Development of BRAF Dimer Inhibitors to Treat Drug Resistant Melanoma

该提案的总体目标是使用RAS-RAF-MEK-ERK(MAPK/ERK)信号通路作为一种 开发小分子抑制剂的模型，该小分子抑制剂专门针对激酶二聚体作为先导化合物 治疗方面的发展。MAPK/ERK信号通路在人类癌症中经常被激活，并且 尤其是激活BRAF V600E突变体(BRAFV600E)约占所有癌症的7%和约50% 恶性黑色素瘤的发生，从而强调BRAF和MAPK/ERK信号转导是 心理治疗。尽管许多黑色素瘤患者接受了高选择性BRAFV600E突变抑制剂(BRAFi)的治疗， 下游MEK抑制剂(Meki)或BRAFi/Meki组合最初有效，几乎都开发药物 联合治疗的中位生存期约一年的耐药性和进展。内在的或后天的 对BRAF抑制剂的耐药性通常是通过MAPK通路的有效激活或重新激活而产生的， 通常是通过上游RAS或下游MEK的突变。在生物化学方面，MAPK的激活是由 药物结合的BRAF或BRAFV600E介导药物诱导的变构激活 野生型RAF亚基(BRAF或CRAF)的激酶二聚体，这一过程被称为‘反式激活’或‘矛盾’ 激活。‘为了测试是否可以通过选择性靶向RAF二聚体来克服反式激活，我们使用了 BRAFV600E和维莫拉非尼作为模型系统开发二价激酶抑制剂以锁定RAF二聚体 不能进行反式激活的非活性构象。这种基于结构的设计工作导致了 维莫拉非尼双分子连接化合物Vem-Bisamamide-2的研制 酰胺链接剂。我们发现维拉非尼与维莫拉非尼具有相似的抑制效力。 BRAFV600E在体外和细胞内，但促进一种不活跃的二聚体BRAFV600E构象，该构象不能 进行转化激活。在相关研究中，我们还将维莫拉非尼与MEK抑制剂G894化学连接， 结果表明，这种化合物比任何一种都能更有效地抑制BRAFi抗性细胞的生长 维莫拉非尼或G894单独使用，或维莫拉非尼/G894联合使用。我们还进行了一次高吞吐量 检测BRAF/MEK相互作用的抑制程度，目的是开发小分子BRAF- MEK二聚体抑制剂。有了这些初步数据，我们现在将(1)开发靶向 与BRAFV600E、BRAF或CRAF的BRAF同源和杂二聚体，以及(2)开发靶向 RAF/MEK复合体。这些研究将导致针对BRAF的小分子抑制剂的开发 我们预计含有BRAF、CRAF、BRAFV600E和MEK的同二聚体和异二聚体将优于 针对黑色素瘤和黑色素瘤中MAPK/ERK信号转导的靶向激酶单体的抑制剂 抗拒治疗。这些铅抑制剂将为开发一种新的 黑色素瘤药物。

The overall goal of this proposal is to use the RAS-RAF-MEK-ERK (MAPK/ERK) signaling pathway as a model to develop small molecule inhibitors that specifically target kinase dimers as lead compounds for therapeutic development. The MAPK/ERK signaling pathway is frequently activated in human cancer and the activating BRAF V600E mutant (BRAFV600E) in particular accounts for about 7% of all cancers and about 50% of malignant melanoma tumors, thus highlighting BRAF and MAPK/ERK signaling as important targets for therapy. Although many melanoma patients treated with highly selective BRAFV600E mutant inhibitors (BRAFi), downstream MEK inhibitors (MEKi) or BRAFi/MEKi combinations, initially respond, nearly all develop drug resistance and progress at a median of about 1 year survival for combination therapy. Intrinsic or acquired resistance to BRAF inhibitors often arise through potent activation or reactivation of the MAPK pathway, typically through mutation of upstream RAS or downstream MEK. Biochemically, MAPK activation is driven by RAF dimerization, whereby drug-bound BRAF or BRAFV600E mediates drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called ‘transactivation’ or ‘paradoxical activation.’ To test if transactivation could be overcome by selectively targeting RAF dimers, we used BRAFV600E and vemurafenib as a model system to develop bivalent kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We showed that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600E both in vitro and in cells, but promotes an inactive dimeric BRAFV600E conformation that is unable to undergo transactivation. In related studies, we also chemically linked vemurafenib to the MEK inhibitor G894, and demonstrated that this compound inhibited growth of BRAFi-resistant cells more effectively than either vemurafenib or G894 alone or a vemurafenib/G894 combination. We have also carried out a high-throughput assay to measure inhibition of the BRAF/MEK interaction with the goal of developing small molecule BRAF- MEK dimerizatioin inhibitors. With this preliminary data on hand, we will now (1) Develop inhibitors that target BRAF homo- and hetero-dimers with BRAFV600E, BRAF or CRAF, and (2) Develop inhibitors that target RAF/MEK complexes. These studies will lead to the development of small molecule inhibitors to target BRAF homo- and hetero-dimers with BRAF, CRAF, BRAFV600E and MEK that we anticipate will be superior to inhibitors that target kinase monomers for inhibiting MAPK/ERK signaling in melanoma and melanoma resistant to therapy. These lead inhibitors will provide a new paradigm for the development of a new family of melanoma drugs.