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

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A new screening method was developed for evaluating effects of calmodulin (CaM) antagonists on the interaction between CaM and its target protein in the Ca2+-signaling pathway. A binding of Ca2+-CaM to the target peptide, M13, derived from myosin light-chain kinase (MLCK) is monitored by surface plasmon resonance (SPR) technique. When a sample solution containing Ca2+-CaM was injected into a flow cell with M13 immobilized on the SPR sensor surface, the SPR signal largely increased and leveled-off within 3min. By adding W-7, a CaM antagonist, into the sample solution, the SPR signal at the equilibrium state decreased. This decrease in the SPR signal is due to the binding of W-7 to Ca2+-CaM, thereby inhibiting the specific interaction between Ca2+-CaM and M13. In the case of other antagonists such as trifluoperazine, prenylamine and bepridil, upon increasing the concentration of these antagonists, the initial rate of the increase in the SPR signals decreased, and the signals reached the same value under the equilibrium state. These IC50 values obtained by the present method were consistent with ones obtained earlier by MLCK activity itself. The present method was thus capable of finding 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 and discussed.