Novel inhibitors of bacterial cell envelope biogenesis for prophylaxis against carbapenem-resistant Enterobacteriaceae

摘要 2017年，世界卫生组织将耐碳青霉烯类肠杆菌科(CRE)列为优先事项1“关键超级细菌”。 由于治疗CRE的疗法寥寥无几，目前任何现有的治疗方法都无法治疗“泛耐药”CRE的风险 抗生素也是存在的，而且是全新的具有新的作用机制(MOA)的药物，对 SoC特工正在苦苦挣扎。我们认为，除了治疗Cre感染的新策略外，更大的 还应考虑从一开始就预防CRE感染。我们的建议 目的是开发一种O-抗原(O-a)生物合成抑制剂，以增强血清介导的杀伤(SMK) 在预防感染的Cre啮齿动物模型中是有效的。我们已经证明了O-a的生物合成 WECA基因是一个在标准生长条件下非必需的基因，对黑斑狼疮的生长和发病是必不可少的。 哺乳动物血清的存在。我们的提案概述了一项开发WECA合成抑制剂的计划 我们之前发现并优化了抑制革兰氏阳性WECA直系同源基因Taro的方法。我们的目标是： 目的1.MOA的验证和检测方法的建立。演示Tarocin直接针对WECA以引发SMK 效果。建立靶向参与(TE)和途径参与(PE)筛选试验。构建等基因集 对大肠杆菌(EC)、肺炎克雷伯菌(Kp)、铜绿假单胞菌(Pa)和金黄色葡萄球菌(A. Baumannii(Ab)引导下的Med Chem SAR。里程碑(M)1.确定最多4个不同的化学目标蛋白 通过WECA过表达，SMK中发生4倍EC50漂移的进展为Lead ID的子系列，剂量- O-a的依赖耗竭，以及映射到WECA的原因药物R突变。 目标2.线索识别。通过经验性地分析SMK与 EC/Kp/Pa/Ab通透性和外排缺陷突变体小组，采用新兴的‘Hergenrother’ GN进入的物理化学规律，并探索铁载体结合的用途。M2。最多可识别2个 不同的Tarocin亚系列与EC和KP WT活性的进展有关(人血清(HS)-MIC &lt；16 ug/ml)，暴露于HS-MIC 4小时，EC TE和PE选择性，EC和Kp为&lt；1×10-9在8X HS- 在10×HS-MIC和HS-MIC90&lt；(n=50)条件下，哺乳动物细胞存活率为90%。 目标3.销售线索优化。优化PK、类药物性质和体外活性。M3。确定3个目标 具有EC/KP WT活性(HS-MIC和lt；1微克/毫升)，暴露于HS-MIC 8的类似物，进展到AIM 4 HRS，EC TE和PE选择性，EC/Kp对于GB 1×10-9，在4倍HS-MIC时，&gt；90%HepG2/HEK293细胞在10倍时存活 HS-MIC、CLEAN CYP/离子通道(&gt；10um)、PanLabs IC50&>10um和HS-MIC90 GB 2ug/ml(n=100株)。 目的4.体内药效演示。展示最多三种含铅优化化合物的功效 Cre菌血症和大腿感染的啮齿动物预防模型。M4。将1个Tarocin类似物标识为Pre- 临床预防候选药物，在24小时治疗后显示细菌负荷对数减少 预防大鼠Cre菌血症和大腿感染模型。

Abstract In 2017, the WHO designated carbapenem-resistant Enterobacteriaceae (CRE) a Priority 1 “critical superbug”. As few therapies remain to treat CRE, the risk of “pan-resistant” CRE untreatable by any currently available antibiotic also exists, and entirely new agents with novel mechanisms of action (MOA) not cross-resistant to SOC agents languish. We believe that in addition to new strategies to treat CRE infections, greater consideration should also be given to preventing CRE infections from occurring in the first place. Our proposal aims to develop an O-antigen (O-a) biosynthetic inhibitory agent that potentiates serum-mediated killing (SMK) and is efficacious in prophylactic CRE rodent models of infection. We have shown that the O-a biosynthesis gene wecA, a nonessential gene under standard growth conditions, is essential for growth and pathogenesis in the presence of mammalian serum. Our proposal outlines a plan to develop synthetic inhibitors of WecA that we previously discovered and optimized to inhibit the Gram-positive WecA ortholog, TarO. Our Aims are: Aim 1. MOA validation and assay development. Demonstrate tarocins directly target WecA to elicit SMK effects. Establish target engagement (TE) and pathway engagement (PE) screening assays. Build isogenic set of permeability and efflux deficient mutants in E. coli (Ec), K. pneumoniae (Kp), P. aeruginosa (Pa), and A. baumannii (Ab) to guide med chem SAR. Milestone (M) 1. Identify up to 4 chemically distinct tarocin subseries for progression to Lead ID with > 4-fold EC50 shift in SMK by WecA overexpression, dose- dependent depletion of O-a, and causal drugR mutations mapping to wecA. Aim 2. Lead Identification. Drive SAR efforts to achieve WT activity by empirically assaying for SMK against a panel of Ec/Kp/Pa/Ab permeability and efflux deficient mutants, employing the emerging ‘Hergenrother physicochemical rules of GN entry’, and exploring the utility of siderophore conjugation. M2. Identify up to 2 distinct tarocin subseries for progression to Lead Opt with Ec and Kp WT activity (Human Serum (HS)-MIC < 16 ug/ml), exposure to cover HS-MIC for 4 hrs, Ec TE and PE selectivity, Ec and Kp FOR < 1 x 10-9 at 8X HS- MIC, > 90% mammalian cell viability at 10 X HS-MIC, and HS-MIC90 < 64 ug/ml (n=50 isolates). Aim 3. Lead Optimization. Optimize PK, drug-like properties, and in vitro activity. M3. Identify 3 tarocin analogs for progression to Aim 4 with Ec/Kp WT activity (HS-MIC < 1 ug/ml), exposure to cover HS-MIC 8 hrs, Ec TE and PE selectivity, Ec/Kp FOR £ 1x 10-9 at 4X HS-MIC, >90% HepG2/HEK293 cell viability at 10X HS-MIC, clean CYP/ion channels (>10 uM), PanLabs IC50 > 10 uM, and HS-MIC90 £ 2 ug/ml (n=100 isolates). Aim 4. In vivo efficacy demonstration. Demonstrate efficacy with up to three lead optimized compounds in rodent prophylaxis models of CRE bacteremia and thigh infections. M4. Identify 1 tarocin analog as a pre- clinical prophylaxis candidate that demonstrates > 3 log reduction of bacterial burden after 24h treatment in prophylactic rat CRE bacteremia and thigh infection models.