Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies

Abstract

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 μmol L (-1) and 164 ± 13.4 μmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

Prion protein-coated magnetic beads: Synthesis, characterization and development of a new ligands screening method

“…It was previously demonstrated that the immobilization of proteins provides in some cases increased stability [36–38], due to the more restricted conformational mobility. A similar increase in stability was observed with the immobilization of PrP C , preventing the conversion of PrP C to PrP Sc and PrP Sc -like isoforms.…”

Schistosomiasis: Setting Routes for Drug Discovery

“…The S. mansoni purine nucleoside phosphorylase (SmPNP) activity and structure have been well characterized [124][125][126]. Selective inhibitors for SmPNP have been tested in enzymatic assays in vitro; however, experiments with parasite larval or adult stages have not been reported yet [127][128][129]. The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) was also considered as a potential drug target [130].…”

Characterization and screening of tight binding inhibitors of xanthine oxidase: an on-flow assay

“…Zonal bioaffinity chromatography has previously been successfully used for ICERs characterization through Michaelis-Menten parameters by means of a 2D LC system. 1,2 Herein, an XO-ICER was prepared and used in the first dimension of a 2D LC system (Figure 3) to not only determine its kinetic parameters but also fully characterize its tight-binding inhibitors. The validated LC method was used to quantify the production of uric acid by the ICERs.…”