FIG 8.

(A) Epitope sequence alignment between M. bovis, M. tuberculosis, M. smegmatis, humans, bovines, and B. subtilis. The region of interest is highlighted in green and found to be unique to mycobacteria. (B) Characterization of in silico screen hits 2 (green line) and 3 (blue line) in oxygen consumption assay with purified recombinant M. tuberculosis cyt-bcc:aa₃ (black line). In the presence of 2,3-dimethyl-[1,4]naphthohydroquinone, the supercomplex reduced oxygen levels by 50%, while hits 2 and 3 decreased respiration of the recombinant M. tuberculosis cyt-bcc:aa₃. (C) Effect of hits on electron transfer in the recombinant M. tuberculosis cyt-bcc:aa₃. cytMycc1 was incubated with recombinant M. tuberculosis cyt-bcc:aa₃ prior to oxidation with potassium ferricyanide. The conditions tested were compound free (black), DMSO (red), cytMycc1 (blue), or hit 2 (green). The heme a (444 nm) and heme b (432 nm) wavelengths showed drastic differences. All experiments were repeated at least once. (D) Effect of cytMycc1 on ATP synthesis of M. smegmatis IMVs. cytMycc1 (blue circles) had a starting concentration of 500 μM and was serially diluted by 2-fold. The starting concentration of Telacebec (red circles) and bedaquiline (BDQ; green circles) was 100 and 80 nM, respectively. The data points are expressed as means ± the standard deviations of triplicates from a representative experiment. The experiment was performed in triplicates and repeated more than once. (E) Binding pose of cytMycc1: the amide (NH₂) group on N-propionamide form H-bonding interactions (black dotted lines) with main chain carbonyl (CO) atoms of residues S176 and C177 and main chain NH atoms of A186 of QcrC. Benzimidazole has strong aromatic interactions with amino acids F180 and close contacts with N176 and G251. The propyl-thio-methyl fragment was in vicinity of QcrA (L362, S361, and C374) residues adjacent to the Fe-S cluster.