ABSTRACT
Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors.
Subject(s)
Antimalarials/pharmacology , Drug Evaluation, Preclinical/methods , Mitochondria/drug effects , Plasmodium falciparum/drug effects , Drug Discovery/methods , Drug Evaluation, Preclinical/instrumentation , Electron Transport Chain Complex Proteins/antagonists & inhibitors , Humans , Inhibitory Concentration 50 , Malaria/drug therapy , Malaria/parasitology , Malaria, Falciparum , Oxygen/metabolism , Plasmodium falciparum/cytologyABSTRACT
Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment.
Subject(s)
Antitubercular Agents/pharmacology , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Animals , Antitubercular Agents/chemistry , Binding Sites , Catalytic Domain , Disease Models, Animal , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/genetics , Enoyl-(Acyl-Carrier-Protein) Reductase (NADH)/metabolism , Enzyme Inhibitors/chemistry , Female , Humans , Mice , Microbial Sensitivity Tests , Microsomes , Models, Molecular , Mutation , Mycobacterium tuberculosis/genetics , Protein Binding , Protein Conformation , Tuberculosis/drug therapy , Tuberculosis/microbiology , Tuberculosis/mortality , Tuberculosis, Multidrug-ResistantABSTRACT
The rising incidence of antimicrobial resistance (AMR) makes it imperative to understand the underlying mechanisms. Mycobacterium tuberculosis (Mtb) is the single leading cause of death from a bacterial pathogen and estimated to be the leading cause of death from AMR. A pyrido-benzimidazole, 14, was reported to have potent bactericidal activity against Mtb. Here, we isolated multiple Mtb clones resistant to 14. Each had mutations in the putative DNA-binding and dimerization domains of rv2887, a gene encoding a transcriptional repressor of the MarR family. The mutations in Rv2887 led to markedly increased expression of rv0560c. We characterized Rv0560c as an S-adenosyl-L-methionine-dependent methyltransferase that N-methylates 14, abolishing its mycobactericidal activity. An Mtb strain lacking rv0560c became resistant to 14 by mutating decaprenylphosphoryl-ß-d-ribose 2-oxidase (DprE1), an essential enzyme in arabinogalactan synthesis; 14 proved to be a nanomolar inhibitor of DprE1, and methylation of 14 by Rv0560c abrogated this activity. Thus, 14 joins a growing list of DprE1 inhibitors that are potently mycobactericidal. Bacterial methylation of an antibacterial agent, 14, catalyzed by Rv0560c of Mtb, is a previously unreported mechanism of AMR.
Subject(s)
Antitubercular Agents/metabolism , Bacterial Proteins/metabolism , Drug Resistance, Bacterial , Mycobacterium tuberculosis/metabolism , Antitubercular Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Benzimidazoles/chemistry , Benzimidazoles/metabolism , Gene Expression Regulation, Bacterial , Methylation , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Molecular Structure , Mutation , Mycobacterium tuberculosis/genetics , Protein Domains , Repressor Proteins/chemistry , Repressor Proteins/genetics , Repressor Proteins/metabolism , S-Adenosylmethionine/metabolismABSTRACT
Tetrahydropyran derivative 1 was discovered in a high-throughput screening campaign to find new inhibitors of mycobacterial InhA. Following initial in-vitro profiling, a structure-activity relationship study was initiated and a focused library of analogs was synthesized and evaluated. This yielded compound 42 with improved antimycobacterial activity and low cytotoxicity. Additionally, the crystal structure of InhA in complex with inhibitor 1 was resolved, to reveal the binding mode and provide hints for further optimization.
Subject(s)
Antitubercular Agents/chemistry , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Oxidoreductases/antagonists & inhibitors , Pyrans/chemistry , Pyrans/pharmacology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Humans , Microbial Sensitivity Tests , Models, Molecular , Mycobacterium tuberculosis/chemistry , Mycobacterium tuberculosis/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Structure-Activity Relationship , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.
Subject(s)
Antitubercular Agents/pharmacology , High-Throughput Screening Assays , Macrophages/drug effects , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/chemistry , Cell Line , Gene Expression , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hep G2 Cells , Humans , Luciferases/genetics , Luciferases/metabolism , Macrophages/microbiology , Microbial Sensitivity Tests , Mycobacterium tuberculosis/growth & development , Structure-Activity RelationshipABSTRACT
As a follow up to the antimycobacterial screening exercise and the release of GSK´s first Tres Cantos Antimycobacterial Set (TCAMS-TB), this paper presents the results of a second antitubercular screening effort of two hundred and fifty thousand compounds recently added to the GSK collection. The compounds were further prioritized based on not only antitubercular potency but also on physicochemical characteristics. The 50 most attractive compounds were then progressed for evaluation in three different predictive computational biology algorithms based on structural similarity or GSK historical biological assay data in order to determine their possible mechanisms of action. This effort has resulted in the identification of novel compounds and their hypothesized targets that will hopefully fuel future TB drug discovery and target validation programs alike.
Subject(s)
Antitubercular Agents/pharmacology , Mycobacterium tuberculosis/drug effects , Algorithms , Cell Line, Tumor , Computational Biology/methods , Drug Design , Drug Discovery/methods , Hep G2 Cells , HumansABSTRACT
Identification of compounds that target metabolically diverse subpopulations of Mycobacterium tuberculosis (Mtb) may contribute to shortening the course of treatment for tuberculosis. This study screened 270,000 compounds from GlaxoSmithKline's collection against Mtb in a nonreplicating (NR) state imposed in vitro by a combination of four host-relevant stresses. Evaluation of 166 confirmed hits led to detailed characterization of 19 compounds for potency, specificity, cytotoxicity, and stability. Compounds representing five scaffolds depended on reactive nitrogen species for selective activity against NR Mtb, and two were stable in the assay conditions. Four novel scaffolds with activity against replicating (R) Mtb were also identified. However, none of the 19 compounds was active against Mtb in both NR and R states. There was minimal overlap between compounds found active against NR Mtb and those previously identified as active against R Mtb, supporting the hypothesis that NR Mtb depends on distinct metabolic pathways for survival.
ABSTRACT
Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide (THPP) and N-benzyl-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran] (Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This 'genetic phenotype' was further confirmed by a 'chemical phenotype', whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice.