Mutation in Transmembrane Domain 8 of Human Urate Transporter 1 Disrupts Uric Acid Recognition and Transport.

Human urate transporter 1 (hURAT1) is the most pivotal therapeutic target for hyperuricemia. Due to a lack of crystal structure information, the atomic structure of URAT1 is not clearly understood. In this study, a multiple sequence alignment was performed, and K393, a positively charged residue in transmembrane domain (TMD) 8, was observed to be highly conserved in organic anion transporters (OATs). K393 was substituted with a positively, negatively, and neutrally charged amino acid via site-directed mutagenesis and then used to transfect HEK293 cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) analyses indicated that mutants of K393 showed mRNA and protein expression levels similar to those in the WT group. The nonpositively charged mutants K393A, K393D, and K393E eliminated 70-80% of 14C-uric acid transport capacity, while the K393H mutant showed slight and the K393R mutant showed no reduced transport capacity compared with the WT group. Binding assays indicated that K393A, K393D, and K393E conferred lowered uric acid binding affinity. As indicated by the K m and V max values obtained from saturation kinetic experiments, K393A, K393D, and K393E showed increased K m values, but K393R and K393H showed K m values similar to those in the WT group. K393 also contributed to a high affinity for benzbromarone (BM) interaction. The inhibitory effects of BM were partly abolished in K393 mutants, with increased IC50 values compared with the WT group. BM also exhibited weaker inhibitory effects on 14C-uric acid binding in K393R and K393H mutants. In an outward homology model of URAT1, K393 was located in the inner part of the transport tunnel, and further molecular docking analysis indicated that uric acid and BM showed possible hydrogen bonds with K393. Mutants K393R and K393H showed possible interactions with uric acid, and positive charges confer high affinity for uric acid as revealed by their surface electrostatic potential. In conclusion, our data provide evidence that K393 is an important residue for the recognition of uric acid or inhibitors by URAT1.

CDER167, a dual inhibitor of URAT1 and GLUT9, is a novel and potent uricosuric candidate for the treatment of hyperuricemia.

Urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) are important targets for the development of uric acid-lowering drugs. We previously showed that the flexible linkers of URAT1 inhibitors could enhance their potency. In this study we designed and synthesized CDER167, a novel RDEA3710 analogue, by introducing a linker (methylene) between the naphthalene and pyridine rings to increase flexibility, and characterized its pharmacological and pharmacokinetics properties in vitro and in vivo. We showed that CDER167 exerted dual-target inhibitory effects on both URAT1 and GLUT9: CDER167 concentration-dependently inhibited the uptake of [14C]-uric acid in URAT1-expressing HEK293 cells with an IC50 value of 2.08 ± 0.31 µM, which was similar to that of RDEA3170 (its IC50 value was 1.47 ± 0.23 µM). Using site-directed mutagenesis, we demonstrated that CDER167 might interact with URAT1 at S35 and F365. In GLUT9-expressing HEK293T cells, CDER167 concentration-dependently inhibited GLUT9 with an IC50 value of 91.55 ± 15.28 µM, whereas RDEA3170 at 100 µM had no effect on GLUT9. In potassium oxonate-induced hyperuricemic mice, oral administration of CDER167 (10 mg·kg-1 · d-1) for 7 days was more effective in lowering uric acid in blood and significantly promoted uric acid excretion in urine as compared with RDEA3170 (20 mg·kg-1 · d-1) administered. The animal experiment proved the safety of CDER167. In addition, CDER167 displayed better bioavailability than RDEA3170, better metabolic stability and no hERG toxicity at 100 µM. These results suggest that CDER167 deserves further investigation as a candidate antihyperuricemic drug targeting URAT1 and GLUT9.

Structural Insights into the Atomistic Mechanisms of Uric Acid Recognition and Translocation of Human Urate Anion Transporter 1.

Background: Human urate transporter 1 (hURAT1) is the most pivotal therapeutic target for treating hyperuricemia. However, the molecular interactions between uric acid and URAT1 are still unknown due to lack of structural details. Methods: In the present study, several methods (homology modeling, sequence alignment, docking, and mutagenesis) were used to explain the atomistic mechanisms of uric acid transport of hURAT1. Results: Residues W357-F365 in the TMD7 and P484-R487 in the TMD11 present in the hURAT1 have unique roles in both binding to the uric acid and causing subsequent structural changes. These residues, located in the transport tunnel, were found to be related to the structural changes, as demonstrated by the reduced V max values and an unaltered expression of protein level. In addition, W357, G361, T363, F365, and R487 residues may confer high affinity for binding to uric acid. An outward-open homology model of hURAT1 revealed a crucial role for these two domains in the conformational changes of hURAT1. F241 and H245 in TMD5, and R477 and R487 in TMD11 may confer high affinity for uric acid, and as the docking analysis suggests, they may also enhance the affinity for the inhibitors. R477 relation to the structural changes was demonstrated by the V max values of the mutants and the contribution of positive charge to the uric acid selectivity. Conclusions: W357-F365 in TMD7, P484-R487 in TMD11, and residues F241, H245, and R477 were found to be critical for the translocation and recognition of uric acid.

Protective effects of S-carvedilol on doxorubicin-induced damages to human umbilical vein endothelial cells and rats.

Doxorubicin (DOX) is a highly active anticancer drug with severe cytotoxicity, which is strongly associated with oxidative stress. Carvedilol (CAR), used as its racemate with S-CAR and R-CAR (1:1), has been previously reported to ameliorate the DOX-induced cytotoxicity. However, the main contributor from CAR of its protective effects has not been clear. Therefore, in this study, we aimed to investigate further the different effects of CAR enantiomers on DOX-induced cytotoxicity in human umbilical vein endothelial cells and rats, respectively. Results indicated that S-CAR could significantly attenuate DOX-induced cell death, apoptotic morphological changes, decrease the mitochondrial membrane potential and oxidative stress responses by increasing the superoxide dismutase and catalase activities, and decreasing malondialdehyde contents and reactive oxygen species levels via the phosphoinositide 3-kinase/AKT/endothelial nitric oxide synthase pathway in vitro. Consistent with the in vitro study, the protective effects of S-CAR on the myocardial tissues and hemodynamics were also detected in rats suffering because of DOX treatment. With the obtained results, we can first conclude that S-CAR provides superior protection to injury induced by DOX relative to that of racemic CAR and R-CAR.

DCPIB, an Inhibitor of Volume-Regulated Anion Channels, Distinctly Modulates K2P Channels.

K2P potassium channels stabilize the resting membrane potential in nearly all cells and have been implicated in several neuronal, cardiovascular, and immune diseases. DCPIB, a known specific and potent inhibitor of volume-regulated anion channels (VRAC), has been reported to activate TREK1 and TREK2 in astrocytes and in vitro recently. In the present study, we demonstrated DCPIB also voltage dependently activated TRAAK besides TREK1/TREK2, showing DCPIB activated all TREK subfamily members. In contrast, the compound potently inhibited several other K2P channels with no voltage dependence, including TRESK, TASK1, and TASK3. DCPIB displayed superior selectivity toward TRESK with an IC50 of 0.14 µM, demonstrating at least 100-fold higher affinity over TREK1/TRAAK channels. Furthermore, the impaired ion selectivity filter region greatly impaired the activating effect of DCPIB on TREK1 but not the inhibitory effect of DCPIB on TRESK. This indicates distinct molecular determinants underlying the effect of DCPIB on TREK1 or TRESK channels. Our results showed that DCPIB played diverse effects on K2P channels and could be a useful tool for further investigating structure-function studies of K2P channels.

Metabolic Epoxidation Is a Critical Step for the Development of Benzbromarone-Induced Hepatotoxicity.

Benzbromarone (BBR) is effective in the treatment of gout; however, clinical findings have shown it can also cause fatal hepatic failure. Our early studies demonstrated that CYP3A catalyzed the biotransformation of BBR to epoxide intermediate(s) that reacted with sulfur nucleophiles of protein to form protein covalent binding both in vitro and in vivo. The present study attempted to define the correlation between metabolic epoxidation and hepatotoxicity of BBR by manipulating the structure of BBR. We rationally designed and synthesized three halogenated BBR derivatives, fluorinated BBR (6-F-BBR), chlorinated BBR (6-Cl-BBR), and brominated BBR (6-Br-BBR), to decrease the potential for cytochrome P450-mediated metabolic activation. Both in vitro and in vivo uricosuric activity assays showed that 6-F-BBR achieved favorable uricosuric effect, while 6-Cl-BBR and 6-Br-BBR showed weak uricosuric efficacy. Additionally, 6-F-BBR elicited much lower hepatotoxicity in mice. Fluorination of BBR offered advantage to metabolic stability in liver microsomes, almost completely blocked the formation of epoxide metabolite(s) and protein covalent binding, and attenuated hepatic and plasma glutathione depletion. Moreover, the structural manipulation did not alter the efficacy of BBR. This work provided solid evidence that the formation of the epoxide(s) is a key step in the development of BBR-induced hepatotoxicity.