Developing High Throughput Assay to Identify Protein Methyltransferase Inhibitors

描述（由申请人提供）：表观遗传调控参与许多生物过程，其错误与包括癌症在内的许多疾病有关。在表观遗传的关键调节因子是蛋白甲基转移酶（pmt）。强有力的证据表明pmt通过甲基化组蛋白和非组蛋白靶点起作用。然而，由于无法描述PMT靶点和描述其下游功能，对PMT生物学作用的充分理解受到了限制。PMT特异性抑制剂可以很容易地劫持PMT相关的表观遗传学，因此作为有价值的化学遗传工具来研究这些过程。然而，缺乏高通量筛选（HTS）方法是鉴定此类小分子实体的一个重大障碍。这种情况如果不加以解决，将极大地限制化学遗传工具在定义、干扰和操纵表观遗传功能方面的应用。本提案的目的是开发可推广的PMT的HTS测定方法，以确定靶向特异性PMT抑制剂。对pmt结构的批判性分析形成了一个中心假设，即pmt独特的底物/辅因子结合口袋可以用于开发靶向抑制剂。这项建议的目标将通过追求两个具体目标来实现。384孔混合测量闪烁接近分析（SPA）已经开发出来，预计将转化为1536孔alphasgreen格式（Aim 1）。这些高温热液相色谱分析将用一个先导化合物文库进行验证。借助反筛选和二级分析（目标1），目标特异性PMT抑制剂有望在体外和体内得到明确的识别和表征（目标2）。这些抑制剂可用于产生pmt特异性低甲基化蛋白质组，用于目标分析，并破坏特异性甲基化以进行功能表征。结合NIH表观基因组学和分子文库与成像的两个路线图任务，开发HTS检测和鉴定PMT抑制剂将垂直推进表观遗传学研究，并促进PMT抑制剂在药理干预中的使用。

DESCRIPTION (provided by applicant): Epigenetic regulations participate in numerous biological processes and their errors have been implicated in many diseases including cancer. Among the key modulators in epigenetic are protein methyltransferases (PMTs). Strong evidence showed that PMTs function through methylating histone and nonhistone targets. Nonetheless, fully understanding the biological roles of PMTs is restricted by the inability to profile the PMT targets and to characterize the downstream functions. PMT-specific inhibitors can readily hijack PMT- involved epigenetics and thus serve as valuable chemical genetic tools to investigate these processes. However, the lack of high throughput screening (HTS) methods represents a significant barrier in identifying such small-molecule entities. The situation, if not addressed, greatly limits the application of chemical genetic tools to define, perturb and manipulate the epigenetic functions. The objective of this proposal is to develop generalizable HTS assays of PMTs for identifying target-specific PMT inhibitors. The critical analysis of PMTs structures formulated the central hypothesis that the distinct substrate/cofactor-binding pockets of PMTs can be exploited for developing target-specific inhibitors. The goal of this proposal will be achieved by pursing two specific aims. A 384-well mix-and-measure scintillation proximity assay (SPA) has been developed and is expected to be transformed into a 1536-well AlphaScreen format (Aim 1). These HTS assays will be then validated with a pilot compound library. With the aid of counter-screening and secondary assays (Aim 1), target-specific PMT inhibitors are expected to be unambiguously identified and characterized in vitro and in vivo (Aim 2). These inhibitors can be used to generate PMT-specific hypomethylation proteome for target profiling and to disrupt specific methylations for function characterization. In conjunction with two NIH Roadmap missions of epigenomics and molecular libraries & imaging, developing the HTS assay and identifying PMT inhibitors will vertically advance epigenetic research and facilitate the use of PMT inhibitors for pharmacological intervention. PUBLIC HEALTH RELEVANCE: Deregulated protein methyltransferases (PMTs) have been implicated in many diseases including cancer. The impact of this proposed research is to develop an efficient approach for identifying PMT inhibitors. These molecules can be used as pharmacological probes to investigate the mechanism of PMT-involved diseases. Additionally, novel therapeutic reagents can be developed from PMT inhibitors.