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

 描述(申请人提供)：需要新的治疗方法来治疗由耐药的革兰氏阴性ESKAPE病原体引起的感染。DNA Ligase LIGA是发现抗菌药物的有效靶点，然而LIGA竞争性抑制物的翻译受到快速出现的耐药突变的阻碍。我们将使用一种创新的底物辅助拴系抑制策略来开发有效的LIGA抑制剂，这种抑制剂在反应途径上与酶中间体(S)形成共价加合物。因此，这些抑制剂将与NAD+失去竞争力，从而绕过了一个主要的耐药机制。我们还将寻求识别在酶上有较长停留时间的抑制剂。这将导致在低药物浓度下增加靶向抑制，从而减少耐药的出现，同时改善化合物的治疗窗口。在提案的R21阶段，我们将在初始建模的指导下合成一个重点关注的恶环化合物文库(~100-150)，并确定这些化合物对肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌和大肠杆菌LIGA的抑制能力。我们将确定对每个病原体的野生型和外排泵突变体的最低抑菌浓度(目标1)。我们将使用酶动力学、X射线结晶学和计算方法(目标2)来建立LIGA抑制的结构-活性关系，以了解更多的化合物合成。这一目标的目的将是确定IC50；1微米Kd；0.1微米(用于E-AMP结合)和MIC；8微克/毫升的非竞争性/非竞争性抑制剂对外排泵菌株的抑制作用。在R33阶段，我们将重点研究对R21阶段LIGA抑制剂最敏感的病原菌(S)，并合成更多对野生型菌株(S)具有更高抗菌活性的化合物。R33阶段的目标是提供具有体内疗效(=1logCFU减少)和适合进一步优化和临床前开发的性质的化合物(S)。活动将包括提高微生物对野生型菌株的活性，评估耐药性 频率和体内外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.