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

基本信息

批准号：
10058819
负责人：
Ronen Marmorstein
金额：
$ 64.36万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
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/10058819
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/antagonist 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

项目摘要

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) 信号通路作为开发专门针对激酶二聚体作为化合物先导的小分子抑制剂的模型 MAPK/ERK 信号通路在人类癌症和癌症中经常被激活。尤其是激活 BRAF V600E 突变体 (BRAFV600E) 约占所有癌症的 7%，约占所有癌症的 50% 恶性黑色素瘤肿瘤，从而强调 BRAF 和 MAPK/ERK 信号作为重要靶标尽管许多黑色素瘤患者接受高选择性 BRAFV600E 突变抑制剂（BRAFi）治疗，下游 MEK 抑制剂 (MEKi) 或 BRAFi/MEKi 组合，最初有反应，几乎都在开发药物联合治疗的耐药性和中位生存期约 1 年的进展。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，通常通过上游 RAS 或下游 MEK 的突变实现，MAPK 激活是由生物化学驱动的。 RAF 二聚化，因此药物结合的 BRAF 或 BRAFV600E 介导药物诱导的变构激活激酶二聚体的野生型 RAF 亚基（BRAF 或 CRAF），这一过程称为“反式激活”或“矛盾” 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 BRAFV600E 和 vemurafenib 作为开发二价激酶抑制剂的模型系统，以将 RAF 二聚体锁定在这种基于结构的设计工作导致了无法进行反式激活的非活性构象。开发 Vem-BisAmide-2，一种含有两个通过双键连接的维罗非尼分子的化合物我们证明 Vem-BisAmide-2 具有与 vemurafenib 相当的抑制效力。 BRAFV600E 在体外和细胞内均存在，但会促进无活性的二聚体 BRAFV600E 构象，该构象无法在相关研究中，我们还将维莫非尼与 MEK 抑制剂 G894 进行化学连接，并证明该化合物比任何一种化合物都能更有效地抑制 BRAFi 耐药细胞的生长单独使用威罗非尼或 G894 或威罗非尼/G894 组合我们还进行了高通量分析。测定 BRAF/MEK 相互作用的抑制作用，目的是开发小分子 BRAF- 有了这些初步数据，我们现在将 (1) 开发针对目标的抑制剂。 BRAF 同源和异源二聚体与 BRAFV600E、BRAF 或 CRAF，以及 (2) 开发靶向抑制剂 RAF/MEK 复合物将导致针对 BRAF 的小分子抑制剂的开发。我们预计 BRAF、CRAF、BRAFV600E 和 MEK 的同源和异源二聚体将优于靶向激酶单体的抑制剂，可抑制黑色素瘤和黑色素瘤中的 MAPK/ERK 信号传导这些先导抑制剂将为新家族的开发提供新的范例。黑色素瘤药物。