title: Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.
abstract: The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.
authors.affiliation: SmithKline Beecham Pharmaceuticals Ltd., Frythe, Welwyn, England.
authors.forename: B A
authors.initials: BA
authors.lastname: Saxty
authors.forename: R
authors.initials: R
authors.lastname: Novelli
authors.forename: R E
authors.initials: RE
authors.lastname: Dolle
authors.forename: L I
authors.initials: LI
authors.lastname: Kruse
authors.forename: D G
authors.initials: DG
authors.lastname: Reid
authors.forename: P
authors.initials: P
authors.lastname: Camilleri
authors.forename: T N
authors.initials: TN
authors.lastname: Wells
mesh_terms: D001275:ATP Citrate (pro-S)-Lyase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D000818:Animals; D002951:Citrates / Q000138:chemical synthesis / Q000494:pharmacology*; D007202:Indicators and Reagents; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D009206:Myocardium / Q000201:enzymology*; D051381:Rats; D013552:Swine
chemical_list: D002951:Citrates; D007202:Indicators and Reagents; C072213:2,2-difluorocitric acid; D001275:ATP Citrate (pro-S)-Lyase; D000154:Aconitate Hydratase