Recent Updates of Natural and Synthetic URAT1 Inhibitors and Novel Screening Methods

Human urate anion transporter 1 (hURAT1) is responsible for the reabsorption of uric acid in the proximal renal tubules and is a promising therapeutic target for treating hyperuricemia. To mitigate the side effects of URAT1-targeted clinical agents such as benzbromarone, there is significant interest in discovering new URAT1 inhibitors and developing technology that can evaluate URAT1 inhibition. This review summarizes the methods for assay of URAT1 inhibition and the progress on the discovery of natural and synthetic URAT1 inhibitors in the past five years.


Introduction
Uric acid is the final product of purine metabolism in the human body [1,2]. In recent years, more people suffer from an unbalanced urate metabolism [3]. Hyperuricemia is characterized by insufficient excretion and the overproduction of uric acid [4] and is defined as serum uric acid concentration of >408 μmol/L or 6.8 mg/dL [5]. e primary clinical characteristic of hyperuricemia is gout [6], which is a common and complex form of arthritis caused by the deposition of monosodium urate crystals in the peripheral joints and surrounding tissues. Gout prevalence ranges from 2.7% to 6.7% in Western countries and from 0.1% to 10% globally, and increases from 0.03% to 0.6% every year [3,7]. Additionally, hyperuricemia is associated with other diseases, including chronic kidney dysfunction [8,9], hyperlipidemia [10], hypertension [11], coronary heart disease [12], diabetes [13], and vascular disease [14,15].
ere are currently three treatments for hyperuricemia: reduction of urate production, increase in urate excretion, and decomposition of urate. Statistics indicate that 90% of hyperuricemia cases are due to impaired renal uric acid excretion and that only 10% are due to excessive production of uric acid [16]. e kidneys and intestines play a major role in uric acid excretion, with more than 70% of urate excretion taking place in the renal pathway. As such, the development of reagents for increasing uric acid excretion has significant therapeutic potential.
Human urate transporter 1 (hURAT1) is a transmembrane protein that consists of 555 amino acids and belongs to the organic anion transporters (OAT) family. URAT1 is expressed on the luminal side of the proximal renal tubule [17] and regulates the absorption of uric acid from the renal tubule to the epithelial cells [18]. As urate is eliminated from the kidneys, approximately 90% of the urate filtered by the glomeruli is reabsorbed back into the bloodstream and only 10% is excreted by the kidneys. e uric acid reabsorption process is primarily controlled by URAT1 and is particularly important to hyperuricemia. Inhibition of URAT1 is currently the main treatment for urate-lowering therapy, in addition to pills such as allopurinol and febuxostat, which block xanthine oxidase (XO) [19,20]. Considering 90% of hyperuricemia cases are due to reduced renal uric acid excretion, the inhibition of URAT1 over XO could be a better approach to treating gout [21]. URAT1 inhibitory drugs currently approved for use in clinical settings include probenecid, benzbromarone, sulfinpyrazone, and lesinurad ( Figure 1) [22][23][24]. However, these medicines have side effects and some degree of toxicity [25]. Probenecid can induce rash, gastrointestinal tract irritation, hypersensitivity, and hemolytic anemia [22]; benzbromarone results in serious hepatotoxicity and bone marrow suppression [26]; sulfinpyrazone can cause nausea, vomiting, abdominal pain, diarrhea, anemia, and skin rashes [27]; lesinurad has dose-dependent nephrotoxicity and can cause adverse cardiovascular events [28]. e use of URAT1 inhibitors is not recommended in patients with severe renal failure, urate kidney stones, or blood dyscrasias [25].
While xanthine oxidase inhibition is still the primary therapy for gout, it can also have serious adverse effects, such as skin rashes, hepatitis, fever, Stevens-Johnson syndrome, nephropathy, fatal liver necrosis, allergic reactions, and cardiovascular events. As such, alternative medicines with fewer side effects are needed to treat this disorder [29][30][31].
Considering these challenges, there is a need for alternative medicines with fewer side effects, and new therapies are needed for patients who cannot sufficiently reduce urate with current therapies. As such, there is an urgent need to discover effective and safe compounds targeting URAT1 to lower serum uric acid [32] and to develop novel techniques validating URAT1 inhibitors. is study summarizes natural and synthetic URAT1 inhibitors in the past five years. Compared with a recently published review [33], this paper outlines the chemical structures, inhibitory effects, and evaluation methods of natural inhibitors in further detail. For synthetic URAT1 inhibitors, we demonstrate different parent structures including lesinurad, verinurad, and febuxostat derivatives, displayed with both in vitro and in vivo data. We also describe classical and recent methods for evaluating URAT1 inhibitors.

Materials and Methods
is review covers the literature published in the last five years and focuses on the most relevant studies assessing cellbased in vitro approaches for evaluating URAT1 inhibition and novel URAT1 inhibitors. e inhibitors are organized according to the origin of the compounds (natural and synthetic) and their chemical structures. e structures of specific compounds with promising results are presented in the figures. All studies were retrieved from the following databases: Web of Science, PubMed, Scopus, and CNKI. e following keywords were used: "URAT1 inhibitors," "hyperuricemia," "screening methods," "and uric acid." e chemical structures in this article were created by ChemDraw.

Recent Development of URAT1 Inhibitors
3.1.1. Natural Compounds with URAT1 Inhibitory Activity. Natural products have received significant attention for their urate-lowering potential [34,35], particularly phenolic compounds, terpenes, and fatty acids, all of which are reported to have antihyperuricemic activity.
In vivo animal studies have demonstrated that 200 mg/kg baicalein can significantly lower urate levels in PO-induced hyperuricemia mice by elevating urate excretion. Docking analysis and site mutation indicate that baicalein interacts with Ser35 and Phe241 of URAT1. Baicalein was also reported to exhibit liver and kidney protection without toxicity [42,43].
To discover novel compounds for hyperuricemia, 107 crude products were screened by uric acid uptake assays on URAT1-HEK293/PDZK1 cells [45]. Of them, the MeOH extract from Cnidii Monnieris Fructus exhibited the highest inhibitory effect. Osthol (compound 7) ( Figure 2) was identified as the active compound in the extract, which noncompetitively inhibits URAT1 with an IC 50  e authors also compared the URAT1 inhibition of different coumarins and found that only compounds with a prenyl group at the 8position of osthol and osthenol (compound 8) displayed URAT1 inhibitory effects at 100 μM, suggesting the important role for this substitution in URAT1 inhibition. BDEO (compound 9) ( Figure 2) is a deoxybenzoins oxime analog and has a structure similar to flavonoids. Its role as a dual inhibitor for lowering urate has been studied, and it is found that it blocked the uptake of uric acid in URAT1-293T cells (K i � 0.14 μM) in a noncompetitive manner and inhibited XO activity (IC 50 � 3.3 μM). In vivo studies demonstrated that BDEO at 5 mg/kg significantly decreased serum urate in PO-induced hyperuricemia mice and exhibited a nontoxic, dose-dependent effect. e administration of 20 mg/kg BDEO has effects comparable to allopurinol or benzbromarone at 10 mg/kg [46].
Additionally, alpinia oxyphylla seed ethanol extract (AE) displays strong URAT1 inhibitory activity. Its URAT1 inhibitory effect at 100 μg/mL is comparable with that of benzbromarone (100 μM), as shown by the uric uptake assay   Evidence-Based Complementary and Alternative Medicine in hURAT1-expressing oocytes, in which only 1 μg/mL AE significantly inhibit URAT1 function. UPLC analysis revealed nootkatone (compound 16) (Figure 3) as the primary bioactive compound in the extract, while in vivo administration of 100 mg/kg nootkatone significantly reduced serum uric acid in PO-induced hyperuricemic rats [52].
(3) Fatty Acids. It is well established that fatty acids (FAs) influence a range of metabolic and inflammatory diseases [53,54] and cancer [55]. A recent study demonstrated the relationship between FAs and hyperuricemia: of 25 FAs tested in vitro [56], nine unsaturated FAs exhibited URAT1 inhibitory effects at 100 μM, but no saturated FAs showed any effects. In particular, three long-chain unsaturated FAs exhibited strong URAT1 inhibitory effects: eicosapentaenoic acid (EPA) (compound 17) (Figure 4), α-linolenic acid (ALA) (compound 18) (Figure 4), and docosahexaenoic acid (DHA) (compound 19) (Figure 4), which had IC 50 values of 6.0, 14.2, and 15.2 μM, respectively. is study could lead to a new kind of URAT1 inhibitor, though the interaction between FAs and URAT1 requires additional study.

Synthetic Compounds
(1) Lesinurad Analogues. Lesinurad (Figure 1) is a selective inhibitor of URAT1 and OAT4 for uric acid reabsorption, though it also displays adverse nephrotoxic effects [25]. Lesinurad is considered a scaffold compound for discovering inhibitors with reduced side effects and improved activities.
(2) Other Synthetic Compounds. CDER167 (compound 27) is an RDEA3170 (compound 28) derivative with the insertion of methylene between the naphthalene and pyridine of RDEA3170 ( Figure 6). It is described as a dual inhibitor of   [62]. Febuxostat ( Figure 6) is a nonpurine selective XO inhibitor and was approved as a first-line drug for treating hyperuricemia and gout in 2009. Zhou found that febuxostat has an inhibitory effect on URAT1 [63]. ey used a fluorescence-based assay and identified the URAT1 inhibitors febuxostat and benzbromarone, which have IC 50 values of 36.1 μM and 14.3 μM, respectively. As such, the authors designed and synthesized a series of "me-too" compounds using febuxostat as the lead to screen URAT1 inhibitors. ey found that compound 4 (compound 29) ( Figure 6) (IC 50 value � 10.8 μM) exhibits a similar URAT1 inhibitory effect as benzbromarone [64].

Technical Methods for Validation of Inhibitors.
Various experimental methods have been used to evaluate URAT1 inhibition. Most research uses in vitro models for cell-based approaches (Table 1). To our knowledge, there is currently no in vivo model that directly evaluates URAT1 inhibition. Many studies focus on URAT1 inhibition expression in the kidneys, but there is a lack of evidence for direct interaction. erefore, hyperuricemia animal models are used to verify the uric-lowering effect and safety of inhibitors [65]. In this review, we focus on the in vitro approaches to evaluating URAT1 inhibition.

Radioisotope-Labeled Uric Acid Uptake Assays.
e radioactive 14 C-labeling method is the most popular method of quantitatively evaluating the URAT1 function ( Figure 7A).
is method directly reveals the uric acid transportation by URAT1 and was established when this protein was first identified [17]. In this assay, the following cell lines are typically used for URAT1-overexpression: Xenopus laevis Oocytes, human epithelial kidney cell, and MDCK cells. When using oocytes, hURAT1-cRNA must be synthesized, injected into the cells, and incubated for 2-3 days. e oocytes are then transferred to a Cl − -free solution containing [ 14 C] uric acid to initiate the uptake of uric acid, while the radioactivity in the oocytes is determined by a liquid scintillation counter [71], considering that the oocyte cell model is complicated and the renal localization of URAT1, kidney cell HEK293, or MDCK cells have been used in recent years to express hURAT1 [67,68]. In this model, the URAT1-expressing plasmid is transiently transfected into cells for 1-2 day protein expression, and the cells are then assayed for [ 14 C] uric acid uptake at certain time points (20 s, 5 min, 10 min) [21,76]. For example, for the inhibition of URAT1 by fatty acids, only 20 s of incubation is needed for uric acid uptake, which helps exclude the impact of fatty acids on the plasma membrane [56].
is method is easy, convenient, and quick. Some studies use hURAT1 stably expressed HEK293 or MDCK cells to obtain a persistent and stable model to evaluate URAT1 inhibitors [48,68,69].
Radioisotope-labeled uric acid uptake assays have been widely used to screen URAT1 inhibitors with high sensitivity and are capable of measuring the IC 50 of these inhibitors  Evidence-Based Complementary and Alternative Medicine ( Figure 8). However, using radioisotopes could be restricted in some laboratories and it is costly to use.

Chromatography-Based
Approach. An ultra performance liquid chromatography (UPLC) method is used for nonradioactive uric acid transport assay to detect the uric acid contents of URAT1-expressing cells. UPLC requires a relatively large amount of uric acid (0.09 μM) and is limited to a maximum concentration of 0.18 μM [77], meaning this method depends on extracellular and intracellular concentrations of uric acid, limiting its practical use. LC-MS/MS is an analysis used in highly selective and sensitive in vitro models of URAT1 inhibition [70] (Figure 7B). Similar to other cell-based methods, hURAT1-expressing cells are incubated with non-radioactive uric acid and the test compounds. e uric acid in the cell is then released and detected via LC-MS/MS. is method is unique in that it uses isotope-labeled 1,3-15N 2 uric acid as an internal standard, which sets the limit of detection (LOD) of uric acid at 50 nM and the limit of quantitation (LOQ) at 200 nM. is approach demonstrates a highly selective and sensitive method of assessing intracellular uric acid and provides a suitable model for the in vitro evaluation of drug candidates targeting URAT1.
In this method, the cells are incubated with a fluorescent substrate for one hour, after which the cells are lysed and assayed using a microplate reader. It is an economical, environmentally friendly, and convenient approach for screening URAT1 inhibitors in vitro. However, because of the low affinity of 6-carboxyfluorescein, the IC 50 values of benzbromarone and lesinurad against URAT1 measured by this method could be 100 times greater than the radioactive methods, meaning that a high concentration of compounds is required to produce the fluorescence signal. e fluorescence method has the advantages of high throughput and easy quantitative analysis and is widely used to detect in vitro activity in recent years [78][79][80]. e primary disadvantage of this approach is its low sensitivity, making the identification of strong fluorescence substrates a priority.

URAT1 Direct Binding Assay.
A URAT1 binding assay was developed to identify the interactions between the proteins and inhibitors [67]. It is a similar procedure to the uric acid uptake assay, where URAT1 is first expressed in cells and URAT-enriched cell membranes are then isolated by two steps of centrifugation. e cell membranes are incubated with a radiolabeled high-affinity URAT1 inhibitor probe (e.g., 3 H-RDEA3170) in the presence and absence of candidate compounds. If the candidate interacts with URAT1 at the same binding site of the radiolabeled probe, the intramembrane accumulated probes will be displaced or its binding to hURAT1 will be weakened [21]. is assay has identified some URAT1 inhibitors, including benzbromarone, sulfinpyrazone, probenecid, and lesinurad. Notably, this assay relies on the same binding site of inhibitors and probes and is ineffective for molecules that bind to other URAT1 sites. As such, this binding assay provides a tool for characterizing the molecular interactions of compounds with URAT1 and provides additional validation of new inhibitors.

Conclusions
URAT1 is a proven key target for promoting uric acid excretion; however, current URAT1 inhibitors have serious side effects. In recent years, many new URAT1 inhibitors have been described and this review summarizes key and novel experimental approaches that contribute to screening URAT1 inhibitors and help validate the urate-lowering effect. Radioisotope-labeled uric acid uptake assays are classical methods of validating URAT1 inhibitors. Given the disadvantages of the radioisotope method, novel identification methods have emerged in recent years. is includes nonradioactive isotope-LC-MS/MS and fluorescence detection, both of which could help explore novel URAT1 inhibitors. However, the sensitivity and efficiency of these methods must be optimized. We also summarized current progress relating to URAT1 inhibitor discovery, including natural products and synthetic compounds. Phenolic compounds are the primary category of natural URAT1 inhibitors, while synthetic inhibitors often use lesinurad as the lead scaffold. is review also demonstrates that many compounds, such as compounds 9, 21, and 27, have dual inhibitory effects on URAT1 and XO or GLUT9, which showed an excellent urate-lowering effect with more safety. erefore, developing dual inhibitors for urate-lowering therapy is a promising area of research, though inhibition mechanisms, pharmacokinetics, and the safety of the novel URAT1 inhibitors all require further study.
Data Availability e data used in the current study are included within this article.

Conflicts of Interest
e authors disclose no conflicts of interest.