Terpene-Based Thiazole Hydrazines as Negative Allosteric Modulators of MRGPRX4: In Vitro Evaluation and Binding Site Analysis

The human MAS-related G-protein-coupled receptors (MRGPRs) represent a promising therapeutic target for managing chronic itch and pain. Among them, MRGPRX4 is activated by bile acids in dorsal root ganglia (DRG) neurons and contributes to the cholestatic itch. Here, we report the design, synthesis, and in vitro evaluation of a focused library of functionalized terpene-based thiazole hydrazines targeting MRGPRX receptors. Functional assays identified two compounds, <b>1C</b> and <b>3G</b>, as negative allosteric modulators (NAMs) of MRGPRX4 with IC₅₀ values of 337 nM and 15.5 μM, respectively, and showed weak activity at MRGPRX2. Computational analysis suggested a putative NAM binding site adjacent to the orthosteric pocket involving key interactions with residues H92^3.22, K96^3.26, and R159^4.62. The resulting site overlaps with the receptor activity-modifying protein 2 (RAMP2) interaction region, where RAMP2 antagonizes MRGPRX4 as a NAM. These findings provide a foundation for developing selective MRGPRX4 NAMs as novel therapeutic agents for chronic itch and pain.