucciarelli, Maria; Forni, Arrigo; Torre, Giovanni; Prati, Fabio
abstract
The Amano PS lipase-catalyzed acetylation of 2-hydroxymethylaziridines 1a–e has been investigated in order to evaluate the effect of ring substituents on the enantioselectivity of the reaction and to assess the stereochemical preference of the enzyme. N-Benzyl-3-substituted cis-aziridines displayed high enantioselectivity and higher E values were found when the bulkiness of the substituent in position 3 was increased. In contrast, the corresponding trans isomers showed only poor enantioselectivity, regardless of the steric hindrance of the substituent at C3. Removal of the N-benzyl group proved to be detrimental to the enantioselectivity. In addition, desymmetrization of meso dimethanolic cis-aziridine 1f was successfully accomplished, and the corresponding monoacetylated product 2f, which is related to a key intermediate used in the total synthesis of the mitomycin antibiotic FR-900482, was obtained in excellent yield and nearly enantiomerically pure form. Moreover, the absolute configuration of enantiomerically pure cis-aziridines was determined by chemical correlation and/or chiroptical techniques, thus showing the stereochemical preference of Amano PS lipase for the 2S enantiomer.
2001
- Energetic, structural, and antimicrobial analyses of beta-lactam side chain recognition by beta-lactamases
[Articolo su rivista]
Caselli, Emilia; Powers, Ra; Blasczcak, Lc; Wu, Cye; Prati, Fabio; Shoichet, Bk
abstract
Penicillins and cephalosporins are among the most widely used and successful antibiotics. The emergence of resistance to these beta -lactams, most often through bacterial expression of beta -lactamases, threatens public health. To understand how beta -lactamases recognize their substrates, it would be helpful to know their binding energies. Unfortunately, these have been difficult to measure because beta -lactams form covalent adducts with beta -lactamases. This has complicated functional analyses and inhibitor design. Results: To investigate the contribution to interaction energy of the key amide (R1) side chain of beta -lactam antibiotics, eight acylglycineboronic acids that bear the side chains of characteristic penicillins and cephalosporins, as well as four other analogs, were synthesized. These transition-state analogs form reversible adducts with serine beta -lactamases. Therefore, binding energies can be calculated directly from K-i values, The K-i values measured span four orders of magnitude against the Group I beta -lactamase AmpC and three orders of magnitude against the Group II beta -lactamase TEM-1. The acylglycineboronic acids have K-i values as low as 20 nM against AmpC and as low as 390 nM against TEM1I. The inhibitors showed little activity against serine proteases, such as chymotrypsin. R1 side chains characteristic of beta -lactam inhibitors did not have better affinity for AmpC than did side chains characteristic of beta -lactam substrates. Two of the inhibitors reversed the resistance of pathogenic bacteria to beta -lactams in cell culture. Structures of two inhibitors in their complexes with AmpC were determined by X-ray Crystallography to 1.90 Angstrom and 1.75 Angstrom resolution; these structures suggest interactions that are important to the affinity of the inhibitors. Conclusions: Acylglycineboronic acids allow us to begin to dissect interaction energies between beta -lactam side chains and beta -lactamases, Surprisingly, there is little correlation between the affinity contributed by R1 side chains and their occurrence in beta -lactum inhibitors or beta -lactam substrates of serine p-lactamases. Nevertheless, presented in acylglycineboronic acids, these side chains can lead to inhibitors with high affinities and specificities, The structures of their complexes with AmpC give a molecular context to their affinities and may guide the design of antiresistance compounds in this series. (C) 2001 Elsevier Science Ltd, All rights reserved.
2001
- Progettazione, Sintesi ed Attività di Acidi 1-Acilamminoalcanboronici Inibitori di beta-Lattamasi
[Abstract in Atti di Convegno]
Caselli, Emilia; Forni, Arrigo; SHOICHET B., K; Prati, Fabio
abstract
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2001
- Structure-based design and in-parallel synthesis of inhibitors of AmpC β-lactamase
[Articolo su rivista]
Tondi, Donatella; Ra, Powers; Caselli, Emilia; Mc, Negri; J., Blazquez; Costi, Maria Paola; Bk, Shoichet
abstract
Background: Group I p-lactamases are a major cause of antibiotic resistance to beta -lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta -lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta -lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta -lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. Results: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K-i 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with iii values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 Angstrom resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Conclusions: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta -lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K-i 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta -lactamase.
2001
- Structure-based design and solid phase synthesis of novel AmpC b-lactamase inhibitors
[Abstract in Atti di Convegno]
Tondi, Donatella; Powers, R.; Caselli, E.; Costi, M. P.; Shoichet, B. K.
abstract
.
2001
- The structures of Ceftazidime and Its Transition State Analog in Complex with AmpC-beta-lactamase: Implication for Resistance Mutation and Inhibitor Design
[Articolo su rivista]
R. A., Powers; Caselli, Emilia; P. J., Focia; Prati, Fabio; B. K., Shoichet
abstract
Third-generation cephalosporins are widely used ‚-lactam antibiotics that resist hydrolysis by‚-lactamases. Recently, mutant ‚-lactamases that rapidly inactivate these drugs have emerged. To investigatewhy third-generation cephalosporins are relatively stable to wild-type class C ‚-lactamases and how mutantenzymes might overcome this, the structures of the class C ‚-lactamase AmpC in complex with the thirdgenerationcephalosporin ceftazidime and with a transition-state analogue of ceftazidime were determinedby X-ray crystallography to 2.0 and 2.3 Å resolution, respectively. Comparison of the acyl-enzyme structuresof ceftazidime and loracarbef, a ‚-lactam substrate, reveals that the conformation of ceftazidime in theactive site differs from that of substrates. Comparison of the structures of the acyl-enzyme intermediateand the transition-state analogue suggests that ceftazidime blocks formation of the tetrahedral transitionstate, explaining why it is an inhibitor of AmpC. Ceftazidime cannot adopt a conformation competent forcatalysis due to steric clashes that would occur with conserved residues Val211 and Tyr221. The X-raycrystal structure of the mutant ‚-lactamase GC1, which has improved activity against third-generationcephalosporins, suggests that a tandem tripeptide insertion in the ø loop, which contains Val211, hascaused a shift of this residue and also of Tyr221 that would allow ceftazidime and other third-generationcephalosporins to adopt a more catalytically competent conformation. These structural differences mayexplain the extended spectrum activity of GC1 against this class of cephalosporins. In addition, thecomplexed structure of the transition-state analogue inhibitor (Ki 20 nM) with AmpC reveals potentialopportunities for further inhibitor design.
2000
- Structure-Based Design and In Parallel Synthesis of Boronic Acid Inhibitors of AmpC 61538;-Lactamase
[Abstract in Atti di Convegno]
Tondi, Donatella; Powers, R.; Caselli, Emilia; Costi, Maria Paola; Shoichet, B. K.
abstract
.
1997
- Stereoselective synthesis of erythro beta-substituted aspartates
[Articolo su rivista]
Antolini, Luciano; Bucciarelli, Maria; Caselli, Emilia; Davoli, Paolo; Forni, Arrigo; Moretti, Irene; Prati, Fabio; Torre, Giovanni
abstract
The nucleophilic ring opening of trans-aziridine-2,3-dicarboxylate 1 and substituted N-acyl-, N-(methoxycarbonyl)-, and N-(methanesulfonyl)aziridine-2,3-dicarboxylates 2-4 allows an easy synthetic approach to beta-hydroxy, beta-amino, beta-(alkylthio), and beta-halogenoaspartates 5-8; in this respect, compounds 2-4 display higher reactivities. The erythro stereochemistry of the synthesized aspartates and the S(N)2-like mechanism of the nucleophilic attack were unambiguously identified by the (2R,3S) X-ray absolute configuration determination of enantiomerically pure beta-amino derivative 9, obtained from (2R,3R)-4, and by its chemical correlation with meso alpha,beta-bis[N-(methanesulfonyl)amino]succinate (10).