Phenylboronic Acids Probing Molecular Recognition against Class A and Class C beta-Lactamases

Worldwide dissemination of pathogens resistant to almost all available antibiotics represent a real problem preventing efficient treatment of infectious diseases. Among antimicrobial used in therapy, \u392-lactam antibiotics represent 40% thus playing a crucial role in the management of infections treatment. We report a small series of phenylboronic acids derivatives (BAs) active against class A carbapenemases KPC-2 and GES-5, and class C cephalosporinases AmpC. The inhibitory profile of our BAs against class A and C was investigated by means of molecular docking, enzyme kinetics and X-ray crystallography. We were interested in the mechanism of recognition among class A and class C to direct the design of broad serine \u392-Lactamases (SBLs) inhibitors. Molecular modeling calculations vs GES-5 and crystallographic studies vs AmpC reasoned, respectively, the ortho derivative 2 and the meta derivative 3 binding affinity. The ability of our BAs to protect \u392-lactams from BLs hydrolysis was determined in biological assays conducted against clinical strains: Fractional inhibitory concentration index (FICI) tests confirmed their ability to be synergic with \u392-lactams thus restoring susceptibility to meropenem. Considering the obtained results and the lack of cytotoxicity, our derivatives represent validated probe for the design of SBLs inhibitors

Crystal structure of Enterobacter cloacae 908R class C beta-lactamase bound to iodo-acetamido-phenyl boronic acid, a transition-state analogue

peer reviewedThe structures of the, class C beta-lactamase from Enterobacter cloacae 908R alone and in complex with a baronic acid transition-state analogue were determined by X-ray crystallography at 2.1 and 2.3 Angstrom, respectively. The structure of the enzyme resembles those of other class C beta-lactamases. The structure of the. complex with the transition-state analogue, iodo-acetamido-phenyl boronic acid, shows that the inhibitor is covalently, bound to the active-site serine (Ser64). Binding of the inhibitor within the active site is compared with previously determined structures of complexes with other class C enzymes. The structure of the boronic acid adduct indicates ways to improve the affinity of this class of inhibitors. This structure of 908R class C beta-lactamase in complex with a transitionstate analogue provides further insights into the mechanism of action of these hydrolases

Targeting Enzymes Involved in Antimicrobial Resistance (AMR) in Gram-negative Bacteria

Antimicrobial resistance (AMR) emerged rapidly after the introduction of the penicillins, the first generation of β-lactam antibiotics, in 1946. Resistance to antibiotics of last resort has highlighted AMR in bacterial pathogens as a pressing therapeutic issue. Gram-negative bacteria manifest high-level resistance to most classes of antibiotics and are the leading cause of severe infectious disease globally. Therefore, reversing their resistant status is of our interest. Among many mechanisms discovered, the expression of drug-inactivating enzymes is the major cause that leads to Gram-negative bacterial AMR. We aim to probe the chemical biology of two proteins associated with drug resistance: Klebsiella pneumoniae carbapenemase (KPC-2) which hydrolyses β-lactam antibiotics and a bacterial glutathione transferase, glutathione transferase (GST-A) which plays roles in antibiotic conjugation and inactivation. Based on the known crystal structures of the proteins (KPC-2: PDB id 3RXX, GST-A: PDB id 1A0F), small molecules were designed and synthesised and tested as inhibitors of the purified enzymes. Promising inhibitors for KPC-2 have been developed with a scaffold containing a 1,4-disubstituted 1,2,3-triazole. In vitro tests indicated that the compounds have a clear SAR and the best inhibitors have nanomolar Ki values. Antibiotic susceptibility tests were used to validate boronic acid KPC-2 inhibitors as potentiators of β-lactam antibiotic activity in cellulo. The compounds showed the successful reversal of resistance to cefotaxime (CTX) and meropenem (MEM) in cellulo in KPC-2 producing Escherichia coli (over 512-fold more sensitive). A small library of glutathione (GSH) analogues was synthesised and tested against GST-A. Binding assays and enzyme kinetics studies suggested that the Gly moiety of GSH is less important than Glu in protein G-site binding, and π-stacking is a critical factor in GST-A H-site binding. We also used susceptibility tests to explore whether GST-A plays a role in antibiotic detoxification and may serve as a target to combat AMR. However, target validation work suggested that GST-A is not essential for E. coli survival and inhibiting the protein may not be a promising approach for drug discovery

Boronic acid inhibitors of the class A beta-lactamase KPC-2

The rapid rise of antimicrobial resistance is one of the greatest challenges currently facing medical science. The most common cause of resistance to β-lactam antibiotics is the expression of β-lactamase enzymes, such as KPC-2. As such the development of novel inhibitors of KPC-2 and related enzymes is of the upmost importance. We report the design and synthesis of novel boronic acid transition state analogs containing a 1,4-substituted 1,2,3-triazole linker based on the known inhibitor 3-nitrophenyl boronic acid and demonstrate that they are promising scaffolds for the development inhibitors of KPC-2 with the ability to recover sensitivity to the antibiotic cefotaxime

Structure-based design and in-parallel synthesis of inhibitors of AmpC beta-lactamase

Background: Group I p-lactamases are a major cause of antibiotic resistance to beta -lactams such as penicillins and cephalosporins. These enzymes are only modestly affected by classic beta -lactam-based inhibitors, such as clavulanic acid. Conversely, small arylboronic acids inhibit these enzymes at sub-micromolar concentrations. Structural studies suggest these inhibitors bind to a well-defined cleft in the group I beta -lactamase AmpC; this cleft binds the ubiquitous R1 side chain of beta -lactams. Intriguingly, much of this cleft is left unoccupied by the small arylboronic acids. Results: To investigate if larger boronic acids might take advantage of this cleft, structure-guided in-parallel synthesis was used to explore new inhibitors of AmpC. Twenty-eight derivatives of the lead compound, 3-aminophenylboronic acid, led to an inhibitor with 80-fold better binding (2; K-i 83 nM). Molecular docking suggested orientations for this compound in the R1 cleft. Based on the docking results, 12 derivatives of 2 were synthesized, leading to inhibitors with iii values of 60 nM and with improved solubility. Several of these inhibitors reversed the resistance of nosocomial Gram-positive bacteria, though they showed little activity against Gram-negative bacteria. The X-ray crystal structure of compound 2 in complex with AmpC was subsequently determined to 2.1 Angstrom resolution. The placement of the proximal two-thirds of the inhibitor in the experimental structure corresponds with the docked structure, but a bond rotation leads to a distinctly different placement of the distal part of the inhibitor. In the experimental structure, the inhibitor interacts with conserved residues in the R1 cleft whose role in recognition has not been previously explored. Conclusions: Combining structure-based design with in-parallel synthesis allowed for the rapid exploration of inhibitor functionality in the R1 cleft of AmpC. The resulting inhibitors differ considerably from beta -lactams but nevertheless inhibit the enzyme well. The crystal structure of 2 (K-i 83 nM) in complex with AmpC may guide exploration of a highly conserved, largely unexplored cleft, providing a template for further design against AmpC beta -lactamase

Triazole-substituted phenylboronic acids as tunable lead inhibitors of KPC-2 antibiotic resistance

Inhibition of β-lactamases is a promising strategy to overcome antimicrobial resistance to commonly used β-lactam antibiotics. Boronic acid derivatives have proven to be effective inhibitors of β-lactamases due to their direct interaction with the catalytic site of these enzymes. We synthesized a series of phenylboronic acid derivatives and evaluated their structure-activity relationships as Klebsiella pneumoniae carbapenemase (KPC-2) inhibitors. We identified potent KPC-2 inhibitors 2e & 6c (Ki = 0.032 μM and 0.038 μM, respectively) that enhance the activity of cefotaxime in KPC-2 expressing Escherichia coli. The measured acid dissociation constants (pKa) of selected triazole-containing phenylboronic acids was broad (5.98–10.0), suggesting that this is an additional property of the compounds that could be tuned to optimize the target interaction and/or the physicochemical properties of the compounds. These findings will help to guide the future development of boronic acid compounds as inhibitors of KPC-2 and other target proteins

Design and synthesis of metallo-β-lactamase inhibitors

Bacterial resistance is a continuously evolving threat to our most common antibiotics; the β-lactams. Most recently, the production of β-lactamase enzymes by bacteria rendered many of our current antibiotics unusable threatening to plunge us into a ‘pre-antibiotic’ era. Metallo-β-lactamases (MBLs) play a key role in bacterial resistance to β-lactam antibiotics by efficiently catalysing the hydrolysis of the β-lactam amide bond. Since their discovery, the clinically relevant MBLs, Verona integrin-encoded metallo-β-lactamase (VIM-2), Imipenemase (IMP-1), New Delhi metallo-β-lactamase (NDM-1) and Sao Paulo metallo-β-lactamase (SPM-1), have spread across the world with cases reported in almost all countries. This thesis describes a number of approaches to the identification of inhibitors of MBLs. A combination of structure-based drug design, chemical synthesis and biological evaluation has been used to rationally identify novel inhibitors of VIM-2, IMP-1, NDM-1 and SPM-1 which have also been co administered with a current β-lactam antibiotic and been found to re-sensitise the bacteria to the antibiotic. Four novel classes of inhibitor have been investigated by taking different approaches to inhibitor discovery. A vHTS screening campaign was conducted to identify potential inhibitors from libraries of known compounds. The campaign identified some weak binding inhibitors. Boronic acid-based inhibitors were identified by using vHTS and de novo design respectively and were synthesised which gave IC50’s in the region of 10 nM against VIM-2 and NDM-1. Additionally, a new computational method for the identification of peptides which bind to enzymes has been developed which found Pro-Cys-Phe to be the most active peptide for binding to NDM-1 with an IC50 of 183 µM. This method could be applied to many other systems. In the final approach, de novo design using SPROUT led to the design of a novel thiol based inhibitor class. The inhibitor class has been co-crystallised in VIM-2 and gives IC50 values in the range of 200 nM. The inhibitor class has also successfully shown a 100 fold recovery of the MIC of meropenem against NDM-1 expressing bacteria

Boronic Acids as Penicillinase Inhibitors

β-lactamases are enzymes produced by bacteria resistant to antibiotics. A common feature on beta lactam antibiotics is the beta-lactam ring. β-lactamases hydrolyze the β-lactam ring leaving the antibiotic inoperative. The advent of bacteria that are resistant to β-lactams has impelled researchers to find inhibitors for β-lactamases that mimic the lactam ring but do not get hydrolyzed. One group of these new antibiotics is the aryl boronic acids. The main reason the boronic acids have been chosen as potential drugs is their lack of toxicity and their easy excretion in the urine. One of the most important structural features of these compounds is their chemical and geometric fitness in the active site of β-lactamases. Boronic acids mimic the tetrahedral intermediate formed in the half-acylation reaction that occurs during the hydrolysis of the β-lactam ring. The major goal of the research presented here was to discover new aryl boronic acids inhibitors of penicillinases from the class A β-lactamases. To accomplish this goal, commercially available boronic acids that are manufactured for the Suzuki reaction were used. These compounds included fluorinated, chlorinated, brominated, carboxylated, nitrophenylated, pinacol-esterified and thiophene-carboxylated aryl boronic acid derivatives. Kinetic evaluations of each class of compounds were performed under pseudo first-order enzymatic reaction conditions and the inhibitory constants (Ki) were reported using nitrocefin as substrate for two enzymes: the in-house expressed β-lactamase BlaC and the β-lactamase from Bacillus cereus 569/H9 (Calbiochem) identified as TEM-116. The structure-activity relationship (SAR) showed that the most potent inhibitors of BlaC β-lactamase were 2-carboxythiophene-5-boronic acid; 3,4,5-trifluorophenylboronic acid; 3-nitrophenytlboronic acid and 2,3,4,5- tetrafluorophenylboronic acid, Ki values of 1.2, 175.7, 213.9 and 228.6 micromolar respectively. In addition, SAR revealed that the most potent inhibitors for Bacillus cereus β-lactamase I were 2-carboxythiophen-5-boronic acid, 3-carboxyphenylboronic acid, 2-carboxythiophene-4-boronic acid, and 3-carboxy-4-fluorophenylboronic acid having Ki values of 1.1, 19.4, 46.5, and 47.1 micromolar respectively. To gain further insight into the molecular interactions between each class of inhibitors and their targeted enzymes docking experiments were performed using Autodock Vina program combined with Sculpt from MDL and followed by the molecular visualization of the protein-ligand complexes using Swiss-PdbViewer and DiscoveryStudio from Accelerys. The results conclusively show that some selective classes of aryl boronic acids are potent competitive inhibitors of BlaC and Bacillus cereus β-lactamase I and that they should be further considered for advanced drug discovery and improvement of treatment against antibiotic resistant bacteria. Furthermore, the discovery that 4,4’-DDT is an inhibitor of Mycobacterium tuberculosis β-lactamase, combined with in silico studies, suggests that further elaboration of this molecule may be one route to new inhibitors

Designing a chemical “toolbox” for the efficient inhibition of metalloenzymes

Metalloenzymes, enzymes that utilise a metal ion within their active site for their enzymatic activity, make up a significant portion of known enzymes. In fact, literature suggests that approximately 40 – 50% of all known enzymes may be classed as metalloenzymes. Many of these are implicated in disease and drug resistance. Despite this, there is a significant lack of small-molecule therapeutics focused upon metalloenzyme inhibition. Metallo-β-lactamases are a family of metalloenzymes that can be found in bacteria. These enzymes cause drug resistance against β-lactam antibiotics, such as penicillin, to emerge in these bacteria. Currently there are no metallo-β-lactamase inhibitors available for clinical use. As such, the World Health Organisation has noted this gap as being a priority for development. This thesis describes attempts to develop new metal-binding functionalities and to extend the evidence of known functionalities in targeting metallo-β-lactamases. What is learned from this work can then be used to inform medicinal chemists of effective metal-binding moieties when developing small-molecule therapeutics for other metalloenzymes. Significant in silico design has been used throughout to aid with the design of molecules based upon targeting Verona integron-encoded metallo-β-lactamase 2. This has led to the extension of knowledge of a previously identified series of thiol-based metallo-β-lactamase inhibitors. Additionally, work on a series of compounds containing a dithiocarboxylate functional group has identified a novel class of inhibitors of these metalloenzymes, showing micromolar activity in assays against a panel of metallo-β-lactamases. Investigation of the stability of the dithiocarboxylate functional group and possible routes of degradation has also been carried out. It is hoped that this work can contribute to the development of potent, selective small-molecule inhibitors of metallo-β-lactamases and other metalloenzymes

High-throughput crystallography reveals boron-containing inhibitors of a Penicillin-binding protein with di- and tricovalent binding modes

The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance