Characterization of a Novel Macrocyclic Molecular Glue That Selectively Inhibits the Nucleoside Transporter ENT1 and Demonstrates Tissue Protection in Ischemic-Reperfusion AKI In Vivo

Background: Acute kidney injury (AKI) from renal ischemia and reperfusion (IR) is a major clinical issue, particularly during the perioperative period, and lacks effective therapy. AKI is characterized by a sudden decline in renal function over hours to days from injuries within renal tissue architecture. We report the in-vitro and in-vivo profile of a novel macrocyclic molecular glue (RAP-0001) that exhibits potent and selective inhibition of equilibrative nucleoside transporter 1 (ENT1). These results and our mechanism of action provide a novel therapeutic strategy to mitigate renal damage following ischemic-reperfusion events that occur in cardiovasclar surgeries (CABG and TAVR). Preclinical molecular genetics (1-4), preclinical and human pharmacological evidence via a novel mechanism of action (5-12), and human clinical genetics (13-14) suggest that inhibition of ENT1 would be an effective approach. Methods: Functional cell-based assays, Biophysical binding assays, X-ray crystallography, In vivo PK and efficacy studies. Results: RAP-001 forms a ternary complex with FKPB12::RAP-0001::ENT1 on the intracellular side of the cell membrane. RAP-0001 is a potent and selective inhibitor of ENT1 forming molecular glue via the formation of a FKPB12::RAP-0001::ENT1 complex. Our X-ray structure of RAP-0001 bound to FKBP-12 shows the contacts RAP-0001 with FKBP12 where the solvent exposed region can contact ENT1. The efficacy of RAP-0001 was demonstrated in a rat ischemic-reperfusion acute kidney injury model where the compound conferred tissue protection as measured by biomarkers and histological data. Conclusion: The data presented support the pusuit of IND enabling studies.