GNS561 acts as a potent anti-fibrotic and pro-fibrolytic agent in liver fibrosis through TGF-β1 inhibition

Abstract

Background: Hepatic fibrosis is the result of chronic liver injury that can progress to cirrhosis and lead to liver failure. Nevertheless, there are no anti-fibrotic drugs licensed for human use. Here, we investigated the anti-fibrotic activity of GNS561, a new lysosomotropic molecule with high liver tropism. Methods: The anti-fibrotic effect of GNS561 was determined in vitro using LX-2 hepatic stellate cells (HSCs) and primary human HSCs by studying cell viability, activity of caspases 3/7, autophagic flux, cathepsin maturation and activity, HSC activation and transforming growth factor-β1 (TGF-β1) maturation and signaling. The contribution of GNS561 lysosomotropism to its anti-fibrotic activity was assessed by increasing lysosomal pH. The potency of GNS561 on fibrosis was evaluated in vivo in a rat model of diethylnitrosamine-induced liver fibrosis. Results: GNS561 significantly decreased cell viability and promoted apoptosis. Disrupting the lysosomal pH gradient impaired its pharmacological effects, suggesting that GNS561 lysosomotropism mediated cell death. GNS561 impaired cathepsin activity, leading to defective TGF-β1 maturation and autophagic processes. Moreover, GNS561 decreased HSC activation and extracellular matrix deposition by downregulating TGF-β1/Smad and mitogen-activated proteine kinase signaling and inducing fibrolysis. Finally, oral administration of GNS561 (15 mg/kg per day) was well tolerated and attenuated diethylnitrosamine-induced liver fibrosis in this rat model (decrease of collagen deposition and of pro-fibrotic markers and increase of fibrolysis). Conclusion: GNS561 is a new potent lysosomotropic compound that could represent a valid medicinal option for hepatic fibrosis treatment through both its anti-fibrotic and its pro-fibrolytic effects. In addition, this study provides a rationale for targeting lysosomes as a promising therapeutic strategy in liver fibrosis.

Systems Pharmacology Study of the Anti-Liver Injury Mechanism of Citri Reticulatae Pericarpium

“…It also suggests that a protein can be affected by several compounds simultaneously. LX-2 cells are prone to proliferate faster, and this can lead to liver fibrosis (Bestion et al, 2020). Our in vitro results suggest that CRP can induce apoptosis in LX-2 cells by regulating targets such as BAX, BCL2, and CASP3, consistent with our finding that the apoptosis pathway (hsa04210) is affected by CRP.…”

“…TGF-β1-induced activation of LX-2 cells predisposes to liver fibrosis, resulting from chronic LI developing into cirrhosis that ultimately leads to liver failure (Bestion et al, 2020;Xiang et al, 2020). We chose hesperidin and naringenin to evaluate our prediction results further to validate changes in several relevant proteins in LX-2 cells with or without TGF-β1 (5 ng/ ml) treatment.…”

The multifaceted role of cathepsins in liver disease

“…In agreement with this study, lysosomal release of CtsB into the cytosol in conjunction with the production of ROS has been reported to partially mediate the activation of NLRP3 upon LPS/ATP stimulation, promoting mouse HSC activation and liver fibrosis (26). In addition, lysosomotropic compound, GNS561, decreases human HSC activation via impairing cathepsin activity, resulting in defective autophagy, maturation of TGF-β1 and decrease cell viability (27). Non-cysteine cathepsins such as aspartate CtsD and serine CtsA have also been J o u r n a l P r e -p r o o f related to autophagy.…”

DEN-Induced Rat Model Reproduces Key Features of Human Hepatocellular Carcinoma

“…By contrast, a classical rat model of DEN-induced HCC is based on chronic exposure to DEN which, by itself, leads to chronic inflammation and fibrosis, followed by HCC after 14–20 weeks [ 5 , 6 , 30 ]. However, while the DEN-induced mouse model of HCC is well described and characterized, there is still limited information about the DEN-induced rat model, despite this model being widely used in preclinical testing to target liver fibrosis and HCC [ 5 , 6 , 30 , 31 , 32 , 33 ]. Here, we performed a characterization, including an RNA-seq analysis, to identify the molecular signatures associated with DEN-induced liver cancer development in rats and compared them with the expression profiles of human HCC.…”