RESUMEN
Clavulanic acid is a medicinally important inhibitor of serine ß-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LCMS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.
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The three human SNM1 metallo-ß-lactamase fold nucleases (SNM1A-C) play key roles in DNA damage repair and in maintaining telomere integrity. Genetic studies indicate that they are attractive targets for cancer treatment and to potentiate chemo- and radiation-therapy. A high-throughput screen for SNM1A inhibitors identified diverse pharmacophores, some of which were shown by crystallography to coordinate to the di-metal ion centre at the SNM1A active site. Structure and turnover assay-guided optimization enabled the identification of potent quinazoline-hydroxamic acid containing inhibitors, which bind in a manner where the hydroxamic acid displaces the hydrolytic water and the quinazoline ring occupies a substrate nucleobase binding site. Cellular assays reveal that SNM1A inhibitors cause sensitisation to, and defects in the resolution of, cisplatin-induced DNA damage, validating the tractability of MBL fold nucleases as cancer drug targets.
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Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The l,d-transpeptidase LdtMt2, which is responsible for the formation of 3 â 3 cross-links in the cell wall peptidoglycan, has been identified as essential for M. tuberculosis virulence. We optimised a high-throughput assay for LdtMt2, and screened a targeted library of â¼10 000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g., ß-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the LdtMt2 catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2 active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC50 value of â¼1 µM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2 and other nucleophilic cysteine enzymes.
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The continuous emergence of multi-drug resistant pathogens co-expressing serine and metallo-carbapenemases seriously threatens the efficacy of carbapenem. Here, we report the first SeCN-derived dual inhibitor of serine and metallo-carbapenemases with IC50 values ranging from 0.0038 to 1.27 µg mL-1. The inhibitor was shown to form covalent bonds with Cys221 of NDM-1 and Ser70 of KPC-2, respectively, achieving selective labelling and cross-class inhibition for carbapenemases. Our results provide a potential strategy to develop clinically useful dual inhibitors targeting serine and metallo-carbapenemases to combat superbugs.
Asunto(s)
Antibacterianos , Proteínas Bacterianas , Antibacterianos/química , beta-Lactamasas/química , Carbapenémicos , Pruebas de Sensibilidad MicrobianaRESUMEN
ß-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine ß-lactamase (SBL)-catalyzed hydrolysis. The scope of ß-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfonederived acylenzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acylenzyme complexes.
Asunto(s)
Compuestos de Azabiciclo , Inhibidores de beta-Lactamasas , Penicilinas , Sulfonas , Triazoles , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/químicaRESUMEN
Carbapenems are important antibacterials and are both substrates and inhibitors of some ß-lactamases. We report studies on the reaction of the unusual carbapenem biapenem, with the subclass B1 metallo-ß-lactamases VIM-1 and VIM-2 and the class A serine-ß-lactamase KPC-2. X-ray diffraction studies with VIM-2 crystals treated with biapenem reveal the opening of the ß-lactam ring to form a mixture of the (2S)-imine and enamine complexed at the active site. NMR studies on the reactions of biapenem with VIM-1, VIM-2, and KPC-2 reveal the formation of hydrolysed enamine and (2R)- and (2S)-imine products. The combined results support the proposal that SBL/MBL-mediated carbapenem hydrolysis results in a mixture of tautomerizing enamine and (2R)- and (2S)-imine products, with the thermodynamically favoured (2S)-imine being the major observed species over a relatively long-time scale. The results suggest that prolonging the lifetimes of ß-lactamase carbapenem complexes by optimising tautomerisation of the nascently formed enamine to the (2R)-imine and likely more stable (2S)-imine tautomer is of interest in developing improved carbapenems.
RESUMEN
Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-ß-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential ß-lactamase stable ß-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.
Asunto(s)
Inhibidores de beta-Lactamasas/farmacología , beta-Lactamas/metabolismo , Animales , Bacterias Gramnegativas/efectos de los fármacos , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Unión Proteica , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas/química , Inhibidores de beta-Lactamasas/metabolismoRESUMEN
Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.
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Electrones , Oxidorreductasas , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Compuestos Férricos , Humanos , Rayos Láser , Oxidorreductasas/química , Oxígeno/química , Penicilinas/química , Penicilinas/metabolismo , Especificidad por SustratoRESUMEN
The effectiveness of ß-lactam antibiotics is increasingly compromised by ß-lactamases. Boron-containing inhibitors are potent serine-ß-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) ß-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by ß-lactamase-driven resistance.
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Antibacterianos/farmacología , Compuestos de Boro/farmacología , Ensayos Analíticos de Alto Rendimiento , Proteínas de Unión a las Penicilinas/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Sitios de Unión/efectos de los fármacos , Compuestos de Boro/síntesis química , Compuestos de Boro/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Proteínas de Unión a las Penicilinas/metabolismo , Relación Estructura-Actividad , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/química , beta-LactamasasRESUMEN
Chiral 3-substituted benzoxaboroles were designed as carbapenemase inhibitors and efficiently synthesised via asymmetric Morita-Baylis-Hillman reaction. Some of the benzoxaboroles were potent inhibitors of clinically relevant carbapenemases and restored the activity of meropenem in bacteria harbouring these enzymes. Crystallographic analyses validate the proposed mechanism of binding to carbapenemases, i.e. in a manner relating to their antibiotic substrates. The results illustrate how combining a structure-based design approach with asymmetric catalysis can efficiently lead to potent ß-lactamase inhibitors and provide a starting point to develop drugs combatting carbapenemases.
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Antibacterianos/síntesis química , Antibacterianos/farmacología , Proteínas Bacterianas/farmacología , Benzoxazoles/síntesis química , Benzoxazoles/farmacología , Diseño de Fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , beta-Lactamasas/farmacología , Antibacterianos/química , Benzoxazoles/química , Técnicas de Química Sintética , EstereoisomerismoRESUMEN
Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.
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Cristalografía por Rayos X/métodos , Enzimas/química , Enzimas/metabolismo , Animales , Proteínas Aviares/química , Proteínas Aviares/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Dominio Catalítico , Pollos , Cristalografía por Rayos X/instrumentación , Diseño de Equipo , Modelos Moleculares , Muramidasa/química , Muramidasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , beta-Lactamasas/química , beta-Lactamasas/metabolismoRESUMEN
Metallo-ß-lactamases (MBLs) can efficiently catalyze the hydrolysis of all classes of ß-lactam antibiotics except monobactams. While serine-ß-lactamase (SBL) inhibitors (e.g., clavulanic acid, avibactam) are established for clinical use, no such MBL inhibitors are available. We report on the synthesis and mechanism of inhibition of N-sulfamoylpyrrole-2-carboxylates (NSPCs) which are potent inhibitors of clinically relevant B1 subclass MBLs, including NDM-1. Crystallography reveals that the N-sulfamoyl NH2 group displaces the dizinc bridging hydroxide/water of the B1 MBLs. Comparison of crystal structures of an NSPC and taniborbactam (VRNX-5133), presently in Phase III clinical trials, shows similar binding modes for the NSPC and the cyclic boronate ring systems. The presence of an NSPC restores meropenem efficacy in clinically derived E. coli and K. pneumoniae blaNDM-1. The results support the potential of NSPCs and related compounds as efficient MBL inhibitors, though further optimization is required for their clinical development.
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Escherichia coli , beta-Lactamasas , Antibacterianos/farmacología , Ácidos Borínicos , Ácidos Carboxílicos , Inhibidores de beta-Lactamasas/farmacologíaRESUMEN
Penems have demonstrated potential as antibacterials and ß-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some ß-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing ß-lactamases, focusing on the class A serine ß-lactamase KPC-2 and the metallo ß-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three ß-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the ß-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with ß-lactamases. They also exemplify how crystal structures of ß-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.
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Antibacterianos/química , Inhibidores de beta-Lactamasas/química , beta-Lactamasas/química , beta-Lactamas/química , Antibacterianos/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Hidrólisis , Klebsiella pneumoniae/enzimología , Unión Proteica , Pseudomonas aeruginosa/enzimología , Stenotrophomonas maltophilia/enzimología , Inhibidores de beta-Lactamasas/metabolismo , beta-Lactamasas/metabolismo , beta-Lactamas/metabolismoRESUMEN
ß-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by ß-lactamases, including the chromosomally encoded class C AmpC serine-ß-lactamases (SBLs). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimize the DBO core. We report kinetic and structural studies, including four high-resolution crystal structures, concerning inhibition of the AmpC serine-ß-lactamase from E. coli (AmpC EC ) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpC EC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (apparent inhibition constant [Kiapp], 0.69 µM) against AmpC EC compared to that of the other DBOs (Kiapp = 5.0 to 7.4 µM) due to an â¼10-fold accelerated carbamoylation rate. However, zidebactam also has an accelerated off-rate, and with sufficient preincubation time, all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpC EC -zidebactam carbamoyl complex compared to those for the other DBOs. The results suggest the carbamoyl complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.
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Escherichia coli , Inhibidores de beta-Lactamasas , Antibacterianos/farmacología , Compuestos de Azabiciclo/farmacología , Proteínas Bacterianas/genética , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/genéticaRESUMEN
Resistance to ß-lactam antibacterials, importantly via production of ß-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) ß-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C ßlactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied ß-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D ßlactamases, our data support the proposal that bicyclic boronates are broad-spectrum ßlactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful ß-lactamase inhibition.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Ácidos Borónicos/farmacología , Inhibidores de beta-Lactamasas/farmacología , Antibacterianos , Proteínas Bacterianas/genética , Ácidos Borónicos/química , Cristalografía por Rayos X , Ciclización , Farmacorresistencia Bacteriana/efectos de los fármacos , Farmacorresistencia Bacteriana/genética , Escherichia coli/enzimología , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/clasificación , beta-Lactamasas/genéticaRESUMEN
OBJECTIVES: To evaluate the potential clinical in vitro efficacy of novel ß-lactam/ß-lactamase-inhibitor combinations - including imipenem-relebactam (IPM-REL) and cefepime-AAI101 (enmetazobactam) (FEP-AAI) - against contemporary multidrug-resistant (MDR) Enterobacteriaceae. METHODS: Agar-based MIC screening against MDR Enterobacteriaceae (n = 264) was used to evaluate the in vitro efficacy of IPM-REL and FEP-AAI, to compare the results with established combinations, and to investigate alternative ß-lactam partners for relebactam (REL) and enmetazobactam (AAI). The inhibition activities of REL, AAI and the comparators avibactam (AVI) and tazobactam, against isolated recombinant ß-lactamases covering representatives from all four Ambler classes of ß-lactamases, were tested using a fluorescence-based assay. RESULTS: Using recombinant proteins, all four inhibitors were highly active against the tested class A serine ß-lactamases (SBLs). REL and AVI showed moderate activity against the Class C AmpC from Pseudomonas aeruginosa and the Class D OXA-10/-48 SBLs, but outperformed tazobactam and AAI. All tested inhibitors lacked activity against Class B metallo-ß-lactamases (MBLs). In the presence of REL and IPM, but not AAI, susceptibility increased against Klebsiella pnuemoniae carbapenemase (KPC)-positive and OXA-48-positive isolates. Both aztreonam-AVI and ceftolozane-tazobactam were more effective than IPM-REL. In all the tested combinations, AAI was a more effective inhibitor of class A ß-lactamases (ESBLs) than the established inhibitors. CONCLUSION: The results lead to the proposal of alternative combination therapies involving REL and AAI to potentiate the use of ß-lactams against clinical Gram-negative isolates expressing a variety of lactamases. They highlight the potential of novel combinations for combating strains not covered by existing therapies.
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Antibacterianos/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/efectos de los fármacos , Inhibidores de beta-Lactamasas/farmacología , Compuestos de Azabiciclo/farmacología , Enterobacteriaceae Resistentes a los Carbapenémicos/aislamiento & purificación , Cefepima/farmacología , Combinación de Medicamentos , Infecciones por Enterobacteriaceae/tratamiento farmacológico , Humanos , Imipenem/farmacología , Pruebas de Sensibilidad Microbiana , Tazobactam/farmacología , Triazoles/farmacología , beta-Lactamasas/metabolismoRESUMEN
19F NMR protein observed spectroscopy is evaluated as a method for analysing protein metal binding using the New Delhi metallo-ß-lactamase 1. The results imply 19F NMR is useful for analysis of different metallated protein states and investigations on equilibrium states in the presence of inhibitors. One limitation is that 19F labelling may affect metal ion binding. The sensitive readout of changes in protein behaviour observed by 19F NMR spectra coupled with the broad scope of tolerated conditions (e.g. buffer variations) means 19F NMR should be further investigated for studying metal ion interactions and the inhibition of metallo-enzymes during drug discovery.
RESUMEN
Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a serine-ß-lactamase (SBL) capable of hydrolysing almost all ß-lactam antibiotics. We compare KPC-2 inhibition by vaborbactam, a clinically-approved monocyclic boronate, and VNRX-5133 (taniborbactam), a bicyclic boronate in late-stage clinical development. Vaborbactam inhibition is slowly reversible, whereas taniborbactam has an off-rate indicating essentially irreversible complex formation and a 15-fold higher on-rate, although both potentiate ß-lactam activity against KPC-2-expressing K. pneumoniae. High resolution X-ray crystal structures reveal closely related binding modes for both inhibitors to KPC-2, with differences apparent only in positioning of the endocyclic boronate ester oxygen. The results indicate the bicyclic boronate scaffold as both an efficient, long-lasting, KPC-2 inhibitor and capable of supporting further iterations that may improve potency against specific enzyme targets and pre-empt the emergence of inhibitor resistant KPC-2 variants.
RESUMEN
ß-Lactamases comprise the most widely used mode of resistance to ß-lactam antibiotics. Cyclic boronates have shown promise as a new class of ß-lactamase inhibitor, with pioneering potential to potently inhibit both metallo- and serine-ß-lactamases. We report studies concerning a bicyclic boronate ester with a thioether rather than the more typical ß-lactam antibiotic "C-6/C-7" acylamino type side chain, which is present in the penicillin/cephalosporin antibiotics. The thioether bicyclic boronate ester was tested for activity against representative serine- and metallo-ß-lactamases. The results support the broad inhibition potential of bicyclic boronate based inhibitors with different side chains, including against metallo-ß-lactamases from B1, B2, and B3 subclasses. Combined with previous crystallographic studies, analysis of a crystal structure of the thioether inhibitor with the clinically relevant VIM-2 metallo-ß-lactamase implies that further SAR work will expand the already broad scope of ß-lactamase inhibition by bicyclic boronates.