Structural and biochemical basis for retinol‐binding protein 4 antagonism by tinlarebant

Background and Purpose Retinol‐binding protein 4 (RBP4) is a member of the lipocalin superfamily that is connected to disease states such as insulin resistance, fatty liver disease and ocular disorders including Stargardt disease. Several retinoid and non‐retinoid antagonists of this protein have been developed for potential clinical use, but none have yet received FDA approval. Tinlarebant, a derivative of the early lead RBP4 antagonist, A1120, was reported to slow macular lesion growth in Stargardt disease patients. However, its precise mechanism of RBP4 antagonism remains undisclosed. Experimental Approach The binding of tinlarebant to RBP4 was studied with fluorescence spectroscopy and crystallography. The impact of tinlarebant occupancy in the RBP4 retinol‐binding pocket on RBP4 complexation with transthyretin (TTR) was studied by size‐exclusion chromatography. The impact of tinlarebant on plasma RBP4 levels was studied in mice. Key Results Fluorescence quenching assays demonstrate that tinlarebant competitively displaces retinol from holo‐RBP4 with an IC 50 of 8.75 ± 0.38 nM. The RBP4‐tinlarebant complex fails to associate with transthyretin in vitro . Administration of single doses of tinlarebant to mice reduced plasma RBP4 levels by up to 93%. The crystal structure shows that tinlarebant impedes RBP4 association with transthyretin through both conformational and steric effects. Conclusion and Implications Tinlarebant is a tight‐binding RBP4 antagonist that lowers RBP4 plasma levels due to disruption of the RBP4‐ transthyretin interaction. The crystal structure shows that tinlarebant integrates multiple aspects of RBP4 interaction and antagonism found in its predecessors. Together, these data illuminate the pharmacology of a clinical inhibitor of the RBP4‐retinol transport system.