Development of Cdk5 Inhibitors

描述（由申请人提供）： 该提议源自在授权R21 NS 45327下获得的数据，该授权R21 NS 45327题为在Tau病理学测定中的化合物鉴定。利用这些资金，我们建立了一个高通量筛选，并发现了抑制Cdk 5的新化合物。体外筛选检测在高量ATP存在下通过纯化的Cdk5抑制重组全长tau磷酸化的化合物。该策略旨在获得不与激酶的ATP口袋结合的“命中”，因此与已知的Cdk5抑制剂不同。从58，000种药物样化合物的初步筛选中，我们有三种具有个位数或亚微摩尔IC50的先导化合物，并且具有不同的作用机制。一种化合物与ATP竞争，并对Cdk5 ATP结合口袋具有高亲和力。第二种化合物也与ATP竞争，与tau是非竞争性的，并且在这类抑制剂中是独特的，取代相邻的氨基酸残基以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，但与tau竞争，因此可能证明比结合保守ATP口袋的化合物更具特异性。实际上，该化合物对Cdk 5的IC 50比对GSK3 β的IC 50低约三倍，对Cdk 5的IC 50比对Cdc 2的IC 50低约六倍。我们建议通过筛选体外IC50&lt;100 nM的额外文库，并在基于细胞的测定和体内测定中EC50 100 mM，将先导化合物的数量增加到20<1 mM and LD50>种。为了提高发现不与ATP竞争的化合物的可能性，我们修改了我们的筛选策略，我们的初步数据支持这样的化合物可以被检测到的想法。基于包括化学易处理性、毒理学、药代动力学和一些ADME在内的几个因素的排序，将选择五种化合物进行化学修饰。来自五个系列中的每一个的优化化合物将在我们的合作小组内开发的神经病理学动物模型中评估疗效。从这些数据中，我们将选择一种或两种化合物进行完整的安全药理学研究。到今年5月底，我们将申请新药调查（IND）的食品和药物管理局（FDA）的人类I期临床试验，以治疗额颞叶痴呆。由于化合物验证需要广泛的专业知识，而这不太可能出现在一个实验室中，因此我们召集了具有共同专业知识的个人来完成目标。

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.