title: Development of the fluorescent biosensor hCalmodulin (hCaM)L39C-monobromobimane(mBBr)/V91C-mBBr, a novel tool for discovering new calmodulin inhibitors and detecting calcium.
abstract: A novel, sensible, and specific fluorescent biosensor of human calmodulin (hCaM), namely hCaM L39C-mBBr/V91C-mBBr, was constructed. The biosensor was useful for detecting ligands with opposing fluorescent signals, calcium ions (Ca(2+)) and CaM inhibitors in solution. Thus, the device was successfully applied to analyze the allosteric effect of Ca(2+) on trifluoroperazine (TFP) binding to CaM (Ca(2+)K(d) = 0.24 μM ± 0.03 with a stoichiometry 4.10 ± 0.15; TFPK(d) ∼ 5.74-0.53 μM depending on the degree of saturation of Ca(2+), with a stoichiometry of 2:1). In addition, it was suitable for discovering additional xanthones (5, 6, and 8) with anti-CaM properties from the fungus Emericella 25379. The affinity of 1-5, 7, and 8 for the complex (Ca(2+))(4)-CaM was excellent because their experimental K(d)s were in the nM range (4-498 nM). Docking analysis predicted that 1-8 bind to CaM at sites I, III, and IV as does TFP.
authors.affiliation: Universidad Nacional Autónoma de México, México DF 04510, México.
authors.forename: Martin
authors.initials: M
authors.lastname: Gonzalez-Andrade
authors.forename: Jose
authors.initials: J
authors.lastname: Rivera-Chavez
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sosa-Peinado
authors.forename: Mario
authors.initials: M
authors.lastname: Figueroa
authors.forename: Rogelio
authors.initials: R
authors.lastname: Rodriguez-Sotres
authors.forename: Rachel
authors.initials: R
authors.lastname: Mata
mesh_terms: D014150:Antipsychotic Agents / Q000032:analysis / Q000737:chemistry; D001665:Binding Sites; D015374:Biosensing Techniques; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D002118:Calcium / Q000032:analysis*; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D032842:Emericella / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D008968:Molecular Conformation; D010330:Patents as Topic; D014268:Trifluoperazine / Q000378:metabolism; D044004:Xanthones / Q000032:analysis / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D014150:Antipsychotic Agents; D001643:Bridged Bicyclo Compounds; D002147:Calmodulin; D005456:Fluorescent Dyes; D008024:Ligands; D044004:Xanthones; C560300:hCaM L39C-mBBr-V91C-mBBr; D014268:Trifluoperazine; D002118:Calcium; C028381:monobromobimane