title: 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.
authors.affiliation: Arthur F. Scott Laboratory of Chemistry, Reed College, 3203 SE Woodstock Blvd, Portland, OR 97202, USA.
authors.forename: Raynard L
authors.initials: RL
authors.lastname: Bateman
authors.forename: Justin
authors.initials: J
authors.lastname: Ashworth
authors.forename: John F
authors.initials: JF
authors.lastname: Witte
authors.forename: L-J
authors.initials: LJ
authors.lastname: Baker
authors.forename: Pullooru
authors.initials: P
authors.lastname: Bhanumoorthy
authors.forename: David E
authors.initials: DE
authors.lastname: Timm
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Hurley
authors.forename: Markus
authors.initials: M
authors.lastname: Grompe
authors.forename: Ronald W
authors.initials: RW
authors.lastname: McClard
mesh_terms: D000818:Animals; D001665:Binding Sites; D040761:Biomimetic Materials / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics*; D002087:Butyrates / Q000097:blood / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics*; D018360:Crystallography, X-Ray; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D015394:Molecular Structure; D009943:Organophosphorus Compounds / Q000097:blood / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics*; D010721:Phosphinic Acids / Q000097:blood / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics*; D011480:Protease Inhibitors / Q000097:blood / Q000138:chemical synthesis / Q000737:chemistry* / Q000493:pharmacokinetics*; D017434:Protein Structure, Tertiary; D013997:Time Factors
chemical_list: C579246:4-((2-carboxyethyl)hydroxyphosphinyl)-3-oxobutyrate; D002087:Butyrates; D009943:Organophosphorus Compounds; D010721:Phosphinic Acids; D011480:Protease Inhibitors; D006867:Hydrolases; C020156:fumarylacetoacetase