Aryal Sadikshya, Wong Cindy Shi Yee, McNabb Harrison J, Junaid Ahmad, Altman Ryan A, Sjögren Benita
Borch Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana.
Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California.
Mol Pharmacol. 2025 May;107(5):100030. doi: 10.1016/j.molpha.2025.100030. Epub 2025 Mar 19.
Regulators of G protein signaling (RGS) proteins negatively regulate signaling through G protein-coupled receptors, 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 show 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, 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 protein-protein interaction 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 toward the development of molecular probes with a defined mechanism to stabilize RGS2 protein levels. SIGNIFICANCE STATEMENT: This study provides a strategy to identify molecules that selectively inhibit RGS2 protein degradation as well as the first example of a compound with the ability to inhibit RGS2 interaction with the E3 ligase component FBXO44. This study provides proof of concept that a small-molecule RGS2-FBXO44 interaction inhibitor will increase RGS2 protein levels. Future development of compounds with this mechanism of action would be clinically useful in pathologies associated with low RGS2 protein levels, including hypertension, heart failure, and asthma.
G蛋白信号调节因子（RGS）蛋白通过G蛋白偶联受体对信号传导起负向调节作用，RGS蛋白功能的降低与多种病理状况有关。然而，由于RGS蛋白缺乏可成药的结合口袋和酶活性，进行治疗干预具有挑战性。相反，靶向控制RGS蛋白表达的机制显示出作为一种替代方法的潜力。在与RGS2蛋白水平降低相关的病理状况（如高血压、心力衰竭和哮喘）中，对RGS2进行药理稳定作用将是一种可行的治疗策略。RGS2通过泛素 - 蛋白酶体系统迅速降解，我们最近鉴定了识别RGS2的E3连接酶。在这个E3连接酶复合物中，仅含F - 盒蛋白44（FBXO44）作为RGS2的底物识别位点，我们推测抑制RGS2 - FBXO44相互作用将导致RGS2水平升高。在此，我们开发了一种检测RGS2与FBXO44之间相互作用的纳米荧光素酶二元技术（NanoBiT）检测方法。该检测方法用于从生命化学公司的蛋白质 - 蛋白质相互作用片段文库中筛选1600种化合物。我们鉴定出一种有前景的命中化合物，即化合物10，它通过直接结合RGS2来抑制RGS2 - FBXO44相互作用，效力为19.6 μM。RGS2蛋白水平的升高依赖于FBXO44，因为siRNA介导的FBXO44敲低减弱了化合物10的作用。总之，化合物10代表了RGS2 - FBXO44相互作用的小分子抑制剂的首个实例，也是朝着开发具有明确机制以稳定RGS2蛋白水平的分子探针迈出的第一步。意义声明：本研究提供了一种鉴定选择性抑制RGS2蛋白降解的分子的策略，以及一种能够抑制RGS2与E3连接酶组分FBXO44相互作用的化合物的首个实例。本研究提供了概念验证，即小分子RGS2 - FBXO44相互作用抑制剂将增加RGS2蛋白水平。具有这种作用机制的化合物的未来开发在与低RGS2蛋白水平相关的病理状况（包括高血压、心力衰竭和哮喘）中具有临床应用价值。
