Lipoprotein synthesis inhibitors for multi-drug resistant Gram-negative therapy

 描述（由申请人提供）：耐药细菌感染的患病率不断增加，突出了对现有药物不敏感的新药物的迫切医疗需求 抵抗机制革兰氏阴性菌很少开发新的药物，它们很少吸收小分子，并将大多数化合物排出到达周质。一个特别有问题的群体是多重耐药（MDR）革兰氏阴性菌，包括鲍氏不动杆菌、铜绿假单胞菌和肺炎克雷伯菌。这些病原体感染的治疗因获得性和内在多药耐药性而变得复杂。本提案的总体目标是通过发现不受现有耐药机制影响的新型抗菌药物，并将其开发为用于治疗MDR革兰氏阴性菌感染的新型治疗或预防药物，来满足这一关键医疗需求。该策略是重点关注一种未开发的基本功能，即脂蛋白生物合成，该功能在革兰氏阴性细菌中是保守的，并且没有同源哺乳动物。在穿过内膜后，脂蛋白前体被脂蛋白二酰基甘油转移酶（Lgt）酰化，其信号肽被脂蛋白信号肽酶裂解 （LspA），并进一步被脂蛋白N-酰基转移酶（Lnt）三酰化。所有这三种酶对于革兰氏阴性病原体中的存活力都是必需的，并且它们的活性定位于内膜的周质侧，这表明抑制剂将不需要穿过内膜。 膜的由于开发针对这些靶标的高通量生物化学筛选的挑战以及鉴定穿透细菌细胞的化合物的需要，在A. LspA和Lgt抑制剂的鲍曼不动杆菌。它们由A.携带Ptac调节的lgt和lspA拷贝代替染色体拷贝的鲍曼不动杆菌菌株。当除去IPTG时，两种菌株停止生长并失去活力以及细胞完整性。基于这些菌株在低浓度诱导剂中对Lgt和LspA抑制剂的超敏性，优化了高通量筛选。两种测定法均在针对5，000种已知的生物活性化合物的中试筛选中一式两份地进行验证，产生Z '因子>0.7和约0.1%的命中率。建立了Lgt和LspA酶活性的中等通量的基于细胞的和生化二级测定以验证命中的靶特异性。在Phae I中，Lgt和LspA HTS测定将应用于> 400，000种化合物，并且将在二次测定中确认和验证命中。将根据结构和纯度、剂量依赖性效价、细胞毒性、由于细胞完整性效应与现有抗菌剂的协同作用以及细菌谱（包括铜绿假单胞菌、A.鲍曼不动杆菌和碳青霉烯类耐药K.肺炎。最有效和选择性的命中将通过ADME性质、作用机制和SAR响应性来优先考虑以产生先导化合物。在第二阶段，我们将对关键支架进行化学优化，并在动物感染模型中评估其PK、毒性和疗效，以产生临床前候选药物。

 DESCRIPTION (provided by applicant): The increasing prevalence of drug-resistant bacterial infections highlights the critical medical need for new agents that are not susceptible to existing resistance mechanisms. Few new agents are in development for Gram-negative bacteria, which take up small molecules sparingly and efflux most compounds that reach the periplasm. A particularly problematic group are the multi-drug-resistant (MDR) Gram-negatives including Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Treatment of infections by these pathogens is complicated by acquired and intrinsic multi-drug resistances. The overall goal of this proposal is to address this critical medical need by discovering novel classes of antibacterials that are not subject to existing resistance mechanisms and developing them into new therapeutic or adjunctive agents for the treatment of MDR Gram-negative infections. The strategy is to focus on an unexploited essential function, lipoprotein biosynthesis which is conserved in Gram-negative bacteria and without homologs mammals. Following translocation across the inner membrane, lipoprotein precursors are acylated by lipoprotein diacylglycerol transferase (Lgt), their signal peptides cleaved off by lipoprotein signal peptidase (LspA), and further triacylated by lipoprotein N-acyl transferase (Lnt). All three enzymes are essential for viability in Gram- negative pathogens and their activity is localized to the periplasmic side of the inner membrane, indicating that inhibitors will not need to cross the inner membrane. Due to the challenges of developing high throughput biochemical screens for these targets and the need for identifying compounds that penetrate bacterial cells, target-biased whole cell screens were built in A. baumannii for both LspA and Lgt inhibitors. These consist of A. baumannii strains carrying Ptac-regulated copies of lgt and lspA in place of the chromosomal copies. Both strains cease growth and lose viability as well as cell integrity when IPTG is removed. High throughput screens were optimized based on the hypersensitivity of these strains to Lgt and LspA inhibitors in low concentrations of inducer. Both assays were validated in pilot screens against 5,000 known bioactive compounds in duplicate, yielding Z'-factors >0.7 and hit rates of ˜0.1%. Moderate throughput cell-based and biochemical secondary assays of the Lgt and LspA enzymatic activities were built to validate the target specificity of hits. In Phae I, the Lgt and LspA HTS assays will be applied to >400,000 compounds, and hits will be confirmed and validated in secondary assays. Validated inhibitors will be prioritized by structure and purity, dose-dependent potency, cytotoxicity, synergy with existing antibacterials due to cell integrity effects, and bacterial spectrum including clinical isolates of P. aeruginosa, A. baumanni and carbapenem-resistant K. pneumoniae. The most potent and selective hits will be prioritized by ADME properties, mechanism of action, and SAR responsiveness to generate lead compounds. In Phase II, we will chemically optimize key scaffolds and evaluate their PK, toxicity, and efficacy in animal infection models to generate preclinical candidates.