Introduction & Objective: Chronic liver injury represents a pathological wound-healing process. However, there is no approved pharmacological therapy for liver fibrosis. Since GABAergic signaling has been suggested to play a beneficial role in cytotoxic liver injury, we aimed to investigate if the hepatoprotective positive allosteric modulator of the GABAA receptor, HK3, could also ameliorate fibrosis in a human 3D spheroid MASH model. Moreover, AI- and chemoinformatic-based next-generation GABAA modulators will be in our focus as MASH therapeutics.
Methods: Cryopreserved primary hepatocytes and non-parenchymal cells from MASH patients were clustered to spheroids and supplemented with 10 μg/ml insulin (control group) or 10 mg/ml insulin and 240 μM palmitic acid (PA)/ oleic acid (OA) (MASH group) at HepaPredict. The CellTiter-Glo Luminescent Assay kit was used to assess viability for up to two weeks with repeated exposure of HK3. Subsequently, the effects of HK3 (1, 3, and 10 µM) for 1 week on intracellular lipid content were quantified using AdipoRed Assay. Secreted cytokines (IL-6, IL-8) or pro-Collagen type 1α1 were measured in the supernatants using Magnetic Luminex Assay or ELISA. The discovery of new hits was performed through generative AI molecule design and virtual screening with AI and docking simulations.
Results: Acute exposure to HK3 did not result in hepatotoxicity at any concentration. HK3 reduced hepatic lipid content (48%, 46%, and 49%) or pro-COL1A1 levels (82%, 73%, and 64%) compared to MASH, respectively. Exposure to 3 µM HK3 led to a strong decrease in IL-6 (55%) and IL-8 (80%) secretion. Iterative, AI-based identification of new hits and their in-depth functional analysis will be presented.
Conclusion: Our thioacrylamide compound promotes the regression of hepatic fibrosis, besides reducing steatosis and inflammation in a human 3D spheroid system. Overall, GABAA receptor modulators represent a first-in-class approach for MASH therapy.
E. Rohbeck: None. L. Koester: None. J. Eckel: None.
