ABSTRACT
OqxB belongs to the RND (Resistance-Nodulation-Division) efflux pump family, recognized widely as a major contributor towards enhancing antimicrobial resistance. It is known to be predominantly present in all Klebsiella spp. and is attributed for its role in increasing resistance against an array of antibiotics like nitrofurantoin, quinolones, ß-lactams and colistin. However, the presence of oqxB encoding this efflux pump is not limited only to Klebsiella spp., but is also found to occur via horizontal gene transfer in other bacterial genera like Escherichia coli, Enterobacter cloacae and Salmonella spp. Recently, we reported the crystal structure of OqxB and its structure-function relationship required for the efflux of fluoroquinolones. Extending these findings further, we characterized the structural architecture of this efflux pump along with identifying some critical amino acids at the substrate binding domain of OqxB. Based on our in silico modelling studies, both hydrophobic residues (F180, L280, L621, F626) and polar residues (R48, E50, E184, R157, R774) were found to be located at this site. The present work reports the importance of these key amino acid residues and the crucial ion-pair interactions at the substrate-binding pocket, thereby establishing their role in OqxB mediated efflux and the resultant resistance development against fluoroquinolones.
Subject(s)
Amino Acids , Fluoroquinolones , Fluoroquinolones/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/metabolism , Nitrofurantoin , Escherichia coli/genetics , Escherichia coli/metabolism , Mutagenesis, Site-Directed , Drug Resistance, Multiple, Bacterial/genetics , Microbial Sensitivity Tests , Bacterial Proteins/metabolismABSTRACT
Using Escherichia coli as the representative biofilm former, we report here the development of an in silico model built by simulating events that transform a free-living bacterial entity into self-encased multicellular biofilms. Published literature on â¼300 genes associated with pathways involved in biofilm formation was curated, static maps were created, and suitably interconnected with their respective metabolites using ordinary differential equations. Precise interplay of genetic networks that regulate the transitory switching of bacterial growth pattern in response to environmental changes and the resultant multicomponent synthesis of the extracellular matrix were appropriately represented. Subsequently, the in silico model was analyzed by simulating time-dependent changes in the concentration of components by using the R and python environment. The model was validated by simulating and verifying the impact of key gene knockouts (KOs) and systematic knockdowns on biofilm formation, thus ensuring the outcomes were comparable with the reported literature. Similarly, specific gene KOs in laboratory and pathogenic E. coli were constructed and assessed. MiaA, YdeO, and YgiV were found to be crucial in biofilm development. Furthermore, qRT-PCR confirmed the elevation of expression in biofilm-forming clinical isolates. Findings reported in this study offer opportunities for identifying biofilm inhibitors with applications in multiple industries. The application of this model can be extended to the health care sector specifically to develop novel adjunct therapies that prevent biofilms in medical implants and reduce emergence of biofilm-associated resistant polymicrobial-chronic infections. The in silico framework reported here is open source and accessible for further enhancements.
Subject(s)
Escherichia coli Infections , Escherichia coli , Bacteria , Biofilms , Computer Simulation , Escherichia coli/genetics , Escherichia coli Infections/microbiology , HumansABSTRACT
We discovered azaindole-based compounds with weak innate activity that exhibit substantial potentiation of antibacterial activities of different antibiotics, viz., rifampicin, erythromycin, solithromycin, and novobiocin in Gram-negative bacteria. In the presence of the azaindole derivatives, these antibiotics exhibited submicromolar minimum inhibitory concentrations (MICs) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The fold improvements in MIC of these antibiotics that were otherwise weak or inactive on their own against these bacteria were also observed against drug-resistant clinical isolates. Our studies indicate that this selective potentiation is probably through destabilization of the outer membrane's integrity, known to be regulated by the lipopolysaccharides (LPS). Thus, the azaindole based compounds described here open opportunities for those antibiotics that are otherwise ineffective due to LPS mediated entry barriers in Gram-negative bacteria.
Subject(s)
Acinetobacter baumannii , Anti-Bacterial Agents , Anti-Bacterial Agents/pharmacology , Gram-Negative Bacteria , Klebsiella pneumoniae , Microbial Sensitivity TestsABSTRACT
OqxB is an RND (Resistance-Nodulation-Division) efflux pump that has emerged as a factor contributing to the antibiotic resistance in Klebsiella pneumoniae. OqxB underwent horizontal gene transfer and is now seen in other Gram-negative bacterial pathogens including Escherichia coli, Enterobacter cloacae and Salmonella spp., further disseminating multi-drug resistance. In this study, we describe crystal structure of OqxB with n-dodecyl-ß-D-maltoside (DDM) molecules bound in its substrate-binding pocket, at 1.85 Å resolution. We utilize this structure in computational studies to predict the key amino acids contributing to the efflux of fluoroquinolones by OqxB, distinct from analogous residues in related transporters AcrB and MexB. Finally, our complementation assays with mutated OqxB and minimum inhibitory concentration (MIC) experiments with clinical isolates of E. coli provide further evidence that the predicted structural features are indeed involved in ciprofloxacin efflux.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Multiple, Bacterial/genetics , Klebsiella pneumoniae/genetics , Membrane Transport Proteins/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Binding Sites/genetics , Crystallography, X-Ray , Klebsiella pneumoniae/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Protein Conformation , Protein Multimerization , Structure-Activity RelationshipABSTRACT
We report a novel benzimidazole (BI) based DprE1 inhibitor that resulted from scaffold morphing of a 1,4-azaindole series. The clinical progression of the 1,4-azaindole series from our previous work validates the potential of exploring newer chemical entities with antimycobacterial activity driven via a noncovalent inhibition of the decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). The representative compounds from the new scaffold reported in this study exhibited an improved solubility and higher free plasma fraction, while retaining potent DprE1 inhibition and antimycobacterial activity. A representative compound from the benzimidazole series demonstrated good efficacy in a murine model of tuberculosis. Furthermore, molecular modeling of the BI scaffold suggests plausible modes of binding in the active site of DprE1 enzyme from Mycobacterium tuberculosis that can be used for further exploration of the series.
ABSTRACT
The mechanism of efflux is a tour-de-force in the bacterial armoury that has thwarted the development of novel antibiotics. We report the discovery of a novel chemical series with potent antibacterial properties that was engineered to overcome efflux liability. Compounds liable to efflux specifically via the Resistance Nodulation and cell Division (RND) pump, AcrAB-TolC were chosen for a hit to lead progression. Using structure-based design, the compounds were optimised to lose their binding to the efflux pump, thereby making them potent on wild-type bacteria. We discovered these compounds to be pro-drugs that require activation in E. coli by specific bacterial nitroreductases NfsA and NfsB. Hit to lead chemistry led to the generation of compounds that were potent on wild-type and multi-drug resistant clinical isolates of E. coli, Shigella spp., and Salmonella spp. These compounds are bactericidal and efficacious in a mouse thigh infection model.
Subject(s)
Anti-Bacterial Agents/chemistry , Drug Resistance, Multiple, Bacterial/drug effects , Escherichia coli Proteins/chemistry , Prodrugs/chemistry , Thiophenes/chemistry , Animals , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Cell Division/drug effects , Drug Resistance, Multiple, Bacterial/genetics , Escherichia coli Proteins/drug effects , Humans , Mice , Microbial Sensitivity Tests , Prodrugs/chemical synthesis , Prodrugs/pharmacology , Protein Conformation/drug effects , Salmonella/chemistry , Salmonella/drug effects , Salmonella/pathogenicity , Shigella/chemistry , Shigella/drug effects , Shigella/pathogenicity , Thiophenes/chemical synthesis , Thiophenes/pharmacologyABSTRACT
The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.
Subject(s)
Adenosine Triphosphate/metabolism , Antitubercular Agents/therapeutic use , Mycobacterium tuberculosis/drug effects , Pyridines/therapeutic use , Quinine/analogs & derivatives , Tuberculosis/drug therapy , Animals , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/pharmacology , Ethers/chemistry , Ethers/pharmacokinetics , Ethers/pharmacology , Ethers/therapeutic use , Humans , Mice , Mice, Inbred BALB C , Models, Molecular , Pyridines/chemistry , Pyridines/pharmacokinetics , Pyridines/pharmacology , Quinine/chemistry , Quinine/pharmacokinetics , Quinine/pharmacology , Quinine/therapeutic use , Tuberculosis/metabolismABSTRACT
Nitroarenes are less preferred in drug discovery due to their potential to be mutagenic. However, several nitroarenes were shown to be promising antitubercular agents with specific modes of action, namely, nitroimidazoles and benzothiazinones. The nitro group in these compounds is activated through different mechanisms, both enzymatic and non-enzymatic, in mycobacteria prior to binding to the target of interest. From a whole-cell screening program, we identified a novel lead nitrobenzothiazole (BT) series that acts by inhibition of decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) of Mycobacterium tuberculosis (Mtb). The lead was found to be mutagenic to start with. Our efforts to mitigate mutagenicity resulted in the identification of 6-methyl-7-nitro-5-(trifluoromethyl)-1,3-benzothiazoles (cBTs), a novel class of antitubercular agents that are non-mutagenic and exhibit an improved safety profile. The methyl group ortho to the nitro group decreases the electron affinity of the series, and is hence responsible for the non-mutagenic nature of these compounds. Additionally, the co-crystal structure of cBT in complex with Mtb DprE1 established the mode of binding. This investigation led to a new non-mutagenic antitubercular agent and demonstrates that the mutagenic nature of nitroarenes can be solved by modulation of stereoelectronic properties.
Subject(s)
Antitubercular Agents/pharmacology , Benzothiazoles/pharmacology , Mutagens/chemistry , Mycobacterium tuberculosis/drug effects , Nitro Compounds/pharmacology , Antitubercular Agents/adverse effects , Antitubercular Agents/chemistry , Benzothiazoles/adverse effects , Benzothiazoles/chemistry , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Structure , Nitro Compounds/adverse effects , Nitro Compounds/chemistry , Stereoisomerism , Structure-Activity RelationshipABSTRACT
We report the discovery of benzothiazoles, a novel anti-mycobacterial series, identified from a whole cell based screening campaign. Benzothiazoles exert their bactericidal activity against Mycobacterium tuberculosis (Mtb) through potent inhibition of decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), the key enzyme involved in arabinogalactan synthesis. Specific target linkage and mode of binding were established using co-crystallization and protein mass spectrometry studies. Most importantly, the current study provides insights on the utilization of systematic medicinal chemistry approaches to mitigate safety liabilities while improving potency during progression from an initial genotoxic hit, the benzothiazole N-oxides (BTOs) to the lead-like AMES negative, crowded benzothiazoles (cBTs). These findings offer opportunities for development of safe clinical candidates against tuberculosis. The design strategy adopted could find potential application in discovery of safe drugs in other therapy areas too.
Subject(s)
Alcohol Oxidoreductases/metabolism , Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins/metabolism , Benzothiazoles/chemistry , Benzothiazoles/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Alcohol Oxidoreductases/antagonists & inhibitors , Bacterial Proteins/antagonists & inhibitors , Drug Design , Humans , Molecular Docking Simulation , Structure-Activity Relationship , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
There are currently 18 drug classes for the treatment of tuberculosis, including those in the development pipeline. An in silico simulation enabled combing the innumerably large search space to derive multidrug combinations. Through the use of ordinary differential equations (ODE), we constructed an in silico kinetic platform in which the major metabolic pathways in Mycobacterium tuberculosis and the mechanisms of the antituberculosis drugs were integrated into a virtual proteome. The optimized model was used to evaluate 816 triplets from the set of 18 drugs. The experimentally derived cumulative fractional inhibitory concentration (∑FIC) value was within twofold of the model prediction. Bacterial enumeration revealed that a significant number of combinations that were synergistic for growth inhibition were also synergistic for bactericidal effect. The in silico-based screen provided new starting points for testing in a mouse model of tuberculosis, in which two novel triplets and five novel quartets were significantly superior to the reference drug triplet of isoniazid, rifampin, and ethambutol (HRE) or the quartet of HRE plus pyrazinamide (HREZ).
Subject(s)
Antitubercular Agents/therapeutic use , Ethambutol/therapeutic use , Isoniazid/therapeutic use , Rifampin/therapeutic use , Tuberculosis/drug therapy , Animals , Mice , Mice, Inbred BALB C , Microbial Sensitivity TestsABSTRACT
DNA topoisomerases perform the essential function of maintaining DNA topology in prokaryotes. DNA gyrase, an essential enzyme that introduces negative supercoils, is a clinically validated target. However, topoisomerase I (Topo I), an enzyme responsible for DNA relaxation has received less attention as an antibacterial target, probably due to the ambiguity over its essentiality in many organisms. The Mycobacterium tuberculosis genome harbors a single topA gene with no obvious redundancy in its function suggesting an essential role. The topA gene could be inactivated only in the presence of a complementing copy of the gene in M. tuberculosis. Furthermore, down-regulation of topA in a genetically engineered strain of M. tuberculosis resulted in loss of bacterial viability which correlated with a concomitant depletion of intracellular Topo I levels. The topA knockdown strain of M. tuberculosis failed to establish infection in a murine model of TB and was cleared from lungs in two months post infection. Phenotypic screening of a Topo I overexpression strain led to the identification of an inhibitor, thereby providing chemical validation of this target. Thus, our work confirms the attractiveness of Topo I as an anti-mycobacterial target.
Subject(s)
Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , DNA Topoisomerases, Type I , Drug Discovery , Mycobacterium tuberculosis/drug effects , Topoisomerase I Inhibitors/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , Gene Expression Regulation, Bacterial , Gene Knockdown Techniques , Genotype , Humans , Microbial Viability , Molecular Targeted Therapy , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/pathogenicity , Phenotype , Time FactorsABSTRACT
Whole cell based screens to identify hits against Mycobacterium tuberculosis (Mtb), carried out under replicating and non-replicating (NRP) conditions, resulted in the identification of multiple, novel but structurally related spiropiperidines with potent antitubercular properties. These compounds could be further classified into three classes namely 3-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[indene-1,4'-piperidine] (abbr. spiroindenes), 4-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[chromene-2,4'-piperidine] (abbr. spirochromenes) and 1'-benzylspiro[indole-1,4'-piperidin]-2(1H)-one (abbr. spiroindolones). Spiroindenes showed ⩾ 4 log10 kill (at 2-12 µM) on replicating Mtb, but were moderately active under non replicating conditions. Whole genome sequencing efforts of spiroindene resistant mutants resulted in the identification of I292L mutation in MmpL3 (Mycobacterial membrane protein Large), required for the assembly of mycolic acid into the cell wall core of Mtb. MIC modulation studies demonstrated that the mutants were cross-resistant to spirochromenes but not to spiroindolones. This Letter describes lead identification efforts to improve potency while reducing the lipophilicity and hERG liabilities of spiroindenes. Additionally, as deduced from the SAR studies, we provide insights regarding the new chemical opportunities that the spiroindolones can offer to the TB drug discovery initiatives.
Subject(s)
Antitubercular Agents/pharmacology , Piperidines/pharmacology , Spiro Compounds/pharmacology , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacokinetics , Bacteria/drug effects , Drug Resistance, Bacterial/genetics , Genome, Bacterial , High-Throughput Screening Assays , Hypoxia , Lipids/chemistry , Matrix Metalloproteinase 13/biosynthesis , Matrix Metalloproteinase 13/genetics , Mice , Microbial Sensitivity Tests , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacokinetics , Structure-Activity RelationshipABSTRACT
A cellular activity-based screen on Mycobacterium tuberculosis (Mtb) H37Rv using a focused library from the AstraZeneca corporate collection led to the identification of 2-phenylindoles and arylsulphonamides, novel antimycobacterial scaffolds. Both the series were bactericidal in vitro and in an intracellular macrophage infection model, active against drug sensitive and drug resistant Mtb clinical isolates, and specific to mycobacteria. The scaffolds showed promising structure-activity relationships; compounds with submicromolar cellular potency were identified during the hit to lead exploration. Furthermore, compounds from both scaffolds were tested for inhibition of known target enzymes or pathways of antimycobacterial drugs including InhA, RNA polymerase, DprE1, topoisomerases, protein synthesis, and oxidative-phosphorylation. Compounds did not inhibit any of the targets suggesting the potential of a possible novel mode of action(s). Hence, both scaffolds provide the opportunity to be developed further as leads and tool compounds to uncover novel mechanisms for tuberculosis drug discovery.
ABSTRACT
The Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor of Escherichia coli and Pseudomonas aeruginosa MurC. However, cellular activity against E. coli or P. aeruginosa was not observed. Compound A was active against efflux pump mutants of both strains. Experiments using an E. coli tolC mutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A ,: indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in the E. coli wild-type cells. Further, overexpression of MurC in the E. coli tolC mutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in the tolC mutant strain. A significant compound A level was not detected in the wild-type E. coli strain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-type E. coli were possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/cytology , Escherichia coli/enzymology , Peptide Synthases/metabolism , Alanine/metabolism , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/metabolism , Escherichia coli/genetics , Escherichia coli Proteins/genetics , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mutation , Peptide Synthases/genetics , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Uridine Diphosphate N-Acetylmuramic Acid/metabolismABSTRACT
A major obstacle in the process of discovery of drugs against Mycobacterium tuberculosis is its extremely slow growth rate and long generation time (â¼20 to 24 h). Consequently, determination of MICs and minimum bactericidal concentrations (MBCs) of potential drug candidates using current methods requires 7 days (resazurin-based MIC assay [REMA]) and 1 month (CFU enumeration), respectively. We employed a synthetic luciferase operon optimized for expression in high-GC-content bacteria and adapted it for use in mycobacteria. Using luminescence-based readouts, we were able to determine the MICs and bactericidal activities of approved tuberculosis (TB) drugs, which correlated well with currently used methods. Although luminescence-based readouts have been used previously to determine the MICs and bactericidal activities of approved TB drugs, in this study we adapted this assay to carry out a pilot screen using a library of 1,114 compounds belonging to diverse chemical scaffolds. We found that MICs derived from a 3-day luminescence assay matched well with REMA-based MIC values. To determine the bactericidal potencies of compounds, a 1:10 dilution of the cultures from the MIC plate was carried out on day 7, and the bactericidal concentrations determined based on time to positivity in 2 weeks were found to be comparable with MBC values determined by the conventional CFU approach. Thus, the luminescent mycobacterium-based approach not only is very simple and inexpensive but also allowed us to generate the information in half the time required by conventional methods.
Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Luminescent Measurements , Microbial Sensitivity Tests , Reproducibility of ResultsABSTRACT
A whole cell based screening effort on a focused library from corporate collection resulted in the identification of biarylmethoxy nicotinamides as novel inhibitors of M. tuberculosis (Mtu) H37Rv. The series exhibited tangible structure-activity relationships, and during hit to lead exploration, a cellular potency of 100 nM was achieved, which is an improvement of >200-fold from the starting point. The series is very specific to Mtu and noncytotoxic up to 250 µM as measured in the mammalian cell line THP-1 based cytotoxicity assay. This compound class retains its potency on several drug sensitive and single drug resistant clinical isolates, which indicate that the compounds could be acting through a novel mode of action.
ABSTRACT
Diarylthiazole (DAT), a hit from diversity screening, was found to have potent antimycobacterial activity against Mycobacterium tuberculosis (Mtb). In a systematic medicinal chemistry exploration, we demonstrated chemical opportunities to optimize the potency and physicochemical properties. The effort led to more than 10 compounds with submicromolar MICs and desirable physicochemical properties. The potent antimycobacterial activity, in conjunction with low molecular weight, made the series an attractive lead (antibacterial ligand efficiency (ALE)>0.4). The series exhibited excellent bactericidal activity and was active against drug-sensitive and resistant Mtb. Mutational analysis showed that mutations in prrB impart resistance to DAT compounds but not to reference drugs tested. The sensor kinase PrrB belongs to the PrrBA two component system and is potentially the target for DAT. PrrBA is a conserved, essential regulatory mechanism in Mtb and has been shown to have a role in virulence and metabolic adaptation to stress. Hence, DATs provide an opportunity to understand a completely new target system for antimycobacterial drug discovery.
Subject(s)
Antitubercular Agents/chemistry , Bacterial Proteins/metabolism , Mycobacterium tuberculosis/drug effects , Protein Kinases/metabolism , Thiazoles/chemistry , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacology , Bacterial Proteins/genetics , Drug Resistance, Bacterial , High-Throughput Screening Assays , Humans , Mice , Microbial Sensitivity Tests , Mutation , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Polymorphism, Single Nucleotide , Protein Kinases/genetics , Small Molecule Libraries , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/pharmacologyABSTRACT
New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-ß-D-ribose 2'-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug.
Subject(s)
Antitubercular Agents/therapeutic use , Indoles/therapeutic use , Pyridines/therapeutic use , Tuberculosis, Pulmonary/drug therapy , Animals , Antitubercular Agents/chemical synthesis , Antitubercular Agents/pharmacokinetics , Dogs , Drug Therapy, Combination , Female , Humans , Indoles/chemical synthesis , Indoles/pharmacokinetics , Male , Mice , Mice, Inbred BALB C , Pyridines/chemical synthesis , Pyridines/pharmacokinetics , RatsABSTRACT
4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of â¼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.
Subject(s)
Amides/chemistry , Antitubercular Agents/chemistry , Bacterial Proteins/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Oxidoreductases/antagonists & inhibitors , Piperidines/chemistry , Quinolones/chemistry , Alcohol Oxidoreductases , Amides/pharmacokinetics , Amides/pharmacology , Animals , Antitubercular Agents/pharmacokinetics , Antitubercular Agents/pharmacology , Catalytic Domain , Cell Line, Tumor , Drug Resistance, Bacterial , Genome, Bacterial , Humans , Kinetics , Microbial Sensitivity Tests , Molecular Docking Simulation , Mutation , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Piperidines/pharmacokinetics , Piperidines/pharmacology , Protein Binding , Quinolones/pharmacokinetics , Quinolones/pharmacology , Rats, Wistar , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A novel pyrazolopyridone class of inhibitors was identified from whole cell screening against Mycobacterium tuberculosis (Mtb). The series exhibits excellent bactericidality in vitro, resulting in a 4 log reduction in colony forming units following compound exposure. The significant modulation of minimum inhibitory concentration (MIC) against a Mtb strain overexpressing the Rv3790 gene suggested the target of pyrazolopyridones to be decaprenylphosphoryl-ß-D-ribose-2'-epimerase (DprE1). Genetic mapping of resistance mutation coupled with potent enzyme inhibition activity confirmed the molecular target. Detailed biochemical characterization revealed the series to be a noncovalent inhibitor of DprE1. Docking studies at the active site suggest that the series can be further diversified to improve the physicochemical properties without compromising the antimycobacterial activity. The pyrazolopyridone class of inhibitors offers an attractive non-nitro lead series targeting the essential and vulnerable DprE1 enzyme for the discovery of novel antimycobacterial agents to treat both drug susceptible and drug resistant strains of Mtb.