Broad spectrum antibacterials selectively targeting an un-drugged site on the ribosome

项目摘要 该项目的最终目标是开发一类新的广谱抗生素，重点是革兰氏。 阴性菌。受天然产品的启发，这门课程针对的是细菌的一个未开发的结合部位。 选择性抑制细菌蛋白质合成的核糖体。随着抗药性的报道不断增加 解决革兰氏阴性菌的一线治疗方法，迫切需要新的治疗方法，但几乎不可能 在开发管道中发现的。因此，任何针对革兰氏阴性细菌的新疗法都将 解决未得到满足的医疗需求。 与嗜热梭菌核糖体结合的天然产物和C-ūRZA类似物的晶体结构 用于指导药物化学的努力。这些晶体结构表明一个高度保守的区域 肽基转移酶中心(Ptc)尚未被任何现有的抗生素利用，实际上是一种可行的 针对经过严格审查的目标--核糖体--的新治疗方法。绑定到PTC的此站点似乎 传达细菌的选择性。以这些络合物为指导的分子模拟初步优化 产生了新的线索，将抗菌活性的范围从单纯的结核分枝杆菌扩大到包括 对大肠杆菌、肺炎克雷伯菌和金黄色葡萄球菌(包括每种细菌的抗药性菌株)都有很强的活性。 这些类似物在NM浓度范围内抑制蛋白质合成，表现出&gt；400的选择性指数 抑制细菌与真核生物的蛋白质合成，对哺乳动物细胞的毒性非常有限。 这个为期五年的R01项目最终将开发出一种新的有效的、广谱的抗菌药物候选药物， 重点关注准备用于IND准备的革兰氏阴性病原体。该抗菌铅系列的研制 将通过以下目标来实现。目标1将优化销售线索系列，以将活动扩展到其他 革兰氏阴性杆菌，尤其是铜绿假单胞菌和鲍曼不动杆菌。目标2将定义行动机制， 用抑菌活性评价类似物的生化/微生物学活性和体外ADME-TOX 和真核蛋白质合成(包括线粒体)，一组代表革兰氏阴性和 革兰氏阳性菌(包括抗药性)。初步的ADME-Tox将在体外进行评估，使用 Vero细胞预测细胞毒性、微粒体稳定性、对HERG和细胞色素P450的抑制、Ames试验 遗传毒性和44个主要分子靶点的体外受体结合分析。AIM 3将使用体内PK 在小鼠大腿感染模型中，通过确定MTD和体内疗效来指导优化 选择一种单一的先导化合物进行推进。目标4将通过确定以下内容来扩大微生物图谱 MIC90，对其他抗生素的耐药性和潜在的交叉耐药性的特征。AIM 5将在 对肺炎感染模型的活体疗效及PK/PD研究。AIM 6将解决CMC问题并制定初步的 用于AIM 7的GLP毒理学研究的cGMP批次。

Project Summary The ultimate goal of this project is developing a new class of broad spectrum antibiotics, focused on Gram- negative bacteria. Inspired by a natural product, this class targets an unexploited binding site of the bacterial ribosome that selectively inhibits bacterial protein synthesis. With ever increasing reports of resistance to frontline therapies addressing Gram-negatives, there is a critical need for new therapies, yet very little can be found in the development pipeline. As a result, any new therapeutic that targets Gram-negative bacteria will address an unmet medical need. Crystal structures of the natural product and Cūrza analogs bound to the T. thermophilus ribosome have been used to guide medicinal chemistry efforts. These crystal structures revealed that a highly conserved region of the peptidyl transferase center (PTC) that has yet to be exploited by any existing antibiotics is in fact a viable new therapeutic approach against a well-vetted target, the ribosome. Binding to this site of the PTC appears to convey bacterial selectivity. Molecular modeling using these complexes as a guide for preliminary optimization has produced new leads that have expanded the antibacterial spectrum of activity from solely Mtb to include potent activity against E. coli, K. pneumoniae and S. aureus (including drug-resistant strains for each of these). These analogs inhibit protein synthesis in the nM concentration range, exhibit selectivity indexes of >400 for inhibition of bacterial versus eukaryotic protein synthesis and have very limited toxicity to mammalian cells. This five-year R01 project will ultimately develop a new potent, broad spectrum antibacterial drug candidate, focusing on Gram-negative pathogens ready for IND preparation. Development of this antibacterial lead series will be accomplished by the following aims. Aim 1 will optimize the lead series to expand activity to additional Gram-negative pathogens, notably P. aeruginosa and A. baumannii. Aim 2 will define mechanism of action, biochemical/microbiological activity and in vitro ADME-Tox of analogs by evaluating activity against bacterial and eukaryotic protein synthesis (including mitochondrial), a panel of bacteria representing Gram-negative and Gram-positive organisms (including antibiotic-resistant). Preliminary ADME-Tox will be evaluated in vitro using Vero cells to predict cytotoxicity, microsomal stability, inhibition of hERG and cytochrome P450s, Ames test for genotoxicity and in an in vitro receptor binding assay of 44 primary molecular targets. Aim 3 will use in vivo PK to guide optimization followed by determining the MTD and in vivo efficacy in a mouse thigh infection model to select a single lead compound for advancement. Aim 4 will expand the microbiological profile by determining MIC90s, characterization of resistance and potential cross-resistance to other antibiotics. Aim 5 will expand in vivo efficacy and PK/PD to pneumonia infection models. Aim 6 will address CMC issues and produce an initial cGMP lot for use in GLP toxicology studies in Aim 7.