Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: kinetics, crystal structure and pharmacokinetics

Abstract

FAH (fumarylacetoacetate hydrolase) catalyses the final step of tyrosine catabolism to produce fumarate and acetoacetate. HT1 (hereditary tyrosinaemia type 1) results from deficiency of this enzyme. Previously, we prepared a partial mimic of the putative tetrahedral intermediate in the reaction catalysed by FAH co-crystallized with the enzyme to reveal details of the mechanism [Bateman, Bhanumoorthy, Witte, McClard, Grompe and Timm (2001) J. Biol. Chem. 276, 15284-15291]. We have now successfully synthesized complete mimics CEHPOBA {4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate} and COPHPAA {3-[(3-carboxy-2-oxopropyl)hydroxyphosphinyl]acrylate}, which inhibit FAH in slow-onset tight-binding mode with K(i) values of 41 and 12 nM respectively. A high-resolution (1.35 A; 1 A=0.1 nm) crystal structure of the FAH.CEHPOBA complex was solved to reveal the affinity determinants for these compounds and to provide further insight into the mechanism of FAH catalysis. These compounds are active in vivo, and CEHPOBA demonstrated a notable dose-dependent increase in SA (succinylacetone; a metabolite seen in patients with HT1) in mouse serum after repeated injections, and, following a single injection (1 mumol/g; intraperitoneal), only a modest regain of FAH enzyme activity was detected in liver protein isolates after 24 h. These potent inhibitors provide a means to chemically phenocopy the metabolic defects of either HT1 or FAH knockout mice and promise future pharmacological utility for hepatocyte transplantation.

A universal system to select gene-modified hepatocytes in vivo

“…In order to apply this system in any genetic liver disease, selection must work in animals that not deficient in FAH. We previously described a small-molecule inhibitor of FAH that was capable of inducing FAA accumulation and exerting selective pressure in vivo in mice (22, 23). In order to demonstrate that the Hpd shRNA could be used in any genetic background, neonatal wild-type C57Bl6 mice were injected with 2×10 11 vg of the promoterless F9 generide construct.…”

RNAi reveals proteins for metabolism and protein processing associated with Langat virus infection in Ixodes scapularis (black-legged tick) ISE6 cells

“…Theoretically, small molecules that disrupt one or more tick proteins could be used to limit virus transmission from the tick to mammalian reservoirs and intermediate hosts. Small molecule inhibitors of FAH [57, 58] and PARP [59, 60] are known but their potential to regulate flavivirus infection in arthropods has not been investigated. In addition, there is precedent for development of transmission blocking vaccines against TBFs.…”

Structural Insight into Substrate Binding and Catalysis of a Novel 2-Keto-3-deoxy-d-arabinonate Dehydratase Illustrates Common Mechanistic Features of the FAH Superfamily

“…This residue is highly conserved throughout the FAH superfamily (Supplementary Data Table 1), and may serve to exclude water from the active center, as has been suggested for FAH. 15 Experiments aimed at deciding between the two possible mechanisms could include kinetic isotope effect studies, and isotope labeling studies to determine the origin of the C3 or C5 protons in the product.…”

“…10,15,25 The dimeric FAH enzyme catalyzes oxo-hydrolytic cleavage of a carbon-carbon bond of the substrate and produces fumarate and acetoacetate as the last step in the mammalian tyrosine catabolism. A genetic loss of enzyme function in humans leads to an autosomal recessive disorder called hereditary tyrosinemia type I (HTI).…”