Photoaffinity labeling probes for development of novel isoform selective HDAC inh

描述（由申请人提供）：该提案旨在通过使用小分子光亲和探针绘制不同HDAC亚型中HDAC配体的结合“指纹”，为设计更具选择性和更有活性的组蛋白脱乙酰酶（HDAC）抑制剂开辟新的方向。由于许多癌症与异常转录活性相关，并且HDAC可以影响转录因子和基因调控，因此这些酶已被鉴定为癌症治疗的有吸引力的靶标。事实上，HDAC的化学抑制剂已显示出抑制肿瘤细胞生长并诱导分化和细胞死亡。至少一种非选择性HDAC抑制剂被FDA批准用于癌症，并且几种非选择性HDAC抑制剂正在临床试验中。多项研究表明，对一种或有限数量的特定HDAC同种型具有选择性的治疗剂可以改善这些化合物的总体功效并降低毒性。虽然一些化合物已经显示出相当有限程度的异构体选择性，但识别选择性抑制剂的问题远未解决。最近在阐明HDAC 7、8和同源HDAC蛋白的晶体结构方面的成功为基于结构的药物设计开辟了可能性。尽管取得了进展，但基于配体和基于结构的方法在设计同种型选择性HDAC抑制剂的适用性方面都受到限制，因为只有很少的同种型选择性抑制剂可用作基于配体的药物设计的起点，或者因为高分辨率三维结构仅可用于11种I类和II类HDAC同种型中的2种。也很少了解HDAC配体的那些结合在HDAC蛋白表面上的部分的结合模式如何影响HDAC抑制剂的活性。显然，需要对这一问题采取新的办法。我们假设，这将是可能的映射的结合模式，在不同的HDAC亚型的配体的小分子光亲和探针（PAP），并使用由此产生的知识来设计亚型选择性HDAC抑制剂作为潜在的治疗癌症。我们在这项建议中的具体目标如下：目标1：设计并合成至少三个系列的光亲和探针（PAP），其由（1）HDAC配体，（2）用于直接参与配体结合的蛋白质残基的光亲和标记（PAL）的芳族叠氮基，和（3）用于连接荧光或生物素标签的脂肪族叠氮化物（成像标签连接基团- imTAG）。目标二：在光亲和标记实验中用可用的重组纯化的I类和II类HDAC评价含有PAL或PAL+imTAG或两种基团的配体，并使用质谱蛋白质组学方法确定蛋白质上的修饰位点。将蛋白质组学结果与建模研究相匹配，并在稍后进行改进，以更好地再现实验结果。目标3：评估最有效的光亲和探针在活Hela细胞中的细胞毒性、选择性和结合“指纹”。目标4。将目标1-3的发现纳入基于结构的药物设计中，并进行多轮CADD、药物化学和生物学测试，以提高HDAC配体的活性和选择性。 公共卫生相关性：该提案旨在通过使用小分子光亲和探针绘制不同HDAC亚型中HDAC配体的结合“指纹”来设计更具选择性和更有活性的组蛋白脱乙酰酶（HDAC）抑制剂。该提案的最终目标是设计适合作为用于癌症和其他HDAC相关疾病和病症的基于HDAC的治疗剂的HDAC抑制剂。

DESCRIPTION (provided by applicant): This proposal is intended to charter new directions for design of more selective and more active histone deacetylase (HDAC) inhibitors by mapping the binding "fingerprints" of the HDAC ligands in the different HDAC isoforms using small molecule photoaffinity probes. Since many cancers are associated with aberrant transcriptional activity, and the HDACs can affect transcription factors and gene regulation, these enzymes have been identified as attractive targets for cancer therapy. Indeed, chemical inhibitors of HDACs have been shown to inhibit tumor cell growth and induce differentiation and cell death. At least one non-selective HDAC inhibitor was approved by FDA for cancer and several non-selective HDAC inhibitors are in clinical trials. Multiple studies have shown that therapeutics selective for one or a limited number of specific HDAC isoforms may improve overall efficacy and lower toxicity of these compounds. While some rather limited degree of isoform selectivity has been shown by a few compounds, the problem of identifying selective inhibitors is far from solved. Recent success in the elucidation of the crystal structure of HDAC7, 8 and homologous HDAC proteins has opened a possibility for structure-based drug design. Despite the progress, both ligand-based and structure-based approaches are limited in their applicability to design of isoform selective HDAC inhibitors either because only few isoform selective inhibitors are available as a starting point for ligand based drug design or because high resolution three-dimensional structures are available only for 2 out of 11 class I and II HDAC isoforms. It is also poorly understood how the binding modes of those portions of the HDAC ligands that bind on the surface of the HDAC protein affect the activity of HDAC inhibitors. Clearly, a new approach to this problem is needed. We hypothesize that it will be possible to map the binding modes available to the ligands in different HDAC isoforms by small molecule photoaffinity probes (PAP) and use the resulting knowledge to design isoform selective HDAC inhibitors as potential therapeutics for cancer. Our specific aims in this proposal are as follow: Aim 1: Design and synthesize at least three series of photoaffinity probes (PAPs) consisting of (1) an HDAC ligand, (2) an aromatic azide group for photoaffinity labeling (PAL) of the protein residues directly involved in the binding of the ligand, and (3) an aliphatic azide (a imaging tag attachment group - imTAG) for attaching a fluorescent or a biotin tag. Aim 2: Evaluate the ligands containing either PAL or PAL+imTAG or both groups in photoaffinity labeling experiments with available recombinant purified class I and class II HDACs and determine the modification sites on the proteins using mass-spectrometry proteomics methods. Match the proteomics results with the modeling studies and refine later to better reproduce the experimental findings. Aim 3. Evaluate the cytotoxicity, selectivity, and binding "fingerprints" of the most potent photoaffinity probes in live Hela cells. Aim 4. Incorporate the findings of Aims 1-3 in the structure-based drug design and perform several rounds of CADD, medicinal chemistry, and biological tests to improve the activity and selectivity of the HDAC ligands. PUBLIC HEALTH RELEVANCE: This proposal is intended to charter new directions for design of more selective and more active histone deacetylase (HDAC) inhibitors by mapping the binding "fingerprints" of the HDAC ligands in the different HDAC isoforms using small molecule photoaffinity probes. The ultimate goal of this proposal is to design HDAC inhibitors suitable as HDAC based therapeutics for cancer and other HDAC relevant diseases and conditions.