DNA Polymerase IIIe, A New Antibiotic Target

DNA 聚合酶 IIIe，新的抗生素靶点

• 批准号: 7038266
• 负责人: George E Wright
• 金额: $ 90.71万
• 依托单位: GLSYNTHESIS, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-11-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7038266
• 关键词: DNA directed DNA polymerase; Enterococcus; Staphylococcus aureus; Streptococcus pneumoniae; Streptococcus pyogenes; X ray crystallography; active sites; antibacterial agents; bacterial proteins; biotechnology; biotherapeutic agent; chemical synthesis; cytotoxicity; drug design /synthesis /production; drug discovery /isolation; drug interactions; drug resistance; drug screening /evaluation; enzyme inhibitors; guanine analog; guinea pigs; high performance liquid chromatography; laboratory mouse; nuclear magnetic resonance spectroscopy; pharmacokinetics

DESCRIPTION (provided by applicant): In phase I we have identified a new class of active-site directed inhibitors of DMA polymerase HIE, a novel replicative enzyme in Gram+ bacteria. The compounds, 7-substituted-N2-(3,4-dichlorobenzyl)guanines "DCBGs", are potent enzyme inhibitors, and selected derivatives have potent and broad activity against clinically relevant Gram+ bacteria. We have identified the first active site directed inhibitors of the related DNA polymerase HIE from Gram- bacteria, i.e. E. coli. In addition, we have established a collaboration to crystallize and solve the structure of a complex between E. fecalis pol IIIE, DNA and one of our inhibitors. Based on the results of phase I, we will pursue designation of a lead antibiotic compound by in vivo testing in systemic and toplical bacterial infection models. We will use both QSAR analysis and structure-based drug design to discover new platform inhibitors of the pol HIE target. The following specific aims will be pursued: 1. scale-up synthesis of lead compound candidates and formulations for evaluation in animal models of bacterial infection; synthesis of 7-substituted analogs with enhanced pol HIE inhibitory and antibacterial activity in vitro. 2. solve the structure of E. fecalis pol IIIE:DNA:inhibitor complexes, and use the coordinates for understanding the basis of inhibition of the enzyme and for further rational, computer-based drug design (collaboration with Dr. Mark Jedrzejas, Children's Hospital of Oakland Research Institute). 3. continue assays of compounds for enzyme inhibition (pol IIIC, pol HIE), antibacterial activity, selectivity, cytotoxicity; incidence of resistance and mechanism(s) of resistance, by cloning and sequencing of targets (by subcontract to Microbiotix Inc.); test candidate drugs against clinical isolates of Gram+ and Gram- bacteria, bactericidal assays, combination studies with marketed antibiotics (by subcontract to UMass Medical School). 4. develop analytical methods for analysis of candidates in animal plasma, drug uptake and distribution by various routes in mice, acute toxicity, in vitro metabolism and stability studies. 5. evaluate candidate drugs for activity in animal models of Gram+ and Gram- infections (if warranted), emphasizing systemic antibiotic-resistant S. aureus, E. fecalis and S. pneumoniae in mice and topical S. aureus infections in guinea pigs.. Potent inhibition of Gram+ pol HIE and pol IIIC may lead to antibiotics with reduced incidence of resistance.

描述（由申请人提供）：在第一阶段，我们已经鉴定出一类新的 DMA 聚合酶 HIE 活性位点定向抑制剂，HIE 是革兰氏+细菌中的一种新型复制酶。这些化合物，7-取代-N2-(3,4-二氯苄基)鸟嘌呤“DCBG”，是有效的酶抑制剂，并且选定的衍生物对临床相关的革兰氏+细菌具有有效和广泛的活性。我们已经从革兰氏细菌（即大肠杆菌）中鉴定出相关 DNA 聚合酶 HIE 的第一个活性位点定向抑制剂。此外，我们还建立了合作关系，以结晶并解析粪肠球菌 pol IIIE、DNA 和我们的一种抑制剂之间的复合物的结构。 根据第一阶段的结果，我们将通过全身和局部细菌感染模型的体内测试来寻找先导抗生素化合物。我们将使用 QSAR 分析和基于结构的药物设计来发现 pol HIE 靶点的新平台抑制剂。将追求以下具体目标： 1. 扩大合成先导化合物候选物和制剂，用于在细菌感染动物模型中进行评估；体外合成具有增强 pol HIE 抑制和抗菌活性的 7-取代类似物。 2. 解析粪肠球菌 pol IIIE:DNA:抑制剂复合物的结构，并使用坐标来了解酶抑制的基础以及进一步合理的、基于计算机的药物设计（与儿童医院的 Mark Jedrzejas 博士合作）奥克兰研究所）。 3. 继续测定化合物的酶抑制作用（pol IIIC、pol HIE）、抗菌活性、选择性、细胞毒性；通过靶标的克隆和测序（通过分包给 Microbiotix Inc.）确定耐药性的发生率和耐药机制；针对革兰氏+和革兰氏-细菌的临床分离株测试候选药物、杀菌测定、与市售抗生素的联合研究（通过分包给麻省大学医学院）。 4. 开发分析方法，用于分析动物血浆中候选药物、小鼠体内各种途径的药物摄取和分布、急性毒性、体外代谢和稳定性研究。 5. 评估候选药物在革兰氏阳性和革兰氏阴性感染动物模型中的活性（如果有必要），重点关注小鼠体内的全身抗生素耐药金黄色葡萄球菌、粪肠球菌和肺炎链球菌以及豚鼠的局部金黄色葡萄球菌感染。 有效抑制 Gram+ pol HIE 和 pol IIIC 可能会导致抗生素耐药性发生率降低。