Profiling interactions of vaborbactam with metallo-β-lactamases

β-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by β-lactamases. β-Lactamases fall into two mechanistic groups: the serine β-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo β-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both β-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-β-lactamases to form covalent complexes that mimic tetrahedral intermediates in catalysis. Vaborbactam has recently been approved for clinical use in combination with the carbapenem meropenem. Here we show that vaborbactam moderately inhibits metallo-β-lactamases from all 3 subclasses (B1, B2 and B3), with a potency of around 20–100 fold below that by which it inhibits its current clinical targets, the Class A serine β-lactamases. This result contrasts with recent investigations of bicyclic boronate inhibitors, which potently inhibit subclass B1 MBLs but which presently lack activity against B2 and B3 enzymes. These findings indicate that cyclic boronate scaffolds have the potential to inhibit the full range of β-lactamases and justify further work on the development of boronates as broad-spectrum β-lactamase inhibitors.     

Peptidyl allyl sulfones: a new class of inhibitors for clan CA cysteine proteases

A new series of peptidyl allyl sulfone inhibitors was discovered while trying to synthesize epoxy sulfone inhibitors from vinyl sulfones using basic oxidizing conditions. The various dipeptidyl allyl sulfones were evaluated with calpain I, papain, cathepsins B and L, cruzain and rhodesain and found to be potent inhibitors. In comparison to the previously developed class of vinyl sulfone inhibitors, the novel dipeptidyl allyl sulfones were more potent inhibitors than the corresponding dipeptidyl vinyl sulfones. It was observed that the stereochemistry of the vinyl sulfone precursor played a role in the potency of the dipeptidyl allyl sulfone inhibitor.     

β-Lactamases and β-Lactamase Inhibitors in the 21st Century

The β-lactams retain a central place in the antibacterial armamentarium. In Gram-negative bacteria, β-lactamase enzymes that hydrolyze the amide bond of the four-membered β-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli) and non-fermenting organisms (e.g., Pseudomonas aeruginosa). β-Lactamases divide into four classes; the active-site serine β-lactamases (classes A, C and D) and the zinc-dependent or metallo-β-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing numbers of crystal structures, in particular for β-lactam complexes, and methods such as neutron diffraction and molecular simulations, have improved understanding of the biochemistry of β-lactam breakdown. A second focus is β-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clinical concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of β-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of β-lactams with diazabicyclooctanone and cyclic boronate serine β-lactamase inhibitors, and of progress and strategies toward clinically useful MBL inhibitors. Despite the long history of β-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new β-lactam:inhibitor combinations and the continuing clinical importance of β-lactams mean that this remains a rewarding research area.     

Inhibition of β-lactamase-I by 6-β-sulfonamidopenicillanic acid sulfones: Evidence for conformational change accompanying the inhibition process

A number of 6-β-sulfonamidopenicillanic acid sulfones were examined for their ability to inhibit Bacillus cereus$\text{569}\text{H}$ β-lactamase I. Among these, 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone was found to be the most potent inhibitor, effecting rapid and irreversible inactivation of the enzyme. Optical rotatory dispersion and differential scanning calorimetry were employed to probe the possible conformational changes accompanying the inactivation of B. cereus$\text{569}\text{H}$ β-lactamase 1 by 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone. Optical rotatory dispersion measurements indicated the presence of approximately 29 and 17% helical structure in the native and inactivated enzyme, respectively. Differential scanning calorimetry determinations revealed that the inactivated enzyme was less thermostable than the native β-lactamase. The temperatures of maximum heat absorption were 48.4(±0.5) and 57.4(±0.1)°C for the inactivated and the native enzyme, respectively. Extensive conformational changes accompanying the interaction of the enzyme with the inhibitor may be responsible for the irreversible loss in the catalytic activity.     

Structure and dynamics of CTX-M enzymes reveal insights into substrate accommodation by extended-spectrum beta-lactamases

Oxyimino-cephalosporin antibiotics, such as ceftazidime, escape the hydrolytic activity of most bacterial β-lactamases. Their widespread use prompted the emergence of the extended-spectrum β-lactamases CTX-Ms, which have become highly prevalent. The C7 β-amino thiazol-oxyimino-amide side chain of ceftazidime has a protective effect against most CTX-M β-lactamases. However, Asp240Gly CTX-M derivatives demonstrate enhanced hydrolytic activity against this compound. In this work, we present the crystallographic structures of Asp240Gly-harboring enzyme CTX-M-16 in complex with ceftazidime-like glycylboronic acid (resolution 1.80 Å) and molecular dynamics simulations of the corresponding acyl-enzyme complex. These experiments revealed breathing motions of CTX-M enzymes and the role of the substitution Asp240Gly in the accommodation of ceftazidime. The substitution Asp240Gly resulted in insertion of the C7β side chain of ceftazidime deep in the catalytic pocket and orchestrated motions of the active serine Ser70, the β3 strand and the omega loop, which favored the key interactions of the residues 237 and 235 with ceftazidime.     

Synthesis and evaluation of boronic acids as inhibitors of Penicillin Binding Proteins of classes A, B and C

In response to the widespread use of β-lactam antibiotics bacteria have evolved drug resistance mechanisms that include the production of resistant Penicillin Binding Proteins (PBPs). Boronic acids are potent β-lactamase inhibitors and have been shown to display some specificity for soluble transpeptidases and PBPs, but their potential as inhibitors of the latter enzymes is yet to be widely explored. Recently, a (2,6-dimethoxybenzamido)methylboronic acid was identified as being a potent inhibitor of Actinomadura sp. R39 transpeptidase (IC(50): 1.3 μM). In this work, we synthesized and studied the potential of a number of acylaminomethylboronic acids as inhibitors of PBPs from different classes. Several derivatives inhibited PBPs of classes A, B and C from penicillin sensitive strains. The (2-nitrobenzamido)methylboronic acid was identified as a good inhibitor of a class A PBP (PBP1b from Streptococcus pneumoniae, IC(50) = 26 μM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC(50) = 138 μM) and a class C PBP (R39 from Actinomadura sp., IC(50) = 0.6 μM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species.     

Metallo-β-lactamase-mediated antimicrobial resistance and progress in inhibitor discovery

Resistance to β-lactam antibiotics is rapidly growing, substantially due to the spread of serine-β-lactamases (SBLs) and metallo-β-lactamases (MBLs), which efficiently catalyse β-lactam hydrolysis. Combinations of a β-lactam antibiotic with an SBL inhibitor have been clinically successful; however, no MBL inhibitors have been developed for clinical use. MBLs are a worrying resistance vector because they catalyse hydrolysis of all β-lactam antibiotic classes, except the monobactams, and they are being disseminated across many bacterial species worldwide. Here we review the classification, structures, substrate profiles, and inhibition mechanisms of MBLs, highlighting current clinical problems due to MBL-mediated resistance and progress in understanding and combating MBL-mediated resistance.     

2-Substituted 4,5-dihydrothiazole-4-carboxylic acids are novel inhibitors of metallo-β-lactamases

Bacterial resistance to β-lactam antibiotics caused by class B metallo-β-lactamases (MBL), especially for certain hospital-acquired, Gram-negative pathogens, poses a significant threat to public health. We report several 2-substituted 4,5-dihydrothiazole-4-carboxylic acids to be novel MBL inhibitors. Structure activity relationship (SAR) and molecular modeling studies were performed and implications for further inhibitor design are discussed.     

The synthesis and evaluation of 6-alkylidene-2'beta-substituted penam sulfones as beta-lactamase inhibitors.

Penicillin sulfones, which structurally incorporate both a 6-position alkylidene substituent and a 2'beta substituent, have been synthesized and evaluated as inhibitors of class C and class A serine beta-lactamases. Incorporation of the 2'beta-substituent generally improves inhibitory activity. Substituents that improve transport across the bacterial cell membrane have also been incorporated.     

Optimization of peptidyl allyl sulfones as clan CA cysteine protease inhibitors

This research investigates the synthesis and inhibitory potency of a series of novel dipeptidyl allyl sulfones as clan CA cysteine protease inhibitors. The structure of the inhibitors consists of a R₁-Phe-R₂-AS-Ph scaffold (AS?=?allyl sulfone). R₁ was varied with benzyloxycarbonyl, morpholinocarbonyl, or N-methylpiperazinocarbonyl substituents. R₂ was varied with either Phe of Hfe residues. Synthesis involved preparation of vinyl sulfone analogues followed by isomerization to allyl sulfones using n-butyl lithium and t-butyl hydroperoxide. Sterics, temperature and base strength were all factors that affected the formation and stereochemistry of the allyl sulfone moiety. The inhibitors were assayed with three clan CA cysteine proteases (cruzain, cathepsin B and calpain I) as well as one serine protease (trypsin). The most potent inhibitor, (E)-Mu-Phe-Hfe-AS-Ph, displayed at least 10-fold selectivity for cruzain over clan CA cysteine proteases cathepsin B and calpain I with a k_obs/[I] of 6080?±?1390?M^?1s^?1.     

Interaction with the S1 beta-pocket of urokinase: 8-heterocycle substituted and 6,8-disubstituted 2-naphthamidine urokinase inhibitors

Several 8-substituted 2-naphthamidine-based inhibitors of the serine protease urokinase plasminogen activator (uPA) are described. Direct attachment of five-membered saturated or unsaturated rings improved inhibitor performance; substitution with sulfones further improved binding profiles. Combination of these substituents or of previously described NH-linked heteroaromatic rings with 6-phenyl amide substituents provided further enhancements to potency and selectivity.     

Antimalarial sulfide, sulfone, and sulfonamide trioxanes

A series of trioxanes featuring sulfide, sulfone, and sulfonamide substituents in diverse positions has been prepared. Structure-activity relationship (SAR) generalizations highlight two major factors controlling the antimalarial potency of these new chemical entities: (1) the proximity of the sulfur-containing substituent to the crucial peroxide bond and (2) the oxidation state of the sulfur-containing substituent. Generally, sulfones are more antimalarially potent than the corresponding sulfides.     

3-mercapto-1,2,4-triazoles and N-acylated thiosemicarbazides as metallo-β-lactamase inhibitors

The production of β-lactamases is an effective strategy by which pathogenic bacteria can develop resistance against β-lactam antibiotics. While inhibitors of serine-β-lactamases are widely used in combination therapy with β-lactam antibiotics, there are no clinically available inhibitors of metallo-β-lactamases (MBLs), and so there is a need for the development of such inhibitors. This work describes the optimisation of a lead inhibitor previously identified by fragment screening of a compound library. We also report that thiosemicarbazide intermediates in the syntheses of these compounds are also moderately potent inhibitors of the IMP-1 MBL from Pseudomonas aeruginosa. The interactions of these inhibitors with the active site of IMP-1 were examined using in silico methods.     

Evaluation of penicillin-based inhibitors of the class A and B beta-lactamases from Bacillus anthracis

Bacillus anthracis contains a class A (Bla1) and class B (Bla2) beta-lactamase, which confer resistance to beta-lactam antibiotics when expressed in Escherichia coli. In an effort to find new beta-lactamase inhibitors, several penicillin derivatives have been evaluated including experimental compounds incorporating a 6-mercaptomethyl group or a 6-pyridylmethylidene group, along with clavulanate and tazobactam, as inhibitors against Bla1 and Bla2. The 6-mercaptomethyl-substituted penicillins showed much greater activity against the zinc-containing Bla2 than Bla1. The compound that incorporated a 6-pyridylmethylidene substituent and a catecholic substituent at the 2' position was the most effective inhibitor of Bla1 with Ki=0.057 microM. Inhibitors containing iron-chelating functional groups have previously been shown to work in combination with antibiotics to inhibit growth of antibiotic-resistant bacteria expressing beta-lactamase. The development of similar compounds, incorporating these types of substituents, may help overcome resistance to currently used antibiotics.     

Structures of the class D carbapenemase OXA-24 from Acinetobacter baumannii in complex with doripenem

The emergence of class D β-lactamases with carbapenemase activity presents an enormous challenge to health practitioners, particularly with regard to the treatment of infections caused by Gram-negative pathogens such as Acinetobacter baumannii. Unfortunately, class D β-lactamases with carbapenemase activity are resistant to β-lactamase inhibitors. To better understand the details of the how these enzymes bind and hydrolyze carbapenems, we have determined the structures of two deacylation-deficient variants (K84D and V130D) of the class D carbapenemase OXA-24 with doripenem bound as a covalent acyl-enzyme intermediate. Doripenem adopts essentially the same configuration in both OXA-24 variant structures, but varies significantly when compared to the non-carbapenemase class D member OXA-1/doripenem complex. The alcohol of the 6α hydroxyethyl moiety is directed away from the general base carboxy-K84, with implications for activation of the deacylating water. The tunnel formed by the Y112/M223 bridge in the apo form of OXA-24 is largely unchanged by the binding of doripenem. The presence of this bridge, however, causes the distal pyrrolidine/sulfonamide group to bind in a drastically different conformation compared to doripenem bound to OXA-1. The resulting difference in the position of the side-chain bridge sulfur of doripenem is consistent with the hypothesis that the tautomeric state of the pyrroline ring contributes to the different carbapenem hydrolysis rates of OXA-1 and OXA-24. These findings represent a snapshot of a key step in the catalytic mechanism of an important class D enzyme, and might be useful for the design of novel inhibitors.     

Studies on the inhibition of AmpC and other β-lactamases by cyclic boronates

BackgroundThe β-lactam antibiotics represent the most successful drug class for treatment of bacterial infections. Resistance to them, importantly via production of β-lactamases, which collectively are able to hydrolyse all classes of β-lactams, threatens their continued widespread use. Bicyclic boronates show potential as broad spectrum inhibitors of the mechanistically distinct serine- (SBL) and metallo- (MBL) β-lactamase families.MethodsUsing biophysical methods, including crystallographic analysis, we have investigated the binding mode of bicyclic boronates to clinically important β-lactamases. Induction experiments and agar-based MIC screening against MDR-Enterobacteriaceae (n = 132) were used to evaluate induction properties and the in vitro efficacy of a bicyclic boronate in combination with meropenem.ResultsCrystallographic analysis of a bicyclic boronate in complex with AmpC from Pseudomonas aeruginosa reveals it binds to form a tetrahedral boronate species. Microbiological studies on the clinical coverage (in combination with meropenem) and induction of β-lactamases by bicyclic boronates further support the promise of such compounds as broad spectrum β-lactamase inhibitors.ConclusionsTogether with reported studies on the structural basis of their inhibition of class A, B and D β-lactamases, biophysical studies, including crystallographic analysis, support the proposal that bicyclic boronates mimic tetrahedral intermediates common to SBL and MBL catalysis.General significanceBicyclic boronates are a new generation of broad spectrum inhibitors of both SBLs and MBLs.     

Characterization of β-lactamase activity using isothermal titration calorimetry

BackgroundHydrolysis of β-lactam antibiotic by β-lactamase is the most common mechanism of β-lactam resistance in clinical isolates. Timely detection and characterization of β-lactamases are therefore of utmost biomedical importance. Conventional spectrophotometric method is time-consuming and cannot provide thermodynamic information on β-lactamases.MethodsA new assay was developed for the study of β-lactamase activity in protein solutions (Metallo-β-lactamase L1) and in clinical bacterial cells, based on heat-flow changes derived from enzymatic hydrolysis of β-lactams using isothermal titration calorimetry.Results(1) The thermokinetic parameters of three antibiotics (penicillin G, cefazolin and imipenem) and the inhibition constant of an azolylthioacetamide inhibitor were determined using the calorimetric assay. The results from the calorimetric assays were consistent with the data from the spectrophotometric assay. (2) The values of heat change in the calorimetric assay using two clinical Escherichia coli strains correlated well with their antibiotic susceptibility results from the broth dilution experiment. The subtypes of β-lactamase were also determined in the calorimetric assay.ConclusionsThe ITC assay is a reliable and fast method to study β-lactamase enzyme kinetics and inhibition. It can also provide thermodynamic information on antibiotic hydrolysis, which has been taken advantage of in this work to study β-lactamase activity in two clinical Escherichia coli isolates.General significanceAs the first calorimetric study of β-lactamase activity, it may provide a new assay to assist biomedical validation of new β-lactamase inhibitors, and also has potential applications on rapid antibiotic susceptibility testing and screening β-lactamase producing bacteria.     

Identification of peptide inhibitors of penicillinase using a phage display library

There is a constant need to identify novel inhibitors to combat β-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM⁻¹) and binding affinity (K_D = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV–vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a K_i of 9.22 μM and a K_i′ of 33.12 μM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of β-lactamases and development of novel inhibitors of β-lactamases.     

α-Substituted norstatines as the transition-state mimic in inhibitors of multiple digestive vacuole malaria aspartic proteases

The impact of moving the P1 side-chain from the β-position to the α-position in norstatine-containing plasmepsin inhibitors was investigated, generating two new classes of tertiary alcohol-comprising α-benzylnorstatines and α-phenylnorstatines. Twelve α-substituted norstatines were designed, synthesized and evaluated for their inhibitory potencies against plasmepsin II and the plasmepsin IV orthologues (PM4) present in the digestive vacuole of all four Plasmodium species causing malaria in man. New synthetic routes were developed for producing the desired α-substituted norstatines as pure stereoisomers. The best compounds provided K_i values in the nanomolar range for all PM4, with a best value of 110 nM in PM4 from Plasmodium ovale. In addition, excellent selectivity over the closely related human aspartic protease Cathepsin D was achieved. The loss of affinity to Plasmodium falciparum PM4, which was experienced upon the move of the P1 substituent, was rationalized by the calculation of inhibitor–protein binding affinities using the linear interaction energy method (LIE).     

Structural insights into the design of inhibitors for the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia

One mechanism by which bacteria can escape the action of β-lactam antibiotics is the production of metallo-β-lactamases. Inhibition of these enzymes should restore the action of these widely used antibiotics. The tetrameric enzyme L1 from Stenotrophomonas maltophilia was used as a model system to determine a series of high-resolution crystal structures of apo, mono and bi-metal substituted proteins as well as protein-inhibitor complexes. Unexpectedly, although the apo structure revealed only few significant structural differences from the holo structure, some inhibitors were shown to induce amino acid side-chain rotations in the tightly packed active site. Moreover, one inhibitor employs a new binding mode in order to interact with the di-zinc center. This structural information could prove essential in the process of elucidation of the mode of interaction between a putative lead compound and metallo-β-lactamases, one of the main steps in structure-based drug design.