Screening calmodulin-binding ligands using intensity-fading matrix-assisted laser desorption/ionization mass spectrometry

Abstract

RATIONALE Calmodulin (CaM) plays an important role in the regulation of metabolism, cytoskeleton and cell proliferation. CaM antagonists are a class of drugs that can bind to CaM and modulate the interactions between CaM and their target biological processes. Screening new calmodulin antagonists and developing novel methods for detecting calmodulin ligands are important for developing novel anti-cancer drugs that bind specifically to CaM. METHODS An intensity-fading matrix-assisted laser desorption/ionization mass spectrometry (IF-MALDI-MS) method for screening calmodulin ligands was established, with the non-binding drug propranolol as the internal control. The experimental sample was prepared by mixing the positive ligand trifluoperazine (or chlorpromazine), propranolol and Ca2+-CaM. The control sample was treated in the same way without the addition of CaM. The experimental and control samples were both analyzed by MALDI-MS. Based on the relative intensity fading (IF) of the ligand to propranolol, the MS conditions were optimized and then used to study the binding of eight alkaloids and calmodulin. Competitive experiments were performed in a similar way by adding two drugs to compare their binding affinities with calmodulin. RESULTS The matrix 2,6-dihydroxyacetophenone (DHAP) was suitable for detecting calmodulin ligands. Compared with propranolol, the relative intensities of six free drugs (berbamine, tetrandrine, papaverine, reserpine, brucine and tetrahydropalmatine) clearly faded after the addition of calmodulin, indicating that they can bind with CaM. The other two alkaloids (strychnine and piperine) had no or weak interaction with the target protein. Based on the data from the competitive binding experiments, the binding affinities of five drugs to calmodulin were obtained in the order: tetrandrine>trifluoperazine>berbamine>chlorpromazine>imipramine. CONCLUSIONS The IF-MALDI-MS method was successfully applied to screen novel calmodulin agents at both qualitative and semiquantitative levels. The new ligands may be novel leads for CaM antagonists. Copyright (c) 2013 John Wiley & Sons, Ltd