title: Inhibition of squalene synthase in vitro by 3-(biphenyl-4-yl)-quinuclidine.
abstract: Squalene synthase (SQS) catalyses a step following the final branch in the pathway of cholesterol biosynthesis. Inhibition of this enzyme, therefore, is an approach for the treatment of atherosclerosis with the potential for low side effects. We have characterised the inhibition of rat liver microsomal SQS by 3-(biphenyl-4-yl)quinuclidine (BPQ). BPQ follows slow binding kinetics in that the rate of accumulation of product decreases with time if the inhibitor is added when the assay is started. Preincubation of BPQ and SQS leads to a biphasic dose-response where accumulation of product is linear with time only for the sensitive phase. When the farnesyl pyrophosphate (FPP) substrate is present at 19.6 microM, approximately 77% of the SQS activity is sensitive to the inhibitor (vOs) and the remainder is insensitive (vOi). The apparent inhibition constants (K'i values) are respectively K'is = 4.5 nM and K'ii = 1300 nM. Similar biphasic behaviour is exhibited by other inhibitors and in microsomes prepared from human and marmoset liver. As the concentration of FPP is reduced below 19.6 microM, there is a decrease in the relative contribution from vOi. Conversely, the value of K'is for BPQ remains constant when the FPP concentration is changed, showing noncompetitive kinetics with respect to this substrate. Possible causes of the observed kinetics are discussed. Inhibition by BPQ is said to follow tight binding kinetics because the value of K'is is similar to the concentration of inhibitor binding sites. Thus, to avoid an artefactual variation in potency when the enzyme concentration is varied, it is necessary to allow for the effects of depletion of free inhibitor. Furthermore, estimates of potency that average activity across the two phases are influenced by the relative contributions of each phase. These contributions differ according to the FPP concentration and the species used as the source of microsomes. Thus, it is necessary to separate the phases to compare measurements made in different experiments. Our observations indicate that careful experimental design and data analysis are required to characterise the kinetics of SQS inhibitors.
authors.affiliation: ZENECA Pharmaceuticals, Macclesfield, Cheshire, U.K.
authors.forename: W H
authors.initials: WH
authors.lastname: Ward
authors.forename: G A
authors.initials: GA
authors.lastname: Holdgate
authors.forename: S
authors.initials: S
authors.lastname: Freeman
authors.forename: F
authors.initials: F
authors.lastname: McTaggart
authors.forename: P A
authors.initials: PA
authors.lastname: Girdwood
authors.forename: R G
authors.initials: RG
authors.lastname: Davidson
authors.forename: K B
authors.initials: KB
authors.lastname: Mallion
authors.forename: G R
authors.initials: GR
authors.lastname: Brown
authors.forename: M A
authors.initials: MA
authors.lastname: Eakin
mesh_terms: D000818:Animals; D001665:Binding Sites; D002144:Callithrix; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D011812:Quinuclidines / Q000378:metabolism / Q000493:pharmacokinetics / Q000494:pharmacology*; D051381:Rats; D012680:Sensitivity and Specificity
chemical_list: C099380:3-(biphenyl-4-yl)quinuclidine; D004791:Enzyme Inhibitors; D011812:Quinuclidines; D013186:Farnesyl-Diphosphate Farnesyltransferase