Fully Synthetic Prodiginines as Probes of Protein-Protein Interactions Regulating

描述（由申请人提供）：在癌细胞中重建细胞凋亡具有相当大的靶向，无毒治疗的前景。我们之前通过“smac模拟物”的开发证明了这一点，小分子破坏蛋白质-蛋白质相互作用，抑制适当的半胱天冬酶激活。几个组织的Smac模拟物已经进入了人体临床试验阶段。这些实验仍在进行中。在本提案中，我们概述了针对第二组蛋白质相互作用的新化学，这些蛋白质相互作用可阻断癌症细胞凋亡并促进耐药性。Bcl-2家族蛋白控制线粒体外膜通透性，调节细胞质释放正常细胞死亡所必需的因子。抗凋亡Bcl-2蛋白的过度表达在化疗和放疗耐药癌症中很常见。因此，Bcl-2系统作为药物开发的靶点被广泛研究。这些努力中最先进的已经进入临床阶段。然而，尽管该机制具有巨大的前景，但意想不到的脱靶效应，特别是Mcl-1对靶向Bcl-XL的药物的耐药性限制了进展。需要具有优异轮廓的新化合物。安莎桥接的prodiginine是细菌产生的细胞毒性色素。Shore发现其中一种天然产物streptorubin B是一种在细胞培养中促进细胞凋亡的分子，据报道是通过与Bcl-2蛋白相互作用实现的。这一观察结果为研制出一种合成的浪子碱obatoclax奠定了基础，这种合成的浪子碱目前正用于治疗淋巴细胞白血病的临床试验。Obatoclax被描述为一种泛Bcl-2拮抗剂，靶向BH3螺旋结合表面，包括Mcl-1上的Noxa结合位点。数据是有希望的，但受到选择性的影响。在金属离子存在的情况下，卵泡藻中的三吡咯生色团产生ROS，并通过跨膜离子传输来中和pH梯度。这两种效应都会损害正常细胞。我们建议研究新的链霉素B的pyrolophane变体，缺乏obatoclax的结构缺陷。基于我们最近完成的亲玫瑰素的全合成，我们概述了合成新分离的海洋细菌产生的抗白血病的浪子碱海洋皂苷A和B的新方法。我们与麦吉尔大学的Gordon Shore合作，在我们的亲玫瑰蛋白合成中发现了体外阻断t-Bid依赖性Mcl-1功能的中间体。我们建议在这些结果的基础上，沿着几个方向推进我们的合成化学。基于计算模型，我们相信天然产物的非桥接杂环核心可以支撑一类新的-螺旋肽模拟物。本文描述了验证这一假设的实验。其中包括选择性地合成针对Mcl-1/Noxa、Bcl-XL /Bim和p53/mdm2相互作用的配体。最后，作为在细胞培养和体内鉴定非Bcl-2效应的手段，我们建议使用基于亲和力的生化方法和最先进的功能基因组筛选来寻找该结构类的其他靶点。

DESCRIPTION (provided by applicant): Reestablishing apoptosis in cancer cells holds considerable promise for targeted, non-toxic therapy. We demonstrated this previously with the development of 'smac mimetics', small molecules that disrupt protein- protein interactions that suppress proper caspase activation. Smac mimetics from several organizations have since advanced to human clinical trials. These experiments remain ongoing. In this proposal we outline new chemistry aimed at a second set of protein-protein interactions that block apoptosis in cancer and contribute to drug resistance. Bcl-2 family proteins gate mitochondrial outer membrane permeability and regulate cytoplasmic release of factors essential for normal cell death. Over-expression of anti-apoptotic Bcl-2 proteins is common in chemotherapy and radiation resistant cancers. As a result, the Bcl-2 system has been intensely studied as a target for drug development. The most advanced of these efforts have reached clinic stages. However, despite tremendous promise for the mechanism, unanticipated off-target effects and, notably, Mcl-1 based resistance to agents targeting Bcl-XL are limiting progress. New compounds with superior profiles are needed. Ansa-bridged prodiginines are cytotoxic pigments produced by bacteria. One of these natural products, streptorubin B, was identified by Shore as a molecule that potentiates apoptosis in cell culture - reportedly through interactions with Bcl-2 proteins. This observation seeded development of obatoclax, a synthetic prodiginine now in clinical trials as therapy for lymphocytic leukemia. Obatoclax is described as a pan Bcl-2 antagonist that targets BH3 helix-binding surfaces, including the Noxa binding site on Mcl-1. Data is promising but tempered by selectivity. The tri-pyrrolic chromophore in obatoclax generates ROS in the presence of metal ions and can neutralize pH gradients via transmembrane ion transport. Both effects damage normal cells. We propose to study new pyrrolophane variants of streptorubin B that lack the structural liabilities of obatoclax. Based on our recently completed total synthesis f roseophilin, we outline new methods to synthesize marineosins A and B - newly isolated anti-leukemic prodiginines produced by marine bacteria. In collaboration with Gordon Shore at McGill University, we have discovered intermediates in our roseophilin synthesis that block t-Bid dependent Mcl-1 functions in vitro. We propose to build on these results by advancing our synthetic chemistry along several lines. Based on computational models, we believe the ansa- bridged heterocyclic cores of the natural products in question can scaffold a new class of �-helix peptidomimetics. Experiments to test this hypothesis are described. These include custom syntheses of ligands targeting the Mcl-1/Noxa, Bcl-XL /Bim and p53/mdm2 interactions selectively. Lastly, as means to identify non Bcl-2 based effects in cell culture and in vivo, we propose to search for additional targets of this structural class using affinity based biochemical methods and state-of-the-art functional genomic screens.