Comparison of in vitro Susceptibility of Mycobacteria Against PA-824 to Identify Key Residues of Ddn, the Deazoflavin-Dependent Nitroreductase from .

OBJECTIVE

PA-824 (Pretomanid), a bicyclic nitroimidazole drug, exhibits significant bactericidal activity toward (MTB) in vitro and in vivo, but not against . Through catalytic bioreduction, deazaflavin-dependent nitroreductase (Ddn) within MTB directly converts PA-824 to potent bactericidal products. This study aimed to identify key MTB Ddn residues involved in PA-824 conversion toward development of in vitro surrogate markers for detection of mycobacterial resistance to PA-824.

METHODS

We evaluated in vitro activity of PA-824 toward MTB and nontuberculous mycobacterial species using antimicrobial susceptibility testing. Ddn amino acid sequence alignments and phylogenetic analysis revealed putative key enzyme active site residues. Candidate MTB Ddn residues required for PA-824 conversion activity were evaluated for loss-of-function using recombinantly cloned Ddn mutant proteins expressed in .

RESULTS

PA-824 minimum inhibitory concentrations of 90% of bacterial growth (MICs) against MTB and were 0.12 mg/L and 8 mg/L, respectively, but >32 mg/L for spp. and . MTB Ddn and Ddn homologous sequences shared the greatest similarity (89.3% amino acid identity). expressing Ddn proteins with Y65L, A76V or Y133F substitutions (but not V75L, Q125K or V148I) were resistant to PA-824.

CONCLUSION

Our data demonstrated that PA-824 exhibited excellent and moderate levels of in vitro activity against MTB and , respectively. Substitutions of Ddn residues Y65, A76 or Y133 conferred mycobacterial resistance to PA-824.