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

• 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
• 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

 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（应用程序提供）：耐药细菌感染的患病率的增加突出了对新药物不容易受到现有的新药物的关键医疗需求 电阻机制。革兰氏阴性细菌的开发很少，它们很少会吸收小分子，而大多数到达周期性的化合物。一个特别有问题的群体是抗多药（MDR）革兰氏阴性菌，包括鲍曼尼杆菌，铜绿假单胞菌和克雷伯氏菌肺炎。这些病原体对感染的治疗变得复杂，并具有内在的多药耐药性。该提案的总体目标是通过发现不受现有抗药性机制的新型抗菌物质来解决这种关键的医疗需求，并将其开发为新的治疗或辅助药物来治疗MDR革兰氏阴性感染。该策略是专注于意外的基本功能，脂蛋白生物合成，该功能在革兰氏阴性细菌中保守，没有同源性哺乳动物。在跨膜易位后，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切开 （LSPA），并通过脂蛋白N-酰基转移酶（LNT）进一步进行三酰基。这三种酶对于革兰氏阴性病原体的生存力都是必不可少的，它们的活性位于内膜的外围侧，表明抑制剂无需跨越内部膜 膜。由于为这些靶标开发高通量生化筛查的挑战，并且需要鉴定渗透细菌细胞的化合物，因此在鲍曼尼（A. baumannii）中为LSPA和LGT抑制剂建立了目标偏置的全细胞筛选。这些由带有PTAC调节的LGT和LSPA副本代替染色体副本的鲍曼尼菌菌株组成。两种菌株都停止了生存力和失去生存力，以及消除IPTG时的细胞完整性。根据这些菌株对LGT和LSPA抑制剂的高敏性，以低浓度的诱导剂对LGT和LSPA抑制剂进行了优化。这两种测定法在驾驶筛查中均以5,000种已知的生物活性化合物的重复验证，产生Z'Factors> 0.7，命中率为约0.1％。建立了LGT和LSPA酶促活性的中等吞吐量细胞和生化次级测定，以验证命中的目标特异性。在PHAE I中，LGT和LSPA HTS测定法将用于> 400,000种化合物，并将在辅助测定中确认和验证。经过验证的抑制剂将通过结构和纯度，剂量依赖性效力，细胞毒性，与现有抗菌作用的协同作用以及因细胞完整性效应引起的现有抗菌素的协同作用，以及包括铜绿假单胞菌，鲍曼A.a。baumanni和carbapenem-carbapenem-抗耐药的K. pneumoniae的临床分离株的细菌光谱。 ADME性质，作用机理以及生成铅化合物的SAR反应能力将优先考虑最潜在和选择性的热门。在第二阶段，我们将化学优化关键支架，并评估其PK，毒性和动物感染模型中的效率，以产生临床前候选。