Novel Inhibitors of DNA Ligase LigA by Substrate-Assisted Tethered Inhibition

 描述（由申请人提供）：需要新型治疗剂来治疗由耐药性革兰氏阴性ESKAPE病原体引起的感染。DNA连接酶LigA是一种有效的抗菌靶标，然而，LigA竞争性抑制剂的翻译受到快速出现的耐药突变的阻碍。我们将使用创新的底物辅助栓系抑制策略来开发LigA的有效抑制剂，其与反应途径上的酶中间体形成共价加合物。因此，这些抑制剂将与NAD+无竞争力，从而避免了主要的耐药机制。我们还将寻求识别在酶上具有长停留时间的抑制剂。这将导致在低药物浓度下增加的靶标抑制，从而减少耐药性的出现，同时改善化合物的治疗窗口。在该提案的R21阶段，我们将在我们的初始建模指导下合成氧杂硼杂环戊烯（~100-150）的集中库，并确定这些化合物抑制来自K的LigA的能力。pneumonia，A.鲍曼不动杆菌、铜绿假单胞菌和E.杆菌我们将确定针对每种病原体的野生型和外排泵突变体的MIC（目的1）。我们将使用酶动力学，X射线晶体学和计算方法（目的2）开发LigA抑制的结构-活性关系，以告知其他化合物的合成。该目标的目的是鉴定对外排泵菌株具有IC 50 < 1 µM Kd < 0.1 µM（对于E-AMP结合）和MIC < 8 µg/ml的非/非竞争性抑制剂。在R33阶段，我们将集中精力研究在R21阶段对LigA抑制剂表现出最大敏感性的病原体，并合成对野生型菌株具有改善的抗菌活性的其他化合物。R33阶段的目的是递送具有体内功效（= 1 log CFU减少）和合适性质的化合物，用于进一步优化和临床前开发。活动将包括提高对野生型菌株的微生物活性， 频率以及体外和体内DMPK。

 DESCRIPTION (provided by applicant): Novel therapeutics are needed to treat infections caused by antibiotic-resistant Gram-negative ESKAPE pathogens. DNA Ligase LigA is a validated target for antibacterial discovery, however the translation of LigA competitive inhibitor has been hindered by the rapid emergence of resistance mutations. We will use an innovative substrate-assisted tethered inhibition strategy to develop potent inhibitors of LigA that form a covalent adduct with enzyme-intermediate(s) on the reaction pathway. These inhibitors will thus be uncompetitive with NAD+, circumventing a major mechanism of resistance. We will also seek to identify inhibitors that have long residence times on the enzyme. This will result in increased target suppression at low drug concentration thereby reducing the emergence of resistance while simultaneously improving the therapeutic window of the compounds. In the R21 phase of the proposal, we will synthesize a focused library of oxaboroles (~100-150) guided by our initial modeling and determine the ability of these compounds to inhibit the LigA from K. pneumonia, A. baumannii P. aeruginosa, and E. coli. We will determine MICs against wild-type and efflux-pump mutants of each pathogen (Aim 1). We will develop structure-activity relationships for LigA inhibition using enzyme kinetics, X-ray crystallography and computational approaches (Aim 2) to inform additional compound synthesis. The objective of this aim will be to identify non/uncompetitive inhibitors with IC50 < 1 µM Kd < 0.1 µM (for E-AMP binding) and MIC < 8 µg/ml against efflux-pump strains. In the R33 phase we will focus our efforts on pathogen(s) that demonstrated the greatest sensitivity to the LigA inhibitors in the R21 phase and synthesize additional compounds with improved antibacterial activity against wild-type strain(s). The objective of the R33 phase is to deliver compound(s) with in vivo efficacy (= 1 log CFU decrease) and suitable properties for further optimization and preclinical development. Activities will include improving microbiological activity against wild-type strains, assessment of resistance frequency and in vitro and in vivo DMPK.