Small-molecule inhibitor of Gαo for GNAO1 encephalopathy.

GNAO1-related neurodevelopmental disorders are caused by mutations in the GNAO1 gene encoding the major neuronal G protein, Gαo. GNAO1 encephalopathies manifest in a range of symptoms, including epilepsy, movement disorder, hypotonia, and developmental delay, affecting >400 patients worldwide to date. A growth in the number of diagnosed cases is expected due to the wider availability of whole genome sequencing. One of the most recurrent pathogenic variants causing GNAO1 encephalopathy is an intronic mutation c.724-8G>A, which results in an in-frame insertion of two amino acid residues, Pro-Gln, after Thr241: Gαo[T241_N242insPQ]. We previously performed in-depth profiling of Gαo[insPQ] using structural, biochemical, and cellular studies. Compared with the wild-type protein, Gαo[insPQ] exhibits faster GTP binding and decreased hydrolysis. Importantly, Gαo[insPQ] is deficient in interacting with regulator of G protein signaling (RGS), GTPase-activating proteins that deactivate Gαo. These defects render Gαo[insPQ] a constitutively active mutant loaded with GTP in the G protein signaling. Patients harboring Gαo[insPQ] variant are in urgent need of novel therapy as they are refractory to available medications. In the present study, we performed a high-throughput screening to find molecules that might suppress the constitutive GTP loading by Gαo[insPQ]. We used a high-diversity chemical library of 54080 compounds, identifying a novel compound, N-[5-(2-methylpropyl)-1,3,4-thiadiazol-2-yl]-1H-1,2,3-benzotriazole-5-carboxamide, that decreases the GTP binding rate of Gαo, likely acting as a competitive inhibitor with higher selectivity to the pathogenic protein. This small-molecule inhibitor of Gαo opens new opportunities to drug discovery towards Gαo-dependent pathologies.