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）和卡非佐米（Kyprolis）作为fda批准的抗癌药物的成功，激发了针对泛素酶和泛素样蛋白修饰系统的上游途径设计新的癌症治疗药物的努力。我们的长期目标是提供抑制NEDD8系统和主要NEDD8靶点cullin-RING泛素连接酶的治疗方法，直接使癌症患者受益。nedd8活化酶（NAE）抑制剂MLN4924是目前这些潜在的新型药物中最先进的。MLN4924有大量已发表的临床前数据，显示出强大的抗癌功效，多项针对血液和晚期非血液恶性肿瘤的1期临床试验正在进行中。考虑到MLN4924通过选择性诱导癌细胞死亡而对实验癌症没有明显的毒性或副作用所提供的明确益处，了解细胞如何对NEDD8系统抑制作出反应仍然具有重要意义。作为实现这一目标的重要一步，也是对PAR-12-145 NCI探索性/发展研究资助计划的回应，本研究的总体目标是利用从检测靶向MLN4924抗性的实验中获得的见解，以确定通过不同于MLN4924的作用机制发挥作用的新NAE抑制剂。通过我们强有力的初步数据，我们设计了一种高度创新的策略，使我们能够独特地描述可能导致MLN4924耐药的靶突变类型。我们还建立了一种基于差示扫描荧光法的新的筛选方法。该研究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中生化变化的多样性足以使癌细胞对MLN4924产生耐药性，而这些生化变化可以通过不涉及酶催化袋的机制抑制NAE的小分子来克服。为了验证这一假设并实现研究的总体目标，我们提出了两个具体目标：1)表征耐药NAE mln4924形式的生化多样性；2)鉴定这些耐药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MLN4924耐药机制之前详细描述其耐药机制的绝佳机会，并利用该信息发现通过不同作用机制起作用的新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.