苦蘵中活性成分Withangulatin-A靶向丝氨酸合成限速酶PHGDH的抗结肠癌机制研究

PHGDH, as the key enzyme in the first step of serine biosynthesis, becomes the target in the research of cancer metabolic reprogramming and anti-tumor drug research. Physalis angulata L as a traditional chinese medicine of Physalis, which is rich in withanolides. The preliminary study of our group showed that Withangulatin A(WGA) had significant anti-tumor effect in colon cancer, which is a withanolide in the Physalis angulata L. Further study showed that WGA could combine with PHGDH and inhibit the enzyme activity of PHGDH. Based on previous research, this project will further confirm the target effect of WGA on PHGDH by recombinant proteins and BLI, then evaluate the effect of WGA on the serine biosynthesis by metabolite analysis to verify the targeting effect of WGA on PHGDH in colon cancer. With the using of pull down, mass spectrum, site-specific mutation, etc, this project will further investigate the binding site and action form of WGA and PHGDH, and evaluate the signal molecules regulated by PHGDH. This project will illuminate the target and anti-tumor mechanism of WGA in colon cancer, which is not only provide the evidence on the anti-tumor effect of Physalis angulata L, but also establish the foundation in the anti-tumor drug development which is based on the withanolides.

PHGDH作为丝氨酸合成途径中的关键限速酶已成为目前肿瘤代谢重编程及抗肿瘤药物研究的新靶标。苦蘵为茄科酸浆属清热解毒中药，其中富含睡茄内酯成分。项目组前期研究发现苦蘵中睡茄内酯Withangulatin A(WGA)具有显著的抗结肠癌作用，能够与PHGDH结合并抑制其酶活。本项目拟在前期研究基础上，通过重组蛋白及生物膜层干涉等实验确证WGA与PHGDH的结合，并以靶标代谢流分析等手段评价WGA对结肠癌细胞中丝氨酸合成途径的调控作用，以明确WGA靶向PHGDH干预丝氨酸合成途径的效应；进一步采用定点突变，质谱鉴定等技术，探究WGA与PHGDH的结合位点及作用形式，并对PHGDH调控的下游分子进行验证，阐明WGA靶向PHGDH的抗结肠癌作用机理。本项目通过发现并阐明WGA的抗结肠癌作用靶标及机理，不仅为阐明中药苦蘵的抗肿瘤功效提供依据，而且为基于睡茄内酯的中药抗肿瘤新药创制奠定基础。

磷酸甘油酸脱氢酶(PHGDH)作为丝氨酸合成途径(SSP)的第一个速率限制酶，在多种肿瘤中极度活跃，能够加速SSP的激活，促进肿瘤发生。然而，迄今为止发现的PHGDH抑制剂很少，尤其是PHGDH的共价抑制剂。苦蘵为茄科酸浆属清热解毒中药，其中富含睡茄内酯成分。项目组前期研究发现苦蘵中睡茄内酯Withangulatin A(WA)具有显著的抗结肠癌作用，然而其具体作用靶点和机制尚不明确。在本研究中，我们发现睡茄内酯类化合物Withangulatin A (WA)是一个特异性的PHGDH共价抑制剂。我们首先合成了WA的亲和探针WP，并利用WP在细胞中进行蛋白亲和实验将直接结合的蛋白沉淀出来。进一步的蛋白质质谱和免疫印迹分析发现，WP与PHGDH相对结合最多。生物膜层干涉实验和LC-MS/MS分析也直接证实了WA能够与PHGDH直接结合，并与PHGDH的295位半胱氨酸残基 (Cys295)选择性共价结合。通过共价修饰Cys295, WA阻断了PHGDH的底物结合域 (SBD)，并发挥变构效应，进一步诱导PHGDH失活并且抑制结肠癌细胞的增殖。进一步研究发现，WA在体内同样能够抑制结肠癌细胞的增殖。当PHGDH敲除后，结肠癌细胞的增殖能力显著降低。而在PHGDH敲除的结肠癌细胞移植瘤模型中，WA并不能进一步抑制结肠癌细胞的增殖，提示WA对结肠癌细胞的增殖抑制具有一定的PHGDH依赖性。本研究表明WA是一种新的PHGDH共价抑制剂，同时鉴定出Cys295是PHGDH的一种新的变构调控位点，本研究表明睡茄内酯类化合物WA在开发靶向PHGDH的抗肿瘤药物方面具有很大潜力。