Targeting the colchicine site in tubulin for advanced melanoma

 描述(申请人提供)：尽管最近靶向治疗和免疫治疗都取得了进展，但转移性黑色素瘤的总体存活率仍然很低。这一更新应用是为了继续我们富有成效的努力，开发新型微管蛋白抑制剂及其靶向递送，以更有效地治疗转移性黑色素瘤。在第一个资助期，我们确定了几种有效的支架，并开发了两种纳米颗粒配方，显著提高了体内对抗黑色素瘤转移的疗效。我们此次更新的目标是使用药物化学、结构生物学和分子生物学的综合方法进行重点铅优化，并优化纳米颗粒配方，以实现高载药量和纳米颗粒的动力学稳定性。目标1：使用两个最好的支架进行有重点的线索优化，并迭代地优化他们的活动。目标1.1。vbl.使用 根据我们已经积累的全面的SAR，我们将对两个最好的支架进行有针对性的铅优化，以进一步改善它们与秋水仙素结合部位的相互作用。目标1.2。我们将使用一组黑色素瘤细胞株筛选新的类似物，包括多药耐药表型和黑色素瘤集落形成。黑素细胞将作为正常细胞的模型来评估选择性。我们将为目标2选择最多20个最佳类比。目标2： 确认作用模式，确定与微管蛋白的分子相互作用，并评估类药物特性。目标2.1。我们将测定它们抑制微管蛋白聚合的能力，定量评估它们与微管蛋白中秋水仙碱的结合，研究它们对 凋亡，并评价其抗血管生成的潜力。目标2.2。我们将通过解算微管蛋白的X射线晶体结构来确定最好的类似物与微管蛋白之间的分子相互作用。这些结果将反馈给目标1，以指导更有效的结构优化。目标2.3。我们将评估选定的新类似物的类药物特性和非靶标效应，以选择最多6个最好的类似物来实现目标3。目标3：优化纳米颗粒配方，确定使用自由药物及其最佳纳米颗粒配方对黑色素瘤生长和转移的体内疗效。目标3.1。我们将使用可水解键将新的类似物连接到聚合物骨架上，然后形成纳米颗粒，以获得更高的载药量和改善纳米颗粒的动力学稳定性。我们将把黑色素瘤肿瘤特异性多肽加入到载药纳米粒中，以进行主动靶向。目的3.2我们将评估体内抗黑色素瘤生长、转移、肿瘤血管生成和潜在毒性的疗效。我们将以Abraxane(纳米制剂紫杉醇)为参照物，比较免费药物和它们的最佳纳米制剂的疗效。目标3.3。对于三种最有效的微管蛋白抑制剂及其纳米粒制剂，我们将在全身给药后测定它们在小鼠体内的生物分布和药动学特征。

 DESCRIPTION (provided by applicant): Despite recent advances in both targeted therapy and immunotherapy, the overall survival for metastatic melanoma remains low. This renewal application is to continue our productive efforts to develop novel tubulin inhibitors and their targeted deliveries for more effective treatment of metastatic melanoma. In the first funding period, we identified several potent scaffolds and developed two nanoparticle formulations that significantly improved in vivo efficacy against melanoma metastasis. Our objectives in this renewal are to perform focused lead optimization using integrated medicinal chemistry, structural biology and molecular biology approaches, and to optimize nanoparticle formulations for high drug loading and nanoparticle kinetic stability. Aim 1: Perform focused lead optimization using the two best scaffolds and iteratively optimize their activities. Aim 1.1. Using the comprehensive SAR we have accumulated, we will perform focused lead optimization with the two best scaffolds to further improve their interactions with the colchicine binding site. Aim 1.2. We will screen new analogs using a panel of melanoma cell lines, including multidrug resistant phenotypes and melanoma colony formation. Melanocytes will serve as a model of normal cells to assess the selectivity. We will select up to the 20 best analogs for Aim 2. Aim 2: Confirm the modes of action, determine molecular interactions with tubulin, and assess drug- like properties. Aim 2.1. We will determine their ability to inhibit tubulin polymerization, quantitatively assess their binding to the colchicine site in tubulin, investigate their effects on apoptosis, and evaluate their anti- angiogenesis potential. Aim 2.2. We will determine molecular interactions between the best analogs and tubulin by solving the X-ray crystal structures of tubulin in complex with a diverse set of potent analogs. These results will feed back to Aim 1 to guide more efficient structural optimization. Aim 2.3. We will assess drug-like properties and off-target effects for the selected new analogs to select up to 6 of the best analogs for Aim 3. Aim 3: Optimize nanoparticle formulations, determine in vivo efficacy against melanoma tumor growth and metastasis using both free drugs and their optimal nanoparticle formulations. Aim 3.1. We will use hydrolyzable bonds to conjugate new analogs to polymer backbones before forming nanoparticles for higher drug loading capacity and improved kinetic stability of nanoparticles. We will incorporate melanoma tumor- specific peptides to the drug loaded nanoparticles for active targeting. Aim 3.2 We will evaluate in vivo efficacy against melanoma growth, metastasis, tumor angiogenesis, and potential toxicities. We will compare the efficacy of free drugs and their optimal nanoparticle formulations, using Abraxane (nanoparticle formulated paclitaxel) as the reference control. Aim 3.3. For the three most efficacious tubulin inhibitors an their nanoparticle formulations, we will determine their bio-distribution and pharmacokinetic profiles in mice after systemic drug administration.