Development of Cdk5 Inhibitors

描述(由申请人提供)： 这项建议源于根据R21 NS45327资助获得的数据，该资助题为Tau病理学检测中的化合物鉴定。有了这些资金，我们建立了高通量筛选，并发现了抑制CDK5的新化合物。体外筛选检测到在高剂量ATP存在下，通过纯化的CDK5抑制重组全长tau磷酸化的化合物。这一策略的目的是获得不与激酶的ATP口袋结合的“HITS”，因此不同于已知的CDK5抑制剂。从最初筛选的58,000种类药物化合物中，我们发现了三种具有个位数或亚微摩尔ICSO的先导化合物，具有不同的作用机制。一种化合物与ATP竞争，对CDK5ATP结合口袋具有高亲和力。第二种化合物也与三磷酸腺苷竞争，与tau非竞争，在这类抑制剂中独一无二，取代邻近的氨基酸残基，为硝基苯基腾出空间。对于我们的策略来说，最重要的是第三种化合物，它不与ATP竞争，但确实与tau竞争，因此可能被证明比结合保守的ATP口袋的化合物更具特异性。事实上，这种化合物对CDK5的IC50比GSK3P低约3倍，对CDK5的IC50比对CdC2低约6倍。我们建议通过筛选额外的体外IC50&lt；100 nm文库，以及基于细胞和体内的EC50&lt；1 mm和LD50&gt；100 mm文库，将先导化合物的数量增加到20个。为了提高发现不与三磷酸腺苷竞争的化合物的可能性，我们修改了筛选策略，我们的初步数据支持可以检测到此类化合物的想法。根据几个因素的排序，包括化学可操作性、毒理学、药代动力学和一些ADME，将选择五种化合物进行化学修饰。来自五个系列中每个系列的优化化合物将在我们合作小组内开发的神经纤维病理动物模型中进行疗效评估。从这些数据中，我们将选择一种或两种化合物来实现完全的安全药理。到第五年年底，我们将向食品和药物管理局(FDA)申请新药调查(IND)，用于治疗额颞叶痴呆的人类I期临床试验。因为化合物验证需要广泛的专业知识，这不太可能出现在单一的实验室中，我们已经召集了集体拥有完成目标所需专业知识的个人。

DESCRIPTION (provided by applicant): This proposal originates from data obtained under grant R21 NS45327 entitled Compound Identification in Assays for Tau Pathology. With those funds we established a high throughput screen and discovered novel compounds that inhibit Cdk5. The in vitro screen detects compounds that inhibit the phosphorylation of recombinant full-length tau by purified Cdk5 performed in the presence of high amounts of ATP. This strategy was designed to obtain 'hits' that do not bind to the ATP pocket of the kinase, and therefore differ from the known Cdk5 inhibitors. From an initial screen of 58,000 drug-like compounds, we have three lead compounds with single digit or sub-micromolar ICso's and with distinct mechanisms of action. One compound is competitive with ATP and has a high affinity for the Cdk5 ATP-binding pocket. The second compound also competes with ATP, is non-competitive with tau, and uniquely among this class of inhibitors, displaces adjacent amino acid residues to make room for the nitrophenyl group. Most importantly for our strategy was the third compound, which did not compete with ATP, but did compete with tau, and therefore may prove to be more specific than compounds which bind the conserved ATP pocket. Indeed, this compound has an approximately three-fold lower IC50 for Cdk5 than for GSK3P and an approximately six-fold lower IC5o for Cdk5 than for Cdc2. We propose to increase the number of lead compounds to twenty by screening additional libraries for in vitro IC50<100 nM, and with EC50<1 mM and LD50>100 mM in cell based and in vivo assays. To enhance the likelihood of finding compounds that do not compete with ATP we have modified our screening strategy and our preliminary data supports the idea that such compounds can be detected. Based on ranking of several factors including chemical tractability, toxicology, pharmacokinetics, and some ADME, five compounds will be selected for chemical modification. The optimized compounds from each of the five series will'be assessed for efficacy in animal models of neurofibrillary pathology developed within our collaborative group. From these data we will select one or two compounds for complete safety pharmacology. By the end of Year 5 we will apply for an Investigation of New Drug (IND) to the Food and Drug Administration (FDA) for a human phase I clinical trial to treat frontotemporal dementia. Because compound validation requires a broad expertise that is unlikely to be present in a single lab we have assembled individuals who collectively have the expertise to complete the aims.