Inhibitors of serine biosynthesis

丝氨酸生物合成抑制剂

• 批准号: 8460831
• 负责人: David M. Sabatini
• 金额: $ 4.73万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460831
• 关键词: 3-phosphoglycerate; Accounting; Active Sites; Affect; Amino Acids; Anabolism; Biological; Biological Assay; Breast; Breast Cancer Cell; Cancer cell line; Cell Line; Cell Survival; Cells; Cellular Assay; Cessation of life; Chemistry; Child; Choline phosphatase; Clinical Chemistry; Collection; Coupled; Development; Dose; Enzymes; Estrogen receptor negative; Evaluation; FDA approved; Fluorescence; Generations; Genes; Glutamates; Goals; Growth; Hospitals; In Vitro; Institutes; Laboratories; Lead; Libraries; Link; Malignant Neoplasms; Measures; Metabolic; Mus; NADH; Nerve Degeneration; Nicotinamide adenine dinucleotide; Oxidoreductase; Pathway interactions; Pharmacologic Substance; Phase; Phenotype; Phosphoglycerate dehydrogenase; Phosphoserine aminotransferase; Production; Program Development; Proliferating; Protein Dephosphorylation; Role; Serine; Structure-Activity Relationship; Testing; Therapeutic; Tumor Cell Line; Validation; Variant; Woman; Work; Xenograft Model; aminoacid biosynthesis; counterscreen; cytotoxicity; drug discovery; high throughput screening; in vivo; inhibitor/antagonist; inorganic phosphate; killings; malignant breast neoplasm; neoplastic cell; new therapeutic target; outcome forecast; overexpression; oxidation; screening; small molecule; transamination; tumor

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.

描述（由申请人提供）：该项目的目标是发现和开发3-磷酸甘油酸脱氢酶（PHGDH）的选择性小分子抑制剂，3-磷酸甘油酸脱氢酶是丝氨酸生物合成途径的第一种酶，我们发现该途径在雌激素中上调。 - 受体阴性 (ER-) 乳腺癌，占乳腺癌的 20-25%，但导致乳腺癌相关死亡的 50%。过度表达 PHGDH 的乳腺癌细胞通过丝氨酸生物合成途径的通量较高，并且对该酶的敲低敏感，表明该途径的抑制剂可能是治疗 ER-乳腺癌的有用候选者。目前还没有小分子抑制剂。我们开发了一种烟酰胺腺嘌呤二核苷酸 (NADH) 连接的 PHGDH 检测方法，并对 1400 种已知的生物活性化合物进行了初步筛选，包括布莱根妇女医院神经退行性疾病药物发现实验室 (LDDN) 的 FDA 批准药物该筛选的 Z' 因子为 0.61，变异系数为 2%。该筛选鉴定出两种剂量依赖性化合物。我们的第一个目标是将该测定转移到 MLPCN 中心，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使用丝氨酸生物合成、氨基酸和代谢流分析的耦合测定，以测量丝氨酸和 ¿ -体外和细胞内丝氨酸生物合成途径产生酮戊二酸，并针对 GAPDH 反筛选这些命中物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这些命中物对肿瘤细胞的选择性细胞毒性来评估这些命中物的生物活性。过表达 PHGDH 并通过丝氨酸生物合成途径具有高通量的细胞系，同时保留 PHGDH 低表达且丝氨酸生物合成低的肿瘤细胞系PHGDH 抑制剂将在过度表达 PHGDH 的小鼠肿瘤异种移植模型中进行体内测试。PHGDH 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。