ABSTRACT
In recent years, xanthine oxidase has emerged as an important target not only for gout but also for cardiovascular and metabolic disorders involving hyperuricemia. Contrary to popular belief, recent clinical trials with uricosurics have demonstrated that enhanced excretion of uric acid is, by itself, not adequate to treat hyperuricemia; simultaneous inhibition of production of uric acid by inhibition of xanthine oxidase is also important. Virtual screening of in-house synthetic library followed by in vitro and in vivo testing led to the identification of a novel scaffold for xanthine oxidase inhibition. In vitro activity results corroborated the results from molecular docking studies of the virtual screening hits. The isocytosine scaffold maintains key hydrogen bonding and pi-stacking interactions in the deep end of the xanthine-binding pocket, which anchors it in an appropriate pose to inhibit binding of xanthine and shows promise for further lead optimization using structure-based drug design approach.
Subject(s)
Computer Simulation , Cytosine/analogs & derivatives , Enzyme Inhibitors/chemistry , Xanthine Oxidase/antagonists & inhibitors , Animals , Cytosine/chemical synthesis , Cytosine/chemistry , Cytosine/pharmacology , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Hyperuricemia/chemically induced , Hyperuricemia/drug therapy , Male , Oxonic Acid/pharmacology , Oxonic Acid/toxicity , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Xanthine Oxidase/metabolismABSTRACT
Macrophage migration inhibitory factor (MIF) is known to contribute to the pathogenesis of inflammatory hyperalgesia and neuropathic pain. Prior studies have shown that Vitamin E treatment is associated with attenuated hyperalgesia and reduced neuropathic pain in rodents. Given these observations, we investigated the possibility that Vitamin E is a MIF inhibitor. Dopachrome tautomerase assays revealed that Vitamin E inhibits the enzymatic activity of purified human recombinant MIF (rhMIF) in a dose-dependent manner (45%, 74%, 92% and 100% inhibition at 3, 10, 30 and 100µM, respectively). Cell-free ELISA based assays showed that Vitamin E binds onto rhMIF thereby blocking its recognition (48% inhibition at 100µM). Circular dichroism studies indicated the Vitamin E has a strong affinity to bind to rhMIF (binding constant 19.52±1.4µM). In silico studies demonstrated that Vitamin E docks well in the active site of MIF with the long aliphatic chain of Vitamin E exhibiting strong van der Waals interactions with MIF. Most importantly, human cell-based assays revealed that Vitamin E significantly inhibits rhMIF-induced production of pro-inflammatory cytokines in a dose-dependent manner (77%, 80%, and 96% inhibition of IL-6 production, respectively, at 10, 30 and 100µM). Taken together, these results demonstrate that Vitamin E inhibits not only the enzymatic activity of MIF but more importantly the biological function of MIF. Our findings suggest that Vitamin E may be attenuating hyperalgesia and reducing neuropathic pain at least in part by inhibiting MIF activity.
