title: An alternative assay to discover potential calmodulin inhibitors using a human fluorophore-labeled CaM protein.
abstract: This article describes the development of a new fluorescent-engineered human calmodulin, hCaM M124C-mBBr, useful in the identification of potential calmodulin (CaM) inhibitors. An hCaM mutant containing a unique cysteine residue at position 124 on the protein was expressed, purified, and chemically modified with the fluorophore monobromobimane (mBBr). The fluorophore-labeled protein exhibited stability and functionality to the activation of calmodulin-sensitive cAMP phosphodiesterase (PDE1) similar to wild-type hCaM. The hCaM M124C-mBBr is highly sensitive to detecting inhibitor interaction given that it showed a quantum efficiency of 0.494, approximately 20 times more than the value for wild-type hCaM, and a large spectral change ( approximately 80% quenching) when the protein is in the presence of saturating inhibitor concentrations. Two natural products previously shown to act as CaM inhibitors, malbrancheamide (1) and tajixanthone hydrate (2), and the well-known CaM inhibitor chlorpromazine (CPZ) were found to quench the hCaM M124C-mBBr fluorescence, and the IC(50) values were comparable to those obtained for the wild-type protein. These results support the use of hCaM M124C-mBBr as a fluorescence biosensor and a powerful analytical tool in the high-throughput screening demanded by the pharmaceutical and biotechnology industries.
authors.affiliation: Facultad de Medicina, Universidad Nacional Autónoma de México, México D.F. 04510, Mexico.
authors.forename: Martín
authors.initials: M
authors.lastname: González-Andrade
authors.forename: Mario
authors.initials: M
authors.lastname: Figueroa
authors.forename: Rogelio
authors.initials: R
authors.lastname: Rodríguez-Sotres
authors.forename: Rachel
authors.initials: R
authors.lastname: Mata
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sosa-Peinado
mesh_terms: D015374:Biosensing Techniques; D001643:Bridged Bicyclo Compounds; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000235:genetics; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D006801:Humans; D026121:Indole Alkaloids / Q000737:chemistry; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D001643:Bridged Bicyclo Compounds; D002147:Calmodulin; D005456:Fluorescent Dyes; D026121:Indole Alkaloids; C529354:malbrancheamide; D010727:Phosphoric Diester Hydrolases; C028381:monobromobimane