Hyperuricemia (HUA) is a metabolic disorder characterized by high blood levels of uric acid (UA) in men and women (>420 μmol/L) [1]. It is caused by excessive production or impaired excretion of UA in human body. HUA is closely associated with complications such as gout, hypertension, chronic kidney diseases, diabetes, and cardiovascular diseases [[2], [3], [4], [5]]. The incidence of HUA has increased rapidly over the past decades with changes in lifestyle and diet. It has shown a tendency to develop at a younger age. Now its treatment is limited to two main UA-lowering strategies: inhibiting UA production and promoting UA excretion [6]. However, some studies have shown that the therapeutic strategies to promote UA excretion are not ideal [7].

XO is a Mo-dependent enzyme involved in catalyzing the degradation of purine nucleotides and the oxidation of hypoxanthine (HPA) to xanthine (XAN) and XAN to UA [8]. XO is found primarily in human liver and kidneys. Increasing activity of XO causes overproduction of UA and subsequently elevates blood UA level, which leads to the development of HUA and even gout. Thus, XO became a promising target for treating HUA, gout, and other related diseases [4]. For example, allopurinol, febuxostat and topiroxostat are XO inhibitors commonly used in clinics. However, these drugs are also associated with serious adverse effects. For example, allopurinol is a common cause of severe cutaneous adverse drug reactions (SCAR), hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (HSS/DRESS) [[9], [10], [11]]. Febuxostat may increase the risk of death from heart-related causes [12].

In recent years, there has been a growing interest in natural products for drug discovery due to the chances of discovering potential drug molecules possessing novel chemical structures and relatively low toxicity [[13], [14], [15], [16]]. Thereby, natural products have become an important source for discovering XO inhibitors. A various naturally occurred XO inhibitors have been reported [16], including flavonoids, chalcones, terpenoids, alkaloids, coumarins, ellagic acids, and glycosides. Among them, flavones and flavonols, such as quercetin, luteolin, apigenin, and myricetin, have received the most attention, and their XO inhibitory activities significantly contribute to the UA-lowering effects [17]. In a randomized, double-blinded, placebo-controlled, cross-over trial, the natural flavonoid quercetin significantly reduced UA levels without additional cardiovascular risk. Furthermore, it did not affect blood pressure or the chemistry of urine and blood after four weeks of administration [18]. These cheerful observations prompted us to identify more biocompatible XO inhibitors with low toxicity from a natural product database.

Traditional methods of drug discovery involving exploratory testing of chemicals in cells and animals are generally costly and time-consuming. Employing computational virtual screening approaches can offer clear molecular insights into ligand-binding mechanisms by identifying the involved amino acid residues, gradually becoming a more accessible and powerful strategy for drug discovery [19]. Molecular docking has been proven to be a fundamental approach for identifying novel ligands. Unfortunately, its ability to discriminate active ligands from inactive compounds for a certain target is often not satisfying [20]. Currently, several methods are available to compensate for this shortcoming, such as developing target-specific scoring functions, integrating ligand-based and pharmacophoric elements within the docking algorithm, and calculating the binding free energy [6,21,22]. For example, the integration of molecular docking and binding free energy calculations has been used for screening microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors for the treatment of inflammatory diseases and cancer [23]. However, to our knowledge, no application of this combined strategy has been observed in screening naturally occurring XO inhibitors.

Therefore, to identify novel druggable XO inhibitors for the treatment of HUA and gout from natural products, a high throughput virtual screening strategy combining molecular docking and the calculation of Prime molecular mechanics with molecular mechanics/generalized born and surface area (MM-GBSA) was performed in the present study (Fig. 1). Its rationality was verified through re-docking and cross-docking. We conducted docking analysis on >19,000 natural products in the natural product database, and ranked them according to docking scores and MM-GBSA dG Bind values. The top 27 hits were selected for XO enzyme inhibiting studies in vitro. The in vitro study results revealed two new xo natural inhibitors with high inhibiting efficacy for the first time: isolicoflavonol (IC50 = 8.45 ± 0.68 μM) and 5,7-dihydroxycoumarin (IC50 = 10.91 ± 0.71 μM). Lastly, we elucidated the inhibiting and binding mechanisms between the two new inhibitors and XO based on the evidence from enzyme kinetic analysis, molecular dynamics simulations and fluorescence spectroscopy method, followed by the evaluation of their druggability and cytotoxicity.