title: Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.
abstract: The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The K(M) value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 ± 29.2 μM and 133 ± 14.9 μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC(50)=40.6 ± 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay.
authors.affiliation: Departamento de Química, Universidade Federal de São Carlos, Cx Postal 676, São Carlos 13565-905, São Paulo, Brazil.
authors.forename: Marcela Cristina
authors.initials: MC
authors.lastname: de Moraes
authors.forename: Rodrigo Gay
authors.initials: RG
authors.lastname: Ducati
authors.forename: Augusto José
authors.initials: AJ
authors.lastname: Donato
authors.forename: Luiz Augusto
authors.initials: LA
authors.lastname: Basso
authors.forename: Diógenes Santiago
authors.initials: DS
authors.lastname: Santos
authors.forename: Carmen Lucia
authors.initials: CL
authors.lastname: Cardoso
authors.forename: Quezia Bezerra
authors.initials: QB
authors.lastname: Cass
mesh_terms: D019149:Bioreactors; D002853:Chromatography, Liquid; D055808:Drug Discovery / Q000379:methods*; D004800:Enzymes, Immobilized / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004867:Equipment Design; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019271:Hypoxanthine / Q000032:analysis / Q000378:metabolism; D007288:Inosine / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D016014:Linear Models; D008956:Models, Chemical; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D012044:Regression Analysis; D015203:Reproducibility of Results
chemical_list: D004800:Enzymes, Immobilized; D006573:Heterocyclic Compounds, 1-Ring; D008024:Ligands; D019271:Hypoxanthine; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase