Importance of the interaction proteinprotein of the CaMPDE1A and CaMMLCK complexes in the development of new anti-CaM drugs

Proteinprotein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in proteinproteinligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin-dependent 3,5-cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands IIspectrin peptide (IIspec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124CmBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaMprotein complex under analysis. For the Ca2+CaM, Ca2+CaMPDE1A, and Ca2+CaMMLCK complexes, CPZ apparent dissociation constants (Kds) were 1.11, 0.28, and 0.55M, respectively; and for MBC Kds were 1.43, 1.10, and 0.61M, respectively. In competition experiments the addition of calmodulin binding peptide 1 (IIspec) to Ca2+hCaM M124CmBBr quenched the fluorescence (Kd=2.55 +/- 1.75pM) and the later addition of MBC (up to 16M) did not affect the fluorescent signal. Instead, the additions of IIspec to a preformed Ca2+hCaM M124CmBBrMBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca2+CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca2+CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands. Copyright (c) 2013 John Wiley & Sons, Ltd.