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

Raynard L. Bateman, Justin Ashworth, John F. Witte, L. J. Baker, Pullooru Bhanumoorthy, David E. Timm, Thomas D. Hurley, Markus Grompe, Ronald W. McClard

Research output: Contribution to journalArticle

10 Scopus citations

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 Ki values of 41 and 12 nM respectively. A high-resolution (1.35 Å; 1 Å = 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 μmol/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.

Original languageEnglish (US)
Pages (from-to)251-260
Number of pages10
JournalBiochemical Journal
Volume402
Issue number2
DOIs
StatePublished - Mar 1 2007

Keywords

  • Crystal structure
  • Fumarylacetoacetate
  • Hepatocyte transplantation
  • Pharmacokineties
  • Transition-state analogue
  • Tyrosinaemia

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Bateman, R. L., Ashworth, J., Witte, J. F., Baker, L. J., Bhanumoorthy, P., Timm, D. E., Hurley, T. D., Grompe, M., & McClard, R. W. (2007). Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: Kinetics, crystal structure and pharmacokinetics. Biochemical Journal, 402(2), 251-260. https://doi.org/10.1042/BJ20060961