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活性位点定向的DMA聚合酶HIE抑制剂，这是革兰氏+细菌中的一种新的复制酶。这些化合物，7-取代-N-(3，4-二氯苯基)鸟嘌呤(DCBGs)，是有效的酶抑制剂，所选的衍生物对临床相关的革兰氏阳性菌具有强大而广泛的活性。我们已经从革兰氏杆菌，即大肠杆菌中鉴定出了第一个活性部位定向的相关DNA聚合酶HIE的抑制物。此外，我们还建立了合作伙伴关系，以结晶和解决粪便埃希氏菌、DNA和我们的一种抑制剂之间的复合体的结构。 基于第一阶段的结果，我们将通过全身和全身细菌感染模型的体内测试来设计一种先导抗生素化合物。我们将同时使用QSAR分析和基于结构的药物设计来发现PolHIE靶标的新平台抑制剂。将致力于实现以下具体目标： 1.用于评价细菌感染动物模型的候选先导化合物和制剂的放大合成；具有增强的PolHIE抑制和体外抗菌活性的7-取代类似物的合成。 2.解决粪肠球菌PolIe：DNA：抑制剂复合体的结构，并利用坐标了解酶的抑制基础，并进一步进行合理的、基于计算机的药物设计(与奥克兰研究所儿童医院Mark Jedrzejas博士合作)。 3.继续检测化合物的酶抑制作用(PolIIIC、PolHIE)、抗菌活性、选择性、细胞毒性；耐药发生率和耐药机制(S)，通过目标克隆和测序(转包给MicroBiotix Inc.)；测试候选药物对革兰氏+和革兰-菌临床分离株的作用；杀菌试验；与市售抗生素的联合研究(转包给美国麻省理工学院)。 4.建立分析方法，用于动物血浆中候选药物的分析、药物在小鼠体内的不同途径的吸收和分布、急性毒性、体外代谢和稳定性研究。 5.评估在革兰氏阳性和革兰氏阳性感染动物模型中的活性候选药物(如果有必要)，重点是小鼠的全身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。 对革兰氏+polHIE和polIIIC的有效抑制可能导致抗生素耐药发生率的降低。

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.