title: FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay: New Internally Quenched Fluorogenic Substrates for High-Throughput Screening.
abstract: In this work, a sensitive and convenient protease-based fluorimetric high-throughput screening (HTS) assay for determining peptidyl-prolyl cis-trans isomerase activity was developed. The assay was based on a new intramolecularly quenched substrate, whose fluorescence and structural properties were examined together with kinetic constants and the effects of solvents on its isomerization process. Pilot screens performed using the Library of Pharmacologically Active Compounds (LOPAC) and cyclophilin A (CypA), as isomerase model enzyme, indicated that the assay was robust for HTS, and that comparable results were obtained with a CypA inhibitor tested both manually and automatically. Moreover, a new compound that inhibits CypA activity with an IC50 in the low micromolar range was identified. Molecular docking studies revealed that the molecule shows a notable shape complementarity with the catalytic pocket confirming the experimental observations. Due to its simplicity and precision in the determination of extent of inhibition and reaction rates required for kinetic analysis, this assay offers many advantages over other commonly used assays.
authors.affiliation: Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy Dipartimento di Farmacia, Università di Napoli "Federico II", Napoli, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Caporale
authors.affiliation: Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy Dipartimento di Farmacia, Università di Napoli "Federico II", Napoli, Italy.
authors.forename: Fabiola
authors.initials: F
authors.lastname: Mascanzoni
authors.affiliation: Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy.
authors.forename: Biancamaria
authors.initials: B
authors.lastname: Farina
authors.affiliation: Molecular Modeling Section, Dipartimento di Scienze del Farmaco, Università di Padova, Padova, Italy.
authors.forename: Mattia
authors.initials: M
authors.lastname: Sturlese
authors.affiliation: Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Università di Napoli, Caserta, Italy.
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Di Sorbo
authors.affiliation: Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Università di Napoli, Caserta, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Fattorusso
authors.affiliation: Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy.
authors.forename: Menotti
authors.initials: M
authors.lastname: Ruvo
authors.affiliation: Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy nunzianna.doti@cnr.it.
authors.forename: Nunzianna
authors.initials: N
authors.lastname: Doti
mesh_terms: D020134:Catalytic Domain; D021984:Cyclophilin A / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D062105:Molecular Docking Simulation / Q000379:methods; D019696:Peptidylprolyl Isomerase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000494:pharmacology; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D012997:Solvents; D021984:Cyclophilin A; D019696:Peptidylprolyl Isomerase
keywords: EDANS–Dabcyl pairs; HTS; chymotrypsin-coupled assay; fluorescence; prolyl-peptidyl isomerases