Identification and Inhibitory Properties of a Novel Ca²⁺/Calmodulin Antagonist

Abstract

We developed a high-throughput yeast-based assay to screen for chemical inhibitors of Ca 2+ / calmodulin-dependent kinase pathways. After screening two small libraries we identified the novel antagonist 125-C9, a substituted ethyleneamine. In vitro kinase assays confirmed that 125-C9 inhibited several CaMKs competitively with Ca 2+ /CaM. This suggested that 125-C9 acted as an antagonist for Ca 2+ /CaM rather than for CaMKs. We confirmed this hypothesis by showing that 125-C9 binds directly to Ca 2+ /CaM using isothermal titration calorimetry. We further characterized 125-C9 binding to Ca 2+ /CaM and compared its properties with those of two well-studied CaM antagonists: trifluoperazine (TFP) and W-13. Isothermal titration calorimetry revealed that 125-C9 binding to CaM is absolutely Ca 2+ -dependent, likely occurs with a stoichiometry of five 125-C9 molecules to one CaM molecule, and involves an exchange of two protons at pH 7.0. Binding of 125-C9 is driven overall by entropy and appears to be competitive with TFP and W-13, which is consistent with occupation of similar binding sites. To test the effects of 125-C9 in living cells, we evaluated mitogen-stimulated re-entry of quiescent cells into proliferation and found similaralthough slightly better -levels of inhibition by 125-C9 than TFP and W-13. Our results not only define a novel Ca 2+ /CaM inhibitor but reveal that chemically unique CaM antagonists can bind CaM by distinct mechanisms but similarly inhibit cellular actions of CaM. KeywordsCalmodulin antagonist; trifluoperazine; W-13; isothermal titration calorimetry; calmodulin binding Calcium (Ca 2+ ) is a major cell signaling transducer that links cell stimuli to specific cell responses. A ten-fold increase in cytoplasmic Ca 2+ levels from 100 nM to 1 μM is a common response to a variety of cell stimuli, resulting in Ca 2+ binding to a variety of calcium binding proteins (1). One of the most prominent calcium binding proteins is calmodulin (CaM), which is evolutionarily conserved amongst all eukaryotic organisms. CaM contains four Ca 2+ binding EF-hand motifs and undergoes conformational changes upon binding of four Ca 2+ molecules. Consequently, two hydrophobic domains of CaM become exposed to the solvent -one on the N-terminal and one on the C-terminal domain -markedly increasing the affinity of Ca 2+ /CaM means001@mc.duke.edu. SUPPORTING INFORMATION AVAILABLE Supporting information includes Extended Experimental Procedures with a description of the synthesis of 125-C9 (Scheme 1S), an example of ITC raw power output of 125-C9 with CaM ( Figure 1S), yeast growth inhibition from the screening process ( Figure 2S), ITC analysis of 125-C9 with CaMKI ( Figure 3S), summary of IC 50 values for 125-C9 on different CaMK (Table 1S), list of χ 2 values for 125-C9 binding to CaM (Table 2S), summary of thermodynamic parameters for 125-C9, TFP and W-13 (Table 3S), and summary of K a values in competition assays (Table 4S). This material is available free of charge via the Internet at http://pubs.acs.org. NIH...

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