Inhibitors of serine biosynthesis

描述（由申请人提供）：本项目的目标是发现和开发丝氨酸生物合成途径的第一种酶3-磷酸甘油酸脱氢酶（PHGDH）的选择性小分子抑制剂。我们发现，这种途径在雌激素受体阴性（ER-）乳腺癌中上调，占乳腺癌的20-25%，但占乳腺癌相关死亡的50%。过表达PHGDH的乳腺癌细胞具有通过丝氨酸生物合成途径的更高通量，并且对该酶的敲低敏感，表明该途径的抑制剂可能是用于治疗ER-乳腺癌的有用候选物。目前还没有PHGDH的小分子抑制剂。我们已经开发了一种烟酰胺腺嘌呤二核苷酸（NADH）连接PHGDH的高通量检测方法，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种已知的生物活性化合物进行了中试筛选，包括FDA批准的药物。该筛选的Z'因子为0.61，变异系数为2%。筛选鉴定了两种剂量依赖性抑制PHGDH的化合物。我们的第一个目标是将该测定转移到MLPCN中心，用于对超过350，000个小分子的MLPCN集合进行高通量筛选。我们的第二个目的是使用丝氨酸生物合成、氨基酸和代谢通量分析的偶联测定来验证这些命中，以测量体外和细胞中丝氨酸生物合成途径对丝氨酸和<$-酮戊二酸产生的抑制，并针对GAPDH反筛选这些命中以消除非特异性脱氢酶抑制剂。我们的第三个目的是通过确定它们对过表达PHGDH并具有通过丝氨酸生物合成途径的高通量的肿瘤细胞系的选择性细胞毒性，同时保留具有低表达PHGDH并具有低丝氨酸生物合成途径通量的肿瘤细胞系，来评估这些命中物的生物活性。PHGDH的抑制剂将在过表达PHGDH的小鼠肿瘤的异种移植模型中进行体内测试。PHGDH的特异性抑制剂的鉴定将允许评估丝氨酸生物合成抑制作为ER-乳腺癌的新治疗靶点。

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.