Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors
Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors
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DOI：
10.1016/j.bmc.2018.02.016
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发表时间：
2018-05-01
影响因子：
3.5
通讯作者：
Boxer, Matthew B.
中科院分区：
文献类型：
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作者：
Rohde, Jason M.;Brimacombe, Kyle R.;Boxer, Matthew B.
包括癌细胞在内的增殖细胞通过外源和葡萄糖代谢获得丝氨酸。磷酸甘油酸脱氢酶（PHGDH）通过催化葡萄糖合成丝氨酸的第一个限速步骤控制这种重要氨基酸通过生物合成途径的通量，并代表肿瘤学中的假定靶点。为了发现PHGDH的抑制剂，开发并优化了一种偶联的生物化学测定法，以高通量筛选人PHGDH的抑制剂。通过将PHGDH活性与两种下游酶（PSAT 1和PSPH）偶联，使反馈抑制最小化，从而显著改善酶周转率。进一步将NADH偶联到心肌黄酶/刃天青系统能够实现红移检测读数，从而最大限度地减少由于化合物自发荧光引起的干扰。使用该方案，筛选了超过400，000种小分子用于PHGDH抑制，并且在命中验证和分类工作之后，鉴定了哌嗪-1-硫脲。经过几轮药物化学和SAR探索，鉴定出两种探针（NCT-502和NCT-503）。这些分子表现出改善的靶向活性和令人鼓舞的ADME性质，使得能够在体外评估PHGDH的生物学重要性及其在PHGDH依赖性癌细胞中丝氨酸命运中的作用。本手稿报告了导致Pacold等人（2016）报告的NCT-502和-503开发的试验开发和药物化学。(C)2018爱思唯尔有限公司版权所有
Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a marked improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling in vitro assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells. This manuscript reports the assay development and medicinal chemistry leading to the development of NCT-502 and -503 reported in Pacold et al. (2016). (C) 2018 Elsevier Ltd. All rights reserved.