Discovery of Glutaryl-CoA Dehydrogenase inhibitors for melanoma and pancreatic cancer

项目摘要 黑色素瘤是一种极具侵袭性的癌症，死亡率很高。它的表型可塑性和异质性 使其能够适应不同的生理环境并击败治疗方法。靶向治疗和 检查点抑制剂在过去十年中变得可用;然而，这些药物仅在一个子集中起作用。 患者和耐药性最终出现，即使在最初的反应。因此，新的目标和临床 用于黑素瘤的方法是未满足的医疗需求。我们将戊二酰辅酶A脱氢酶（GCDH）鉴定为 一个这样的目标。GCDH表达与黑色素瘤患者的侵袭性癌症和低生存率相关。 我们的数据表明，GCDH敲低导致黑色素瘤细胞凋亡。黑素瘤 似乎是唯一敏感的有毒戊二酸代谢产物所造成的GCDH的缺陷，因为抑制 脱氢酶E1和含转酮醇酶结构域1（DHTKD 1）酶，催化前述 在分解代谢途径中的反应并将2-氧代己二酸转化为戊二酰辅酶A， 凋亡胰腺癌细胞，而不是其他癌症或正常细胞，分享这种过度依赖GCDH， 在GCDH敲低后发生细胞凋亡。我们假设，小分子干扰GCDH， 通过细胞凋亡导致黑色素瘤细胞消失。我们建议鉴定GCDH的化学探针 以进一步验证该酶作为黑色素瘤的分子靶点。在一个试点屏幕上，我们建立并验证了 本文提出的所有测定。我们将执行大规模HTS，命中确认和优化， 验证黑色素瘤和其他癌症的一组不同细胞系中鉴定的命中。鉴定的化合物 将提供理想的药理学工具，研究GCDH在黑色素瘤和其他疾病中的病理生理学。 癌症，和赖氨酸代谢的分子机制，在黑色素瘤的责任，以及提供潜在的 未来治疗的起点。这个为期四年的项目将追求以下具体目标， 与PAR-20-271中的预期一致：AIM 1生成GCDH蛋白，优化条件并进行 筛选以鉴定靶向GCDH的化合物。优化命中验证的三级检测。主要分析将 靶结合活性和抑制，三级测定将监测细胞靶结合和蛋白水平。 AIM 2使用一组二级检测进行命中选择、确认和分析。功能命中配置文件 将使用代表性酰基辅酶A脱氢酶的抑制测定和 通过使用蛋白质热位移与底物类似物的作用研究和酶动力学研究， 与底物的竞争概况。AIM 3使用生物化学方法进行命中验证和支架优先级排序 和细胞分析。将在细胞内蛋白质稳定性试验中确认GCDH靶标结合。影响 在黑色素瘤细胞中使用内源性标记的GCDH评价细胞蛋白水平。最好的脚手架 在细胞测定中得到验证，以监测细胞凋亡、细胞活力和对黑素瘤的特异性， 胰腺癌

PROJECT SUMMARY Melanoma is an extremely aggressive cancer with high mortality. Its phenotypic plasticity and heterogeneity enable it to adapt to diverse physiological settings and defeat treatment approaches. Targeted therapies and check point inhibitors became available in the past decade; however, these drugs work in only a subset of patients and drug resistance eventually emerges, even in initial responders. Therefore, new targets and clinical approaches for melanoma are an unmet medical need. We identified glutaryl-CoA dehydrogenase (GCDH) as one such target. GCDH expression correlates with aggressive cancers and low survival in melanoma patients. Our data demonstrates that GCDH knockdown results in apoptotic cell death in melanoma cells. Melanomas seem uniquely sensitive to toxic glutarate metabolites resulting from GCDH deficiencies, since suppression of the Dehydrogenase E1 and Transketolase Domain Containing 1 (DHTKD1) enzyme, catalyzing the preceding reaction in the catabolic pathway and converting 2-oxoadipate to glutaryl-CoA, rescues melanoma cells from apoptosis. Pancreatic cancer cells, but not other cancers nor normal cells, share this overreliance on GCDH and undergo apoptosis upon GCDH knockdown. We hypothesize that small-molecules interferring with GCDH will result in the obliteration of melanoma cells through apoptosis. We propose to identify chemical probes of GCDH to further validate the enzyme as a molecular target for melanoma. In a pilot screen, we established and validated all of the assays proposed herein. We will perform large-scale HTS, hit confirmation and optimization, and validate the identified hits in a panel of diverse cell lines for melanoma and other cancers. Compounds identified will provide desirable pharmacological tools to study the pathophysiology of GCDH in melanoma and other cancers, and the molecular mechanisms of lysine metabolism liability in melanoma, as well as provide potential starting points for future therapeutic treatments. This 4-year project will pursue the following Specific Aims, consistent with the expectations in PAR-20-271: AIM 1 Generate GCDH protein, optimize conditions and perform screening to identify compounds targeting GCDH. Optimize tertiary assays for hit validation. Primary assays will target binding activity and inhibition, and tertiary assays will monitor cellular target engagement and protein level. AIM 2 Perform hit selection, confirmation and profiling using a panel of secondary assays. Functional hit profiles will be established using inhibition assays for a representative of acyl-CoA dehydrogenases and mechanism of action studies by using protein thermal shift with substrate analogs and enzyme kinetic studies to establish competition profiles with the substrates. AIM 3 Perform hit validation and scaffold prioritization using biochemical and cellular assays. GCDH target engagement will be confirmed in an in-cell protein stability assay. Effects on cellular protein levels will be evaluated using endogenous-tagged GCDH in melanoma cells. Best scaffolds will be validated in cellular assays to monitor apoptosis, cell viability, and specificity towards melanoma and pancreatic cancer.