Inhibitors of serine biosynthesis

描述（由申请人提供）：该项目的目标是发现和开发3-磷酸甘油脱氢酶（PHGDH）的选择性小分子抑制剂，PHGDH是丝氨酸生物合成途径的第一个酶。我们发现这一途径在雌激素受体阴性（ER-）乳腺癌中表达上调，雌激素受体阴性占乳腺癌的20-25%，但占乳腺癌相关死亡的50%。过表达PHGDH的乳腺癌细胞通过丝氨酸生物合成途径具有更高的通量，并且对该酶的敲低敏感，这表明该途径的抑制剂可能是治疗ER-乳腺癌的有用候选物。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退行性疾病药物发现实验室（LDDN）对1400种已知生物活性化合物进行了试点筛选，包括fda批准的药物。该筛选的Z′因子为0.61，变异系数为2%。筛选确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这个实验转移到MLPCN中心，对MLPCN收集的超过35万个小分子进行高通量筛选。我们的第二个目标是利用丝氨酸生物合成、氨基酸和代谢通量分析的耦合分析来验证这些命中，以测量丝氨酸生物合成途径在体外和细胞中对丝氨酸和-酮戊二酸盐产生的抑制作用，并对这些命中进行反筛选，以消除非特异性脱氢酶抑制剂。我们的第三个目标是通过确定它们对高表达PHGDH和高丝氨酸生物合成途径通量的肿瘤细胞系的选择性细胞毒性来评估这些命中的生物活性，同时保留低表达PHGDH和低丝氨酸生物合成途径通量的肿瘤细胞系。PHGDH抑制剂将在过表达PHGDH的小鼠肿瘤异种移植模型中进行体内测试。鉴定特定的PHGDH抑制剂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.