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

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 cata...

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Veröffentlicht in:Biochemical journal 2007-03, Vol.402 (2), p.251-260
Hauptverfasser: Bateman, Raynard L, Ashworth, Justin, Witte, John F, Baker, L-J, Bhanumoorthy, Pullooru, Timm, David E, Hurley, Thomas D, Grompe, Markus, McClard, Ronald W
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container_end_page 260
container_issue 2
container_start_page 251
container_title Biochemical journal
container_volume 402
creator Bateman, Raynard L
Ashworth, Justin
Witte, John F
Baker, L-J
Bhanumoorthy, Pullooru
Timm, David E
Hurley, Thomas D
Grompe, Markus
McClard, Ronald W
description 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.
doi_str_mv 10.1042/BJ20060961
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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. 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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. 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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.</abstract><cop>England</cop><pub>Portland Press Ltd</pub><pmid>17064256</pmid><doi>10.1042/BJ20060961</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record>
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subjects Animals
Binding Sites
Biomimetic Materials - chemical synthesis
Biomimetic Materials - chemistry
Biomimetic Materials - pharmacokinetics
Butyrates - blood
Butyrates - chemical synthesis
Butyrates - chemistry
Butyrates - pharmacokinetics
Crystallography, X-Ray
Humans
Hydrolases - antagonists & inhibitors
Hydrolases - chemistry
Hydrolases - metabolism
Kinetics
Male
Mice
Mice, Inbred C57BL
Models, Molecular
Molecular Structure
Organophosphorus Compounds - blood
Organophosphorus Compounds - chemical synthesis
Organophosphorus Compounds - chemistry
Organophosphorus Compounds - pharmacokinetics
Phosphinic Acids - blood
Phosphinic Acids - chemical synthesis
Phosphinic Acids - chemistry
Phosphinic Acids - pharmacokinetics
Protease Inhibitors - blood
Protease Inhibitors - chemical synthesis
Protease Inhibitors - chemistry
Protease Inhibitors - pharmacokinetics
Protein Structure, Tertiary
Time Factors
title Slow-onset inhibition of fumarylacetoacetate hydrolase by phosphinate mimics of the tetrahedral intermediate: kinetics, crystal structure and pharmacokinetics
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