56. Discovery of RGS2-FBXO44 interaction inhibitors using a cell-based NanoBit assay

Aryal, S.; Wong, C. S. Y.; McNabb, H. J.; Junaid, A.; Altman, R. A.; Sjögren, B.

Molecular Pharmacology 2025, https://doi.org/10.1016/j.molpha.2025.100030

Regulators of G protein Signaling (RGS) proteins negatively regulate signaling through G protein-coupled receptors (GPCRs), and reduced RGS protein function is involved in numerous pathologies. However, therapeutic intervention is challenging, as RGS proteins lack druggable binding pockets and enzymatic activity. Instead, targeting mechanisms that control RGS protein expression shows promise as an alternative. Pharmacological stabilization of RGS2 would be a feasible therapeutic strategy in pathologies associated with reduced RGS2 protein levels, such as hypertension, heart failure and asthma. RGS2 is rapidly degraded through the ubiquitin-proteasomal system (UPS), and we recently identified the E3 ligase that recognizes RGS2. F-box Only Protein 44 (FBXO44) acts as the substrate recognition site for RGS2 in this E3 ligase complex, and we hypothesize that inhibiting the RGS2-FBXO44 interaction will lead to enhanced RGS2 levels. Here, we developed a NanoLuc® Binary Technology (NanoBiT) assay that detects the interaction between RGS2 and FBXO44. This assay was used to screen 1600 compounds from the Life Chemicals PPI fragment library. We identified a promising hit, denoted compound 10, that inhibits the RGS2-FBXO44 interaction with a potency of 19.6 μM, through direct binding to RGS2. The resulting increase in RGS2 protein levels is dependent on FBXO44, as siRNA mediated FBXO44 knockdown attenuates the effect of compound 10. Altogether, compound 10 represents the first example of a small molecule inhibitor of the RGS2-FBXO44 interaction and a first step towards the development of molecular probes with a defined mechanism to stabilize RGS2 protein levels.