BACKGROUND: Fibroblast activiation protein alpha (FAP) is considered a diagnostic and prognostic biomarker for various types of cancer. FAP shares substrate specificity with prolyl oligopeptidase (PREP), studied in (neuro)inflammation and neurodegeneration as well as cancer. Current assays inadequately discriminate between FAP and PREP and there is need for an assay that reliably quantitates the FAP/PREP activity ratio in plasma. METHODS: FAP and PREP activities were measured in human EDTA-plasma in presence of well characterized PREP and FAP inhibitors. RESULTS: A combined kinetic assay was developed in conditions to optimally measure FAP as well as PREP activity with Z-Gly-Pro-AMC as substrate. Limit of detection was 0.009 U/L and limit of quantitation was 0.027 U/L for the combined FAP-PREP assay. Within-run coefficient of variation was 3% and 4% and between-run precision was 7% and 12% for PREP and FAP, respectively. Accuracy was demonstrated by comparison with established end-point assays. Hemolysis interferes with the assay with 1.5 g/L hemoglobin as cut-off value. PREP (but not FAP) activity can increase upon lysis of platelets and red blood cells during sample preparation. CONCLUSION: With this new assay, on average 67% of the Z-Gly-Pro-AMC converting activity in plasma can be attributed to FAP.
Our docking program, Fitted, implemented in our computational platform, Forecaster, has been modified to carry out automated virtual screening of covalent inhibitors. With this modified version of the program, virtual screening and further docking-based optimization of a selected hit led to the identification of potential covalent reversible inhibitors of prolyl oligopeptidase activity. After visual inspection, a virtual hit molecule together with four analogues were selected for synthesis and made in one-five chemical steps. Biological evaluations on recombinant POP and FAPalpha enzymes, cell extracts, and living cells demonstrated high potency and selectivity for POP over FAPalpha and DPPIV. Three compounds even exhibited high nanomolar inhibitory activities in intact living human cells and acceptable metabolic stability. This small set of molecules also demonstrated that covalent binding and/or geometrical constraints to the ligand/protein complex may lead to an increase in bioactivity.
Title: Structures of prolyl oligopeptidase substrate/inhibitor complexes. Use of inhibitor binding for titration of the catalytic histidine residue Fulop V, Szeltner Z, Renner V, Polgar L Ref: Journal of Biological Chemistry, 276:1262, 2001 : PubMed
Structure determination of the inactive S554A variant of prolyl oligopeptidase complexed with an octapeptide has shown that substrate binding is restricted to the P4-P2' region. In addition, it has revealed a hydrogen bond network of potential catalytic importance not detected in other serine peptidases. This involves a unique intramolecular hydrogen bond between the P1' amide and P2 carbonyl groups and another between the P2' amide and Nepsilon2 of the catalytic histidine 680 residue. It is argued that both hydrogen bonds promote proton transfer from the imidazolium ion to the leaving group. Another complex formed with the product-like inhibitor benzyloxycarbonyl-glycyl-proline, indicating that the carboxyl group of the inhibitor forms a hydrogen bond with the Nepsilon2 of His(680). Because a protonated histidine makes a stronger interaction with the carboxyl group, it offers a possibility of the determination of the real pK(a) of the catalytic histidine residue. This was found to be 6.25, lower than that of the well studied serine proteases. The new titration method gave a single pK(a) for prolyl oligopeptidase, whose reaction exhibited a complex pH dependence for k(cat)/K(m), and indicated that the observed pK(a) values are apparent. The procedure presented may be applicable for other serine peptidases.
