Screening method for antagonists that inhibit the binding of calmodulin toa target peptide using surface plasmon resonance

A new screening method was developed for evaluating effects of calmodulin ( CaM) antagonists on the interaction between CaM and its target protein in t he Ca2+-signaling pathway. A binding of Ca2+-CaM to the target peptide, M13 , derived from myosin light-chain kinase (MLCK) is monitored by surface pla smon resonance (SPR) technique. When a sample solution containing Ca2+-CaM was injected into a flow cell with M13 immobilized on the SPR sensor surfac e, the SPR signal largely increased and leveled-off within 3 min. By adding W-7, a CaM antagonist, into the sample solution, the SPR signal at the equ ilibrium state decreased. This decrease in the SPR signal is due to the bin ding of W-7 to Ca2+-CaM, thereby inhibiting the specific interaction betwee n Ca2+-CaM and M13. In the case of other antagonists such as trifluoperazin e, prenylamine and bepridil, upon increasing the concentration of these ant agonists, the initial rate of the increase in the SPR signals decreased, an d the signals reached the same value under the equilibrium state. These IC5 0 values obtained by the present method were consistent with ones obtained earlier by MLCK activity itself. The present method was thus capable of fin ding antagonists inhibiting the interaction between Ca2+-CaM and MLCK. The applicability of the present method for evaluating the effect of endocrine disrupting chemicals toward the Ca2+-signaling pathway was also examined an d discussed. (C) 2001 Elsevier Science BN. All rights reserved.