Purine nucleoside phosphorylase-coated magnetic particles for a new ligand screening assays

Purine nucleoside phosphorylase (PNP) plays a central role in purine recycling and salvage pathway and has been considered an attractive chemotherapeutic target for several diseases. In this work, PNP from Mycobacterium tuberculosis (MtPNP) was covalently immobilized on magnetic particles and employed in the development of activity- and affinity-based assays. In the activity-based assays, the quantification of the formed hypoxanthine was conducted by LC-DAD to monitor the MtPNP activity. The immobilized MtPNP retained its catalytic activity for over six months. The KM values for the substrates inosine and phosphate were assessed (88.7 ± 9.24 μmol.L-1 and 1853 ± 52.87 μmol.L-1, respectively). In the inhibition assays, a fourth-generation Immucillin derivative (DI4G), a known inhibitor of other PNPs, was used to validate the use of this assay for screening purposes. It was determined IC50 value for this inhibitor of 9.14 ± 1.2 nmol.L-1, with concentration-dependent response through a competitive inhibition mechanism. Ligand fishing assay, an affinity-based screening model, was developed using DI4G as a probe to modulate the optimal experimental conditions. An UHPLC-MS/MS method was developed and validated to quantify isolated DI4G. The established ligand fishing assay conditions were: 12.5 μg of the MtPNP-coated magnetic beads were incubated with the probe for 1 minute, and the retained ligands were eluted with 5 mM ammonium acetate pH 7.4: acetonitrile (8:2) for 1 minute. Therefore, this study demonstrates the versatility of using magnetic particles as a solid support for the immobilization of proteins of clinical interest for the development of new screening assays.