DNA Polymerase IIIe, A New Antibiotic Target

描述（由申请人提供）：在第一阶段，我们已经确定了一类新的DMA聚合酶HIE（革兰氏阳性菌中的一种新型复制酶）的活性位点定向抑制剂。所述化合物，7-取代的-N2-（3，4-二氯苄基）鸟嘌呤“DCBG”，是有效的酶抑制剂，并且所选择的衍生物对临床相关的革兰氏阳性细菌具有有效和广泛的活性。我们首次从革兰氏细菌中鉴定出了相关DNA聚合酶HIE的活性位点定向抑制剂，即E.杆菌此外，我们已经建立了一个合作，以结晶和解决之间的E。粪便聚合物，DNA和我们的抑制剂。 基于第一阶段的结果，我们将通过在全身和局部细菌感染模型中的体内测试来寻求指定一种先导抗生素化合物。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。将努力实现以下具体目标： 1.用于在细菌感染的动物模型中评估的先导化合物候选物和制剂的按比例放大合成;具有增强的pol HIE抑制和体外抗菌活性的7-取代的类似物的合成。 2.解决了E. fecalis pol IIIE：DNA：抑制剂复合物，并使用坐标来理解酶抑制的基础，并进一步合理地进行基于计算机的药物设计（与奥克兰研究所儿童医院的Mark Jedrzejas博士合作）。 3.继续测定化合物的酶抑制（pol IIIC、pol HIE）、抗菌活性、选择性、细胞毒性;通过靶点克隆和测序（由Microbiotix Inc.提供）测定耐药性发生率和耐药性机制;针对革兰氏阳性和革兰氏阴性细菌的临床分离株测试候选药物，杀菌试验，与市售抗生素的联合研究（由马萨诸塞大学医学院负责）。 4.开发分析方法，用于分析动物血浆中的候选物、小鼠中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.评价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（如有必要），强调系统性耐药链球菌。aureus、E. fecalis和S. pneumoniae的小鼠和局部S.豚鼠的金黄色葡萄球菌感染。 对革兰氏+ pol HIE和pol IIIC的有效抑制可能导致抗生素耐药性发生率降低。

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.