Characterization and suppression of resistance to NEDD8 E1 inhibition

描述（由申请人提供）：蛋白酶体抑制剂硼替佐米（Velcade（R））和卡非佐米（Kyprolis（R））作为FDA批准的癌症药物的成功，使设计靶向上游途径的新癌症治疗方法的努力更加努力，这些上游途径涉及泛素和泛素样蛋白修饰系统的酶。我们的长期目标是提供抑制NEDD 8系统和主要NEDD 8靶向cullin-RING遍在蛋白连接酶的疗法，以直接使癌症患者受益。NEDD 8激活酶（NAE）抑制剂MLN 4924是目前这些潜在新型药物中最先进的。MLN 4924具有广泛的已发表临床前数据，证明其具有较强的抗癌疗效，目前正在进行几项针对血液学和晚期非血液学恶性肿瘤的I期临床试验。鉴于MLN 4924通过选择性诱导癌细胞死亡而提供的明确获益，且对实验性癌症无明显毒性或副作用，因此了解细胞如何对NEDD 8系统抑制作出反应仍然非常重要。作为实现这一目标的重要一步，并响应PAR-12-145 NCI探索性/开发性研究资助计划，本拟定研究的总体目标是利用从检查MLN 4924靶向耐药性的实验中获得的见解，鉴定通过与MLN 4924不同的作用机制发挥作用的新型NAE抑制剂。通过我们强大的初步数据，我们设计了一种高度创新的策略，使我们能够独特地描述MLN 4924耐药性可能的靶向突变类型。我们还产生了一个新的筛选分析的基础上差示扫描荧光法。这使用配体竞争策略来评估结合NAE的分子的作用机制。我们的中心假设是，NAE中足以使癌细胞对MLN 4924产生耐药性的生物化学变化多样性可被通过不涉及酶催化口袋的机制抑制NAE的小分子克服。为检验这一假设并实现研究的总体目标，提出了两个特定目的：1）表征MLN 4924耐药形式NAE的生化多样性和2）鉴别这些耐药NAE复合物的小分子抑制剂。影响：这项工作具有高度创新性，具有直接的翻译相关性，因为它提供了在复发患者中报告MLN 4924耐药机制之前详细描述MLN 4924耐药机制的绝佳机会，并利用这些信息发现通过不同作用机制发挥作用的新NAE抑制剂。总的来说，这些努力将大大有助于为癌症患者提供急需的新治疗选择。

DESCRIPTION (provided by applicant): The successes of proteasome inhibitors bortezomib (Velcade(R)) and carfilzomib (Kyprolis(R)) as FDA-approved cancer drugs have invigorated efforts to devise new cancer therapeutics targeting upstream pathways involving enzymes of ubiquitin and ubiquitin-like protein modification systems. Our long-term goal is to provide therapies that inhibit the NEDD8 system and the major NEDD8 targets cullin-RING ubiquitin ligases to directly benefit cancer patients. The NEDD8-activating enzyme (NAE) inhibitor MLN4924 is currently the most advanced of these potential new classes of drugs. MLN4924 has extensive published pre-clinical data demonstrating strong anti-cancer efficacy and several Phase 1 clinical trials are in progress for hematologic and advanced non-hematologic malignancies. Given the clear benefit MLN4924 provides by selectively inducing cancer cell death with no noted toxicities or side-effects on experimental cancers, it remains highly significant to understand how cells respond to NEDD8 system inhibition. As an important step towards this goal and in response to PAR-12-145 NCI Exploratory/Developmental Research Grant Program, the overall objective of this proposed study is to use insight derived from experiments examining on-target MLN4924 resistance to identify new NAE inhibitors that function through mechanisms of action distinct from MLN4924. Through our strong preliminary data, we have devised a highly innovative strategy that uniquely positions us to describe the types of on-target mutations possible for MLN4924 resistance. We have also generated a new screening assay based on differential scanning fluorimetry. This uses a ligand competition strategy to evaluate the mechanism of action of molecules that bind NAE. Our central hypothesis is that the diversity of biochemical changes in NAE sufficient for cancer cell resistance to MLN4924 can be overcome by small molecules that inhibit NAE through mechanisms not involving the enzyme's catalytic pocket. To test this hypothesis and achieve the overall objective of the research, two Specific Aims are proposed: 1) to characterize the biochemical diversity of MLN4924-resistant forms of NAE and 2) to identify small molecule inhibitors of these drug-resistant NAE complexes. IMPACT: This work is highly innovative with direct translational relevance due to the extraordinary opportunity it provides to describe MLN4924 resistance mechanisms in detail before they are reported in relapsed patients and to use this information to discover new NAE inhibitors that function through distinct mechanisms of action. Collectively, these efforts will significantly contribute to providing much needed new treatment options for cancer patients.