Zhang Fuzhen, Li Shanshan, Wen Shuan, Zhang Tingting, Shang Yuanyuan, Huo Fengmin, Xue Yi, Li Ling, Pang Yu
Biosafety Level 3 Laboratory, School of Public Health, Southern Medical University, Guangzhou, People's Republic of China.
National Clinical Laboratory on Tuberculosis, Beijing Key Laboratory on Drug-Resistant Tuberculosis Research, Beijing Chest Hospital, Capital Medical University, Beijing Tuberculosis and Thoracic Tumor Institute, Beijing, People's Republic of China.
Infect Drug Resist. 2020 Mar 11;13:815-822. doi: 10.2147/IDR.S240716. eCollection 2020.
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.
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 .
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.
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.
PA - 824（pretomanid）是一种双环硝基咪唑药物，在体外和体内均对结核分枝杆菌（MTB）表现出显著的杀菌活性，但对[未提及的细菌种类]无活性。通过催化生物还原，MTB内的脱氮黄素依赖性硝基还原酶（Ddn）直接将PA - 824转化为强效杀菌产物。本研究旨在鉴定参与PA - 824转化的MTB Ddn关键残基，以开发用于检测分枝杆菌对PA - 824耐药性的体外替代标志物。
我们使用抗菌药物敏感性试验评估了PA - 824对MTB和非结核分枝杆菌菌种的体外活性。Ddn氨基酸序列比对和系统发育分析揭示了假定的关键酶活性位点残基。使用在[未提及的表达系统]中表达的重组克隆Ddn突变蛋白，评估PA - 824转化活性所需的候选MTB Ddn残基的功能丧失情况。
PA - 824对MTB和[未提及的细菌种类]的90%细菌生长最低抑菌浓度（MICs）分别为0.12 mg/L和8 mg/L，但对[未提及的细菌种类] spp.和[未提及的细菌种类]大于32 mg/L。MTB Ddn和[未提及的细菌种类] Ddn同源序列具有最大的相似性（氨基酸同一性为89.3%）。表达Y65L、A76V或Y133F替代（但不是V75L、Q125K或V148I）的Ddn蛋白的[未提及的细菌种类]对PA - 824耐药。
我们的数据表明，PA - 824分别对MTB和[未提及的细菌种类]表现出优异和中等水平的体外活性。Ddn残基Y65、A7
