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

用于多重耐药革兰氏阴性治疗的脂蛋白合成抑制剂

基本信息

批准号：
8905244
负责人：
Tomas Maira-Litran
金额：
$ 28.85万
依托单位：
MICROBIOTIX, INC
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-02-01 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8905244
关键词：
1,2-diacylglycerol Acinetobacter baumannii Address Anabolism Animals Anti-Bacterial Agents Antibiotics Bacteria Bacterial Infections Biochemical Biological Assay Calculi Cells Cellular Assay Chemical Structure Cleaved cell Clinical Development Diglycerides Dimethyl Sulfoxide Dose Drug Kinetics Drug resistance Enzymes Essential Genes Excretory function Genes Goals Gram-Negative Bacteria Gram-Negative Bacterial Infections Growth Homologous Gene Hypersensitivity In Vitro Infection Inhibitory Concentration 50 Isopropyl Thiogalactoside Klebsiella pneumonia bacterium Lead Libraries Lipoproteins Mammals Medical Membrane Membrane Proteins Metabolism Modeling Multi-Drug Resistance New Agents Organism Pathway interactions Peptide Signal Sequences Pharmaceutical Preparations Phase Plasmids Prevalence Process Property Protease Inhibitor Pseudomonas aeruginosa Research Resistance Series Side Specificity Structure Structure-Activity Relationship Tars Temperature Testing Time Toxic effect Transferase absorption acyl group assay development base carbapenem resistance cytotoxicity drug development globomycin high throughput screening improved in vivo inhibitor/antagonist novel novel therapeutics pathogen periplasm pre-clinical preclinical study promoter public health relevance resistance mechanism scaffold screening signal peptidase small molecule therapy resistant

项目摘要

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.

描述（由申请人提供）：耐药细菌感染的日益流行凸显了对不易受现有药物影响的新药物的迫切医疗需求针对革兰氏阴性菌开发的新药物很少，这些细菌很少吸收小分子，并排出大多数到达周质的化合物，其中包括鲍曼不动杆菌在内的多重耐药（MDR）革兰氏阴性菌。、铜绿假单胞菌和肺炎克雷伯菌感染的治疗因获得性和内在的多重耐药性而变得复杂。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治疗耐多药革兰氏阴性菌感染的新治疗剂或辅助药物来满足这一关键的医疗需求。该策略的重点是关注未开发的基本功能——脂蛋白。脂蛋白前体在革兰氏阴性细菌中保守，在哺乳动物中没有同源物。在跨过内膜移位后，脂蛋白前体被脂蛋白二酰基甘油转移酶酰化。 (Lgt)，它们的信号肽被脂蛋白信号肽酶切割掉 (LspA)，并被脂蛋白 N-酰基转移酶 (Lnt) 进一步三酰化。所有三种酶对于革兰氏阴性病原体的生存能力都是必需的，并且它们的活性位于内膜的周质侧，表明抑制剂不需要。穿越内在由于开发这些靶点的高通量生化筛选的挑战以及识别穿透细菌细胞的化合物的需要，在鲍曼不动杆菌中建立了 LspA 和 Lgt 抑制剂的靶向全细胞筛选。当 IPTG 被优化时，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会停止生长并失去活力以及细胞完整性。基于这些菌株对低浓度诱导剂中的 Lgt 和 LspA 抑制剂的超敏性，两种测定均在针对 5,000 种已知生物活性化合物的预筛选中进行了验证，产生的 Z' 因子 >0.7，命中率约为 0.1%。对 Lgt 和 LspA 酶活性进行基于细胞和生化的二次测定，以验证 Phae I 中命中的目标特异性。 Lgt 和 LspA HTS 测定将应用于超过 400,000 种化合物，并且将在二次测定中确认和验证已验证的抑制剂，将根据结构和纯度、剂量依赖性效力、细胞毒性以及由于细胞完整性效应而与现有抗菌药物的协同作用进行优先排序。和细菌谱，包括铜绿假单胞菌、鲍曼不动杆菌和耐碳青霉烯类肺炎克雷伯菌的临床分离株。将根据 ADME 特性、作用机制和 SAR 反应性优先生成先导化合物。在第二阶段，我们将对关键支架进行化学优化，并评估其在动物感染模型中的 PK、毒性和功效，以生成临床前候选药物。