Hotspot pocket-based discovery of urea transporter selective inhibitors

Urea Transporter (UT) inhibitors are a promising class of diuretics, as selective inhibitors targeting UT-A subtypes have demonstrated considerable therapeutic potential. Herein, we employ a two-round progressive hotspot pocket-based virtual screening approach combined with biological validation to identify M353-0039 as a highly potent and selective inhibitor of UT-A2. We conduct cryo-electron microscopy to solve the structures of UT-A2 bound with the two inhibitors, M353-0039 and E822-1968, at the resolution of 2.7 Å and 2.9 Å respectively, and elucidate the structural mechanism underlying the superior efficacy and selectivity of M353-0039. Compared with the inhibitor HQA2 and E822-1968, M353-0039 occupies a deeper binding pocket and forms more interactions with UT-A2, thus leading to greater inhibitory potency. We demonstrate that the selectivity of M353-0039 is driven by the nonconserved residues C285 and G322 within the "T-T" subpocket of UT-A2. Finally, we validate the selective effects of M353-0039 in inhibiting UT-A2 function both in mouse models and hepatic cell. These findings not only identify a selective inhibitor as a tool that can be applied to elucidate the unique physiological roles of UT-A2 but also provide an available method for efficiently developing UT-A-selective inhibitors with potent activity as the next-generation diuretics.