Development of BRAF Dimer Inhibitors to Treat Drug Resistant Melanoma

开发 BRAF 二聚体抑制剂来治疗耐药性黑色素瘤

• 批准号: 10317051
• 负责人: Ronen Marmorstein
• 金额: $ 63.15万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-03 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10317051
• 关键词: Amides; BRAF gene; Biochemical; Biological Assay; Biological Models; Cell Line; Cells; Chemicals; Combined Modality Therapy; Complex; Crystallization; Data; Development; Dimerization; Drug Kinetics; Drug resistance; Family; Goals; Growth; Hand; Homo; Human; In Vitro; Lead; Link; MAP Kinase Gene; MEKs; Malignant Neoplasms; Measures; Mediating; Methodology; Modeling; Molecular; Molecular Conformation; Mutation; Normal tissue morphology; Oncogenic; Pathway interactions; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phosphotransferases; Play; Process; Ras/Raf; Resistance; Signal Pathway; Signal Transduction; Structure; Testing; Transactivation; X-Ray Crystallography; base; biophysical analysis; cancer therapy; design; dimer; efficacy evaluation; high throughput screening; inhibitor; kinase inhibitor; melanoma; monomer; mutant; novel; novel therapeutic intervention; preclinical development; scaffold; screening; small molecule; small molecule inhibitor; targeted treatment; therapeutic development; therapeutic lead compound; therapeutic target; therapy resistant; treatment strategy; tumor

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.

该提案的总体目标是使用RAS-RAF-MEK-ERK（MAPK/ERK）信号通路作为一种新的信号通路。 开发特异性靶向激酶二聚体的小分子抑制剂作为先导化合物的模型， 治疗发展MAPK/ERK信号通路在人类癌症中频繁激活， 活化BRAF V600 E突变体（BRAFV 600 E）特别地占所有癌症的约7%和约50%。 因此，BRAF和MAPK/ERK信号转导是恶性黑色素瘤的重要靶点。 疗法尽管许多黑色素瘤患者用高选择性BRAFV 600 E突变体抑制剂（BRAFi）治疗， 下游MEK抑制剂（MEKi）或BRAFi/MEKi组合，最初响应，几乎所有开发药物 联合治疗的中位生存期约为1年。内在的还是后天的 对BRAF抑制剂的抗性通常通过MAPK途径的有效激活或再激活而产生， 通常通过上游RAS或下游MEK的突变。在生物化学上，MAPK激活是由 RAF二聚化，由此药物结合的BRAF或BRAFV 600 E介导药物诱导的RAF的变构活化。 激酶二聚体的野生型RAF亚基（BRAF或CRAF），这一过程被称为“反式激活”或“矛盾” 激活。为了测试选择性靶向RAF二聚体是否可以克服反式激活，我们使用 BRAFV 600 E和维罗非尼作为模型系统开发二价激酶抑制剂以将RAF二聚体锁定在一个细胞中。 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 开发Vem-双酰胺-2，一种含有两个维罗非尼分子的化合物， 酰胺连接基。我们表明，Vem-双酰胺-2具有与维罗非尼相当的抑制效力， 在体外和细胞中，BRAFV 600 E都是无活性的，但促进了不能与BRAFV 600 E结合的无活性二聚体BRAFV 600 E构象。 进行反式激活。在相关研究中，我们还将维罗非尼与MEK抑制剂G894化学连接， 并证明这种化合物比任何一种化合物都更有效地抑制BRAFi抗性细胞的生长， 单独的维罗非尼或G894或维罗非尼/G894组合。我们还进行了高通量的 测量BRAF/MEK相互作用的抑制的测定，目的是开发小分子BRAF- MEK二聚化抑制剂。有了这些初步数据，我们现在将（1）开发靶向 BRAF同源和异源二聚体与BRAFV 600 E、BRAF或CRAF，和（2）开发靶向 RAF/MEK复合物。这些研究将导致开发针对BRAF的小分子抑制剂 与BRAF、CRAF、BRAFV 600 E和MEK的同源和异源二聚体，我们预期将上级 靶向激酶单体用于抑制黑素瘤和黑素瘤中MAPK/ERK信号传导的抑制剂 对治疗有抵抗力这些先导抑制剂将为开发新的药物家族提供新的范例。 黑色素瘤药物