National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.
SLAS Discov. 2020 Jan;25(1):43-56. doi: 10.1177/2472555219873559. Epub 2019 Sep 14.
Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a "tough target" to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.
延胡索酸水合酶 (FH) 是一种代谢酶，属于三羧酸循环，可逆地催化延胡索酸水合生成苹果酸。基因的突变与延胡索酸水合酶缺乏症 (FHD)、遗传性平滑肌瘤病和肾细胞癌 (HLRCC) 以及其他疾病有关。目前，尚无用于研究人 FH 的高质量小分子探针。为了解决这个问题，我们开发了一种定量高通量筛选 (qHTS) FH 测定法，并对来自内部文库的总共 57,037 种化合物进行了剂量反应筛选。虽然没有确认 FH 的抑制剂，但鉴定出一系列苯并吡咯并嘧啶二酮类化合物是人类 FH 的激活剂。这些化合物不是 FH 的底物，在苹果酸脱氢酶对照筛选中无活性，并且没有检测到可检测的氧化还原活性。两种来自该系列的化合物与人类 FH 的结合通过微尺度热泳法得到证实。在这次筛选活动中，低命中率证实了 FH 是一个难以调节的“难靶点”，并且这里报道的人 FH 的小分子激活剂可能作为进一步优化和开发为人类 FH 的细胞探针和潜在药物候选物的起点。
