FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay: New Internally Quenched Fluorogenic Substrates for High-Throughput Screening(155 visite) Caporale A, Mascanzoni F, Farina B, Sturlese M, Di Sorbo G, Fattorusso R, Ruvo M, Doti N
Impact factor: 2.444, Impact factor a 5 anni: 2.454
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Parole chiave: Edans-Dabcyl Pairs, Hts, Chymotrypsin-Coupled Assay, Fluorescence, Prolyl-Peptidyl Isomerases,
*** IBB - CNR *** Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy Dipartimento di Farmacia, Universita di Napoli "Federico II", Napoli, Italy., Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy., Molecular Modeling Section, Dipartimento di Scienze del Farmaco, Universita di Padova, Padova, Italy., Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy Dipartimento di Scienze e Tecnologie Ambientali Biologiche e Farmaceutiche, Seconda Universita di Napoli, Caserta, Italy., Istituto di Biostrutture e Bioimmagini-CNR and CIRPEB, Napoli, Italy firstname.lastname@example.org.,
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.
153 Records (146 escludendo Abstract e Conferenze). Impact factor totale: 590.935 (573.221 escludendo Abstract e Conferenze). Impact factor a 5 anni totale: 612.47 (593.403 escludendo Abstract e Conferenze).