Critchley Ian A, Green Louis S, Young Casey L, Bullard James M, Evans Ron J, Price Melissa, Jarvis Thale C, Guiles Joseph W, Janjic Nebojsa, Ochsner Urs A
Replidyne, Inc., Louisville, CO 80027, USA.
J Antimicrob Chemother. 2009 May;63(5):954-63. doi: 10.1093/jac/dkp041. Epub 2009 Mar 3.
The aim of this study was to characterize the antimicrobial profile of REP3123, a novel inhibitor of methionyl-tRNA synthetase (MetRS) in development for the treatment of Clostridium difficile infection.
The spectrum of activity of REP3123 was determined by susceptibility testing of C. difficile and non-target organisms. The mode of action was studied by enzyme inhibition assays, macromolecular synthesis assays, target overexpression and selection of spontaneous resistant mutants.
REP3123 was active against a collection of 108 clinical isolates of C. difficile and against epidemic, moxifloxacin-resistant BI/NAP1/027 strains (MIC range=0.5-1 mg/L and MIC(90) = 1 mg/L). The spectrum of activity included clinically important aerobic Gram-positive cocci such as Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Enterococcus faecium (MIC(90)s < 1 mg/L), but REP3123 was not active against most Gram-negative bacteria. REP3123 targeted C. difficile MetRS with a calculated inhibition constant (K(i)) of 0.020 nM, and selectivity was >1000-fold over human mitochondrial and cytoplasmic MetRS. The specific mode of action within bacterial cells was demonstrated by macromolecular synthesis assays that showed inhibition of protein synthesis by REP3123, and by metS overexpression, which resulted in a 16-fold increase in MIC for REP3123. Spontaneous REP3123-resistant mutants of C. difficile (MICs, 4-128 mg/L) arose with frequencies of 10(-8)-10(-9) and harboured distinct point mutations within the metS gene, resulting in 13 different amino acid substitutions. Most of the MetRS substitutions caused reduced catalytic efficiency and a growth fitness burden.
REP3123 demonstrated a favourable microbiological profile and was found to target C. difficile with high specificity and selectivity.
本研究旨在表征REP3123的抗菌谱，REP3123是一种正在开发的用于治疗艰难梭菌感染的新型甲硫氨酰 - tRNA合成酶（MetRS）抑制剂。
通过对艰难梭菌和非靶标生物的药敏试验确定REP3123的活性谱。通过酶抑制试验、大分子合成试验、靶标过表达和自发抗性突变体的筛选研究其作用模式。
REP3123对108株艰难梭菌临床分离株以及流行的、耐莫西沙星的BI/NAP1/027菌株有活性（MIC范围 = 0.5 - 1 mg/L，MIC90 = 1 mg/L）。活性谱包括临床上重要的需氧革兰氏阳性球菌，如金黄色葡萄球菌、化脓性链球菌、粪肠球菌和屎肠球菌（MIC90 < 1 mg/L），但REP3123对大多数革兰氏阴性菌无活性。REP3123靶向艰难梭菌MetRS，计算得出的抑制常数（K(i)）为0.020 nM，对人线粒体和细胞质MetRS的选择性大于1000倍。大分子合成试验表明REP3123在细菌细胞内抑制蛋白质合成，metS过表达导致REP3123的MIC增加16倍，从而证明了其在细菌细胞内的具体作用模式。艰难梭菌的自发REP3123抗性突变体（MICs为4 - 128 mg/L）出现频率为10(-8) - 10(-9)，在metS基因内存在不同的点突变，导致13种不同的氨基酸替代。大多数MetRS替代导致催化效率降低和生长适应性负担。
REP3123表现出良好的微生物学特征，并且发现其对艰难梭菌具有高度特异性和选择性。
