title: Importance of the interaction protein-protein of the CaM-PDE1A and CaM-MLCK complexes in the development of new anti-CaM drugs.
abstract: Protein-protein 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 protein-protein-ligands 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 αII-spectrin peptide (αII-spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C-mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM-protein complex under analysis. For the Ca(2+) -CaM, Ca(2+) -CaM-PDE1A, and Ca(2+) -CaM-MLCK complexes, CPZ apparent dissociation constants (Kds ) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC Kds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII-spec) to Ca(2+) -hCaM M124C-mBBr quenched the fluorescence (Kd  = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII-spec to a preformed Ca(2+) -hCaM M124C-mBBr-MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca(2+) -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 Ca(2+) -CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands.
authors: Instituto Nacional de Medicina Genómica, Secretaría de Salud, México DF, 14610, México. mgonzalez@inmegen.gob.mx
authors: Martin
authors: M
authors: González-Andrade
authors: Rachel
authors: R
authors: Mata
authors: Abraham
authors: A
authors: Madariaga-Mazón
authors: Rogelio
authors: R
authors: Rodríguez-Sotres
authors: Luis
authors: L
authors: Del Pozo-Yauner
authors: Alejandro
authors: A
authors: Sosa-Peinado
mesh_terms: D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000737:chemistry*; D002746:Chlorpromazine / Q000737:chemistry*; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1 / Q000737:chemistry*; D055808:Drug Discovery; D006801:Humans; D026121:Indole Alkaloids / Q000737:chemistry*; D062105:Molecular Docking Simulation; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
chemical_list: D002147:Calmodulin; D026121:Indole Alkaloids; C529354:malbrancheamide; D009219:Myosin-Light-Chain Kinase; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; C517265:PDE1A protein, human; D002118:Calcium; D002746:Chlorpromazine