ABSTRACT
ß-Lactams are the most successful antibacterials, yet their use is threatened by resistance, importantly as caused by ß-lactamases. ß-Lactamases fall into two mechanistic groups: the serine ß-lactamases that utilise a covalent acyl-enzyme mechanism and the metallo ß-lactamases that utilise a zinc-bound water nucleophile. Achieving simultaneous inhibition of both ß-lactamase classes remains a challenge in the field. Vaborbactam is a boronate-based inhibitor that reacts with serine-ß-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-ß-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 ß-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 ß-lactamases and justify further work on the development of boronates as broad-spectrum ß-lactamase inhibitors.
Subject(s)
Anti-Bacterial Agents/therapeutic use , Boronic Acids/therapeutic use , beta-Lactamases/therapeutic use , Anti-Bacterial Agents/pharmacology , Boronic Acids/pharmacology , Humans , beta-Lactamases/pharmacologyABSTRACT
Clavulanic acid and avibactam are clinically deployed serine ß-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo ß-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine ß-lactamase) but not L1 (metallo ß-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like ß-lactams plus nonclassical ß-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.
Subject(s)
Stenotrophomonas maltophilia/metabolism , beta-Lactamase Inhibitors/metabolism , Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/metabolism , Azabicyclo Compounds/pharmacology , Aztreonam , Bacterial Proteins/metabolism , Ceftazidime , Crystallography, X-Ray/methods , Drug Resistance, Bacterial , Microbial Sensitivity Tests , beta-Lactamase Inhibitors/chemistry , beta-Lactamases/genetics , beta-Lactamases/metabolismABSTRACT
ß-Lactamase-mediated resistance is a growing threat to the continued use of ß-lactam antibiotics. The use of the ß-lactam-based serine-ß-lactamase (SBL) inhibitors clavulanic acid, sulbactam, and tazobactam and, more recently, the non-ß-lactam inhibitor avibactam has extended the utility of ß-lactams against bacterial infections demonstrating resistance via these enzymes. These molecules are, however, ineffective against the metallo-ß-lactamases (MBLs), which catalyze their hydrolysis. To date, there are no clinically available metallo-ß-lactamase inhibitors. Coproduction of MBLs and SBLs in resistant infections is thus of major clinical concern. The development of "dual-action" inhibitors, targeting both SBLs and MBLs, is of interest, but this is considered difficult to achieve due to the structural and mechanistic differences between the two enzyme classes. We recently reported evidence that cyclic boronates can inhibit both serine- and metallo-ß-lactamases. Here we report that cyclic boronates are able to inhibit all four classes of ß-lactamase, including the class A extended spectrum ß-lactamase CTX-M-15, the class C enzyme AmpC from Pseudomonas aeruginosa, and class D OXA enzymes with carbapenem-hydrolyzing capabilities. We demonstrate that cyclic boronates can potentiate the use of ß-lactams against Gram-negative clinical isolates expressing a variety of ß-lactamases. Comparison of a crystal structure of a CTX-M-15:cyclic boronate complex with structures of cyclic boronates complexed with other ß-lactamases reveals remarkable conservation of the small-molecule binding mode, supporting our proposal that these molecules work by mimicking the common tetrahedral anionic intermediate present in both serine- and metallo-ß-lactamase catalysis.
Subject(s)
Anti-Bacterial Agents/pharmacology , Boronic Acids/pharmacology , Enterobacteriaceae/drug effects , beta-Lactam Resistance/drug effects , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/chemistry , Amino Acid Motifs , Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Boronic Acids/chemical synthesis , Cloning, Molecular , Crystallography, X-Ray , Cyclization , Enterobacteriaceae/enzymology , Enterobacteriaceae/genetics , Enterobacteriaceae/growth & development , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Kinetics , Models, Molecular , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity , Thermodynamics , beta-Lactam Resistance/genetics , beta-Lactamase Inhibitors/chemical synthesis , beta-Lactamases/genetics , beta-Lactamases/metabolism , beta-Lactams/pharmacologyABSTRACT
In recent years bacterial resistance has been observed against many of our current antibiotics, for instance most worryingly against the cephalosporins which are typically the last line of defence against many bacterial infections. Additionally the failure of high throughput screening in the discovery of new antibacterial drug leads has led to a decline in the number of antibacterial agents reaching the market. Alternative methods of drug discovery including structure based drug design are needed to meet the threats caused by the emergence of resistance. In this review we explore the latest advancements in the identification of new antibacterial agents through the use of a number of structure based drug design programs.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacterial Infections/drug therapy , Drug Design , Drug Discovery , Animals , Humans , Structure-Activity RelationshipABSTRACT
This study describes a novel series of UDP-N-acetylglucosamine acyltransferase (LpxA) inhibitors that was identified through affinity-mediated selection from a DNA-encoded compound library. The original hit was a selective inhibitor of Pseudomonas aeruginosa LpxA with no activity against Escherichia coli LpxA. The biochemical potency of the series was optimized through an X-ray crystallography-supported medicinal chemistry program, resulting in compounds with nanomolar activity against P. aeruginosa LpxA (best half-maximal inhibitory concentration (IC50) <5 nM) and cellular activity against P. aeruginosa (best minimal inhibitory concentration (MIC) of 4 µg/mL). Lack of activity against E. coli was maintained (IC50 > 20 µM and MIC > 128 µg/mL). The mode of action of analogues was confirmed through genetic analyses. As expected, compounds were active against multidrug-resistant isolates. Further optimization of pharmacokinetics is needed before efficacy studies in mouse infection models can be attempted. To our knowledge, this is the first reported LpxA inhibitor series with selective activity against P. aeruginosa.
Subject(s)
Acyltransferases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , Pseudomonas aeruginosa/drug effects , Anti-Bacterial Agents/chemistry , Crystallography, X-Ray , Drug Resistance, Bacterial/drug effects , Enzyme Inhibitors/chemistry , Escherichia coli/enzymology , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
Novel antimicrobials are urgently needed to combat drug-resistant bacteria and to overcome the inherent difficulties in treating biofilm-associated infections. Studying plants and other natural materials used in historical infection remedies may enable further discoveries to help fill the antibiotic discovery gap. We previously reconstructed a 1,000-year-old remedy containing onion, garlic, wine, and bile salts, known as 'Bald's eyesalve', and showed it had promising antibacterial activity. In this current paper, we have found this bactericidal activity extends to a range of Gram-negative and Gram-positive wound pathogens in planktonic culture and, crucially, that this activity is maintained against Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes in a soft-tissue wound biofilm model. While the presence of garlic in the mixture can explain the activity against planktonic cultures, garlic has no activity against biofilms. We have found the potent anti-biofilm activity of Bald's eyesalve cannot be attributed to a single ingredient and requires the combination of all ingredients to achieve full activity. Our work highlights the need to explore not only single compounds but also mixtures of natural products for treating biofilm infections and underlines the importance of working with biofilm models when exploring natural products for the anti-biofilm pipeline.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteria/drug effects , Bacterial Infections/prevention & control , Biofilms/drug effects , Garlic/chemistry , Onions/chemistry , Plant Extracts/pharmacology , Bacteria/growth & development , Bacterial Infections/microbiology , Biofilms/growth & development , Humans , Microbial Sensitivity Tests , Plant Extracts/isolation & purificationABSTRACT
Resistance to ß-lactam antibacterials, importantly via production of ß-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) ß-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C ßlactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied ß-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D ßlactamases, our data support the proposal that bicyclic boronates are broad-spectrum ßlactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful ß-lactamase inhibition.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Boronic Acids/pharmacology , beta-Lactamase Inhibitors/pharmacology , Anti-Bacterial Agents , Bacterial Proteins/genetics , Boronic Acids/chemistry , Crystallography, X-Ray , Cyclization , Drug Resistance, Bacterial/drug effects , Drug Resistance, Bacterial/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Microbial Sensitivity Tests , beta-Lactamases/classification , beta-Lactamases/geneticsABSTRACT
Aminoacyl-tRNA synthetases are ubiquitous and essential enzymes for protein synthesis and also a variety of other metabolic processes, especially in bacterial species. Bacterial aminoacyl-tRNA synthetases represent attractive and validated targets for antimicrobial drug discovery if issues of prokaryotic versus eukaryotic selectivity and antibiotic resistance generation can be addressed. We have determined high-resolution X-ray crystal structures of the Escherichia coli and Staphylococcus aureus seryl-tRNA synthetases in complex with aminoacyl adenylate analogues and applied a structure-based drug discovery approach to explore and identify a series of small molecule inhibitors that selectively inhibit bacterial seryl-tRNA synthetases with greater than 2 orders of magnitude compared to their human homologue, demonstrating a route to the selective chemical inhibition of these bacterial targets.
Subject(s)
Enzyme Inhibitors/pharmacology , Escherichia coli/enzymology , Molecular Probes/chemistry , Serine-tRNA Ligase/antagonists & inhibitors , Staphylococcus aureus/enzymology , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Molecular Structure , Serine-tRNA Ligase/chemistryABSTRACT
BACKGROUND: The ß-lactam antibiotics represent the most successful drug class for treatment of bacterial infections. Resistance to them, importantly via production of ß-lactamases, which collectively are able to hydrolyse all classes of ß-lactams, threatens their continued widespread use. Bicyclic boronates show potential as broad spectrum inhibitors of the mechanistically distinct serine- (SBL) and metallo- (MBL) ß-lactamase families. METHODS: Using biophysical methods, including crystallographic analysis, we have investigated the binding mode of bicyclic boronates to clinically important ß-lactamases. Induction experiments and agar-based MIC screening against MDR-Enterobacteriaceae (nâ¯=â¯132) were used to evaluate induction properties and the in vitro efficacy of a bicyclic boronate in combination with meropenem. RESULTS: Crystallographic 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 ß-lactamases by bicyclic boronates further support the promise of such compounds as broad spectrum ß-lactamase inhibitors. CONCLUSIONS: Together with reported studies on the structural basis of their inhibition of class A, B and D ß-lactamases, biophysical studies, including crystallographic analysis, support the proposal that bicyclic boronates mimic tetrahedral intermediates common to SBL and MBL catalysis. GENERAL SIGNIFICANCE: Bicyclic boronates are a new generation of broad spectrum inhibitors of both SBLs and MBLs.
Subject(s)
Anti-Bacterial Agents/pharmacology , Boronic Acids/pharmacology , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Anti-Bacterial Agents/chemistry , Boronic Acids/chemistry , Crystallography, X-Ray , Models, Molecular , Molecular Structure , Pseudomonas aeruginosa/enzymology , beta-Lactamase Inhibitors/chemistryABSTRACT
Zinc ion-dependent ß-lactamases (MBLs) catalyze the hydrolysis of almost all ß-lactam antibiotics and resist the action of clinically available ß-lactamase inhibitors. We report how application of in silico fragment-based molecular design employing thiol-mediated metal anchorage leads to potent MBL inhibitors. The new inhibitors manifest potent inhibition of clinically important B1 subfamily MBLs, including the widespread NDM-1, IMP-1, and VIM-2 enzymes; with lower potency, some of them also inhibit clinically relevant Class A and D serine-ß-lactamases. The inhibitors show selectivity for bacterial MBL enzymes compared to that for human MBL fold nucleases. Cocrystallization of one inhibitor, which shows potentiation of Meropenem activity against MBL-expressing Enterobacteriaceae, with VIM-2 reveals an unexpected binding mode, involving interactions with residues from conserved active site bordering loops.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Computer Simulation , Drug Design , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/metabolism , Drug Evaluation, Preclinical , Models, Molecular , Protein Conformation , Structure-Activity Relationship , beta-Lactamases/chemistryABSTRACT
Drug reprofiling is emerging as an effective paradigm for discovery of cancer treatments. Herein, an antipsychotic drug is immobilised using the Magic Tag® chemical genomics tool and screened against a T7 bacteriophage displayed library of polypeptides from Drosophila melanogaster, as a whole genome model, to uncover an interaction with a section of 17-ß-HSD10, a proposed prostate cancer target. A computational study and enzyme inhibition assay with full length human 17-ß-HSD10 identifies risperidone as a drug reprofiling candidate. When formulated with rumenic acid, risperidone slows proliferation of PC3 prostate cancer cells in vitro and retards PC3 prostate cancer tumour growth in vivo in xenografts in mice, presenting an opportunity to reprofile risperidone as a cancer treatment.
Subject(s)
17-Hydroxysteroid Dehydrogenases/antagonists & inhibitors , 3-Hydroxyacyl CoA Dehydrogenases/antagonists & inhibitors , Adenocarcinoma/drug therapy , Antineoplastic Agents/pharmacology , Antipsychotic Agents/pharmacology , Drosophila melanogaster/drug effects , Drug Repositioning/methods , Enzyme Inhibitors/pharmacology , Genomics/methods , Prostatic Neoplasms/drug therapy , Risperidone/pharmacology , 17-Hydroxysteroid Dehydrogenases/chemistry , 17-Hydroxysteroid Dehydrogenases/genetics , 17-Hydroxysteroid Dehydrogenases/metabolism , 3-Hydroxyacyl CoA Dehydrogenases/chemistry , 3-Hydroxyacyl CoA Dehydrogenases/genetics , 3-Hydroxyacyl CoA Dehydrogenases/metabolism , Adenocarcinoma/enzymology , Adenocarcinoma/genetics , Animals , Antineoplastic Agents/chemistry , Antipsychotic Agents/chemistry , Bacteriophage T7/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drosophila melanogaster/enzymology , Drosophila melanogaster/genetics , Drug Compounding , Enzyme Inhibitors/chemistry , Gene Library , Humans , Linoleic Acids, Conjugated/chemistry , Male , Mice, Nude , Molecular Docking Simulation , Molecular Targeted Therapy , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Protein Conformation , Risperidone/chemistry , Structure-Activity Relationship , Time Factors , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
ß-Lactamases enable resistance to almost all ß-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as 'transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-ß-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent ß-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-ß-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.
Subject(s)
Boronic Acids/pharmacology , Enzyme Inhibitors/pharmacology , Penicillin-Binding Proteins/antagonists & inhibitors , Serine/metabolism , beta-Lactamases/chemistry , Boronic Acids/chemistry , Cyclization , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , HEK293 Cells , Humans , Microbial Sensitivity Tests , Penicillin-Binding Proteins/metabolism , Structure-Activity Relationship , beta-Lactamases/metabolismABSTRACT
The development of resistance to all current antibiotics in the clinic means there is an urgent unmet need for novel antibacterial agents with new modes of action. One of the best ways of finding these is to identify new essential bacterial enzymes to target. The advent of a number of in silico tools has aided classical methods of discovering new antibacterial targets, and these programs are the subject of this review. Many of these tools apply a cheminformatic approach, utilizing the structural information of either ligand or protein, chemogenomic databases, and docking algorithms to identify putative antibacterial targets. Considering the wealth of potential drug targets identified from genomic research, these approaches are perfectly placed to mine this rich resource and complement drug discovery programs.
Subject(s)
Anti-Bacterial Agents/chemistry , Computational Biology , Drug Discovery/methods , Drug Delivery Systems , Drug Design , Models, BiologicalABSTRACT
Parathyroid hormone (PTH)(1-34), given once daily, increases bone mass in a variety of animal models and humans with osteoporosis. However, continuous PTH infusion has been shown to cause bone loss. To determine the pharmacokinetic profile of PTH(1-34) associated with anabolic and catabolic bone responses, PTH(1-34) pharmacokinetic and serum biochemical profiles were evaluated in young male rats using dosing regimens that resulted in either gain or loss of bone mass. Once-daily PTH(1-34) or 6 PTH(1-34) injections within 1 h, for a total daily dose of 80 microg/kg, induced equivalent increases in proximal tibia bone mass. In contrast, 6 PTH(1-34) injections/day over 6 h for a total dose of 80 microg/kg/day or 3 injections/day over 8 h for a total of 240 microg/kg/day decreased tibia bone mass. The PTH(1-34) pharmacokinetics of the different treatment regimens were distinctive. The magnitude of the maximum serum concentrations (Cmax) of PTH(1-34) and area under the curve (AUC) did not predict the catabolic bone outcome. Compared to the anabolic pharmacokinetic profile of a transient increase in PTH(1-34) with rapid decreases in serum calcium and phosphate, the catabolic regimen was associated with PTH(1-34) concentrations remaining above baseline values during the entire 6-h dosing period with a trend toward an increase in serum calcium and a prolonged decrease in phosphate. The pharmacokinetic profiles suggest that the anabolic or catabolic response of bone to PTH(1-34) is determined primarily by the length of time each day that serum concentrations of PTH(1-34) remain above baseline levels of endogenous PTH and only secondarily by the Cmax or AUC of PTH(1-34) achieved.
Subject(s)
Bone Remodeling/drug effects , Parathyroid Hormone/pharmacology , Peptide Fragments/pharmacology , Tibia/drug effects , Tibia/metabolism , Animals , Calcium/blood , Dose-Response Relationship, Drug , Humans , Injections, Subcutaneous , Male , Phosphates/blood , Rats , Rats, Sprague-DawleyABSTRACT
Metallo-ß-lactamases (MBLs) are a growing threat to the use of almost all clinically used ß-lactam antibiotics. The identification of broad-spectrum MBL inhibitors is hampered by the lack of a suitable screening platform, consisting of appropriate substrates and a set of clinically relevant MBLs. We report procedures for the preparation of a set of clinically relevant metallo-ß-lactamases (i.e., NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification of suitable fluorogenic substrates (umbelliferone-derived cephalosporins). The fluorogenic substrates were compared to chromogenic substrates (CENTA, nitrocefin, and imipenem), showing improved sensitivity and kinetic parameters. The efficiency of the fluorogenic substrates was exemplified by inhibitor screening, identifying 4-chloroisoquinolinols as potential pan MBL inhibitors.
Subject(s)
Metals/chemistry , beta-Lactamases/analysis , Enzyme Inhibitors/pharmacology , Magnetic Resonance Spectroscopy , Mass Spectrometry , beta-Lactamase Inhibitors , beta-Lactamases/chemistryABSTRACT
Osteoprotegerin (OPG), a secreted member of the tumor necrosis factor receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Because OPG functions physiologically as a locally generated (paracrine) factor, we used high-throughput screening to identify small molecules that enhance the activity of the promoter of the human OPG gene. We found three structurally unrelated compounds that selectively increased OPG gene transcription, OPG mRNA levels, and OPG protein production and release by osteoblastic cells. Structural analysis of one compound, a benzamide derivative, led to the identification of four related molecules, which are also OPG inducers. The most potent of these compounds, Cmpd 5 inhibited osteoclast formation and parathyroid hormone-induced calvarial bone resorption. In vivo, Cmpd 5 completely blocked resorptive activity (serum calcium, osteoclast number) in parathyroid hormone-treated rats. Furthermore, Cmpd 5 reduced the ability of a rat breast cancer to metastasize to bone. Finally, the compound also prevented bone loss in a rat adjuvant arthritis model. These results provide proof of the concept that low molecular weight compounds can enhance OPG production in ways that can result in effective therapies.