Competitive and slow-binding inhibition of calcineurin by drug x immunophilin complexes.
The calcium- and calmodulin-activated protein phosphatase calcineurin (CN) is the target for the immunosuppressive drugs FK506 and cyclosporin A (CsA) when bound to their intracellular receptor proteins, the immunophilins known as FK506-binding protein (FKBP) and cyclophilin A (CypA), respectively. Investigation of the reaction kinetics for inhibition of CN using progress curves of [33P]phosphopeptide hydrolysis revealed slow-binding inhibition by the FK506 . FKBP complex. Final steady-state velocities were extracted by curve fitting over a range of substrate and inhibitor concentrations; the data fit well to a simple competitive inhibition model with a Ki of 14 nM for the FK506 . FKBP complex. The FKBP complex with L-732,531, an analog of FK506 containing a hydroxyethylindole substituent, was significantly more potent than FK506 x FKBP and was investigated in greater detail. The hyperbolic dependencies of the initial velocities and the first-order rate constants for the approach to steady state upon the concentration of L-732,531 x FKBP were consistent with a two-step inhibition mechanism in which the initial E x I complex slowly isomerizes to a more stable E x I* form. The reverse isomerization rate constant with L-732,531 . FKBP was markedly slower than that with FK506 x FKBP and is likely responsible for the higher affinity of the former for CN. Inhibition of CN by the CsA x CypA complex was not time-dependent, but the data did conform to a competitive inhibition model like FK506 x FKBP. These results are consistent with the hypothesis that both classes of drug x immunophilin complexes interact with a common locus on CN which excludes phosphopeptide binding in the enzyme's active site.
Department of Molecular Design and Diversity, Merck Research Laboratories, Rahway, New Jersey, 07065, USA. Scott_Salowe@Merck.com
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Hermes
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D019703:Calcineurin / Q000378:metabolism; D065095:Calcineurin Inhibitors; D002352:Carrier Proteins / Q000378:metabolism* / Q000494:pharmacology; D016572:Cyclosporine / Q000378:metabolism / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism* / Q000494:pharmacology; D006360:Heat-Shock Proteins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007166:Immunosuppressive Agents / Q000378:metabolism* / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D022021:Tacrolimus Binding Proteins
D065095:Calcineurin Inhibitors; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007166:Immunosuppressive Agents; C076383:L 685818; C110188:32-O-(1-hydroxyethylindol-5-yl)ascomycin; D016572:Cyclosporine; D019703:Calcineurin; D022021:Tacrolimus Binding Proteins; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus