Characterization and screening of tight binding inhibitors of xanthine oxidase : an on-flow assay.
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2015
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Abstract
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 mM 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 Ki value of 0.29 mM, which was obtained from the Morrison equation. This
report describes the first on-flow characterization of tight binders of xanthine oxidase
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RODRIGUES, M. V. N. et al. Characterization and screening of tight binding inhibitors of xanthine oxidase: an on-flow assay. RSC Advances: an international journal to further the chemical sciences, v. 5, p. 37533-37538, 2015. Disponível em: <https://pubs.rsc.org/en/content/articlelanding/2015/ra/c5ra01741f#!divAbstract>. Acesso em: 05 ago. 2017.