Abstract：
In bacteria, peptidyl-tRNA hydrolase (Pth, E.C. 3.1.1.29) is a ubiquitous and essential enzyme for preventing the accumulation of peptidyl-tRNA and sequestration of tRNA. Pth is an esterase that cleaves the ester bond between peptide and tRNA. Here, we present the crystal structure of Pth from Enterococcus faecium (EfPth) at a resolution of 1.92 Å. The two molecules in the asymmetric unit differ in the orientation of sidechain of N66, a conserved residue of the catalytic site. Enzymatic hydrolysis of substrate α-N-BODIPY-lysyl-tRNALys (BLT) by EfPth was characterized by Michaelis-Menten parameters KM 163.5 nM and Vmax 1.9 nM/s. Compounds having pyrrolinone scaffold were tested for inhibition of Pth and one compound, 1040-C, was found to have IC50 of 180 nM. Antimicrobial activity profiling was done for 1040-C. It exhibited equipotent activity against drug-susceptible and resistant S. aureus (MRSA and VRSA) and Enterococcus (VSE and VRE) with MICs 2-8 μg/mL. 1040-C synergized with gentamicin and the combination was effective against the gentamicin resistant S. aureus strain NRS-119. 1040-C was found to reduce biofilm mass of S. aureus to an extent similar to Vancomycin. In a murine model of infection, 1040-C was able to reduce bacterial load to an extent comparable to Vancomycin.
摘要：
在细菌中，肽基-tRNA水解酶(Pth，E.C.3.1.1.29）是一种普遍存在的必需酶，用于防止肽基-tRNA的积累和tRNA的螯合。Pth是切割肽和tRNA之间的酯键的酯酶。这里,我们显示了屎肠球菌（EfPth）的Pth的晶体结构，分辨率为1.92µ。不对称单元中的两个分子在N66的侧链方向上不同，N66是催化位点的保守残基。通过EFPth对底物α-N-BODIPY-赖氨酰-tRNALys（BLT）的酶水解，其特征在于Michaelis-Menten参数KM163.5nM和Vmax1.9nM/s。测试了具有吡咯烷酮支架的化合物对Pth和一种化合物的抑制作用，1040-C,发现IC50为180nM。对1040-C进行抗菌活性分析。它对药物敏感和耐药的金黄色葡萄球菌（MRSA和VRSA）和肠球菌（VSE和VRE）表现出等效活性，MIC为2-8μg/mL。1040-C与庆大霉素协同作用,并且该组合对庆大霉素抗性金黄色葡萄球菌菌株NRS-119有效。发现1040-C将金黄色葡萄球菌的生物膜质量降低至与万古霉素相似的程度。在鼠感染模型中，1040-C能够将细菌负荷降低至与万古霉素相当的程度。
