Identification of activators of human fumarate hydratase by quantitative high-throughput screening

Abstract

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 FH gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis, renal cell cancer (HLRCC), and other diseases. Currently there are no high-quality small molecule probes for studying human fumarate hydratase. 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-pyrrolopyrimidine-diones were identified as activators of human fumarate hydratase. These compounds were not substrates of fumarate hydratase, were inactive in a malate dehydrogenase counter screen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human fumarate hydratase 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 fumarate hydratase reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates. Introduction:Fumarate hydratase (FH) is an important metabolic enzyme in the Krebs-cycle, which reversibly catalyzes the conversion of fumarate to malate (hydration). It is encoded by the FH gene, which produces two isoforms of protein by alternative splicing, a cytosolic form and a mitochondrial form. The mitochondrial form of FH contains a N-terminal mitochondrial targeting sequence, which is removed in the mitochondrion to generate the same protein as that in the cytoplasm.Despite decades of research, a detailed understanding of the mechanism of FH's biochemical activity remains a matter of debate. The active enzyme is a homo-tetramer in which each active site is made up of residues at the interface of three of the four subunits 1 . FH participates in ATP production in mitochondria and the metabolism of amino acids and fumarate in cytoplasm.In humans, mutations of FH gene lead to defects of the enzymatic activity either by indirectly compromising the integrity of the protein's core architecture or directly affecting residues within the active site that regulate catalytic activity of the enzyme 2 . Mutations of FH gene have been mainly associated with two heritable diseases: fumarate hydratase deficiency, and hereditary leiomyomatosis and renal cell cancer (HLRCC). Homozygous germline mutations in FH gene cause autosomal recessive fumarate hydratase deficiency syndrome. It is characterized by early-onset hypotonia, profound psychomotor retardation, seizures, facial dysmorphism and brain abnormalities 3 . Mutations of FH gene severely impair the activity of enzyme which leads to the defect of energy production and the accumulation of fumarate, which is believed to be the cause of clinical symptoms 4 . Heterozygous germline mutati...