ABSTRACT
Many drug candidates fail in clinical trials owing to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug-target complex has a longer half-life than off-target-drug complexes. However, though slow-binding inhibition kinetics are a key feature of many marketed drugs, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters, including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.
Subject(s)
Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Threonine/analogs & derivatives , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacokinetics , Kinetics , Mice, Inbred Strains , Microbial Sensitivity Tests , Models, Biological , Molecular Structure , Protein Binding , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/enzymology , Threonine/chemistry , Threonine/pharmacokinetics , Threonine/pharmacology , Time FactorsABSTRACT
We characterized the inhibition of Neisseria gonorrhoeae type II topoisomerases gyrase and topoisomerase IV by AZD0914 (AZD0914 will be henceforth known as ETX0914 (Entasis Therapeutics)), a novel spiropyrimidinetrione antibacterial compound that is currently in clinical trials for treatment of drug-resistant gonorrhea. AZD0914 has potent bactericidal activity against N. gonorrhoeae, including multidrug-resistant strains and key Gram-positive, fastidious Gram-negative, atypical, and anaerobic bacterial species (Huband, M. D., Bradford, P. A., Otterson, L. G., Basrab, G. S., Giacobe, R. A., Patey, S. A., Kutschke, A. C., Johnstone, M. R., Potter, M. E., Miller, P. F., and Mueller, J. P. (2014) In Vitro Antibacterial Activity of AZD0914: A New Spiropyrimidinetrione DNA Gyrase/Topoisomerase Inhibitor with Potent Activity against Gram-positive, Fastidious Gram-negative, and Atypical Bacteria. Antimicrob. Agents Chemother. 59, 467-474). AZD0914 inhibited DNA biosynthesis preferentially to other macromolecules in Escherichia coli and induced the SOS response to DNA damage in E. coli. AZD0914 stabilized the enzyme-DNA cleaved complex for N. gonorrhoeae gyrase and topoisomerase IV. The potency of AZD0914 for inhibition of supercoiling and the stabilization of cleaved complex by N. gonorrhoeae gyrase increased in a fluoroquinolone-resistant mutant enzyme. When a mutation, conferring mild resistance to AZD0914, was present in the fluoroquinolone-resistant mutant, the potency of ciprofloxacin for inhibition of supercoiling and stabilization of cleaved complex was increased greater than 20-fold. In contrast to ciprofloxacin, religation of the cleaved DNA did not occur in the presence of AZD0914 upon removal of magnesium from the DNA-gyrase-inhibitor complex. AZD0914 had relatively low potency for inhibition of human type II topoisomerases α and ß.
Subject(s)
Anti-Bacterial Agents/pharmacology , Barbiturates/pharmacology , DNA Gyrase/metabolism , DNA Topoisomerase IV/antagonists & inhibitors , DNA, Bacterial/chemistry , Spiro Compounds/pharmacology , Topoisomerase II Inhibitors/pharmacology , Ciprofloxacin/pharmacology , Clinical Trials as Topic , DNA/chemistry , DNA/metabolism , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , DNA Topoisomerase IV/metabolism , DNA, Bacterial/metabolism , Drug Resistance, Bacterial/drug effects , Escherichia coli/drug effects , Escherichia coli/enzymology , Escherichia coli/genetics , Fluoroquinolones/pharmacology , Gene Expression , Humans , Isoxazoles , Morpholines , Mutation , Neisseria gonorrhoeae/drug effects , Neisseria gonorrhoeae/enzymology , Neisseria gonorrhoeae/genetics , Oxazolidinones , Species SpecificityABSTRACT
Deciphering the mode of action (MOA) of new antibiotics discovered through phenotypic screening is of increasing importance. Metabolomics offers a potentially rapid and cost-effective means of identifying modes of action of drugs whose effects are mediated through changes in metabolism. Metabolomics techniques also collect data on off-target effects and drug modifications. Here, we present data from an untargeted liquid chromatography-mass spectrometry approach to identify the modes of action of eight compounds: 1-[3-fluoro-4-(5-methyl-2,4-dioxo-pyrimidin-1-yl)phenyl]-3-[2-(trifluoromethyl)phenyl]urea (AZ1), 2-(cyclobutylmethoxy)-5'-deoxyadenosine, triclosan, fosmidomycin, CHIR-090, carbonyl cyanidem-chlorophenylhydrazone (CCCP), 5-chloro-2-(methylsulfonyl)-N-(1,3-thiazol-2-yl)-4-pyrimidinecarboxamide (AZ7), and ceftazidime. Data analysts were blind to the compound identities but managed to identify the target as thymidylate kinase for AZ1, isoprenoid biosynthesis for fosmidomycin, acyl-transferase for CHIR-090, and DNA metabolism for 2-(cyclobutylmethoxy)-5'-deoxyadenosine. Changes to cell wall metabolites were seen in ceftazidime treatments, although other changes, presumably relating to off-target effects, dominated spectral outputs in the untargeted approach. Drugs which do not work through metabolic pathways, such as the proton carrier CCCP, have no discernible impact on the metabolome. The untargeted metabolomics approach also revealed modifications to two compounds, namely, fosmidomycin and AZ7. An untreated control was also analyzed, and changes to the metabolome were seen over 4 h, highlighting the necessity for careful controls in these types of studies. Metabolomics is a useful tool in the analysis of drug modes of action and can complement other technologies already in use.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Metabolome/drug effects , Metabolomics , Acyltransferases/antagonists & inhibitors , Acyltransferases/genetics , Acyltransferases/metabolism , Adenosine/metabolism , Adenosine/pharmacology , Anti-Bacterial Agents/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/metabolism , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Ceftazidime/metabolism , Ceftazidime/pharmacology , Cell Wall/chemistry , Cell Wall/drug effects , Cell Wall/metabolism , Chromatography, Liquid , DNA, Bacterial/antagonists & inhibitors , DNA, Bacterial/biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Fosfomycin/analogs & derivatives , Fosfomycin/metabolism , Fosfomycin/pharmacology , Gene Expression , HEK293 Cells , Humans , Hydroxamic Acids/metabolism , Hydroxamic Acids/pharmacology , Mass Spectrometry , Nucleoside-Phosphate Kinase/antagonists & inhibitors , Nucleoside-Phosphate Kinase/genetics , Nucleoside-Phosphate Kinase/metabolism , Pyrimidines/metabolism , Pyrimidines/pharmacology , Terpenes/antagonists & inhibitors , Terpenes/metabolism , Threonine/analogs & derivatives , Threonine/metabolism , Threonine/pharmacology , Triclosan/metabolism , Triclosan/pharmacologyABSTRACT
In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.
Subject(s)
Drug Resistance, Bacterial/drug effects , Escherichia coli/drug effects , Morpholines/pharmacology , Naphthyridines/pharmacology , Pseudomonas aeruginosa/drug effects , Topoisomerase II Inhibitors/pharmacology , Bacterial Proteins/genetics , DNA Gyrase/genetics , Drug Resistance, Bacterial/genetics , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Microbial Sensitivity Tests , Mutation , Pseudomonas aeruginosa/geneticsABSTRACT
Sulbactam is a class A ß-lactamase inhibitor with intrinsic whole-cell activity against certain bacterial species, including Acinetobacter baumannii. The clinical use of sulbactam for A. baumannii infections is of interest due to increasing multidrug resistance in this pathogen. However, the molecular drivers of its antibacterial activity and resistance determinants have yet to be precisely defined. Here we show that the antibacterial activities of sulbactam vary widely across contemporary A. baumannii clinical isolates and are mediated through inhibition of the penicillin-binding proteins (PBPs) PBP1 and PBP3, with very low frequency of resistance; the rare pbp3 mutants with high levels of resistance to sulbactam are attenuated in fitness. These results support further investigation of the potential clinical utility of sulbactam.
Subject(s)
Acinetobacter baumannii/drug effects , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/physiology , Sulbactam/pharmacology , Penicillin-Binding Proteins/antagonists & inhibitorsABSTRACT
The objective of this study was to investigate the risk of attenuated efficacy due to adaptive resistance for the siderophore-conjugated monocarbam SMC-3176 in Pseudomonas aeruginosa by using a pharmacokinetic/pharmacodynamic (PK/PD) approach. MICs were determined in cation-adjusted Mueller-Hinton broth (MHB) and in Chelex-treated, dialyzed MHB (CDMHB). Spontaneous resistance was assessed at 2× to 16× the MIC and the resulting mutants sequenced. Efficacy was evaluated in a neutropenic mouse thigh model at 3.13 to 400 mg/kg of body weight every 3 h for 24 h and analyzed for association with free time above the MIC (fT>MIC). To closer emulate the conditions of the in vivo model, we developed a novel assay testing activity mouse whole blood (WB). All mutations were found in genes related to iron uptake: piuA, piuC, pirR, fecI, and pvdS. Against four P. aeruginosa isolates, SMC-3176 displayed predictable efficacy corresponding to the fT>MIC using the MIC in CDMHB (R(2) = 0.968 to 0.985), with stasis to 2-log kill achieved at 59.4 to 81.1%. Efficacy did not translate for P. aeruginosa isolate JJ 4-36, as the in vivo responses were inconsistent with fT>MIC exposures and implied a threshold concentration that was greater than the MIC. The results of the mouse WB assay indicated that efficacy was not predictable using the MIC for JJ 4-36 and four additional isolates, against which in vivo failures of another siderophore-conjugated ß-lactam were previously reported. SMC-3176 carries a risk of attenuated efficacy in P. aeruginosa due to rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems. Substantial in vivo testing is warranted for compounds using the siderophore approach to thoroughly screen for this in vitro-in vivo disconnect in P. aeruginosa.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azetidines/pharmacology , Drug Resistance, Bacterial/genetics , Pseudomonas aeruginosa/metabolism , Siderophores/pharmacology , Sulfonamides/pharmacology , Animals , Anti-Bacterial Agents/pharmacokinetics , Azetidines/pharmacokinetics , Female , Iron/metabolism , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , Oligopeptides/metabolism , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Siderophores/pharmacokinetics , Sulfonamides/pharmacokinetics , beta-Lactamases/metabolismABSTRACT
Avibactam is a ß-lactamase inhibitor that is in clinical development, combined with ß-lactam partners, for the treatment of bacterial infections comprising gram-negative organisms. Avibactam is a structural class of inhibitor that does not contain a ß-lactam core but maintains the capacity to covalently acylate its ß-lactamase targets. Using the TEM-1 enzyme, we characterized avibactam inhibition by measuring the on-rate for acylation and the off-rate for deacylation. The deacylation off-rate was 0.045 min(-1), which allowed investigation of the deacylation route from TEM-1. Using NMR and MS, we showed that deacylation proceeds through regeneration of intact avibactam and not hydrolysis. Other than TEM-1, four additional clinically relevant ß-lactamases were shown to release intact avibactam after being acylated. We showed that avibactam is a covalent, slowly reversible inhibitor, which is a unique mechanism of inhibition among ß-lactamase inhibitors.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azabicyclo Compounds/pharmacology , Gram-Negative Bacterial Infections/drug therapy , beta-Lactamase Inhibitors , Acylation/drug effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/metabolism , Drug Discovery/methods , Kinetics , Magnetic Resonance Spectroscopy , Mass Spectrometry , Molecular Structure , beta-LactamasesABSTRACT
Avibactam is a non-ß-lactam ß-lactamase inhibitor with a spectrum of activity that includes ß-lactamase enzymes of classes A, C, and selected D examples. In this work acylation and deacylation rates were measured against the clinically important enzymes CTX-M-15, KPC-2, Enterobacter cloacae AmpC, Pseudomonas aeruginosa AmpC, OXA-10, and OXA-48. The efficiency of acylation (k2/Ki) varied across the enzyme spectrum, from 1.1 × 10(1) m(-1)s(-1) for OXA-10 to 1.0 × 10(5) for CTX-M-15. Inhibition of OXA-10 was shown to follow the covalent reversible mechanism, and the acylated OXA-10 displayed the longest residence time for deacylation, with a half-life of greater than 5 days. Across multiple enzymes, acyl enzyme stability was assessed by mass spectrometry. These inhibited enzyme forms were stable to rearrangement or hydrolysis, with the exception of KPC-2. KPC-2 displayed a slow hydrolytic route that involved fragmentation of the acyl-avibactam complex. The identity of released degradation products was investigated, and a possible mechanism for the slow deacylation from KPC-2 is proposed.
Subject(s)
Azabicyclo Compounds/chemistry , Escherichia coli/drug effects , beta-Lactamases/chemistry , Anti-Bacterial Agents/chemistry , Drug Resistance, Bacterial , Enterobacter cloacae/metabolism , Enzyme Inhibitors/chemistry , Escherichia coli/metabolism , Hydrolysis , Kinetics , Magnetic Resonance Spectroscopy , Mass Spectrometry , Microbial Sensitivity Tests , Plasmids/metabolism , Pseudomonas aeruginosa/metabolism , Time FactorsABSTRACT
The need for new antibiotics that address serious Gram-negative infections is well recognized. Our efforts with a series of novel bacterial type II topoisomerase inhibitors (NBTIs) led to the discovery of NBTI 5463, an agent with improved activity over other NBTIs against Gram-negative bacteria, in particular against Pseudomonas aeruginosa (F. Reck, D. E. Ehmann, T. J. Dougherty, J. V. Newman, S. Hopkins, G. Stone, N. Agrawal, P. Ciaccio, J. McNulty, H. Barthlow, J. O'Donnell, K. Goteti, J. Breen, J. Comita-Prevoir, M. Cornebise, M. Cronin, C. J. Eyermann, B. Geng, G. R. Carr, L. Pandarinathan, X. Tang, A. Cottone, L. Zhao, N. Bezdenejnih-Snyder, submitted for publication). In the present work, NBTI 5463 demonstrated promising activity against a broad range of Gram-negative pathogens. In contrast to fluoroquinolones, the compound did not form a double-strand DNA cleavable complex with Escherichia coli DNA gyrase and DNA, but it was a potent inhibitor of both DNA gyrase and E. coli topoisomerase IV catalytic activities. In studies with P. aeruginosa, NBTI 5463 was bactericidal. Resistant mutants arose at a low rate, and the mutations were found exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux system. Levofloxacin-selected resistance mutations in GyrA did not result in decreased susceptibility to NBTI 5463. Animal infection studies demonstrated that NBTI 5463 was efficacious in mouse models of lung, thigh, and ascending urinary tract infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Morpholines/pharmacology , Naphthyridines/pharmacology , Topoisomerase II Inhibitors/pharmacology , DNA Gyrase/metabolism , DNA Topoisomerase IV/metabolism , Escherichia coli/drug effects , Escherichia coli/enzymology , Fluoroquinolones/pharmacology , Gram-Negative Bacteria/drug effects , Microbial Sensitivity TestsABSTRACT
Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pKa and logD) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.
Subject(s)
DNA Topoisomerases, Type II/metabolism , Pseudomonas aeruginosa/drug effects , Topoisomerase II Inhibitors/pharmacology , Animals , Chemistry, Physical , Dogs , Dose-Response Relationship, Drug , Drug Resistance, Bacterial/drug effects , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/metabolism , Guinea Pigs , Humans , Mice , Microbial Sensitivity Tests , Molecular Structure , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/metabolism , Rats , Structure-Activity Relationship , Topoisomerase II Inhibitors/chemical synthesis , Topoisomerase II Inhibitors/chemistryABSTRACT
Mucopolysaccharidosis II (MPS II) is a rare lysosomal storage disease characterized by deficient activity of iduronate-2-sulfatase (I2S), leading to pathological accumulation of glycosaminoglycans (GAGs) in tissues. We used iduronate-2-sulfatase knockout (Ids KO) mice to investigate if liver-directed recombinant adeno-associated virus vectors (rAAV8-LSP-hIDSco) encoding human I2S (hI2S) could cross-correct I2S deficiency in Ids KO mouse tissues, and we then assessed the translation of mouse data to non-human primates (NHPs). Treated mice showed sustained hepatic hI2S production, accompanied by normalized GAG levels in somatic tissues (including critical tissues such as heart and lung), indicating systemic cross-correction from liver-secreted hI2S. Brain GAG levels in Ids KO mice were lowered but not normalized; higher doses were required to see improvements in brain histology and neurobehavioral testing. rAAV8-LSP-hIDSco administration in NHPs resulted in sustained hepatic hI2S production and therapeutic hI2S levels in cross-corrected somatic tissues but no hI2S exposure in the central nervous system, perhaps owing to lower levels of liver transduction in NHPs than in mice. Overall, we demonstrate the ability of rAAV8-LSP-hIDSco to cross-correct I2S deficiency in mouse somatic tissues and highlight the importance of showing translatability of gene therapy data from rodents to NHPs, which is critical for supporting translation to clinical development.
ABSTRACT
BACKGROUND: Myostatin antagonists are being developed as therapies for Duchenne muscular dystrophy due to their strong hypertrophic effects on skeletal muscle. Engineered follistatin has the potential to combine the hypertrophy of myostatin antagonism with the anti-inflammatory and anti-fibrotic effects of activin A antagonism. METHODS: Engineered follistatin was administered to C57BL/6 mice for 4 weeks, and muscle mass and myofiber size was measured. In the mdx model, engineered follistatin was dosed for 12 weeks in two studies comparing to an Fc fusion of the activin IIB receptor or an anti-myostatin antibody. Functional measurements of grip strength and tetanic force were combined with tissue analysis for markers of necrosis, inflammation, and fibrosis to evaluate improvement in dystrophic pathology. RESULTS: In wild-type and mdx mice, dose-dependent increases in muscle mass and quadriceps myofiber size were observed for engineered follistatin. In mdx, increases in grip strength and tetanic force were combined with improvements in muscle markers for necrosis, inflammation, and fibrosis. Improvements in dystrophic pathology were greater for engineered follistatin than the anti-myostatin antibody. CONCLUSIONS: Engineered follistatin generated hypertrophy and anti-fibrotic effects in the mdx model.
Subject(s)
Activins/antagonists & inhibitors , Follistatin/therapeutic use , Muscular Dystrophies/drug therapy , Myostatin/antagonists & inhibitors , Animals , Follistatin/administration & dosage , Hand Strength , Male , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle Contraction , Recombinant Proteins/administration & dosage , Recombinant Proteins/therapeutic useABSTRACT
Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of ß-lactamases, enzymes that inactivate ß-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new ß-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine ß-lactamase inhibitors that potently inhibit clinically relevant class A, C and D ß-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of ß-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.
Subject(s)
Acinetobacter baumannii/drug effects , Azabicyclo Compounds/chemistry , Azabicyclo Compounds/pharmacology , Gram-Negative Bacteria/drug effects , beta-Lactamase Inhibitors/chemistry , beta-Lactamase Inhibitors/pharmacology , Acinetobacter Infections/drug therapy , Acinetobacter Infections/microbiology , Animals , Azabicyclo Compounds/therapeutic use , Azabicyclo Compounds/toxicity , Carbapenems/pharmacology , Dogs , Drug Design , Drug Evaluation, Preclinical , Drug Resistance, Multiple, Bacterial , Enterobacteriaceae/drug effects , Gram-Negative Bacterial Infections/drug therapy , Humans , Mice , Models, Molecular , Penicillin-Binding Proteins/antagonists & inhibitors , Rats , Sulbactam/chemistry , Sulbactam/pharmacology , beta-Lactamase Inhibitors/therapeutic use , beta-Lactamase Inhibitors/toxicity , beta-Lactamases/metabolism , beta-Lactams/pharmacologyABSTRACT
UDP-N-acetylmuramyl-l-alanine ligase (MurC) is an essential bacterial enzyme involved in peptidoglycan biosynthesis and a target for the discovery of novel antibacterial agents. As a result of a high-throughput screen (HTS) against a chemical library for inhibitors of MurC, a series of benzofuran acyl-sulfonamides was identified as potential leads. One of these compounds, Compound A, inhibited Escherichia coli MurC with an IC(50) of 2.3 microM. Compound A exhibited time-dependent, partially reversible inhibition of E. coli MurC. Kinetic studies revealed a mode of inhibition consistent with the compound acting competitively with the MurC substrates ATP and UDP-N-acetyl-muramic acid (UNAM) with a K(i) of 4.5 microM against ATP and 6.3 microM against UNAM. Fluorescence binding experiments yielded a K(d) of 3.1 microM for the compound binding to MurC. Compound A also exhibited high-affinity binding to bovine serum albumin (BSA) as evidenced by a severe reduction in MurC inhibition upon addition of BSA. This finding is consistent with the high lipophilicity of the compound. Advancement of this compound series for further drug development will require reduction of albumin binding.
Subject(s)
Benzofurans/pharmacology , Escherichia coli/metabolism , Peptide Synthases/antagonists & inhibitors , Sulfonamides/pharmacology , Uridine Diphosphate N-Acetylmuramic Acid/analogs & derivatives , Uridine Diphosphate N-Acetylmuramic Acid/metabolism , Benzofurans/chemistry , Data Interpretation, Statistical , Sulfonamides/chemistryABSTRACT
The Class D (or OXA-type) ß-lactamases have expanded to be the most diverse group of serine ß-lactamases with a highly heterogeneous ß-lactam hydrolysis profile and are typically resistant to marketed ß-lactamase inhibitors. Class D enzymes are increasingly found in multidrug resistant (MDR) Acinetobacter baumannii, Pseudomonas aeruginosa, and various species of the Enterobacteriaceae and are posing a serious threat to the clinical utility of ß-lactams including the carbapenems, which are typically reserved as the drugs of last resort. Avibactam, a novel non-ß-lactam ß-lactamase inhibitor, not only inhibits all class A and class C ß-lactamases but also has the promise of inhibition of certain OXA enzymes, thus extending the antibacterial activity of the ß-lactam used in combination to the organisms that produce these enzymes. X-ray structures of OXA-24 and OXA-48 in complex with avibactam revealed the binding mode of this inhibitor in this diverse class of enzymes and provides a rationale for selective inhibition of OXA-48 members. Additionally, various subunits of the OXA-48 structure in the asymmetric unit provide snapshots of different states of the inhibited enzyme. Overall, these data provide the first structural evidence of the exceptionally slow reversibility observed with avibactam in class D ß-lactamases. Mechanisms for acylation and deacylation of avibactam by class D enzymes are proposed, and the likely extent of inhibition of class D ß-lactamases by avibactam is discussed.
Subject(s)
Azabicyclo Compounds/pharmacology , Bacterial Proteins/classification , Bacterial Proteins/metabolism , Escherichia coli Proteins/metabolism , beta-Lactamase Inhibitors/pharmacology , beta-Lactamases/classification , beta-Lactamases/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Binding Sites , Crystallization , Escherichia coli Proteins/genetics , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Conformation , beta-Lactamases/geneticsABSTRACT
To identify new agents for the treatment of multi-drug-resistant Pseudomonas aeruginosa, we focused on siderophore-conjugated monocarbams. This class of monocyclic ß-lactams are stable to metallo-ß-lactamases and have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker, siderophore, and oxime portion of the molecules were examined. Through these efforts, a series of pyrrolidinone-based monocarbams with good P. aeruginosa cellular activity (P. aeruginosa MIC90 = 2 µg/mL), free fraction levels (>20% free), and hydrolytic stability (t1/2 ≥ 100 h) were identified. To differentiate the lead compounds and enable prioritization for in vivo studies, we applied a semi-mechanistic pharmacokinetic/pharmacodynamic model to enable prediction of in vivo efficacy from in vitro data.
Subject(s)
Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/pharmacokinetics , Drug Discovery , Monobactams/pharmacology , Monobactams/pharmacokinetics , Pseudomonas Infections/drug therapy , Pseudomonas aeruginosa/drug effects , Siderophores/metabolism , Animals , Humans , Male , Monobactams/chemistry , Pseudomonas Infections/microbiology , Rats , Rats, Wistar , Structure-Activity Relationship , beta-Lactamases/chemistryABSTRACT
Among the targets for the development of new antibacterial agents, bacterial topoisomerases remain a vibrant area of discovery. A structurally diverse set of inhibitors that bind to the adenosine 5'-triphosphate (ATP) site of type II topoisomerases have been disclosed recently. Seven compounds with this mechanism are highlighted, focusing on antibacterial potency and spectrum, as well as examples of in vivo efficacy against pathogens including Staphylococcus aureus and Mycobacterium tuberculosis. Five compounds from two structural classes are exemplified that are inhibitors that bind to the catalytic site of DNA gyrase and topoisomerase IV. The pharmacokinetic and pharmacodynamic properties of these molecules, derived from in vivo efficacy against Gram-positive and Gram-negative pathogens, define the potential for these agents with broad-spectrum and targeted-spectrum clinical utilities.
Subject(s)
Anti-Bacterial Agents/pharmacology , Topoisomerase I Inhibitors/pharmacology , Topoisomerase II Inhibitors/pharmacology , Adenosine Triphosphate/metabolism , Animals , Bacterial Proteins/metabolism , DNA Topoisomerases/metabolismABSTRACT
The NAD-dependent DNA ligase is an excellent target for the discovery of antibacterial agents with a novel mode of action. In this work the DNA ligase from Streptococcus pneumoniae was investigated for its steady-state kinetic parameters and inhibition by compounds with an adenosine substructure. Inhibition by substrate DNA that was observed in the enzyme turnover experiments was verified by direct binding measurements using isothermal titration calorimetry (ITC). The substrate-inhibited enzyme form was identified as deadenylated DNA ligase. The binding potencies of 2-(butylsulfanyl) adenosine and 2-(cyclopentyloxy) adenosine were not significantly affected by the presence of the enzyme-bound DNA substrate. Finally, a mutant protein was prepared that was known to confer resistance to the adenosine compounds' antibacterial activity. The mutant protein was shown to have little catalytic impairment yet it was less susceptible to adenosine compound inhibition.
Subject(s)
Adenosine/pharmacology , Anti-Bacterial Agents/pharmacology , DNA Ligases/metabolism , Enzyme Inhibitors/pharmacology , Streptococcus pneumoniae/enzymology , Adenosine/chemistry , Anti-Bacterial Agents/chemistry , Base Sequence , Calorimetry , DNA Ligases/antagonists & inhibitors , DNA Ligases/genetics , DNA Primers , Drug Discovery , Enzyme Inhibitors/chemistry , Kinetics , Mutagenesis, Site-DirectedABSTRACT
Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. N-Linked amino piperidines, such as 7a, generally show potent antibacterial activity, including against quinolone-resistant isolates, but suffer from hERG inhibition (IC(50) = 44 µM for 7a) and QT prolongation in vivo. We now disclose the finding that new analogues of 7a with reduced pK(a) due to substitution with an electron-withdrawing substituent in the piperidine moiety, such as R,S-7c, retained the Gram-positive activity of 7a but showed significantly less hERG inhibition (IC(50) = 233 µM for R,S-7c). This compound exhibited moderate clearance in dog, promising efficacy against a MRSA strain in a mouse infection model, and an improved in vivo QT profile as measured in a guinea pig in vivo model. As a result of its promising activity, R,S-7c was advanced into phase I clinical studies.