1. INTRODUCTION
Uric acid, a critical antioxidant species in human physiology, has attracted substantial attention for its dual roles in health and disease. Although uric acid is essential for neutralizing reactive oxygen species, elevated uric acid levels—a condition termed hyperuricemia—pose a risk of gout, and are associated with various metabolic and cardiovascular disorders [1–3]. The unmet need of managing uric acid levels has led to intensive exploration of related transport mechanisms, particularly those of the GLUT9 and URAT1 urate transporters.
The GLUT9 and URAT1 transporters are critical for urate homeostasis, and their dysregulation has been implicated in hyperuricemia and gout. GLUT9’s high urate transport capacity has made it a potential biological target for novel anti-hyperuricemic drugs [4]. GLUT9 exhibits a pronounced preference for urate over glucose, with transport activity toward urate 45–60 times higher than that toward glucose [5]. Similarly, URAT1 is responsible for urate reabsorption in the kidneys—an aspect with clinical relevance for the development of treatments [6]. Mutations in the URAT1 gene can decrease urate reabsorption and subsequently lead to hypouricemia. Although both transporters have meaningful roles in hyperuricemia disorder, their detailed molecular mechanisms in urate transport and recognition have been elusive.
2. TECHNICAL CHALLENGES AND BOTTLENECKS
Structural biology studies of membrane transport proteins, such as GLUT9 and URAT1, have faced several challenges, including the instability of these proteins during isolation, the difficulty in obtaining high-resolution structures, and the high complexity of their interactions with substrates or inhibitors. Overcoming these hurdles will be crucial for understanding the transport mechanisms and achieving rational design of drugs targeting these proteins.
3. GLUT9: STRUCTURAL ELUCIDATION AND THERAPEUTIC POTENTIAL
An innovative study by Shen et al. [7] has revealed the molecular underpinnings of urate recognition by GLUT9, thus offering novel insights into the selectivity of urate transport. In general, cryo-EM human GLUT9 structures in complex with urate have revealed that the Tyr327, Asn333, and Trp336 residues are critical in the binding process, through forming hydrogen bonding interactions with urate that are essential for substrate recognition ( Figure 1 ). Additionally, the roles of Leu75, Ile209, and Leu332 in forming hydrophobic interactions with urate have been shown to further stabilize urate within the binding pocket. This study is particularly relevant, because it has provided the first high-resolution structures of GLUT9 (PDB code: 8Y65), thereby revealing previously unknown molecular details regarding urate binding.
Cryo-EM structures of human GLUT9-urate complex (PDB code: 8Y65). The figures are generated by PyMOL 3.0 (www.pymol.org).
The identification of these key residues is a substantial advancement enabling a clear understanding of how GLUT9 differentiates urate from other substrates such as glucose. This knowledge may provide a rationale for the development of specific inhibitors targeting GLUT9, which may be instrumental in treating gout and hyperuricemia.
4. URAT1: UNRAVELING THE TRANSPORT MECHANISM
He et al. [8] have presented a detailed structural analysis of URAT1 (PDB code: 8WJQ), focusing on the roles of specific amino acid residues in urate transport and substrate recognition ( Figure 2 ). That study identified a conserved arginine residue (Arg477 in URAT1, corresponding to Arg473 in organic anion transporter 4 (OAT4)) that is essential for chloride-mediated inhibition. This finding represents a substantial departure from prior understanding, by providing molecular evidence of how chloride ions modulate the transport activity of URAT1. Furthermore, the URAT1-urate complex structure has revealed the binding mode of urate, in which Phe364 is a key residue contributing to the higher urate transport activity than observed in other OAT transporters. That study has also revealed the importance of other residues, such as Ser35 and Gln473, in urate binding to URAT1, thus offering a comprehensive view of the molecular interactions within this transporter.
Cryo-EM structures of human URAT1 (R477S)-urate complex (PDB code: 8WJQ). The figures are generated by PyMOL 3.0 (www.pymol.org).
These discoveries have been critical in only advancing knowledge of URAT1’s transport mechanism but also providing potential targets for the development of novel anti-hyperuricemic drugs, which may meet clinical needs for safer and more effective treatments for gout and other related disorders. The identification of these key residues and their roles in substrate recognition and transport represent a major advance in the field, thereby opening new avenues for therapeutic intervention.
5. INNOVATIONS AND IMPLICATIONS
Both studies used cutting-edge cryo-EM techniques to provide the first high-resolution structures of GLUT9 and URAT1 in complex with urate. These advances not only have illuminated the molecular foundations of urate recognition and transport, but also may pave the way to the design of more selective and potent inhibitors. Notably, the reported structure pertain to the complex with the R477S mutant rather than wild-type URAT1 [8]. Although the R477S mutant might exhibit structural divergences from the wild-type, it has provided invaluable insights into the urate recognition and binding patterns. This mutated construct has served as a valuable surrogate, offering a new perspective into the molecular underpinnings of urate’s interactions within the URAT1 transporter framework.
6. INSPIRATION FOR DRUG DISCOVERY
The findings from both studies have provided valuable insights for pharmaceutical companies. For instance, the structural details of GLUT9’s binding pocket and URAT1’s chloride-sensitivity inhibition may guide the development of drugs with fewer adverse effects and higher efficacy. Moreover, understanding of the specific interactions between transporters and their substrates or inhibitors can facilitate drug design and achieve more effective modulation of urate levels.
Our research group has long been investigating uric acid lowering and anti-gout drugs, and has also identified several novel URAT1/GLUT9 inhibitors. Relevant results have been published in high-quality medicinal chemistry journals [9–11], and a candidate drug has been approved for clinical trials. However, the binding mode of these drug candidates with URTA1/GLUT9 remains unclear, thus preventing the rational design of new drugs. These two important achievements will greatly aid in new drug development. Structural biology techniques can help analyze the binding modes of existing drug candidates and uric acid transporters, and subsequently guide rational drug design.
Whereas traditional methods for drug design, including ligand-based and target-based strategies, have been a cornerstone of prior research, they pose certain limitations to fostering innovation. To overcome these constraints, medicinal chemists should harness the advances in artificial intelligence technology, by using sophisticated techniques such as machine learning and deep learning to perform comprehensive analyses of structural data for URAT1 and GLUT9. Leveraging these advanced computational approaches will enable the design of drugs with novel structures and mechanisms. For instance, through the precise targeting of proteins such as xanthine oxidase, URAT1, and GLUT9, in conjunction with the development of dual-target and multi-target drugs, these strategies hold immense potential for opening new avenues in drug discovery.
7. CONCLUSION
The structural elucidation of GLUT9 and URAT1 marks a major leap in the field of urate transport biology. These landmark studies in the molecular mechanisms of urate recognition and transport may provide a foundation for the development of targeted therapies. Moreover, these discoveries portend a promising future for urate transporter research and drug development.