ABSTRACT
Tuberculosis is a major global cause of both mortality and financial burden mainly in low and middle-income countries. Given the significant and ongoing rise of drug-resistant strains of Mycobacterium tuberculosis within the clinical setting, there is an urgent need for the development of new, safe and effective treatments. Here the development of a drug-like series based on a fused dihydropyrrolidino-pyrimidine scaffold is described. The series has been developed against M. tuberculosis lysyl-tRNA synthetase (LysRS) and cellular studies support this mechanism of action. DDD02049209, the lead compound, is efficacious in mouse models of acute and chronic tuberculosis and has suitable physicochemical, pharmacokinetic properties and an in vitro safety profile that supports further development. Importantly, preliminary analysis using clinical resistant strains shows no pre-existing clinical resistance towards this scaffold.
Subject(s)
Lysine-tRNA Ligase , Mycobacterium tuberculosis , Tuberculosis , Animals , Lysine-tRNA Ligase/chemistry , Lysine-tRNA Ligase/genetics , Lysine-tRNA Ligase/pharmacology , Mice , Mycobacterium tuberculosis/genetics , Tuberculosis/drug therapyABSTRACT
Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis (Mtb) identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against clinical isolates of Mtb. Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and in vitro microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.
Subject(s)
Antitubercular Agents/chemistry , Pyrimidinones/chemistry , Animals , Antitubercular Agents/metabolism , Antitubercular Agents/pharmacology , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Half-Life , Humans , Iron/metabolism , Male , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Microsomes/metabolism , Mutation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/isolation & purification , Pyrazoles/chemistry , Pyrimidinones/metabolism , Pyrimidinones/pharmacology , Rats , Structure-Activity RelationshipABSTRACT
A potent, noncytotoxic indazole sulfonamide was identified by high-throughput screening of >100,000 synthetic compounds for activity against Mycobacterium tuberculosis (Mtb). This noncytotoxic compound did not directly inhibit cell wall biogenesis but triggered a slow lysis of Mtb cells as measured by release of intracellular green fluorescent protein (GFP). Isolation of resistant mutants followed by whole-genome sequencing showed an unusual gene amplification of a 40 gene region spanning from Rv3371 to Rv3411c and in one case a potential promoter mutation upstream of guaB2 (Rv3411c) encoding inosine monophosphate dehydrogenase (IMPDH). Subsequent biochemical validation confirmed direct inhibition of IMPDH by an uncompetitive mode of inhibition, and growth inhibition could be rescued by supplementation with guanine, a bypass mechanism for the IMPDH pathway. Beads containing immobilized indazole sulfonamides specifically interacted with IMPDH in cell lysates. X-ray crystallography of the IMPDH-IMP-inhibitor complex revealed that the primary interactions of these compounds with IMPDH were direct pi-pi interactions with the IMP substrate. Advanced lead compounds in this series with acceptable pharmacokinetic properties failed to show efficacy in acute or chronic murine models of tuberculosis (TB). Time-kill experiments in vitro suggest that sustained exposure to drug concentrations above the minimum inhibitory concentration (MIC) for 24 h were required for a cidal effect, levels that have been difficult to achieve in vivo. Direct measurement of guanine levels in resected lung tissue from tuberculosis-infected animals and patients revealed 0.5-2 mM concentrations in caseum and normal lung tissue. The high lesional levels of guanine and the slow lytic, growth-rate-dependent effect of IMPDH inhibition pose challenges to developing drugs against this target for use in treating TB.
Subject(s)
Antitubercular Agents/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Sulfonamides/pharmacology , Animals , Drug Design , Drug Discovery , Drug Resistance, Bacterial , Gene Expression Regulation, Bacterial/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Humans , Mice , Mice, Inbred C57BL , Molecular Structure , Mutation , Protein Conformation , Rabbits , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , Tuberculosis/drug therapyABSTRACT
The pharmacokinetic based optimisation of a novel series of indole-2-carboxamide antagonists of the cannabinoid CB(1) receptor is disclosed. Compound 24 was found to be a highly potent and selective cannabinoid CB(1) antagonist with high predicted human oral bioavailability.
Subject(s)
Indoles/pharmacokinetics , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Administration, Oral , Biological Availability , Humans , Indoles/administration & dosage , Indoles/chemistry , Structure-Activity RelationshipABSTRACT
The discovery and structure-activity relationship of a novel series of indole-2-carboxamide antagonists of the cannabinoid CB(1) receptor is disclosed. Compound 26i was found to be a high potency, selective cannabinoid CB(1) antagonist.