Hydralazine (HYZ) is one of the oldest FDA-approved vasodilators and has been used for more than 70 years to treat hypertensive crises and preeclampsia. Despite its long history, its primary molecular target and mechanisms of action have remained unclear. Pleiotropic physiological effects of HYZ are associated with significant side effects like lupus-like autoimmune syndrome, which suggests that it interacts with multiple cellular proteins. Various enzymes like prolyl hydroxylase domain (PHD) enzymes, aldehyde oxidase, glutamine oxaloacetate transaminase 1, protein kinase A, and Keap1-Nrf2 complex, have been proposed as potential targets. But none of these explain the vasodilatory activity of HYZ. The regulatory role of Ca²⁺ signaling in vascular tone and the function of RGS proteins suppresses GPCR-mediated Gαq signaling. There is a need to detect the missing molecular link between intracellular signaling changes, HYZ exposure, and vasodilation.
The aim of this study was to detect the direct molecular target of HYZ in live cells and tissues and to determine how the target engagement produces the known physiological effects of the drug. It also evaluated whether the target of HYZ represents a clinically relevant vulnerability in other diseases, specifically glioblastoma, where related signaling pathways are dysregulated.
To achieve these goals, the researchers used HYZyne, an alkyne-tagged activity-based probe, engineered to retain the reactive pharmacophore of HYZ. HYZyne covalently labels HYZ-binding proteins. It enables chemoproteomic identification in cultured cells and mouse tissues. Molecular targets were validated using both in vivo labelling and in vitro biochemical analysis. Quantitative Western blotting, BRET-based G protein assay, and Ca²⁺ imaging with Fura-2 were used to assess functional consequences of target inhibition. Therapeutic potential in glioblastoma was evaluated through cancer cell proliferation assays, senescence markers, and gene expression analyses.
HYZyne profiling demonstrated that HYZ directly and selectively targets the oxygen-sensing enzyme ADO (2-aminoethanethiol dioxygenase) across all biological systems tested. HYZ covalently modifies His112, which is a key Fe(II)-binding residue necessary for ADO catalysis. ADO inhibition stabilizes its substrates, like RGS4, RGS5, and RGS16, in multiple cell types. Accumulated RGS proteins inhibit GPCR-mediated Gαq signaling, increase GTPase-accelerating protein (GAP) activity, and reduce intracellular Ca²⁺ release following receptor activation. These effects occur at the HYZ concentrations consistent with its known vasodilatory activity. HYZ is >100-fold more selective for ADO than PHD enzymes. It does not behave as a general Fe(II) chelator, differentiating it from the investigated metal binding components.
In glioblastoma models, HYZ produces potent cytostatic growth inhibition with hallmarks of cellular senescence, including morphological changes and upregulation of p21, IL6, IL8, CCL20, and MMP3 without inducing cytotoxicity. HYZyne was delivered in mouse brains, which confirmed that ADO is the principal target in neural tissue.
These findings establish a definitive molecular mechanism underlying longstanding therapeutic benefits. By inhibiting ADO, HYZ stabilizes RGS proteins, thereby suppressing Gαq-driven Ca²⁺ mobilization and promoting vasodilation. This mechanism also provides a biochemical explanation for HYZ’s efficacy in preeclampsia and its association with lupus-like symptoms, as impaired GPCR signaling and decreased ERK phosphorylation may contribute to autoimmune dysregulation in T cells. Furthermore, ADO inhibition represents a novel therapeutic approach for glioblastoma as ADO expression and hypotaurine accumulation increase tumor aggressiveness. This implies that optimized HYZ-derived compounds could serve as a brain-penetrating anticancer medicine.
Overall, this study found that ADO is a primary molecular target of hydralazine. HYZ modulates GPCR-mediated Ca²⁺ signaling through ADO inhibition and consequent RGS protein stabilization. It also explained its vasodilatory properties and offered new paths for drug refinement. HYZ emerges as a promising basis for glioblastoma therapy. HYZyne provides a robust chemical tool for investigating ADO biology and advancing targeted drug development in cardiovascular, neurological, and oncologic diseases.
Reference: Shishikura K, Moskowitz DM, Yang J, et al. Hydralazine inhibits cysteamine dioxygenase to treat preeclampsia and senesce glioblastoma. Sci Adv. 2025;11:eadx7687. doi:10.1126/sciadv.adx7687
