Department of Biochemistry and Molecular Medicine, Faculty of Medicine, Université de Montréal, 2900 Boulevard Édouard-Montpetit, Montreal, QC, H3T 1J4, Canada.
Sci Rep. 2017 Nov 2;7(1):14907. doi: 10.1038/s41598-017-14953-1.
The increasing frequency of antimicrobial resistance is a problem of global importance. Novel strategies are urgently needed to understand and inhibit antimicrobial resistance gene transmission that is mechanistically related to bacterial virulence functions. The conjugative transfer of plasmids by type IV secretion systems is a major contributor to antimicrobial resistance gene transfer. Here, we present a structure-based strategy to identify inhibitors of type IV secretion system-mediated bacterial conjugation. Using differential scanning fluorimetry we screened a fragment library and identified molecules that bind the essential TraE protein of the plasmid pKM101 conjugation machinery. Co-crystallization revealed that fragments bind two alternative sites of the protein and one of them is a novel inhibitor binding site. Based on the structural information on fragment binding we designed novel small molecules that have improved binding affinity. These molecules inhibit the dimerization of TraE, bind to both inhibitor binding sites on TraE and inhibit the conjugative transfer of plasmid pKM101. The strategy presented here is generally applicable for the structure-based design of inhibitors of antimicrobial resistance gene transfer and of bacterial virulence.
抗菌药物耐药性日益加剧是一个具有全球重要意义的问题。目前迫切需要新的策略来理解和抑制与细菌毒力功能相关的抗菌药物耐药性基因的传播。通过 IV 型分泌系统的质粒共轭转移是导致抗菌药物耐药性基因转移的主要原因。在这里，我们提出了一种基于结构的策略，用于鉴定抑制 IV 型分泌系统介导的细菌共轭的抑制剂。我们使用差示扫描荧光法筛选了一个片段文库，并鉴定出与质粒 pKM101 共轭机制中必需的 TraE 蛋白结合的分子。共结晶揭示了片段结合两个蛋白质的替代位点，其中一个是新的抑制剂结合位点。基于对片段结合的结构信息，我们设计了具有改善结合亲和力的新型小分子。这些分子抑制 TraE 的二聚化，与 TraE 上的两个抑制剂结合位点结合，并抑制质粒 pKM101 的共轭转移。本文提出的策略通常适用于基于结构的抗菌药物耐药性基因转移和细菌毒力抑制剂的设计。
