Keywords: Fret, Hts, Proteases, Small Molecules, Trypsin Inhibitors, Diagnosis, Energy Transfer, Enzyme Activity, Fluorescence, Naphthalene, Peptides, Enzyme Inhibition, Alpha 1 Antitrypsin, Proteinase Inhibitor, Serine Proteinase, Article, Assay, Chemical Structure, Controlled Study, Excitation, Fluorescence Resonance Energy Transfer, Fluorometry, Ic 50, Molecular Docking, Spectral Sensitivity, Fluorescent Dyes, Naphthalenesulfonates, P-Dimethylaminoazobenzene, Protease Inhibitors,
Affiliations: *** IBB - CNR ***
Istituto di Biostrutture e Bioimmagini, IBB-CNR, Via Mezzocannone 16, 80134 Naples, Italy
Department of Physics, Sapienza Università di Roma, P.le Aldo Moro, 5, 00185 Rome, Italy
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Identification of Protease Inhibitors by a Fast Fluorimetric Assay
Anomalous protease activities are associated with many diseases. Great efforts are paid for selecting specific protease modulators for therapeutic approaches. We have selected new modulators of enzyme activity by an homogeneous assay based on a doubly labeled small peptide used as substrate of trypsin. The substrate incorporates the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS) at one end and an EDANS-quenching moiety (Dabcyl, (4-(4-dimethylaminophenylazo)-benzoic acid)) on the other end. Following cleavage by trypsin, the peptide-EDANS product is released interrupting the fluorescence resonance energy transfer effect and yielding bright fluorescence, which can be detected using excitation wavelengths at 335-345 nm and emission wavelengths at 485-510 nm. The method optimized, tested by detecting the strong inhibiting effect of alpha 1-antitrypsin on trypsin activity, has been developed on 384 multi-well plates in a volume of 10 mu L, using an automated platform. From the screening of a chemical library, four compounds that inhibit trypsin activity with IC(50)s in the micromolar range have been identified. Interestingly, the most active compound (M4) shows a chemical structure recapitulating that of other more potent inhibitors with thiourea and halogenated centers. Molecular docking studies show that M4 is a competitive inhibitor recognizing most residues within or nearby the catalytic pocket.
Identification of Protease Inhibitors by a Fast Fluorimetric Assay
Santulli G, Cipolletta E, Sorriento D, Del Giudice C, Anastasio A, Monaco S, Maione AS, Condorelli G, Puca A, Trimarco B, Illario M, Iaccarino G * CaMK4 gene deletion induces hypertension(348 views) J Am Heart Assoc Journal Of The American Heart Association (ISSN: 2047-9980), 2012; 1(4): N/D-N/D. Impact Factor:2.882 ViewExport to BibTeXExport to EndNote