Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

Interdomain flexibility and interfacial integrity of ß-lactamase inhibitory protein (BLIP) modulate its binding to class A ß-lactamases.

ß-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αß domains conjugated by an interdomain loop and can effectively bind and inactivate class A ß-lactamases, which are responsible for resistance of bacteria to ß-lactam antibiotics. The varied ability of BLIP to bind different ß-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A ß-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A ß-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.

Deep Sequencing of a Systematic Peptide Library Reveals Conformationally-Constrained Protein Interface Peptides that Disrupt a Protein-Protein Interaction.

Disrupting protein-protein interactions is difficult due to the large and flat interaction surfaces of the binding partners. The BLIP and BLIP-II proteins are unrelated in sequence and structure and yet each potently inhibit ß-lactamases. High-throughput oligonucleotide synthesis was used to construct a 12,470-member library containing overlapping linear and cyclic peptides ranging in size from 6 to 21 amino acids that scan through the sequences of BLIP and BLIP-II. Phage display affinity selections and deep sequencing revealed that, despite the differences in interaction surfaces with ß-lactamases, rapid enrichment of consensus peptide regions originating from both BLIP and BLIP-II contact residues in the binding interface occurred. BLIP and BLIP-II peptides that were enriched by affinity selection were shown to bind ß-lactamases and disrupt the BLIP/ß-lactamase interaction. The results suggest that peptides that bind at and disrupt PPI interfaces can be identified through systematic peptide library construction, affinity selection, and deep sequencing.

Prospects for Antibacterial Discovery and Development.

The rising prevalence of multidrug-resistant bacteria is an urgent health crisis that can only be countered through renewed investment in the discovery and development of antibiotics. There is no panacea for the antibacterial resistance crisis; instead, a multifaceted approach is called for. In this Perspective we make the case that, in the face of evolving clinical needs and enabling technologies, numerous validated antibacterial targets and associated lead molecules deserve a second look. At the same time, many worthy targets lack good leads despite harboring druggable active sites. Creative and inspired techniques buoy discovery efforts; while soil screening efforts frequently lead to antibiotic rediscovery, researchers have found success searching for new antibiotic leads by studying underexplored ecological niches or by leveraging the abundance of available data from genome mining efforts. The judicious use of "polypharmacology" (i.e., the ability of a drug to alter the activities of multiple targets) can also provide new opportunities, as can the continued search for inhibitors of resistance enzymes with the capacity to breathe new life into old antibiotics. We conclude by highlighting available pharmacoeconomic models for antibacterial discovery and development while making the case for new ones.

Trends and correlation between antibacterial consumption and carbapenem resistance in gram-negative bacteria in a tertiary hospital in China from 2012 to 2019.

BACKGROUND: To investigate the trends and correlation between antibacterial consumption and carbapenem resistance in Gram-negative bacteria from 2012 to 2019 in a tertiary-care teaching hospital in southern China. METHODS: This retrospective study included data from hospital-wide inpatients collected between January 2012 and December 2019. Data on antibacterial consumption were expressed as defined daily doses (DDDs)/1000 patient-days. Antibacterials were classified according to the Anatomical Therapeutic Chemical (ATC) classification system. The trends in antimicrobial usage and resistance were analyzed by linear regression, while Pearson correlation analysis was used for assessing correlations. RESULTS: An increasing trend in the annual consumption of tetracyclines, ß-lactam/ß-lactamase inhibitor (BL/BLI) combinations, and carbapenems was observed (P < 0.05). Carbapenem resistance in Acinetobacter baumannii (A. baumannii) significantly increased (P < 0.05) from 18% in 2012 to 60% in 2019. Moreover, significant positive correlations were found between resistance to carbapenems in A. baumannii (P < 0.05) and Escherichia coli (E. coli; P < 0.05) and consumption of carbapenems, while the resistance rate of A. baumannii to carbapenems was positively correlated with cephalosporin/ß-lactamase inhibitor (C/BLI) combinations (P < 0.01) and tetracyclines usage (P < 0.05). We also found that use of quinolones was positively correlated with the resistance rate of Burkholderia cepacia (B. cepacia) to carbapenems (P < 0.05), and increasing uses of carbapenems (P < 0.01) and penicillin/ß-Lactamase inhibitor (P/BLI) combinations (P < 0.01) were significantly correlated with reduced resistance of Enterobacter cloacae (E. cloacae) to carbapenems. CONCLUSION: These results revealed significant correlations between consumption of antibiotics and carbapenem resistance rates in Gram-negative bacteria. Implementing proper management strategies and reducing the unreasonable use of antibacterial drugs may be an effective measure to reduce the spread of carbapenem-resistant Gram-negative bacteria (CRGN), which should be confirmed by further studies.

Structure-guided optimization of D-captopril for discovery of potent NDM-1 inhibitors.

ß-lactam antibiotics have long been the mainstay for the treatment of bacterial infections. New Delhi metallo-ß-lactamase 1 (NDM-1) is able to hydrolyze nearly all ß-lactam antibiotics and even clinically used serine-ß-lactamase inhibitors. The wide and rapid spreading of NDM-1 gene among pathogenic bacteria has attracted extensive attention, therefore high potency NDM-1 inhibitors are urgently needed. Here we report a series of structure-guided design of D-captopril derivatives that can inhibit the activity of NDM-1 in vitro and at cellular levels. Structural comparison indicates the mechanisms of inhibition enhancement and provides insights for further inhibitor optimization.

Mass spectrometric identification and quantification of the antibiotic clavulanic acid in broiler chicken plasma and meat as a necessary analytical tool in finding ways to increase the effectiveness of currently used antibiotics in the treatment of broiler chickens.

Clavulanic acid is a molecule with antimicrobial effect used in several livestock species treatment. Its inclusion in the treatment of infectious diseases of broilers requires determination of pharmacokinetic and pharmacodynamic parameters in order to determine the appropriate dosage for broilers and ensure safety of chicken products for human health. The present study describes the optimisation of analytical LC-MS/MS method for identification and quantification of clavulanic acid in broiler chicken plasma and meat. The limit of detection and the limit of quantification for the developed method were 3.09 µg·L-1 and 10.21 µg·L-1 for plasma and 2.57 µg·kg-1 and 8.47 µg·kg-1 for meat. The recoveries of the developed plasma and tissue extraction procedure were > 105.7% and > 95.6%, respectively. The achieved coefficient of variation of within-run precision ranged from 2.8 to 10.9% for plasma and from 6.5 to 8.5% for meat. The pharmacokinetic experiment was performed in 112 Ross broiler chickens assigned into time interval groups ranging from 10 min to 24 h in accredited animal facilities. Administered dose of clavulanic acid was 2.5 mg·kg-1 according to the manufacturer's recommendations. The pharmacokinetic parameters obtained from the experiment are as follows: Cmax = 1.82 ± 0.91 mg·L-1, Tmax = 0.25 h, T1/2 = 0.87 h, Kel = 0.80 ± 0.04 h-1, AUC0-∞ = 2.17 mg·h ·L-1.

4-Alkyl-1,2,4-triazole-3-thione analogues as metallo-ß-lactamase inhibitors.

In Gram-negative bacteria, the major mechanism of resistance to ß-lactam antibiotics is the production of one or several ß-lactamases (BLs), including the highly worrying carbapenemases. Whereas inhibitors of these enzymes were recently marketed, they only target serine-carbapenemases (e.g. KPC-type), and no clinically useful inhibitor is available yet to neutralize the class of metallo-ß-lactamases (MBLs). We are developing compounds based on the 1,2,4-triazole-3-thione scaffold, which binds to the di-zinc catalytic site of MBLs in an original fashion, and we previously reported its promising potential to yield broad-spectrum inhibitors. However, up to now only moderate antibiotic potentiation could be observed in microbiological assays and further exploration was needed to improve outer membrane penetration. Here, we synthesized and characterized a series of compounds possessing a diversely functionalized alkyl chain at the 4-position of the heterocycle. We found that the presence of a carboxylic group at the extremity of an alkyl chain yielded potent inhibitors of VIM-type enzymes with Ki values in the µM to sub-µM range, and that this alkyl chain had to be longer or equal to a propyl chain. This result confirmed the importance of a carboxylic function on the 4-substituent of 1,2,4-triazole-3-thione heterocycle. As observed in previous series, active compounds also preferentially contained phenyl, 2-hydroxy-5-methoxyphenyl, naphth-2-yl or m-biphenyl at position 5. However, none efficiently inhibited NDM-1 or IMP-1. Microbiological study on VIM-2-producing E. coli strains and on VIM-1/VIM-4-producing multidrug-resistant K. pneumoniae clinical isolates gave promising results, suggesting that the 1,2,4-triazole-3-thione scaffold worth continuing exploration to further improve penetration. Finally, docking experiments were performed to study the binding mode of alkanoic analogues in the active site of VIM-2.

Serendipitous discovery of aryl boronic acids as ß-lactamase inhibitors.

High throughput screening for ß-lactamase inhibitors afforded biphenyl hits such as 1. Hit confirmation and X-ray soaking experiments with Pseudomonas Aeruginosa AmpC enzyme led to the identification of an aryl boronic acid-serine complex 4, which was formed from phenyl boronic acid 8 (an impurity in compound 1) and ethylene glycol (the cryoprotectant in the soaking experiment).

Structural studies of triazole inhibitors with promising inhibitor effects against antibiotic resistance metallo-ß-lactamases.

Metallo-ß-lactamases (MBLs) are an emerging cause of bacterial antibiotic resistance by hydrolysing all classes of ß-lactams except monobactams, and the MBLs are not inhibited by clinically available serine-ß-lactamase inhibitors. Two of the most commonly encountered MBLs in clinical isolates worldwide - the New Delhi metallo-ß-lactamase (NDM-1) and the Verona integron-encoded metallo-ß-lactamase (VIM-2) - are included in this study. A series of several NH-1,2,3-triazoles was prepared by a three-step protocol utilizing Banert cascade reaction as the key step. The inhibitor properties were evaluated in biochemical assays against the MBLs VIM-2, NDM-1 and GIM-1, and VIM-2 showed IC50 values down to nanomolar range. High-resolution crystal structures of four inhibitors in complex with VIM-2 revealed hydrogen bonds from the triazole inhibitors to Arg228 and to the backbone of Ala231 or Asn233, along with hydrophobic interactions to Trp87, Phe61 and Tyr67. The inhibitors show reduced MIC in synergy assays with Pseudomonas aeruginosa and Escherichia coli strains harbouring VIM enzymes. The obtained results will be useful for further structural guided design of MBL inhibitors.

Relevance of preclinical rodent pharmacokinetics in the selection of a companion antibiotic for combining with beta-lactamase inhibitor.

Recent approvals of beta-lactamase inhibitor (BLI) drug in combination with cephalosporins/penems have provided the right impetus for novel BLIs. One important research question, hitherto not addressed, is pertaining to the relevance of preclinical pharmacokinetics for pairing the antibiotic with existing/novel BLI.Two BLI combination drugs: (a) approved (i.e. ceftazidime/avibactam); (b) clinical development (i.e. cefepime/zidebactam) were explored to provide insights to address the research question.Individual intravenous dosing of ceftazidime, avibactam, cefepime and zidebactam was done at 1 mg/kg by intravenous route in Balb/c mice and Wistar rats. Serial blood samples were collected and analysed by LC-MS/MS method.Examination of the ratios of pharmacokinetic parameters (CL, VSS and T1/2) for individual drugs in combinations (for instance, CL (ceftazidime)/CL (avibactam); CL (cefepime)/CL (zidebactam)) suggested that the pharmacokinetic data gathered in rats were generally within 0.5- to 2-fold; but mouse data revealed larger disparity for VSS (0.11- to 8.25-fold) or CL (0.49- to 4.03-fold).The observed ratio for CL/VSS observed in rats agreed with corresponding human ratios for the pairwise comparison of the individual drugs in the combinations.Retrospectively, current pharmacokinetic findings suggest rat pharmacokinetic data may aid the combination of BLI with an appropriate antibiotic.

Permeation of ß-Lactamase Inhibitors through the General Porins of Gram-Negative Bacteria.

Modern medicine relies upon antibiotics, but we have arrived to the point where our inability to come up with new effective molecules against resistant pathogens, together with the declining private investment, is resulting in the number of untreatable infections increasing worldwide at worrying pace. Among other pathogens, widely recognized institutions have indicated Gram-negative bacteria as particularly challenging, due to the presence of the outer membrane. The very first step in the action of every antibiotic or adjuvant is the permeation through this membrane, with small hydrophilic drugs usually crossing through protein channels. Thus, a detailed understanding of their properties at a molecular level is crucial. By making use of Molecular Dynamics simulations, we compared the two main porins of four members of the Enterobacteriaceae family, and, in this paper, we show their shared geometrical and electrostatic characteristics. Then, we used metadynamics simulations to reconstruct the free energy for permeation of selected diazobicyclooctans through OmpF. We demonstrate how porins features are coupled to those of the translocating species, modulating their passive permeation. In particular, we show that the minimal projection area of a molecule is a better descriptor than its molecular mass or the volume. Together with the magnitude and orientation of the electric dipole moment, these are the crucial parameters to gain an efficient compensation between the entropic and enthalpic contributions to the free energy barrier required for permeation. Our results confirm the possibility to predict the permeability of molecules through porins by using a few molecular parameters and bolster the general model according to which the free energy increase is mostly due to the decrease of conformational entropy, and this can be compensated by a favorable alignment of the electric dipole with respect to the channel intrinsic electric field.

New Conformations of Acylation Adducts of Inhibitors of ß-Lactamase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to ß-lactam antibiotics due to the production of the extended spectrum ß-lactamase BlaC. ß-Lactam/ß-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the ß-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other ß-lactamases are discussed.

Evolution of New Delhi metallo-ß-lactamase (NDM) in the clinic: Effects of NDM mutations on stability, zinc affinity, and mono-zinc activity.

Infections by carbapenem-resistant Enterobacteriaceae are difficult to manage owing to broad antibiotic resistance profiles and because of the inability of clinically used ß-lactamase inhibitors to counter the activity of metallo-ß-lactamases often harbored by these pathogens. Of particular importance is New Delhi metallo-ß-lactamase (NDM), which requires a di-nuclear zinc ion cluster for catalytic activity. Here, we compare the structures and functions of clinical NDM variants 1-17. The impact of NDM variants on structure is probed by comparing melting temperature and refolding efficiency and also by spectroscopy (UV-visible, 1H NMR, and EPR) of di-cobalt metalloforms. The impact of NDM variants on function is probed by determining the minimum inhibitory concentrations of various antibiotics, pre-steady-state and steady-state kinetics, inhibitor binding, and zinc dependence of resistance and activity. We observed only minor differences among the fully loaded di-zinc enzymes, but most NDM variants had more distinguishable selective advantages in experiments that mimicked zinc scarcity imposed by typical host defenses. Most NDM variants exhibited improved thermostability (up to ∼10 °C increased Tm ) and improved zinc affinity (up to ∼10-fold decreased Kd, Zn2). We also provide first evidence that some NDM variants have evolved the ability to function as mono-zinc enzymes with high catalytic efficiency (NDM-15, ampicillin: kcat/Km = 5 × 106 m-1 s-1). These findings reveal the molecular mechanisms that NDM variants have evolved to overcome the combined selective pressures of ß-lactam antibiotics and zinc deprivation.

Structural Insights into the Inhibition of the Extended-Spectrum ß-Lactamase PER-2 by Avibactam.

The diazabicyclooctane (DBO) avibactam (AVI) reversibly inactivates most serine-ß-lactamases. Previous investigations showed that inhibition constants of AVI toward class A PER-2 are reminiscent of values observed for class C and D ß-lactamases (i.e., k2/K of ≈103 M-1 s-1) but lower than other class A ß-lactamases (i.e., k2/K = 104 to 105 M-1 s-1). Herein, biochemical and structural studies were conducted with PER-2 and AVI to explore these differences. Furthermore, biochemical studies on Arg220 and Thr237 variants with AVI were conducted to gain deeper insight into the mechanism of PER-2 inactivation. The main biochemical and structural observations revealed the following: (i) both amino-acid substitutions in Arg220 and the rich hydrophobic content in the active site hinder the binding of catalytic waters and acylation, impairing AVI inhibition; (ii) movement of Ser130 upon binding of AVI favors the formation of a hydrogen bond with the sulfate group of AVI; and (iii) the Thr237Ala substitution alters the AVI inhibition constants. The acylation constant (k2/K) of PER-2 by AVI is primarily influenced by stabilizing hydrogen bonds involving AVI and important residues such as Thr237 and Arg220. (Variants in Arg220 demonstrate a dramatic reduction in k2/K) We also observed that displacement of Ser130 side chain impairs AVI acylation, an observation not made in other extended-spectrum ß-lactamases (ESBLs). Comparatively, relebactam combined with a ß-lactam is more potent against Escherichia coli producing PER-2 variants than ß-lactam-AVI combinations. Our findings provide a rationale for evaluating the utility of the currently available DBO inhibitors against unique ESBLs like PER-2 and anticipate the effectiveness of these inhibitors toward variants that may eventually be selected upon AVI usage.

Molecular Basis of Class A ß-Lactamase Inhibition by Relebactam.

ß-Lactamase production is the major ß-lactam resistance mechanism in Gram-negative bacteria. ß-Lactamase inhibitors (BLIs) efficacious against serine ß-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum ß-lactamases L2 (inhibition constant 3 µM) and CTX-M-15 (21 µM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 µM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates ß-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-ß-lactam combinations.

Selecting for Fast Protein-Protein Association As Demonstrated on a Random TEM1 Yeast Library Binding BLIP.

Protein-protein interactions mediate the vast majority of cellular processes. Though protein interactions obey basic chemical principles also within the cell, the in vivo physiological environment may not allow for equilibrium to be reached. Thus, in vitro measured thermodynamic affinity may not provide a complete picture of protein interactions in the biological context. Binding kinetics composed of the association and dissociation rate constants are relevant and important in the cell. Therefore, changes in protein-protein interaction kinetics have a significant impact on the in vivo activity of the proteins. The common protocol for the selection of tighter binders from a mutant library selects for protein complexes with slower dissociation rate constants. Here we describe a method to specifically select for variants with faster association rate constants by using pre-equilibrium selection, starting from a large random library. Toward this end, we refine the selection conditions of a TEM1-ß-lactamase library against its natural nanomolar affinity binder ß-lactamase inhibitor protein (BLIP). The optimal selection conditions depend on the ligand concentration and on the incubation time. In addition, we show that a second sort of the library helps to separate signal from noise, resulting in a higher percent of faster binders in the selected library. Fast associating protein variants are of particular interest for drug development and other biotechnological applications.

Structural/mechanistic insights into the efficacy of nonclassical ß-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates.

Clavulanic acid and avibactam are clinically deployed serine ß-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo ß-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine ß-lactamase) but not L1 (metallo ß-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like ß-lactams plus nonclassical ß-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.

Kinetics of Sulbactam Hydrolysis by ß-Lactamases, and Kinetics of ß-Lactamase Inhibition by Sulbactam.

Sulbactam is one of four ß-lactamase inhibitors in current clinical use to counteract drug resistance caused by degradation of ß-lactam antibiotics by these bacterial enzymes. As a ß-lactam itself, sulbactam is susceptible to degradation by ß-lactamases. I investigated the Michaelis-Menten kinetics of sulbactam hydrolysis by 14 ß-lactamases, representing clinically widespread groups within all four Ambler classes, i.e., CTX-M-15, KPC-2, SHV-5, and TEM-1 for class A; IMP-1, NDM-1, and VIM-1 for class B; Acinetobacter baumannii ADC-7, Pseudomonas aeruginosa AmpC, and Enterobacter cloacae P99 for class C; and OXA-10, OXA-23, OXA-24, and OXA-48 for class D. All of the ß-lactamases were able to hydrolyze sulbactam, although they varied widely in their kinetic constants for the reaction, even within each class. I also investigated the inactivation kinetics of the inhibition of these enzymes by sulbactam. The class A ß-lactamases varied widely in their susceptibility to inhibition, the class C and D enzymes were very weakly inhibited, and the class B enzymes were essentially or completely unaffected. In addition, we measured the sulbactam turnover number, the sulbactam/enzyme molar ratio required for complete inhibition of each enzyme. Class C enzymes had the lowest turnover numbers, class A enzymes varied widely, and class D enzymes had very high turnover numbers. These results are valuable for understanding which ß-lactamases ought to be well inhibited by sulbactam. Moreover, since sulbactam has intrinsic antibacterial activity against Acinetobacter species pathogens, these results contribute to understanding ß-lactamase-mediated sulbactam resistance in Acinetobacter, especially due to the action of the widespread class D enzymes.

Inhibition of ß-lactamases of mycobacteria by avibactam and clavulanate.

Objectives: Mycobacterium tuberculosis and Mycobacterium abscessus produce broad-spectrum class A ß-lactamases, BlaC and Bla Mab , which are inhibited by clavulanate and avibactam, respectively. BlaC differs from Bla Mab at Ambler position 132 in the conserved motif SDN (SDG versus SDN, respectively). Here, we investigated whether this polymorphism could account for the inhibition specificity of ß-lactamases from slowly and rapidly growing mycobacteria. Methods: Enzyme kinetics were determined to assess the impact of the substitutions G 132 N in BlaC and N 132 G in Bla Mab on ß-lactamase inhibition by clavulanate and avibactam. The stability of acylenzymes was evaluated by MS. The impact of the substitutions on the antibacterial activity of drug combinations was determined based on production of the ß-lactamases in Escherichia coli . Results: The substitution G 132 N increased 140-fold the efficacy of BlaC inhibition by avibactam and abolished clavulanate inhibition due to acylenzyme hydrolysis. Bla Mab efficiently hydrolysed clavulanate, but the substitution N 132 G led to a 5600-fold reduction in the hydrolysis rate constant k cat due to stabilization of Bla Mab -clavulanate covalent adducts. The N 132 G substitution also led to a 610-fold reduction in the efficacy of Bla Mab carbamylation by avibactam. Testing resistance to the amoxicillin/clavulanate and amoxicillin/avibactam combinations revealed that modifications in the catalytic properties of the ß-lactamases resulted in opposite shifts from susceptibility to resistance and vice versa. Conclusions: G 132 N and N 132 G had opposite effects on the inhibition of BlaC and Bla Mab , indicating that these substitutions might lead to acquisition of resistance to either of the ß-lactamase inhibitors, but not to both of them.