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We report results of 12 ns, all-atom molecular dynamics simulation (MDS) and Poisson-Boltzmann free energy calculations (PBFE) on calmodulin (CaM) bound to two molecules of trifluoperazine (TFP) and of N-(3,3, diphenylpropyl)- N'-[1- R-(3,4-bis-butoxyphenyl)-ethyl]-propylenediamine (DPD). X-ray data show very similar structures for the two complexes, yet the antagonists significantly differ with respect to their CaM binding affinities, the neutral DPD is much more potent. The goal of the study was to unravel the reason why TFP is less potent although its positive charge should facilitate binding. The electrostatic energy terms in CHARMM and binding free energy terms of the PBFE approach showed TFP a better antagonist, while inspection of hydrophobic contacts supports DPD binding. Detailed inspection of the amino acid contributions of PBFE calculations unravel that steric reasons oppose the favorable binding of TFP. Structural conditions are given for a successful drug design strategy, which may benefit also from charge-charge interactions.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin., (Copyright 2000 Academic Press.)