FRET-Protease-Coupled Peptidyl-Prolyl cis-trans Isomerase Assay

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 IC 50 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.

Structural and biochemical insights of CypA and AIF interaction

“…We have tested this hypothesis using a CypA peptide substrate 25 and performing binding experiments between wild type and R55A mutant protein and the substrate in the presence and absence of the AIF(370-394) and CsA. Data showed that, in line with the large difference in affinity (57 nM for the substrate and 6 μM for the AIF peptide), AIF(370-394), used at a 10-fold excess, is unable to prevent the binding of both CypA and CypA R55A to the substrate, but recognition is abolished in the presence of the strong inhibitor CsA (Supplementary Fig.…”

“…His-tagged CypA (hereafter CypA) and the corresponding R55A mutant, were expressed and purified as previously reported 25 . The protein concentration was determined by reading the absorbance at 280 nm with a NanoDrop200c UV-Vis spectrophotometer and using a theoretical molar extinction coefficient of 8730 M −1 cm −1 .…”