Restoration of Tumor Suppression Activity in Malignant Melanoma

描述(申请人提供)：在恶性黑色素瘤(MM)中，我们已经证明，肿瘤标记物S100B的水平升高直接与野生型(Wt)p53结合，解离P53四聚体，增强Hdm2依赖的P53泛素化，并下调P53依赖的肿瘤抑制功能；因此，重要的是开发S100B抑制剂(SBiXs；X=化合物数)来恢复这种致命癌症中活跃的P53。作为药物设计项目的原则证明，用小干扰反义RNA(SiRNAS100B)和几个SBiX抑制S100B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。我们推测，低分子量化合物可以更高的亲和力结合钙离子负载的S100B上明确的P53结合部位，并特异性地抑制钙依赖的S100B-P53相互作用，以模拟siRNAS100B的作用。在最后的授权期内，38份出版物、10份专利申请和20份蛋白质数据库意见书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，使用几种SBiX的动物模型研究和使用SBi1的人类临床试验也在进行中。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实现：在目标1中，计算机辅助药物设计(CADD)将与高通量筛选方法(即核磁共振、结合和细胞分析)相结合，以发现/设计与S100B结合的新化合物，并以比已发现的更低的浓度和更高的特异性抑制S100B-P53的相互作用。在3D结构和计算机辅助药物设计(CADD)引线优化方法的指导下，将通过化学修饰对有希望的引线进行优化。在目标2中，将利用核磁共振光谱和/或X射线结晶学来确定S100B-SBiX络合物的三维结构，以进一步表征S100B上的结合表面，从而可以设计和合成改进的SBiX。将使用现有的热力学结合和生物分析(如目标1)测试新的类似物，检查最有希望的化合物在黑色素瘤小鼠模型中抑制/消除肿瘤生长的能力(目标3)。体内数据对于将我们的设计/合成努力集中在先导化合物或具有体内有效性的化合物类别上将是重要的，它们将被用来为未来进行更多的人类临床试验设定优先事项。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的P53活性。SBiX还可能对治疗其他S100B和wt p53升高的癌症具有治疗价值，如星形细胞瘤、肾脏肿瘤和某些形式的白血病，因此这将在未来的努力中探索。

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.