Inhibitors Of Penicillin-binding Proteins Research Articles

A Combined QSAR and Molecular Docking Approach for Identifying Pyrimidine Derivatives as Penicillin Binding Protein Inhibitors

Background:Antimicrobial resistance has been rising continuously in the past few years due to the overuse and exploitation of existing antimicrobials. This has motivated the search for a novel scaffold that has the capability of rapid antimicrobial action. The hybridized pyrimidines have attracted us due to their widespread biological activities, such as anti-bacterial and antifungal activities.Objective:The present study incorporates a series of pyrimidine-based antimicrobial agents for the 2D quantitative structure-activity relationship analysis (2D QSAR) and docking analysis.Methods:The exploration of the chemical structures in combination with the biological activity in CPMLR led to the detection of six descriptors (Constitutional descriptors, Topological descriptors, Modified Burden Eigenvalues and 2D autocorrelations) for modeling the activity. The resulted QSAR model has been validated using combinatorial protocol in multiple linear regression (CP-MLR) and partial least squares (PLS) analysis.Methods:The exploration of the chemical structures in combination with the biological activity in CPMLR led to the detection of six descriptors (Constitutional descriptors, Topological descriptors, Modified Burden Eigenvalues and 2D autocorrelations) for modeling the activity. The resulted QSAR model has been validated using a combinatorial protocol in multiple linear regression (CP-MLR) and partial least squares (PLS) analysis.Results:The best QSAR model displays the r2 t value of 0.594, Q2 LOO value of 0.779, Q2 L5O value of 0.767. Further docking study was executed using Autodock Vina against Penicillin-binding protein (PBP2a).Conclusion:From the results, Compounds 4, 11and 24 were found to possess a good binding affinity towards PBP2a.

Combined Structural Analysis and Molecular Dynamics Reveal Penicillin-Binding Protein Inhibition Mode with β-Lactones.

β-Lactam antibiotics comprise one of the most widely used therapeutic classes to combat bacterial infections. This general scaffold has long been known to inhibit bacterial cell wall biosynthesis by inactivating penicillin-binding proteins (PBPs); however, bacterial resistance to β-lactams is now widespread, and new strategies are urgently needed to target PBPs and other proteins involved in bacterial cell wall formation. A key requirement in the identification of strategies to overcome resistance is a deeper understanding of the roles of the PBPs and their associated proteins during cell growth and division, such as can be obtained with the use of selective chemical probes. Probe development has typically depended upon known PBP inhibitors, which have historically been thought to require a negatively charged moiety that mimics the C-terminus of the PBP natural peptidoglycan substrate, d-Ala-d-Ala. However, we have identified a new class of β-lactone-containing molecules that interact with PBPs, often in an isoform-specific manner, and do not incorporate this C-terminal mimetic. Here, we report a series of structural biology experiments and molecular dynamics simulations that we utilized to evaluate specific binding modes of this novel PBP inhibitor class. In this work, we obtained <2 Å resolution X-ray structures of four β-lactone probes bound to PBP1b from Streptococcus pneumoniae. Despite their diverging recognition modes beyond the site of covalent modification, these four probes all efficiently labeled PBP1b, as well as other PBPs from S. pneumoniae. From these structures, we analyzed protein-ligand interactions and characterized the β-lactone-bound active sites using in silico mutagenesis and molecular dynamics. Our approach has clarified the dynamic interaction profile in this series of ligands, expanding the understanding of PBP inhibitor binding.

Rational design of a new antibiotic class for drug-resistant infections.

The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including β-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of β-lactamases, the primary resistance mechanism associated with β-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPEpathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.

Structures of Mycobacterium tuberculosis Penicillin-Binding Protein 3 in Complex with Five β-Lactam Antibiotics Reveal Mechanism of Inactivation.

Because of β-lactamase-mediated resistance, β-lactam antibiotics were long considered ineffective drugs for tuberculosis (TB) treatment. However, some β-lactams, including meropenem and faropenem, are being re-evaluated in patients infected with TB. Penicillin-binding protein (PBP) 3, or ftsI, is an essential transpeptidase in Mycobacterium tuberculosis (Mtb) required for cell division, and thus it is an important drug target. Structures of apo MtbPBP3 and of complexes with five β-lactams, including meropenem and faropenem, reveal how they cause inactivation via formation of hydrolytically stable acyl-enzyme complexes. The structures reveal unique features of the antibiotic interactions, both in terms of differences in their binding to MtbPBP3 and in comparison with structures of other PBPs and serine β-lactamases, including the tautomerization status of the carbapenem-derived acyl-enzyme complexes. The results suggest that rather than hoping PBP inhibitors developed for other infections will work against TB, work should focus on developing PBP inhibitors specialized for treating TB. SIGNIFICANCE STATEMENT: The structures of Mycobacterium tuberculosis penicillin-binding protein 3, an essential protein in M. tuberculosis, in complex with a number of widely used β-lactam antibiotics (e.g., meropenem, aztreonam, and amoxicillin) were solved. These data provide new insights for next-generation rational approaches to design tuberculosis (TB)-specific β-lactam or nonlactam antibiotics. This manuscript is a seminal article in the field of anti-TB drug discovery and suitable for the broad readership.

Morphological Deconvolution of Beta-Lactam Polyspecificity in E. coli.

Beta-lactams comprise one of the earliest classes of antibiotic therapies. These molecules covalently inhibit enzymes from the family of penicillin-binding proteins (PBPs), which are essential in construction of the bacterial cell wall. As a result, beta-lactams cause striking changes to cellular morphology, the nature of which varies by the range of PBPs simultaneously engaged in the cell. The traditional method of exploring beta-lactam polyspecificity is a gel-based binding assay which is low-throughput and typically isrun ex situ in cell extracts. Here, we describe a medium-throughput, image-based assay combined with machine learning methods to automatically profile the activity of beta-lactams in E. coli cells. By testing for morphological change across a panel of strains with perturbations to individual PBP enzymes, our approach automatically and quantifiably relates different beta-lactam antibiotics according to their preferences for individual PBPs in cells. We show the potential of our approach for guiding the design of novel inhibitors toward different PBP-binding profiles by predicting the mechanisms of two recently reported PBP inhibitors.

Structural Basis for E. coli Penicillin Binding Protein (PBP) 2 Inhibition, a Platform for Drug Design.

Penicillin-binding proteins (PBPs) are the targets of the β-lactams, the most successful class of antibiotics ever developed against bacterial infections. Unfortunately, the worldwide and rapid spread of large spectrum β-lactam resistance genes such as carbapenemases is detrimental to the use of antibiotics in this class. New potent PBP inhibitors are needed, especially compounds that resist β-lactamase hydrolysis. Here we describe the structure of the E. coli PBP2 in its Apo form and upon its reaction with 2 diazabicyclo derivatives, avibactam and CPD4, a new potent PBP2 inhibitor. Examination of these structures shows that unlike avibactam, CPD4 can perform a hydrophobic stacking on Trp370 in the active site of E. coli PBP2. This result, together with sequence analysis, homology modeling, and SAR, allows us to propose CPD4 as potential starting scaffold to develop molecules active against a broad range of bacterial species at the top of the WHO priority list.

Slt, MltD, and MltG of Pseudomonas aeruginosa as Targets of Bulgecin A in Potentiation of β-Lactam Antibiotics.

The interplay between the activities of lytic transglycosylases (LTs) and penicillin-binding proteins (PBPs) is critical for the health of the bacterial cell wall. Bulgecin A (a natural-product inhibitor of LTs) potentiates the activity of β-lactam antibiotics (inhibitors of PBPs), underscoring this intimate mechanistic interdependence. Bulgecin A in the presence of an appropriate β-lactam causes bulge deformation due to the formation of aberrant peptidoglycan at the division site of the bacterium. As Pseudomonas aeruginosa, a nefarious human pathogen, has 11 LT paralogs, the answer as to which LT activity correlates with β-lactam potentiation is important and is currently unknown. Growth of P. aeruginosa PAO1 strains harboring individual transposon-insertion mutants at each of the 11 genes for LTs, in the presence of the β-lactam antibiotic ceftazidime or meropenem, implicated the gene products of slt, mltD, and mltG (of the 11), in bulge formation and potentiation. Hence, the respective enzymes would be the targets of inhibition by bulgecin A, which was indeed documented. We further demonstrated by imaging in real time and by SEM that cell lysis occurs by the structural failure of this bulge. Upon removal of the β-lactam antibiotic prior to lysis, P. aeruginosa experiences delayed recovery from the elongation and bulge phenotype in the presence of bulgecin A. These observations argue for a collaborative role for the target LTs in the repair of the aberrant cell wall, the absence of activities of which in the presence of bulgecin A results in potentiation of the β-lactam antibiotic.

In Silico Design and Enantioselective Synthesis of Functionalized Monocyclic 3-Amino-1-carboxymethyl-β-lactams as Inhibitors of Penicillin-Binding Proteins of Resistant Bacteria.

As a complement to the renowned bicyclic β-lactam antibiotics, monocyclic analogues provide a breath of fresh air in the battle against resistant bacteria. In that framework, the present study discloses the in silico design and unprecedented ten-step synthesis of eleven nocardicin-like enantiomerically pure 2-{3-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-2-oxoazetidin-1-yl}acetic acids starting from serine as a readily accessible precursor. The capability of this novel class of monocyclic 3-amino-β-lactams to inhibit penicillin-binding proteins (PBPs) of various (resistant) bacteria was assessed, revealing the potential of α-benzylidenecarboxylates as interesting leads in the pursuit of novel PBP inhibitors. No deactivation by representative enzymes belonging to the four β-lactamase classes was observed, while weak inhibition of class C β-lactamase P99 was demonstrated.

Design, synthesis and characterization of peptidyl boronate analogues as effective antimicrobial agents

Antibiotic-resistant penicillin binding protein (PBPs) production is one of the reasons why bacteria develop resistance to β-lactam antibiotics, and this needs to be tackled in the continual battle to produce effective antibiotics. The transition state analogue of boronic acid inhibitors mimicking the structures and interactions of good penicillin substrates are known to be potent β-lactamase inhibitors. We have recently identified boronic acids as a selective scaffold for Actinomadura sp. strain R39 DD-peptidase (PBP). Here, we report the synthesis and biological evaluation of a comprehensive set of 16 boronic acid analogues and establish their structure–activity relationships as well as their potential for use as PBP inhibitors. The docking studies of all the synthesized compounds were carried out in Molegro Virtual Docker. Of the 16 compounds synthesized, compound 8e exhibits the highest binding affinity (187.5 kcal/mol) followed by compounds 8f, 8d, 8a, 8g, 8c and 8i. The minimum inhibitory concentration (MIC) of all the 16 synthesised compounds 6a–6f, 8a–8i, 10 and 11 were tested against two Gram-positive (S. aureus, and S. pyogene) and eight Gram-negative (E. coli, C. koseri, K. oxytoca, K. pneumoniae, S. dysentriae, S. typhi, C. fruendi, and P. aeruginosa) bacteria. The biological evaluation of the synthesized boronic acid analogues were found to be effective in all cases, although it is more pronounced for pyrazine-containing dipeptidyl boronic acid (8e, MIC = 0.062 mg/ml) and hence considered as an effective inhibitor, thereby highlighting the importance of phenylalanine as linker and heterocyclic aromatic carboxylic acid as terminal moieties. This study exemplifies that pyrazine-containing dipeptidyl boronic acid (8e) as a PBP inhibitor could open up new avenues in the development of PBP-inhibiting molecules that eventually display bactericidal effects on distinct bacterial species.

Oxazin-5-Ones as a Novel Class of Penicillin Binding Protein Inhibitors: Design, Synthesis and Structure Activity Relationship.

Penicillin binding proteins (PBPs) are normal constituents of bacterial which are absent in mammalian cells. The theoretical binding modes of known oxazin-5-ones toward the protein were used as a guide to synthesis new inhibitors. Structural studies of protein-ligand complexes revealed that conformational discrepancies of the derivatives in the protein’s binding site gave rise to the variation in their inhibition constant which ranged from 68.58 μM to 2.04 mM. Biological assay results further confirmed the antibiotic potencies of the studied compounds. Although the outcome of biological screening does not parallel computational predictions, the results obtained from both methods suggest that the oxazin-5-one derivatives are potential PBP inhibitors, hence interesting antibiotic lead agents.

Virtual high throughput screening of carbapenem derivatives as new generation carbapenemase and penicillin binding protein inhibitors: a hunt to save drug of last resort.

The advent of carbapenem resistance by the production of β-lectamases and mutated penicillin binding proteins (PBPs) has challenged the treatment of Enterobacteriaceae. Hence there is an urgent need to establish drugs that can fit in the pipeline by overcoming those situations. The working hypothesis of the work is based on two facts, i.e., i) design of inhibitors against mutated PBPs to which present drugs cannot bind efficiently to kill pathogen by inhibiting cell wall formation, ii) design of molecules that can bind with β-lectamases with high affinity, so that they can supplement available drugs preventing their unwanted hydrolysis. In this work, over thousands of thienamycin (first natural carbapenem) derivatives were generated and out of which non-toxic 273 molecules were used for further study. Out of which, only few followed the first hypothesis and rest obeyed the second. Ligand L5 strictly followed the first hypothesis and L1-L4 followed to a satisfactory level. Molecular dynamic simulation was performed to check post-docking stability of the pharmacophores.

Discovery of a New Class of Non-β-lactam Inhibitors of Penicillin-Binding Proteins with Gram-Positive Antibacterial Activity

Infections caused by hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA) are a serious global public-health concern, as MRSA has become broadly resistant to many classes of antibiotics. We disclose herein the discovery of a new class of non-β-lactam antibiotics, the oxadiazoles, which inhibit penicillin-binding protein 2a (PBP2a) of MRSA. The oxadiazoles show bactericidal activity against vancomycin- and linezolid-resistant MRSA and other Gram-positive bacterial strains, in vivo efficacy in a mouse model of infection, and have 100% oral bioavailability.

New Antibacterial Agents: Patent Applications Published in 2011

This article reviews 101 patent applications published in 2011 that disclosed small-molecule antibacterials and reported bacterial growth inhibition, in which the compounds were not similarly disclosed to be toxic to fungal or mammalian cells. The patent applications were analyzed according to their biological target and/or antibacterial class. Protein synthesis inhibitors included ligands of the 50S ribosome subunit (oxazolidinones, macrolides/ketolides and pleuromutilins), the 30S ribosome subunit (aminoglycosides and tetracyclines) and nonribosomal targets. DNA synthesis inhibitors included ligands of topoisomerase type II and type IV. Inhibitors directed at the bacterial cell envelope included those that act on cell envelope synthesis (LpxC inhibitors, penicillin-binding protein inhibitors and glycopeptides) as well as membrane disruptors (lantibiotics). Other antibacterial targets included cell division (FtsZ and WalR) and fatty acid biosynthesis (FabH/I). Compounds for which the targets are unknown or undisclosed are also covered, as are compounds aimed at overcoming resistance mechanisms (efflux inhibitors, β-lactamase inhibitors).

Binding of (5S)-Penicilloic Acid to Penicillin Binding Protein 3

β-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.

2-Nitrobenzyl Esters of Penam and Cephem Derivatives as Inhibitors of Penicillin-Binding Proteins

Ace in the pack: 2-Nitrobenzyl esters of penams, which contain the characteristic core of penicillin-based antibiotics, are acylating inhibitors of penicillin-binding proteins R39 and BlaR-CTD. Docking experiments are in agreement with the experimental data and show that the 2-nitro group of the esters could play the role of a carboxylate group in interactions with the proteins.

Development of New Drugs for an Old Target — The Penicillin Binding Proteins

The widespread use of β-lactam antibiotics has led to the worldwide appearance of drug-resistant strains. Bacteria have developed resistance to β-lactams by two main mechanisms: the production of β-lactamases, sometimes accompanied by a decrease of outer membrane permeability, and the production of low-affinity, drug resistant Penicillin Binding Proteins (PBPs). PBPs remain attractive targets for developing new antibiotic agents because they catalyse the last steps of the biosynthesis of peptidoglycan, which is unique to bacteria, and lies outside the cytoplasmic membrane. Here we summarize the “current state of the art” of non-β-lactam inhibitors of PBPs, which have being developed in an attempt to counter the emergence of β-lactam resistance. These molecules are not susceptible to hydrolysis by β-lactamases and thus present a real alternative to β-lactams. We present transition state analogs such as boronic acids, which can covalently bind to the active serine residue in the catalytic site. Molecules containing ring structures different from the β-lactam-ring like lactivicin are able to acylate the active serine residue. High throughput screening methods, in combination with virtual screening methods and structure based design, have allowed the development of new molecules. Some of these novel inhibitors are active against major pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and thus open avenues new for the discovery of novel antibiotics.

High-Throughput Screening for Novel Inhibitors of Neisseria gonorrhoeae Penicillin-Binding Protein 2

The increasing prevalence of N. gonorrhoeae strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of N. gonorrhoeae PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of N. gonorrhoeae. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of N. gonorrhoeae. It also provides a set of lead compounds for optimization of anti-gonococcal agents.

4-quinolones as noncovalent inhibitors of high molecular mass penicillin-binding proteins.

Penicillin-binding proteins (PBPs) are important bacterial enzymes that carry out the final steps of bacterial cell wall assembly. Their DD-transpeptidase activity accomplishes the essential peptide cross-linking step of the cell wall. To date, all attempts to discover effective inhibitors of PBPs, apart from β-lactams, have not led to new antibiotics. Therefore, the need for new classes of efficient inhibitors of these enzymes remains. Guided by a computational fragment-based docking procedure, carried out on Escherichia coli PBP5, we have designed and synthesized a series of 4-quinolones as potential inhibitors of PBPs. We describe their binding to the PBPs of E. coli and Bacillus subtilis. Notably, these compounds bind quite tightly to the essential high molecular mass PBPs.

Putting a Stop to l,d-Transpeptidases

In this issue of Structure, Lecoq et al. investigated the structural and dynamic basis for the unexpected inhibition of peptidoglycan-crosslinking l,d-transpeptidases by carbapenem antibiotics. In addition to defining a neutral thiol-imidazole catalytic triad, their studies revealed extensive induced motions upon formation of a long-live covalent drug-enzyme complex.

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 (IC50: 1.3μM). 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, IC50=26μM), a class B PBP (PBP2xR6 from Streptococcus pneumoniae, IC50=138μM) and a class C PBP (R39 from Actinomadura sp., IC50=0.6μM). This work opens new avenues towards the development of molecules that inhibit PBPs, and eventually display bactericidal effects, on distinct bacterial species.