RESUMEN
Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.
Asunto(s)
Mycobacterium tuberculosis , Tomografía de Emisión de Positrones , Trehalosa , Tuberculosis , Animales , Mycobacterium tuberculosis/metabolismo , Tomografía de Emisión de Positrones/métodos , Trehalosa/metabolismo , Tuberculosis/diagnóstico por imagen , Tuberculosis/microbiología , Tuberculosis/metabolismo , Humanos , Ratones , Radioisótopos de Flúor , Fluorodesoxiglucosa F18/metabolismo , Fluorodesoxiglucosa F18/química , Radiofármacos/metabolismo , Modelos Animales de Enfermedad , FemeninoRESUMEN
Linezolid is a drug with proven human antitubercular activity whose use is limited to highly drug-resistant patients because of its toxicity. This toxicity is related to its mechanism of actionâlinezolid inhibits protein synthesis in both bacteria and eukaryotic mitochondria. A highly selective and potent series of oxazolidinones, bearing a 5-aminomethyl moiety (in place of the typical 5-acetamidomethyl moiety of linezolid), was identified. Linezolid-resistant mutants were cross-resistant to these molecules but not vice versa. Resistance to the 5-aminomethyl molecules mapped to an N-acetyl transferase (Rv0133) and these mutants remained fully linezolid susceptible. Purified Rv0133 was shown to catalyze the transformation of the 5-aminomethyl oxazolidinones to their corresponding N-acetylated metabolites, and this transformation was also observed in live cells of Mycobacterium tuberculosis. Mammalian mitochondria, which lack an appropriate N-acetyltransferase to activate these prodrugs, were not susceptible to inhibition with the 5-aminomethyl analogues. Several compounds that were more potent than linezolid were taken into C3HeB/FeJ mice and were shown to be highly efficacious, and one of these (9) was additionally taken into marmosets and found to be highly active. Penetration of these 5-aminomethyl oxazolidinone prodrugs into caseum was excellent. Unfortunately, these compounds were rapidly converted into the corresponding 5-alcohols by mammalian metabolism which retained antimycobacterial activity but resulted in substantial mitotoxicity.
Asunto(s)
Antituberculosos , Mycobacterium tuberculosis , Oxazolidinonas , Profármacos , Profármacos/farmacología , Profármacos/química , Antituberculosos/farmacología , Antituberculosos/química , Mycobacterium tuberculosis/efectos de los fármacos , Oxazolidinonas/farmacología , Oxazolidinonas/química , Animales , Pruebas de Sensibilidad Microbiana , Ratones , Humanos , Linezolid/farmacología , Linezolid/química , Farmacorresistencia Bacteriana , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismoRESUMEN
Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - can act as a mechanism-based enzyme reporter in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-specific processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-specific, clinical diagnostic candidate. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either bespoke radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.
RESUMEN
The extent to which a drug accumulates in Mycobacterium tuberculosis (Mtb) and its host cell can affect treatment efficacy. We describe protocols measuring drug accumulation in Mtb, macrophages, and Mtb-infected macrophages. The method leverages drug extraction from the cellular lysate and drug-level quantification by liquid chromatography-mass spectrometry. The general methodology has broad applicability and can quantify drug accumulation in other cell types, while being extended to quantification of drug metabolites formed within the cell under study. For complete details on the use and execution of this protocol, please refer to Lavin et al. (2021).1.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Tuberculosis/tratamiento farmacológico , Tuberculosis/metabolismo , Macrófagos/metabolismo , Espectrometría de Masas , Cromatografía LiquidaRESUMEN
Rifamycin antibiotics are a valuable class of antimicrobials for treating infections by mycobacteria and other persistent bacteria owing to their potent bactericidal activity against replicating and non-replicating pathogens. However, the clinical utility of rifamycins against Mycobacterium abscessus is seriously compromised by a novel resistance mechanism, namely, rifamycin inactivation by ADP-ribosylation. Using a structure-based approach, we rationally redesign rifamycins through strategic modification of the ansa-chain to block ADP-ribosylation while preserving on-target activity. Validated by a combination of biochemical, structural, and microbiological studies, the most potent analogs overcome ADP-ribosylation, restored their intrinsic low nanomolar activity and demonstrated significant in vivo antibacterial efficacy. Further optimization by tuning drug disposition properties afforded a preclinical candidate with remarkable potency and an outstanding pharmacokinetic profile.
Asunto(s)
Mycobacterium , Rifamicinas , Antibacterianos/farmacología , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Rifamicinas/farmacología , Rifamicinas/química , ADP-RibosilaciónRESUMEN
Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances. Here, we describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6-fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2-oxa-8-azaspiro[4.5]decan-3-yl)methanol], which was developed to target both ATP-binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae, including drug-resistant strains. A phosphate pro-drug, JSF-2659, was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram-positive organisms, and JSF-2659 was shown in a deep tissue model of vancomycin-resistant Staphylococcus aureus (VRSA) and a model of Clostridioides difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel preclinical drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.
Asunto(s)
Gonorrea , Staphylococcus aureus Resistente a Meticilina , Profármacos , Adenosina Trifosfato , Animales , Antibacterianos/química , Antibacterianos/farmacología , Girasa de ADN/genética , Farmacorresistencia Bacteriana , Femenino , Gonorrea/tratamiento farmacológico , Metanol/farmacología , Staphylococcus aureus Resistente a Meticilina/metabolismo , Pruebas de Sensibilidad Microbiana , Neisseria gonorrhoeae , Fosfatos/farmacología , Profármacos/farmacología , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Rickettsia is a genus of Gram-negative bacteria that has for centuries caused large-scale morbidity and mortality. In recent years, the resurgence of rickettsial diseases as a major cause of pyrexias of unknown origin, bioterrorism concerns, vector movement, and concerns over drug resistance is driving a need to identify novel treatments for these obligate intracellular bacteria. Utilizing an uvGFP plasmid reporter, we developed a screen for identifying anti-rickettsial small molecule inhibitors using Rickettsia canadensis as a model organism. The screening data were utilized to train a Bayesian model to predict growth inhibition in this assay. This two-pronged methodology identified anti-rickettsial compounds, including duartin and JSF-3204 as highly specific, efficacious, and noncytotoxic compounds. Both molecules exhibited in vitro growth inhibition of R. prowazekii, the causative agent of epidemic typhus. These small molecules and the workflow, featuring a high-throughput phenotypic screen for growth inhibitors of intracellular Rickettsia spp. and machine learning models for the prediction of growth inhibition of an obligate intracellular Gram-negative bacterium, should prove useful in the search for new therapeutic strategies to treat infections from Rickettsia spp. and other obligate intracellular bacteria.
Asunto(s)
Aprendizaje Automático , Teorema de Bayes , PlásmidosRESUMEN
Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase of Staphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response in Mycobacterium tuberculosis (Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin to Mtb DnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused on Mtb DnaE1.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Antibacterianos/farmacología , Microscopía por Crioelectrón , ADN Polimerasa Dirigida por ADN , Humanos , Mycobacterium tuberculosis/genética , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
We present the application of Bayesian modeling to identify chemical tools and/or drug discovery entities pertinent to drug-resistant Staphylococcus aureus infections. The quinoline JSF-3151 was predicted by modeling and then empirically demonstrated to be active against in vitro cultured clinical methicillin- and vancomycin-resistant strains while also exhibiting efficacy in a mouse peritonitis model of methicillin-resistant S. aureus infection. We highlight the utility of an intrabacterial drug metabolism (IBDM) approach to probe the mechanism by which JSF-3151 is transformed within the bacteria. We also identify and then validate two mechanisms of resistance in S. aureus: one mechanism involves increased expression of a lipocalin protein, and the other arises from the loss of function of an azoreductase. The computational and experimental approaches, discovery of an antibacterial agent, and elucidated resistance mechanisms collectively hold promise to advance our understanding of therapeutic regimens for drug-resistant S. aureus.
Asunto(s)
Staphylococcus aureus Resistente a Meticilina , Preparaciones Farmacéuticas , Infecciones Estafilocócicas , Animales , Teorema de Bayes , Ratones , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureusRESUMEN
Sensing and response to environmental cues, such as pH and chloride (Cl-), is critical in enabling Mycobacterium tuberculosis (Mtb) colonization of its host. Utilizing a fluorescent reporter Mtb strain in a chemical screen, we have identified compounds that dysregulate Mtb response to high Cl- levels, with a subset of the hits also inhibiting Mtb growth in host macrophages. Structure-activity relationship studies on the hit compound "C6," or 2-(4-((2-(ethylthio)pyrimidin-5-yl)methyl)piperazin-1-yl)benzo[d]oxazole, demonstrated a correlation between compound perturbation of Mtb Cl- response and inhibition of bacterial growth in macrophages. C6 accumulated in both bacterial and host cells, and inhibited Mtb growth in cholesterol media, but not in rich media. Subsequent examination of the Cl- response of Mtb revealed an intriguing link with bacterial growth in cholesterol, with increased transcription of several Cl--responsive genes in the simultaneous presence of cholesterol and high external Cl- concentration, versus transcript levels observed during exposure to high external Cl- concentration alone. Strikingly, oral administration of C6 was able to inhibit Mtb growth in vivo in a C3HeB/FeJ murine infection model. Our work illustrates how Mtb response to environmental cues can intersect with its metabolism and be exploited in antitubercular drug discovery.
Asunto(s)
Antituberculosos/farmacología , Desarrollo de Medicamentos , Mycobacterium tuberculosis/efectos de los fármacos , Animales , Antituberculosos/química , Cloruros/metabolismo , Colesterol/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Macrófagos/microbiología , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/crecimiento & desarrollo , Relación Estructura-ActividadRESUMEN
Published Mycobacterium tuberculosis ß-ketoacyl-ACP synthase KasA inhibitors lack sufficient potency and/or pharmacokinetic properties. A structure-based approach was used to optimize existing KasA inhibitor DG167. This afforded indazole JSF-3285 with a 30-fold increase in mouse plasma exposure. Biochemical, genetic, and X-ray studies confirmed JSF-3285 targets KasA. JSF-3285 offers substantial activity in an acute mouse model of infection and in the corresponding chronic infection model, with efficacious reductions in colony-forming units at doses as low as 5 mg/kg once daily orally and improvement of the efficacy of front-line drugs isoniazid or rifampicin. JSF-3285 is a promising preclinical candidate for tuberculosis.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis/tratamiento farmacológico , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/metabolismo , Animales , Antituberculosos/química , Antituberculosos/uso terapéutico , Proteínas Bacterianas/metabolismo , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Ratones , Modelos Moleculares , Mycobacterium tuberculosis/enzimologíaRESUMEN
The triazine antitubercular JSF-2019 was of interest due to its in vitro efficacy and the nitro group shared with the clinically relevant delamanid and pretomanid. JSF-2019 undergoes activation requiring F420H2 and one or more nitroreductases in addition to Ddn. An intrabacterial drug metabolism (IBDM) platform was leveraged to demonstrate the system kinetics, evidencing formation of NOâ and a des-nitro metabolite. Structure-activity relationship studies focused on improving the solubility and mouse pharmacokinetic profile of JSF-2019 and culminated in JSF-2513, relying on the key introduction of a morpholine. Mechanistic studies with JSF-2019, JSF-2513, and other triazines stressed the significance of achieving potent in vitro efficacy via release of intrabacterial NOâ along with inhibition of InhA and, more generally, the FAS-II pathway. This study highlights the importance of probing IBDM and its potential to clarify mechanism of action, which in this case is a combination of NOâ release and InhA inhibition.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Triazinas/química , Animales , Antituberculosos/farmacocinética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana/efectos de los fármacos , Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/metabolismo , Femenino , Semivida , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/metabolismo , Óxido Nítrico/metabolismo , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas/metabolismo , Triazinas/farmacocinética , Triazinas/farmacologíaRESUMEN
Mechanisms of magnesium homeostasis in Mycobacterium tuberculosis are poorly understood. Here, we describe the characterization of a pyrimidinetrione amide scaffold that disrupts magnesium homeostasis in the pathogen by direct binding to the CorA Mg2+/Co2+ transporter. Mutations in domains of CorA that are predicted to regulate the pore opening in response to Mg2+ ions conferred resistance to this scaffold. The pyrimidinetrione amides were cidal against the pathogen under both actively replicating and nonreplicating conditions in vitro and were efficacious against the organism during macrophage infection. However, the compound lacked efficacy in infected mice, possibly due to limited exposure. Our results indicate that inhibition of Mg2+ homeostasis by CorA is an attractive target for tuberculosis drug discovery and encourage identification of improved CorA inhibitors.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/metabolismo , Magnesio/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/metabolismo , Antibacterianos/química , Antibacterianos/farmacocinética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/genética , Homeostasis/efectos de los fármacos , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
Nicotinamide adenine dinucleotide (NAD+) synthetase catalyzes the last step in NAD+ biosynthesis. Depletion of NAD+ is bactericidal for both active and dormant Mycobacterium tuberculosis (Mtb). By inhibiting NAD+ synthetase (NadE) from Mtb, we expect to eliminate NAD+ production which will result in cell death in both growing and nonreplicating Mtb. NadE inhibitors have been investigated against various pathogens, but few have been tested against Mtb. Here, we report on the expansion of a series of urea-sulfonamides, previously reported by Brouillette et al. Guided by docking studies, substituents on a terminal phenyl ring were varied to understand the structure-activity-relationships of substituents on this position. Compounds were tested as inhibitors of both recombinant Mtb NadE and Mtb whole cells. While the parent compound displayed very weak inhibition against Mtb NadE (IC50=1000µM), we observed up to a 10-fold enhancement in potency after optimization. Replacement of the 3,4-dichloro group on the phenyl ring of the parent compound with 4-nitro yielded 4f, the most potent compound of the series with an IC50 value of 90µM against Mtb NadE. Our modeling results show that these urea-sulfonamides potentially bind to the intramolecular ammonia tunnel, which transports ammonia from the glutaminase domain to the active site of the enzyme. This hypothesis is supported by data showing that, even when treated with potent inhibitors, NadE catalysis is restored when treated with exogenous ammonia. Most of these compounds also inhibited Mtb cell growth with MIC values of 19-100µg/mL. These results improve our understanding of the SAR of the urea-sulfonamides, their mechanism of binding to the enzyme, and of Mtb NadE as a potential antitubercular drug target.
Asunto(s)
Amida Sintasas/antagonistas & inhibidores , Antituberculosos/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Amida Sintasas/metabolismo , Antituberculosos/síntesis química , Antituberculosos/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/metabolismo , Relación Estructura-ActividadRESUMEN
In this issue of Cell Chemical Biology, Ruecker et al. (2017) show that fumarase depletion in Mycobacterium tuberculosis leads to fumarate, a TCA cycle intermediate, accumulation, causing succination of a range of thiol-containing metabolites and proteins. Fumarate is bactericidal to the pathogen, and its accumulation may enhance the bactericidal effector mechanisms of other TCA cycle intermediates that accumulate due to activation of infected macrophages.
Asunto(s)
Mycobacterium tuberculosis , Fumarato Hidratasa/deficiencia , Fumaratos , Errores Innatos del Metabolismo , Hipotonía Muscular , Oxidación-Reducción , Trastornos PsicomotoresRESUMEN
An adenylated sulfoximine transition-state analogue 1, which inhibits human asparagine synthetase (hASNS) with nanomolar potency, has been reported to suppress the proliferation of an l-asparagine amidohydrolase (ASNase)-resistant MOLT-4 leukemia cell line (MOLT-4R) when l-asparagine is depleted in the medium. We now report the synthesis and biological activity of two new sulfoximine analogues of 1 that have been studied as part of systematic efforts to identify compounds with improved cell permeability and/or metabolic stability. One of these new analogues, an amino sulfoximine 5 having no net charge at cellular pH, is a better hASNS inhibitor (K(I)(∗)=8 nM) than 1 and suppresses proliferation of MOLT-4R cells at 10-fold lower concentration (IC(50)=0.1mM). More importantly, and in contrast to the lead compound 1, the presence of sulfoximine 5 at concentrations above 0.25 mM causes the death of MOLT-4R cells even when ASNase is absent in the culture medium. The amino sulfoximine 5 exhibits different dose-response behavior when incubated with an ASNase-sensitive MOLT-4 cell line (MOLT-4S), supporting the hypothesis that sulfoximine 5 exerts its effect by inhibiting hASNS in the cell. Our work provides further evidence for the idea that hASNS represents a chemotherapeutic target for the treatment of leukemia, and perhaps other cancers, including those of the prostate.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Asparaginasa/metabolismo , Aspartatoamoníaco Ligasa/antagonistas & inhibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Amidohidrolasas/metabolismo , Asparagina/metabolismo , Aspartatoamoníaco Ligasa/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Iminas/química , Iminas/farmacología , Modelos Moleculares , Compuestos de Azufre/química , Compuestos de Azufre/farmacologíaRESUMEN
The class C beta-lactamase of Enterobacter cloacae P99 is known to catalyze the hydrolysis of certain acyclic (thio)esters. Previous experiments have employed thioglycolate, m-hydroxybenzoate, and phenylphosphate leaving groups. The relative effectiveness of these leaving groups has now been quantitatively assessed by employment of a series of compounds with common acyl groups, and found to rank in the order phenylphosphate >m-hydroxybenzoate >thioglycolate. Structural models suggest that these leaving groups interact during acylation principally with Tyr 150, Lys 315, and Thr 316 of the beta-lactamase active site. The positions of the leaving group carboxylates in these models is compared with those in published crystal structures of complexes of class C beta-lactamases with beta-lactams. The particular effectiveness of the acyl phosphate indicates the positions of two oxyanions that strongly interact with the active site. This information should be useful in the design of inhibitors of class C beta-lactamases.