Rational Design of Benzobisheterocycle Metallo-ß-Lactamase Inhibitors: A Tricyclic Scaffold Enhances Potency against Target Enzymes.

Antimicrobial resistance is a global public health threat. Metallo-ß-lactamases (MBLs) inactivate ß-lactam antibiotics, including carbapenems, are disseminating among Gram-negative bacteria, and lack clinically useful inhibitors. The evolving bisthiazolidine (BTZ) scaffold inhibits all three MBL subclasses (B1-B3). We report design, synthesis, and evaluation of BTZ analogues. Structure-activity relationships identified the BTZ thiol as essential, while carboxylate is replaceable, with its removal enhancing potency by facilitating hydrophobic interactions within the MBL active site. While the introduction of a flexible aromatic ring is neutral or detrimental for inhibition, a rigid (fused) ring generated nM benzobisheterocycle (BBH) inhibitors that potentiated carbapenems against MBL-producing strains. Crystallography of BBH:MBL complexes identified hydrophobic interactions as the basis of potency toward B1 MBLs. These data underscore BTZs as versatile, potent broad-spectrum MBL inhibitors (with activity extending to enzymes refractory to other inhibitors) and provide a rational approach to further improve the tricyclic BBH scaffold.

Correction: 2-Mercaptomethyl-thiazolidines use conserved aromatic-S interactions to achieve broad-range inhibition of metallo-ß-lactamases.

[This corrects the article DOI: 10.1039/D0SC05172A.].

Tautomer-Specific Deacylation and Ω-Loop Flexibility Explain the Carbapenem-Hydrolyzing Broad-Spectrum Activity of the KPC-2 ß-Lactamase.

KPC-2 (Klebsiella pneumoniae carbapenemase-2) is a globally disseminated serine-ß-lactamase (SBL) responsible for extensive ß-lactam antibiotic resistance in Gram-negative pathogens. SBLs inactivate ß-lactams via a mechanism involving a hydrolytically labile covalent acyl-enzyme intermediate. Carbapenems, the most potent ß-lactams, evade the activity of many SBLs by forming long-lived inhibitory acyl-enzymes; however, carbapenemases such as KPC-2 efficiently deacylate carbapenem acyl-enzymes. We present high-resolution (1.25-1.4 Å) crystal structures of KPC-2 acyl-enzymes with representative penicillins (ampicillin), cephalosporins (cefalothin), and carbapenems (imipenem, meropenem, and ertapenem) obtained utilizing an isosteric deacylation-deficient mutant (E166Q). The mobility of the Ω-loop (residues 165-170) negatively correlates with antibiotic turnover rates (kcat), highlighting the role of this region in positioning catalytic residues for efficient hydrolysis of different ß-lactams. Carbapenem-derived acyl-enzyme structures reveal the predominance of the Δ1-(2R) imine rather than the Δ2 enamine tautomer. Quantum mechanics/molecular mechanics molecular dynamics simulations of KPC-2:meropenem acyl-enzyme deacylation used an adaptive string method to differentiate the reactivity of the two isomers. These identify the Δ1-(2R) isomer as having a significantly (7 kcal/mol) higher barrier than the Δ2 tautomer for the (rate-determining) formation of the tetrahedral deacylation intermediate. Deacylation is therefore likely to proceed predominantly from the Δ2, rather than the Δ1-(2R) acyl-enzyme, facilitated by tautomer-specific differences in hydrogen-bonding networks involving the carbapenem C-3 carboxylate and the deacylating water and stabilization by protonated N-4, accumulating a negative charge on the Δ2 enamine-derived oxyanion. Taken together, our data show how the flexible Ω-loop helps confer broad-spectrum activity upon KPC-2, while carbapenemase activity stems from efficient deacylation of the Δ2-enamine acyl-enzyme tautomer.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Penicillanic Acid Sulfones Inactivate the Extended-Spectrum ß-Lactamase CTX-M-15 through Formation of a Serine-Lysine Cross-Link: an Alternative Mechanism of ß-Lactamase Inhibition.

ß-Lactamases hydrolyze ß-lactam antibiotics and are major determinants of antibiotic resistance in Gram-negative pathogens. Enmetazobactam (formerly AAI101) and tazobactam are penicillanic acid sulfone (PAS) ß-lactamase inhibitors that differ by an additional methyl group on the triazole ring of enmetazobactam, rendering it zwitterionic. In this study, ultrahigh-resolution X-ray crystal structures and mass spectrometry revealed the mechanism of PAS inhibition of CTX-M-15, an extended-spectrum ß-lactamase (ESBL) globally disseminated among Enterobacterales. CTX-M-15 crystals grown in the presence of enmetazobactam or tazobactam revealed loss of the Ser70 hydroxyl group and formation of a lysinoalanine cross-link between Lys73 and Ser70, two residues critical for catalysis. Moreover, the residue at position 70 undergoes epimerization, resulting in formation of a d-amino acid. Cocrystallization of enmetazobactam or tazobactam with CTX-M-15 with a Glu166Gln mutant revealed the same cross-link, indicating that this modification is not dependent on Glu166-catalyzed deacylation of the PAS-acylenzyme. A cocrystal structure of enmetazobactam with CTX-M-15 with a Lys73Ala mutation indicates that epimerization can occur without cross-link formation and positions the Ser70 Cß closer to Lys73, likely facilitating formation of the Ser70-Lys73 cross-link. A crystal structure of a tazobactam-derived imine intermediate covalently linked to Ser70, obtained after 30 min of exposure of CTX-M-15 crystals to tazobactam, supports formation of an initial acylenzyme by PAS inhibitors on reaction with CTX-M-15. These data rationalize earlier results showing CTX-M-15 deactivation by PAS inhibitors to involve loss of protein mass, and they identify a distinct mechanism of ß-lactamase inhibition by these agents. IMPORTANCE ß-Lactams are the most prescribed antibiotic class for treating bacterial diseases, but their continued efficacy is threatened by bacterial strains producing ß-lactamase enzymes that catalyze their inactivation. The CTX-M family of ESBLs are major contributors to ß-lactam resistance in Enterobacterales, preventing effective treatment with most penicillins and cephalosporins. Combining ß-lactams with ß-lactamase inhibitors (BLIs) is a validated route to overcome such resistance. Here, we describe how exposure to enmetazobactam and tazobactam, BLIs based on a penicillanic acid sulfone (PAS) scaffold, leads to a protein modification in CTX-M-15, resulting in irremediable inactivation of this most commonly encountered member of the CTX-M family. High-resolution X-ray crystal structures showed that PAS exposure induces formation of a cross-link between Ser70 and Lys73, two residues critical to ß-lactamase function. This previously undescribed mechanism of inhibition furthers our understanding of ß-lactamase inhibition by classical PAS inhibitors and provides a basis for further, rational inhibitor development.

A multiscale approach to predict the binding mode of metallo beta-lactamase inhibitors.

Antibiotic resistance is a major threat to global public health. ß-lactamases, which catalyze breakdown of ß-lactam antibiotics, are a principal cause. Metallo ß-lactamases (MBLs) represent a particular challenge because they hydrolyze almost all ß-lactams and to date no MBL inhibitor has been approved for clinical use. Molecular simulations can aid drug discovery, for example, predicting inhibitor complexes, but empirical molecular mechanics (MM) methods often perform poorly for metalloproteins. Here we present a multiscale approach to model thiol inhibitor binding to IMP-1, a clinically important MBL containing two catalytic zinc ions, and predict the binding mode of a 2-mercaptomethyl thiazolidine (MMTZ) inhibitor. Inhibitors were first docked into the IMP-1 active site, testing different docking programs and scoring functions on multiple crystal structures. Complexes were then subjected to molecular dynamics (MD) simulations and subsequently refined through QM/MM optimization with a density functional theory (DFT) method, B3LYP/6-31G(d), increasing the accuracy of the method with successive steps. This workflow was tested on two IMP-1:MMTZ complexes, for which it reproduced crystallographically observed binding, and applied to predict the binding mode of a third MMTZ inhibitor for which a complex structure was crystallographically intractable. We also tested a 12-6-4 nonbonded interaction model in MD simulations and optimization with a SCC-DFTB QM/MM approach. The results show the limitations of empirical models for treating these systems and indicate the need for higher level calculations, for example, DFT/MM, for reliable structural predictions. This study demonstrates a reliable computational pipeline that can be applied to inhibitor design for MBLs and other zinc-metalloenzyme systems.

Imitation of ß-lactam binding enables broad-spectrum metallo-ß-lactamase inhibitors.

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.

Crystallography and QM/MM Simulations Identify Preferential Binding of Hydrolyzed Carbapenem and Penem Antibiotics to the L1 Metallo-ß-Lactamase in the Imine Form.

Widespread bacterial resistance to carbapenem antibiotics is an increasing global health concern. Resistance has emerged due to carbapenem-hydrolyzing enzymes, including metallo-ß-lactamases (MßLs), but despite their prevalence and clinical importance, MßL mechanisms are still not fully understood. Carbapenem hydrolysis by MßLs can yield alternative product tautomers with the potential to access different binding modes. Here, we show that a combined approach employing crystallography and quantum mechanics/molecular mechanics (QM/MM) simulations allow tautomer assignment in MßL:hydrolyzed antibiotic complexes. Molecular simulations also examine (meta)stable species of alternative protonation and tautomeric states, providing mechanistic insights into ß-lactam hydrolysis. We report the crystal structure of the hydrolyzed carbapenem ertapenem bound to the L1 MßL from Stenotrophomonas maltophilia and model alternative tautomeric and protonation states of both hydrolyzed ertapenem and faropenem (a related penem antibiotic), which display different binding modes with L1. We show how the structures of both complexed ß-lactams are best described as the (2S)-imine tautomer with the carboxylate formed after ß-lactam ring cleavage deprotonated. Simulations show that enamine tautomer complexes are significantly less stable (e.g., showing partial loss of interactions with the L1 binuclear zinc center) and not consistent with experimental data. Strong interactions of Tyr32 and one zinc ion (Zn1) with ertapenem prevent a C6 group rotation, explaining the different binding modes of the two ß-lactams. Our findings establish the relative stability of different hydrolyzed (carba)penem forms in the L1 active site and identify interactions important to stable complex formation, information that should assist inhibitor design for this important antibiotic resistance determinant.

2-Mercaptomethyl Thiazolidines (MMTZs) Inhibit All Metallo-ß-Lactamase Classes by Maintaining a Conserved Binding Mode.

Metallo-ß-lactamase (MBL) production in Gram-negative bacteria is an important contributor to ß-lactam antibiotic resistance. Combining ß-lactams with ß-lactamase inhibitors (BLIs) is a validated route to overcoming resistance, but MBL inhibitors are not available in the clinic. On the basis of zinc utilization and sequence, MBLs are divided into three subclasses, B1, B2, and B3, whose differing active-site architectures hinder development of BLIs capable of "cross-class" MBL inhibition. We previously described 2-mercaptomethyl thiazolidines (MMTZs) as B1 MBL inhibitors (e.g., NDM-1) and here show that inhibition extends to the clinically relevant B2 (Sfh-I) and B3 (L1) enzymes. MMTZs inhibit purified MBLs in vitro (e.g., Sfh-I, Ki 0.16 µM) and potentiate ß-lactam activity against producer strains. X-ray crystallography reveals that inhibition involves direct interaction of the MMTZ thiol with the mono- or dizinc centers of Sfh-I/L1, respectively. This is further enhanced by sulfur-π interactions with a conserved active site tryptophan. Computational studies reveal that the stereochemistry at chiral centers is critical, showing less potent MMTZ stereoisomers (up to 800-fold) as unable to replicate sulfur-π interactions in Sfh-I, largely through steric constraints in a compact active site. Furthermore, in silico replacement of the thiazolidine sulfur with oxygen (forming an oxazolidine) resulted in less favorable aromatic interactions with B2 MBLs, though the effect is less than that previously observed for the subclass B1 enzyme NDM-1. In the B3 enzyme L1, these effects are offset by additional MMTZ interactions with the protein main chain. MMTZs can therefore inhibit all MBL classes by maintaining conserved binding modes through different routes.

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography.

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.

2-Mercaptomethyl-thiazolidines use conserved aromatic-S interactions to achieve broad-range inhibition of metallo-ß-lactamases.

Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-ß-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all ß-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., K i = 0.44 µM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(ii) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than d/l-captopril. Unexpectedly, MMTZ binding features a thioether-π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur-π interactions can be exploited for general ligand design in medicinal chemistry.

Faropenem reacts with serine and metallo-ß-lactamases to give multiple products.

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.

Catalytic mechanism of the colistin resistance protein MCR-1.

The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyses phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calculations on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via transfer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an additional Zn2+. The calculations suggest the involvement of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addition. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases.

Natural variants modify Klebsiella pneumoniae carbapenemase (KPC) acyl-enzyme conformational dynamics to extend antibiotic resistance.

Class A serine ß-lactamases (SBLs) are key antibiotic resistance determinants in Gram-negative bacteria. SBLs neutralize ß-lactams via a hydrolytically labile covalent acyl-enzyme intermediate. Klebsiella pneumoniae carbapenemase (KPC) is a widespread SBL that hydrolyzes carbapenems, the most potent ß-lactams; known KPC variants differ in turnover of expanded-spectrum oxyimino-cephalosporins (ESOCs), for example, cefotaxime and ceftazidime. Here, we compare ESOC hydrolysis by the parent enzyme KPC-2 and its clinically observed double variant (P104R/V240G) KPC-4. Kinetic analyses show that KPC-2 hydrolyzes cefotaxime more efficiently than the bulkier ceftazidime, with improved ESOC turnover by KPC-4 resulting from enhanced turnover (kcat), rather than altered KM values. High-resolution crystal structures of ESOC acyl-enzyme complexes with deacylation-deficient (E166Q) KPC-2 and KPC-4 mutants show that ceftazidime acylation causes rearrangement of three loops; the Ω, 240, and 270 loops, which border the active site. However, these rearrangements are less pronounced in the KPC-4 than the KPC-2 ceftazidime acyl-enzyme and are not observed in the KPC-2:cefotaxime acyl-enzyme. Molecular dynamics simulations of KPC:ceftazidime acyl-enyzmes reveal that the deacylation general base E166, located on the Ω loop, adopts two distinct conformations in KPC-2, either pointing "in" or "out" of the active site; with only the "in" form compatible with deacylation. The "out" conformation was not sampled in the KPC-4 acyl-enzyme, indicating that efficient ESOC breakdown is dependent upon the ordering and conformation of the KPC Ω loop. The results explain how point mutations expand the activity spectrum of the clinically important KPC SBLs to include ESOCs through their effects on the conformational dynamics of the acyl-enzyme intermediate.

Resistance to the "last resort" antibiotic colistin: a single-zinc mechanism for phosphointermediate formation in MCR enzymes.

MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the "last-resort" antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.

Cyclic boronates as versatile scaffolds for KPC-2 ß-lactamase inhibition.

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.

Discovery of New and Potent InhA Inhibitors as Antituberculosis Agents: Structure-Based Virtual Screening Validated by Biological Assays and X-ray Crystallography.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosis is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski's rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 µg/mL against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 µM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.

Mechanistic Insights into ß-Lactamase-Catalysed Carbapenem Degradation Through Product Characterisation.

ß-Lactamases are a major threat to the clinical use of carbapenems, which are often antibiotics of last resort. Despite this, the reaction outcomes and mechanisms by which ß-lactamases degrade carbapenems are still not fully understood. The carbapenem bicyclic core consists of a ß-lactam ring fused to a pyrroline ring. Following ß-lactamase-mediated opening of the ß-lactam, the pyrroline may interconvert between an enamine (2-pyrroline) form and two epimeric imine (1-pyrroline) forms; previous crystallographic and spectroscopic studies have reported all three of these forms in the contexts of hydrolysis by different ß-lactamases. As we show by NMR spectroscopy, the serine ß-lactamases (KPC-2, SFC-1, CMY-10, OXA-23, and OXA-48) and metallo-ß-lactamases (NDM-1, VIM-1, BcII, CphA, and L1) tested all degrade carbapenems to preferentially give the Δ2 (enamine) and/or (R)-Δ1 (imine) products. Rapid non-enzymatic tautomerisation of the Δ2 product to the (R)-Δ1 product prevents assignment of the nascent enzymatic product by NMR. The observed stereoselectivity implies that carbapenemases control the form of their pyrroline ring intermediate(s)/product(s), thereby preventing pyrroline tautomerisation from inhibiting catalysis.

Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-ß-Lactamases.

The bicyclic boronate VNRX-5133 (taniborbactam) is a new type of ß-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-ß-lactamases (SBLs) and some clinically important metallo-ß-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1 and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds to the class D SBL OXA-10 and MBL NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography, we observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen onto the boron of the bicyclic core. Different side-chain binding modes for bicyclic boronates for SBLs and MBLs imply scope for side-chain optimization. The results further support the "high-energy-intermediate" analogue approach for broad-spectrum ß-lactamase inhibitor development and highlight the ability of boron inhibitors to interchange between different hybridization states/binding modes.

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.