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

Autor(es)

Rodrigues, M. V. N. ^{[1, 2]} ; Correa, R. S. ^[1] ; Vanzolini, K. L. ^[1] ; Santos, D. S. ^[3] ; Batista, A. A. ^[1] ; Cass, Q. B. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Carlos, Dept Quim, BR-13565905 Sao Carlos, SP - Brazil

^[2] Univ Estadual Campinas, Ctr Pluridisciplinar Pesquisas Quim Biol & Agr, BR-13140000 Paulinia, SP - Brazil

^[3] Pontificia Univ Catolica Rio Grande do Sul, Ctr Pesquisas Biol Mol & Func, Inst Nacl Ciencia & Tecnol TB, BR-90619900 Porto Alegre, RS - Brazil

• Tipo de documento: Artigo Científico
• Fonte: RSC ADVANCES; v. 5, n. 47, p. 37533-37538, 2015.
• Citações Web of Science: 6

Resumo

Xanthine oxidase (XO) is an enzyme in the purine salvage pathway that catalyzes the oxidation of hypoxanthine to xanthine with subsequent production of uric acid from the xanthine oxidation, and it has been considered an important target of newly developed inhibitors. Based on the advantages of using immobilized capillary enzyme reactors (ICERs) in a 2D LC system as a tool for screening new enzymatic ligands, this work validated an XO-ICER using allopurinol as a positive control. Despite the complex interaction between XO and allopurinol due its tight binding nature, it was possible to recognize the inhibitory kinetics parameters through Morrison's equation. The tight binding nature of inhibition was established by varying the IC50 values according to the substrate concentration. The kinetic inhibitory profile of allopurinol was used to validate the XO-ICER. Then, the XO-ICER was used to screen specific ruthenium derivatives. The selected compound, 4CBALO, an allopurinol ruthenium derivative, exhibited 100% inhibition at 200 mu M compared to 86% inhibition from allopurinol at the same concentration. The inhibitory effect on the immobilized XO was reversible after the elution of the compound, with immediate recovery of the ICER activity. Additionally, 4CBALO behaved as a selective and competitive tight binder of xanthine oxidase with a true K-i value of 0.29 mu M, which was obtained from the Morrison equation. This report describes the first on-flow characterization of tight binders of xanthine oxidase. (AU)

• Processo FAPESP: 13/01710-1 - Ligantes enzimáticos: novos modelos de triagem
• Beneficiário: Quezia Bezerra Cass
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 09/08131-1 - Complexos de rutênio com ligantes de interesse biológico: aspectos químicos, estruturais e avaliação de suas citotoxicidades em células tumorais
• Beneficiário: Rodrigo de Souza Corrêa
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/26559-4 - Modificações estruturais em complexos de Ru(II) biologicamente ativos para o desenho de novos candidatos a metalofármacos
• Beneficiário: Rodrigo de Souza Corrêa
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado