CAJ | 학술논문

维吉尼亚霉素乙酰转移酶D(VatD)通过灭活链阳霉素A而在链阳霉素耐药性的产生中起重要作用.本研究采用虚拟筛选技术寻找VatD的抑制剂,此VatD抑制剂可以和链阳霉素联合使用,从而提供新的治疗耐药菌感染的方法.作者首次应用基于结构的虚拟筛选方法(分子对接)从含300 000化合物的商业化数据库中筛选对抗VatD底物结合位点的化合物,从200个评分最高的化合物中选取26个测定对VatD酶活性的抑制作用.将构建的质粒pRSET B/vatD转染宿主细胞E.coli(TrxB)用于过表达,纯化的VatD对维吉尼亚霉素M1表现乙酰转移酶活性.26个化合物中有3个对VatD表现抑制作用,IC50分别为168.6,91.0和55.2 μmol·L-1.其他化合物在反应体系中不溶解和/或对酶活性的抑制作用很小(IC50＞200 μmol·L-1).本文首次设计VatD的小分子化合物抑制剂,发现了3个有活性的化合物,希望其可作为先导化合物进一步发展为新的对抗链阳霉素耐药性的药物.
유길니아매소을선전이매D(VatD)통과멸활련양매소A이재련양매소내약성적산생중기중요작용.본연구채용허의사선기술심조VatD적억제제,차VatD억제제가이화련양매소연합사용,종이제공신적치료내약균감염적방법.작자수차응용기우결구적허의사선방법(분자대접)종함300 000화합물적상업화수거고중사선대항VatD저물결합위점적화합물,종200개평분최고적화합물중선취26개측정대VatD매활성적억제작용.장구건적질립pRSET B/vatD전염숙주세포E.coli(TrxB)용우과표체,순화적VatD대유길니아매소M1표현을선전이매활성.26개화합물중유3개대VatD표현억제작용,IC50분별위168.6,91.0화55.2 μmol·L-1.기타화합물재반응체계중불용해화/혹대매활성적억제작용흔소(IC50＞200 μmol·L-1).본문수차설계VatD적소분자화합물억제제,발현료3개유활성적화합물,희망기가작위선도화합물진일보발전위신적대항련양매소내약성적약물.
Virginiamycin acetyltransferase D (VatD) plays a vital rule in streptogramins resistance by chemically inactivating streptogramin A. Therefore, it is desirable to discover novel small molecular weight inhibitors of VatD via state-of-the-art virtual screening techniques. This "cocktail" strategy by combining VatD inhibitor with streptogramins may provide new therapeutic opportunity for resistant bacteria infections. Structure-based virtual screening method (molecular docking) was applied to rank and score a chemical database containing 300 000 commercially available compounds against the VatD substrate binding site. Twenty six out of the 200 top scored compounds from the docking calculation were selected and submitted to the VatD enzymatic inhibition assay. The plasmid pRSET B/vatD was constructed and transformed into E.coli (trxB) host cells for over-expression, and VatD enzyme was purified and validated by showing acetyltransferase activity to Virginiamycin M1. Three out of these 26 tested compounds showed enzymatic inhibition on VatD with IC50 168.6, 91.0 and 55.2 μmol·L-1, separately. Other compounds could not be dissolved in the system and/or had little effect on the enzyme (IC50>200 μmol·L-1). To our knowledge, it is first time that small molecular weight organic compounds were identified as VatD inhibitors. It is expected that the VatD inhibitors identified at present study could serve as lead compounds for the further development of the novel therapeutic agents to overcome streptogramins resistance.