IngentaConnect Competitive and Slow-Binding Inhibition of Calcineurin by Drug Im...

Abstract:

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 33Pphosphopeptide 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 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 FKBP were consistent with a two-step inhibition mechanism in which the initial E I complex slowly isomerizes to a more stable E I form. The reverse isomerization rate constant with L-732,531 FKBP was markedly slower than that with FK506 FKBP and is likely responsible for the higher affinity of the former for CN. Inhibition of CN by the CsA CypA complex was not time-dependent, but the data did conform to a competitive inhibition model like FK506 FKBP. These results are consistent with the hypothesis that both classes of drug immunophilin complexes interact with a common locus on CN which excludes phosphopeptide binding in the enzymes active site. Copyright 1998 Academic Press.

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Competitive and Slow-Binding Inhibition of Calcineurin by Drug Immunophilin Complexes