Triple Mycobacterial ATP-synthase mutations impedes Bedaquiline binding: Atomistic and structural perspectives

Top-Down Identification and Sequence Analysis of Small Membrane Proteins Using MALDI-MS/MS

“…The outer surface of the rotor c -ring comprises the access site for ions and is at the same time the target of the antituberculosis drug bedaquiline. The mode of action of bedaquiline has been described to bind at the mycobacteria’s c -ring ion binding site and block its rotational mechanism leading to a complete standstill of the ATP synthase motor function in this pathogen, killing M. tuberculosis bacteria due to a lack of cellular ATP supply. ,, While bedaquiline binds highly specifically to ATP synthases in Mycobacteria , both in vitro selected and the recent appearance of single point mutations have shown to diminish the drug’s high activity to kill M. tuberculosis in multidrug resistant cases and last line treatment approaches. ,− Notably, in all these cases, the structural environment around the binding pocket and an essential ion binding glutamate E61 were identified to be affected in these bedaquiline resistant mutants, in line with initial findings when bedaquiline was discovered and point mutations were screened in M. smegmatis (e.g., D32V) . In clinical M. tuberculosis isolates, showing elevated bedaquiline minimal inhibitory concentrations (MICs), these positions were identified in the c subunit: G25S, D28G, D28N, E61D, A63P, and A63V .…”

“…Here we present this workflow in more detail and showcase the sequencing power on several target proteins. We then apply our technique to the c subunit of the mycobacterial ATP synthase (∼8.5 kDa), which is the target of the 2012 FDA-approved antimycobacterial drug bedaquiline used to treat multidrug- and extensively drug-resistant tuberculosis. , In some cases, single point-mutations in the c subunit are responsible for antibiotic drug resistance, and direct, PCR-free sequence determination from bacterial lysate would allow immediate decision if bedaquiline or other drugs should be used for antibiotic treatment. ,− We show that our approach allows direct identification and sequence analysis of wild type and variants, including positional tracking of 1 Da single point mutations leading to antibiotic resistance, in a workflow taking less than 4 h directly from microgram amounts of bacterial cells.…”

Calcium ATPase signaling: A must include mechanism in the Radar of therapeutics development against Tuberculosis

“…One of the factors associated with it is persisting nature of mycobacteria in intraphagosomal environment in dormancy stage [3][4]. Although in this environment, mycobacteria must suffer with nutrient deficiency, toxic metabolites, reactive oxygen species (ROS), and so on, but this also helps in prolonging the period of dormancy and developing latent TB [5][6][7]. Applied Biochemistry Highlights r Calcium is a universal secondary messenger involved in various signaling cascades of eukaryotes and prokaryotes.…”

Frequent acquisition of bedaquiline resistance by epidemic extensively drug-resistant Mycobacterium tuberculosis strains in Russia during long-term treatment

“…Frameshift mutations in Rv0678 significantly change its amino acid sequence and/or truncate the protein, arguably resulting in the loss of function and bedaquiline efflux. The effects of other mutations on protein structures were assessed using in silico structural analysis of electrostatic potential around mutated amino acids and accessible surface area, then analysed in light of previous studies [3,8,9] (Supplementary Table S2, Fig. 1(b)).…”