Application of a Fluorescent Biosensor in Determining the Binding of 5-HT to Calmodulin

Abstract

Here, we show the utility of the fluorescent biosensor hCaM-M124C-mBBr in detecting and determining the affinity of serotonin (5-HT). We obtained a Kd of 5-HT (0.71 μm) for the first time, the same order of magnitude as most anti-CaM drugs. This data can contribute to understanding the direct and indirect modulation of CaM on its binding proteins when the 5-HT concentration varies in different tissues or explain some of the side effects of anti-CaM drugs. On the other hand, molecular modeling tools help the rational design of biosensors and adequately complement the experimental results. For example, the docking study indicates that 5-HT binds at the same site as chlorpromazine (site 1) with a theoretical Ki of 2.84 μM; while the molecular dynamics simulations indicate a stability of the CaM–5-HT complex with a theoretical ΔG of −4.85 kcal mol−1, where the enthalpy contribution is greater. Thus, the combination of biotechnology and bioinformatics helps in the design and construction of more robust biosensors.

Calmodulin-Targeting Molecules from Ageratina grandifolia

“…Extensive chromatography of a CaM inhibitor acetone extract (Figure S1) from the aerial parts of A. grandifolia afforded four new compounds, including 2-hydroxy-α-truxillic acid (2), (3R,4S)-2,2-dimethyl-3-hydroxy-4-(1-angeloyloxy)-6-acetyl-7methoxychromane (3), N-tricosanoyltyramine (4), and grandifolamide (5). In addition, the known compounds 2,2dimethyl-3R-hydroxy-4S-(1-angeloyloxy)-6-acetyl-chromane (1), 15,16 quercetagetin-7-O-β-D-glucopyranoside (6), 18 sonorol (7), 19 3,5-diprenyl-4-hydroxyacetophenone (8), 20 demethoxyencecalin (9), 21 O-methylencecalinol (10), 22 encecalin (11), 23 encecalinol (12), 23 spathulenol (13), 24 dehydroespeletone ( 14), 25 and p-hydroxybenzoic acid (15) 26 were isolated. All of these compounds were characterized by comparing their spectroscopic data with those previously reported.…”

“…Each globular domain contains two Ca 2+ -binding sites. Upon binding Ca 2+ ions, extensive hydrophobic surfaces of CaM become exposed for interacting with its target proteins (>100), thus mediating many physiological processes such as apoptosis, intracellular movement, inflammation, metabolism, proliferation, short- and long-term memory, and smooth muscle contraction, to mention a few. − It is essential to remark that CaM is a molecular target of compounds with therapeutic value for treating sulfur-mustard-induced skin lesions, cancer, and depression, among others. − …”

Bisindolylmaleimides New Ligands of CaM Protein

“…The estimated K ds are in the nM order, with the following affinity order: BIM-VII > BIM-XI > BIM-IV > BIM-X > BIM-II > CPZ ( Table 1 ); which makes this series of compounds desirable to be considered as possible anti-CaM drugs. Many of the inhibitors reported so far are in the micromolar range, such as KAR-2 (5 mM) [ 28 ], Imipramine (14 mM) [ 29 ], Serotonin (0.71 mM), Chlorpromazine (0.97 mM) [ 26 ], Trifluoperazine (1 mM), W7 (7 mM) [ 30 ] and Lubeluzole (2.9 mM) [ 31 ]; only some peptides are in the nanomolar range [ 32 ]. The stoichiometry of the compounds is from the ratio of 1:2 to 1:4, which is mainly attributed to the size of each compound.…”

“…In many cases, slight modifications in chemical structure can significantly alter a compound’s ability to bind to CaM and inhibit its activity. A direct detection tool developed by our group is the calmodulin biosensor ( h CaM-M124C- mBBr ), which we have used to detect the binding of various ligands [ 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. On the other hand, theoretical and computational studies, such as cheminformatics, docking, and molecular dynamics simulations (MD), are considered tools of great value to complement the experimental data.…”