Inhibitors of serine biosynthesis

描述(申请人提供)：该项目的目标是发现和开发选择性的小分子3-磷酸甘油脱氢酶(PHGDH)抑制剂，该酶是丝氨酸生物合成途径的第一种酶。我们发现，这一途径在雌激素受体阴性(ER-)乳腺癌中上调，这种乳腺癌占乳腺癌的20%-25%，但导致50%的乳腺癌相关死亡。高表达PHGDH的乳腺癌细胞通过丝氨酸生物合成途径的通量较高，并且对该酶的敲除敏感，表明该途径的抑制剂可能是治疗ER乳腺癌的有用候选药物。目前还没有小分子的PHGDH抑制剂。我们已经开发了一种烟酰胺腺嘌呤二核苷酸(NADH)连接的PHGDH高通量检测方法，并在布里格姆妇女医院的神经变性药物发现实验室(LDDN)对1400种已知生物活性化合物进行了中试筛选，其中包括FDA批准的药物。该筛的Z‘因子为0.61，变异系数为2%。筛选鉴定出两种化合物，它们呈剂量依赖性地抑制PHGDH。我们的第一个目标是将这一分析转移到MLPCN中心，以便对超过350,000个小分子的MLPCN集合进行高通量筛选。我们的第二个目标是通过丝氨酸生物合成、氨基酸和代谢通量分析的耦合分析来验证这些HITS，以测量在体外和细胞内丝氨酸生物合成途径对丝氨酸和β-酮戊二酸产生的抑制，并对这些HITS进行反筛选，以消除非特异性脱氢酶抑制剂。我们的第三个目标是评估这些HITS的生物活性，方法是确定它们对高表达PHGDH和通过丝氨酸生物合成途径具有高通量的肿瘤细胞株的选择性细胞毒性，而不是对PHGDH低表达和丝氨酸生物合成途径通量低的肿瘤细胞株。PHGDH的抑制剂将在体内测试过表达PHGDH的小鼠肿瘤的异种移植模型。PHGDH特异性抑制剂的鉴定将使丝氨酸生物合成抑制作为ER乳腺癌的新治疗靶点的评估成为可能。

DESCRIPTION (provided by applicant): The goal of this project is the discovery and development of selective small molecule inhibitors of 3- phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the serine biosynthetic pathway. We have found that this pathway is upregulated in estrogen-receptor negative (ER-) breast cancers, which account for 20-25% of breast cancers but are responsible for 50% of breast cancer-related deaths. Breast cancer cells that overexpress PHGDH have higher flux through the serine biosynthesis pathway and are sensitive to knockdown of this enzyme, indicating that inhibitors of this pathway may be useful candidates for the treatment of ER- breast cancer. At present there are no small molecule inhibitors of PHGDH. We have developed a nicotinamide adenine dinucleotide (NADH)-linked high-throughput assay for PHGDH and carried out a pilot screen of 1400 known bioactive compounds, including FDA-approved pharmaceuticals, at the Laboratory for Drug Discovery in Neurodegeneration (LDDN) at the Brigham and Women's Hospital. This screen had a Z' factor of 0.61 and a coefficient of variation of 2%. The screen identified two compounds that dose-dependently inhibit PHGDH. Our first aim is to transfer this assay to an MLPCN center for a high- throughput screen of the MLPCN collection of over 350,000 small molecules. Our second aim is validation of these hits using a coupled assay of serine biosynthesis, amino acid and metabolic flux analysis to measure inhibition of serine and ¿-ketoglutarate production by the serine biosynthesis pathway in vitro and in cells, and counterscreening of these hits against GAPDH to eliminate non-specific dehydrogenase inhibitors. Our third aim is assessment of the biological activity of these hits by determining their selective cytotoxicity towards a tumor cell line that overexpresses PHGDH and has high flux through the serine biosynthesis pathway, while sparing a tumor cell line that has low expression of PHGDH and has low serine biosynthesis pathway flux. Inhibitors of PHGDH will be tested in vivo in xenograft models of mouse tumors that overexpress PHGDH. Identification of specific inhibitors of PHGDH will permit the evaluation of serine biosynthesis inhibition as a novel therapeutic target for ER- breast cancer.