Restoration of Tumor Suppression Activity in Malignant Melanoma

描述（由申请人提供）：在恶性黑色素瘤（MM）中，我们发现肿瘤标志物S100B水平升高，直接与野生型（wt） p53结合，解离p53四聚体，增强hdm2依赖性p53的泛素化，并下调p53依赖性肿瘤抑制功能；因此，开发S100B抑制剂（SBiXs； X=化合物数）来恢复这种致命癌症中的活性p53是很重要的。作为药物设计项目的原理证明，用小干扰反义RNA （siRNAS100B）和几种sbix抑制S100B被证明可以恢复野生型p53及其下游基因产物的水平，这是诱导恶性黑色素瘤细胞生长停滞和凋亡所必需的。我们假设可以设计低分子量化合物，以更高的亲和力结合Ca2+负载的S100B上明确的p53结合位点，并特异性抑制Ca2+依赖的S100B-p53相互作用，以模拟siRNAS100B的作用。在上一个授权期内，38篇出版物，10个专利申请和20个蛋白质数据库提交描述了我们在解决这些目标/假设方面的进展。由于我们的进展，几种sbix的动物模型研究和SBi1的人体临床试验也正在进行中。然而，重要的是我们设计和/或合成新的和改进的sbix，具有更高的亲和力和更好的特异性来抑制S100B。这将通过以下具体目标来实现：在目标1中，计算机辅助药物设计（CADD）结合高通量筛选方法（即NMR，结合和细胞分析）将用于发现/设计结合S100B并以较低浓度和更高特异性抑制S100B-p53相互作用的新化合物。在3D结构和计算机辅助药物设计（CADD）先导物优化方法的指导下，通过化学修饰对有希望的先导物进行优化。在Aim 2中，S100B- sbix配合物的三维结构将通过核磁共振波谱和/或x射线晶体学来确定，以进一步表征Ca2+S100B的结合表面，从而可以设计和合成改进的sbix。测试新的类似物将使用现有的热力学结合和生物测定（如在第1部分），并在黑色素瘤小鼠模型中检测最有希望的化合物抑制/消除肿瘤生长的能力（如在第3部分）。体内数据将对我们的设计/合成工作重点放在先导化合物或在体内有效的化合物类别上非常重要，它们将用于确定未来要进行的其他人体临床试验的优先顺序。我们的目标是发现/合成和/或改进现有的sbix，以最佳地恢复人类恶性黑色素瘤中p53的活性。sbix也可能对其他S100B和wt p53升高的癌症有治疗价值，如星形细胞瘤、肾肿瘤和某些形式的白血病，因此这将在未来的努力中进行探索。

DESCRIPTION (provided by applicant): In malignant melanoma (MM), we have shown that elevated levels of the tumor marker, S100B, binds directly to wild-type (wt) p53, dissociates the p53 tetramer, enhances hdm2-dependent ubiquitination of p53, and down-regulates p53-dependent tumor suppression functions; therefore, it is important to develop S100B inhibitors (SBiXs; X=compound number) to restore active p53 in this deadly cancer. As a proof of principle for a drug design project, inhibiting S100B with small interfering antisense RNA (siRNAS100B) and several SBiXs was shown to restore wild-type p53 levels and its downstream gene products, as necessary to induce cell growth arrest and apoptosis in malignant melanoma. We hypothesize that low molecular weight compounds can be designed to bind the well-defined p53 binding site on Ca2+ loaded S100B with higher affinity and specifically inhibit the Ca2+dependent S100B-p53 interaction to mimic the siRNAS100B effects. In the last granting period, thirty-eight publications, ten patent applications, and twenty protein data base submissions describe our progress at addressing these goals/hypotheses. Animal model studies with several SBiXs and a human clinical trial with SBi1 are also ongoing as a result of our progress. However, it is important that we engineer and/or synthesis new and improved SBiXs with higher affinity and better specificity towards inhibiting S100B. This will be achieved with the following specific aims: In Aim 1, computer aided drug design (CADD) combined with high-throughput screening methods (i.e. NMR, binding, and cellular assays) will be used to discover/design new compounds that bind S100B and inhibit the S100B-p53 interaction at lower concentrations and with higher specificity than those already discovered. Optimization of promising leads will be performed via chemical modifications as guided by 3D structural and computer aided drug design (CADD) lead optimization approaches. In Aim 2, 3D structures of S100B-SBiX complexes will be determined using NMR spectroscopy and/or X-ray crystallography to further characterize the binding surface on Ca2+S100B, so improved SBiXs can be designed and synthesized. Testing new analogues will be performed using existing thermodynamic binding and biological assays (as in Aim 1) with the most promising compounds examined for their ability to suppress/eliminate tumor growth in melanoma mouse models (in Aim 3). The in vivo data will be important for focusing our design/synthesis efforts on lead compounds or classes of compounds that have efficacy in vivo, and they will be used to set priorities for additional human clinical trials to be done in the future. It is our goal to discover/synthesize and/or to improve existing SBiXs to optimally restore p53 activity in human malignant melanoma. SBiXs may also have therapeutic value for treating other cancers with elevated S100B and wt p53 such as astrocytomas, renal tumors, and some forms of leukemia, so this will be explored in future endeavors.