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

项目总结 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和失败的治疗方法。靶向治疗和 在过去的十年里，检查点抑制剂已经出现；然而，这些药物只对 患者和耐药性最终会出现，即使是在最初的应答者中也是如此。因此，新的靶点和临床 治疗黑色素瘤的方法是一个尚未得到满足的医疗需求。我们鉴定了戊二酰辅酶A脱氢酶(GCDH)是 一个这样的目标。GCDH的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基因敲除导致黑色素瘤细胞的凋亡细胞死亡。黑色素瘤 似乎对GCDH缺乏引起的毒性戊二酸代谢物唯一敏感，因为抑制 脱氢酶E1和转酮醇酶结构域包含1(DHTKD1)酶，催化前述 分解代谢途径中的反应并将2-氧己二酸转化为戊二酰辅酶A，将黑色素瘤细胞从 细胞凋亡。胰腺癌细胞，但不是其他癌症或正常细胞，分享这种过度依赖GCDH和 GCDH基因敲除后发生细胞凋亡。我们假设干扰GCDH的小分子将 导致黑色素瘤细胞通过细胞凋亡而消失。我们建议鉴定GCDH的化学探针 以进一步验证该酶作为治疗黑色素瘤的分子靶点。在试点屏幕中，我们建立并验证了 本文提出的所有化验方法。我们将进行大规模的HTS，命中确认和优化，以及 在黑色素瘤和其他癌症的不同细胞系的面板中验证已识别的匹配。已鉴定的化合物 将为研究GCDH在黑色素瘤和其他疾病中的病理生理学提供理想的药理学工具 癌症，以及黑色素瘤赖氨酸代谢易感性的分子机制，以及提供潜在的 未来治疗的起点。这一为期4年的项目将实现以下具体目标， 与PAR-20-271中的预期一致：目标1产生GCDH蛋白，优化条件并执行 筛选以GCDH为靶点的化合物。优化HIT验证的三次化验。一次化验将 靶结合活性和抑制，以及三级检测将监测细胞靶参与和蛋白质水平。 目标2使用一组二次化验进行命中选择、确认和分析。功能命中配置文件 将利用抑制试验建立具有代表性的酰基辅酶A脱氢酶及其机制 通过使用底物类似物的蛋白质热位移和酶动力学研究来进行作用研究 与衬底的竞争概况。目标3使用生物化学执行命中验证和脚手架优先级 和细胞分析。GCDH靶向参与将在细胞内蛋白质稳定性分析中得到确认。对……的影响 黑色素瘤细胞中的细胞蛋白水平将使用内源性标记的GCDH进行评估。最好的脚手架 在细胞检测中得到验证，以监测细胞凋亡、细胞活力和对黑色素瘤的特异性 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.