Developing High Throughput Assay to Identify Protein Methyltransferase Inhibitors

描述(由申请人提供)：表观遗传调控参与了许多生物学过程，其错误与包括癌症在内的许多疾病有关。表观遗传学中的关键调控因子是蛋白质甲基转移酶(PMT)。强有力的证据表明，PMTs通过甲基化组蛋白和非组蛋白靶标发挥作用。然而，由于不能描述PMT靶标和表征下游功能，充分理解PMT的生物学作用受到限制。PMT特异性抑制剂可以很容易地劫持PMT相关的表观遗传学，从而成为研究这些过程的有价值的化学遗传学工具。然而，缺乏高通量筛选(HTS)方法是识别此类小分子实体的一个重要障碍。这种情况如果得不到解决，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。这项建议的目的是开发可推广的PMT的HTS检测方法，以识别靶向特异性PMT抑制剂。对PMTs结构的关键分析形成了一个中心假设，即PMTs的不同底物/辅因子结合口袋可以被用来开发靶向特异性抑制剂。这项提议的目标将通过追求两个具体目标来实现。已经开发了384孔混合测量闪烁邻近分析(SPA)，预计将转换为1536孔AlphaScreen格式(目标1)。然后，这些HTS分析将通过一个中试化合物文库进行验证。借助于反筛选和二次检测(目标1)，靶向特异性PMT抑制剂有望在体外和体内被明确地识别和表征(目标2)。这些抑制剂可用于生成PMT特异的低甲基化蛋白质组，用于靶向分析，并用于干扰特定的甲基化以进行功能表征。结合美国国立卫生研究院表观基因组学和分子库与成像的两个路线图任务，开发HTS分析和识别PMT抑制剂将垂直推进表观遗传学研究，并促进将PMT抑制剂用于药理学干预。 公共卫生相关性：非调节蛋白甲基转移酶(PMTs)与包括癌症在内的许多疾病有关。这项拟议的研究的影响是开发一种有效的方法来识别PMT抑制剂。这些分子可作为药理探针研究PMT相关疾病的发病机制。此外，从PMT抑制剂可以开发出新的治疗试剂。

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.