Functional Analysis of HOXA13 Small Molecule Antagonists

项目描述（由申请人提供）：本项目将专注于HOXA13小分子抑制剂的开发和体内表征，作为减少肿瘤血管化的治疗方法。在胚胎发生过程中，HOX蛋白通过调节基因簇来指导组织和器官的发育，这些基因簇的产物控制组织规范和分化。我们已经确定HOXA13调控了胚胎中血管发育所必需的一组促血管基因。更重要的是，我们已经确定了许多相同的血管前基因在肝癌的血管发育中受到HOXA13的调节。由于HOXA13以多效性方式调节血管发育，我假设HOXA13转录功能的破坏将通过同时影响肿瘤血管形成所需的一组基因的表达来有效抑制肿瘤血管形成。认识到HOXA13的转录功能需要DNA结合，我们开发了一个小分子筛选来鉴定能够阻止HOXA13与其靶DNA序列相互作用的化合物。从这个筛选中，我们发现了化合物B (CpdB)，这是一个小分子，可以部分抑制HOXA13调节一组血管前基因的能力。通过仔细修改CpdB的初始结构，我假设可以提高这个小分子的功效，从而抑制HOXA13在肿瘤发生中的功能。为了验证这一假设，将实现以下具体目标：目标1。确定CpdB对HOXA13的特异性。目标2。定量CpdB对HOXA13的亲和力，确定HOXA13 DNA结合域内哪些氨基酸残基与CpdB接触。目标3。确定HOXA13与CpdB的构效关系，优化CpdB效价，提高CpdB的溶解度。目标4。确定CpdB类似物在肿瘤发生过程中干扰hoxa13调控的血管前基因表达的功效。公共卫生相关性：拟议的研究将开发新鉴定的一系列HOXA13小分子拮抗剂，因为它们具有抑制血管生成的潜力。这些发现的应用将转化为防止肿瘤血管化和生长的新疗法。

DESCRIPTION (provided by applicant): This project will focus on the development and in vivo characterization of small molecule inhibitors of HOXA13 as a therapy for reducing tumor vascularization. During embryogenesis, HOX proteins direct tissue and organ development by regulating clusters of genes whose products control tissue specification and differentiation. We have established that HOXA13 regulates a cluster of pro-vascular genes necessary for vascular development in the developing embryo. More importantly, we have determined that many of the same provascular genes are regulated by HOXA13 in the developing vasculature of hepatocarcinomas. Because HOXA13 functions in a pleiotropic manner to regulate vascular development, I hypothesize that the disruption of HOXA13's transcriptional function would effectively inhibit tumor vascularization by simultaneously affecting the expression of a cohort genes required for tumor vasculogenesis. Recognizing that DNA binding is required for HOXA13's transcriptional function, we developed a small molecule screen to identify compounds capable of preventing HOXA13 from interacting with its target DNA sequence. From this screen, we identified Compound B (CpdB), a small molecule that can partially inhibit HOXA13's ability to regulate a group of provascular genes. Through careful modifications to CpdB's initial structure, I hypothesize that the efficacy of this small molecule can be improved to inhibit HOXA13 function in developing tumors. To test this hypothesis, the following specific aims will be accomplished: Aim 1. Determine the specificity of CpdB for HOXA13. Aim 2. Quantitate the affinity of CpdB for HOXA13 and determine which amino acid residues within the HOXA13 DNA binding domain are contacted by CpdB. Aim 3. Determination of structure activity relationships for HOXA13 and CpdB, optimization of CpdB potency, and improvement of CpdB solubility. Aim 4. Determine the efficacy of CpdB analogs to perturb HOXA13-regulated provascular gene expression in developing tumors. PUBLIC HEALTH RELEVANCE: The proposed investigations will develop a newly identified series of HOXA13 small molecule antagonists for their potential to inhibit vasculogenesis. Applications of these findings will translate into new therapies to prevent tumor vascularization and growth.