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.