FXR cyclic peptide antagonists as a novel strategy for MASH treatment

The farnesoid X receptor (FXR) is a bile acid nuclear receptor that plays a crucial role in regulating bile acid and sterol metabolism. Bile acids are natural ligands of FXR, which bind to the ligand-binding pocket (LBP) in the ligand-binding domain (LBD) of FXR, inducing conformational changes that recruit co-activators and activate transcriptional activity. Current endogenous or synthetic FXR antagonists, such as glyco-ursodeoxycholic acid, target the LBP by competitively blocking the binding of FXR-activating bile acids like chenodeoxycholic acid, thereby inhibiting FXR activity. Studies have confirmed that intestinal FXR antagonists can effectively improve metabolic disorders, particularly in treating metabolic dysfunction-associated steatohepatitis (MASH). However, traditional FXR antagonists are mostly indirect regulators of FXR activity, and their inhibition is often influenced by the proportion of bile acids with antagonistic and agonistic properties in the bile acid pool, leading to weak and unstable effects.

Cyclic peptides, which combine characteristics of both small molecules and proteins, have emerged as significant molecules for regulating protein–protein interactions (PPIs). Recent studies have confirmed their efficacy in influencing metabolism and gut microbiota composition. Moreover, cyclic peptides exhibit strong lipophilicity, making them less likely to penetrate the intestinal epithelium and more suitable for intestine-targeted drug development. Their inherent structural stability also ensures resistance to proteolytic degradation, enhancing their stability and action duration in the intestine. Therefore, developing FXR-antagonistic cyclic peptides holds promise for creating intestinal-targeted FXR antagonists.

A recent study published in Life Metabolism introduced a novel cyclic peptide, DC646, which specifically binds to the co-activator binding site of FXR, thereby blocking co-activator recruitment, reducing intestinal ceramide production, and promoting glucagon-like peptide-1 (GLP-1) release. This approach holds potential for MASH treatment while maintaining a good safety profile.

In this study, the authors first used an AlphaScreen assay to identify a series of tryptophan-based cyclic peptide compounds with FXR-antagonistic activity, specifically those modified with a C4-position morpholine group. By optimizing the peptide's size and side chain structure, they identified DC646 as the most potent FXR antagonist, with an inhibition rate exceeding 70%. Molecular docking simulations and site-directed mutagenesis of key amino acid residues (Q296 and K303) further confirmed that DC646 directly disrupts FXR-LBD and co-activator interactions by occupying the co-activator binding site (AF2). Additionally, the authors designed and synthesized a biotinylated cyclic peptide probe, BIOTIN-DC646, to validate its role as a PPI inhibitor using co-immunoprecipitation experiments.

FXR is primarily expressed in the liver and intestine. Hepatic FXR may act as a tumor suppressor in hepatocellular carcinoma (HCC) and is also associated with liver injury and cholestasis. Therefore, avoiding hepatic FXR antagonism is critical when developing intestine-targeted FXR antagonists. In organoid and target selectivity experiments, DC646 effectively reversed the expression of GW4064-induced FXR target gene small heterodimer partner (Shp) in an FXR-dependent manner without significantly affecting other nuclear receptor target genes, demonstrating its high target specificity.

In vivo experiments showed that DC646 selectively inhibited FXR signaling in the intestines of MASH model mice without affecting hepatic FXR signaling. Tissue distribution studies further confirmed the strong intestine-targeting properties of DC646, as it was nearly undetectable in the liver. Compared to endogenous intestinal FXR antagonist GUDCA and FXR agonist OCA, DC646 exhibited superior efficacy in alleviating hepatic lipid metabolism disorders, inflammation, and fibrosis.

To further investigate the mechanism underlying DC646's effects on MASH, the authors conducted pharmacodynamic evaluations in Fxr-deficient mice. The reduction in alanine aminotransferase (ALT) levels and anti-steatosis effects observed in wild-type (WT) mice treated with DC646 were absent in Fxr-deficient mice, confirming that DC646's beneficial effects on steatosis and MASH mitigation are mediated through its specific targeting of intestinal FXR. Additionally, in high-fat diet (HFD) mouse models, DC646 was shown to inhibit intestinal FXR, reduce intestinal ceramide production, and induce GLP-1 expression, further elucidating its beneficial mechanisms in metabolic dysfunction-associated fatty liver disease (MAFLD). Moreover, subacute toxicity studies demonstrated that DC646 has a tolerable dose 20 times higher than the minimum effective dose (200 mg/kg), indicating a favorable safety profile.

In conclusion, this study developed a novel cyclic peptide antagonist that targets intestinal FXR and disrupts FXR-co-activator interactions, serving as a promising therapeutic approach for MASH with a strong safety profile.