Class-Selective Drug Detection:  Fluorescently-Labeled Calmodulin as the Biorecognition Element for Phenothiazines and Tricyclic Antidepressants

Abstract

A small-scale, homogeneous, rapid sensing system for phenothiazines and tricyclic antidepressants (TCAs) has been developed by employing fluorescently labeled mutant calmodulin (CaM) as the recognition element. A calmodulin mutant containing a unique cysteine residue at position 109 on the protein was expressed in Escherichia coli. Following purification, the environment-sensitive, thiol-specific fluorophores N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), and 4-[N-(2-(iodoacetoxy)ethyl)-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole (IANBD ester) were coupled to the C109 site of the mutant protein. The response of labeled CaM in the presence of calcium to increasing concentrations of chlorpromazine hydrochloride (CPZ), as well as other phenothiazines and structurally related antipsychotics and antidepressants, was investigated. Fluorescence measurements were performed on benchtop and microtiter plate fluorometers. The responses were characterized as a change in the signal intensity of the labeled protein upon ligand binding, and the stability of the system was monitored over a nine-month period. The assay showed specificity for the phenothiazine and TCA classes of drugs, with limits of detection in the micromolar range. Selectivity studies indicated negligible response of the biosensing system to structurally unrelated compounds. This work represents a proof-of-concept assay for rapid, homogeneous detection of drugs employing binding proteins as the biorecognition element.

Fluorescence, circular dichroism, NMR, and docking studies of the interaction of the alkaloid malbrancheamide with calmodulin

“…The regulatory properties of CaM can be inhibited by a wide range of drugs and peptides, which modify its function by blocking its interaction with other proteins. Such is the case of the antipsychotics phenotiazines trifluoperazine (TFP) and chlorpromazine (CPZ), which bind to CaM through hydrophobic interactions with high affinity and known stoichiometry [7][8][9] . Other compounds, however, bind to CaM with lower specificity and uncertain stoichiometry 10,11 .…”

Fluorescent Biosensors for Protein Interactions and Drug Discovery

“…In this sense several CaM has been used a protein target to interact with several protein by fluorescent attachment, for example: interaction between calmodulin (CaM) and a CaMbinding peptide of the ryanodine receptor (CaMBP) and its sub-fragments F1, has been measured by the mutant Thr31Cys with the fluorescent group badan attached (Sharma, Deo et al 2005). A mutant of CaM coupled to three different environment-sensitive fluorophores (MDCC, acrylodan, and IANBD ester) was detected the CaM interaction with phenothiazines and related tryclic antidepressants (Douglass, Salins et al 2002). Recently Gonález-Andrade and col, has been designed a alternative biosensing assay for CaM inhibitors by chemical modification of bromobimane at position 124 (Gonzalez-Andrade, et al 2009;Figueroa, et al 2010), that allowed to determine the IC 50 …”

Anti-calmodulins and Tricyclic Adjuvants in Pain Therapy Block the TRPV1 Channel

“…Ca 2+ , among others, selectively interacts with the so-called “EF-hand”/Ca 2+ -binding protein motifs located either intracellularly or extracellularly [17]. Dose-dependent interaction of Ca 2+ with calmodulin elicits a robust conformational change that exposes hidden hydrophobic domains required for subsequent effects on down-stream protein targets [18], [19]. Ca 2+ - calmodulin complex, formed upon entry of Ca 2+ to the cytosol resulting in elevation of intracellular free calcium [Ca 2+ ] i can turn on/off different enzymes and ion channels.…”