Discovery of a peripheral 5HT2A antagonist as a clinical candidate for metabolic dysfunction-associated steatohepatitis

Abstract

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) is currently the leading cause of chronic liver disease worldwide. Metabolic Dysfunction-Associated Steatohepatitis (MASH), an advanced form of MASLD, can progress to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Based on recent findings by our team that liver 5HT_2A knockout male mice suppressed steatosis and reduced fibrosis-related gene expression, we developed a peripheral 5HT_2A antagonist, compound 11c for MASH. It shows good in vitro activity, stability, and in vivo pharmacokinetics (PK) in rats and dogs. Compound 11c also shows good in vivo efficacy in a diet-induced obesity (DIO) male mice model and in a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) male mice model, effectively improving histologic features of MASH and fibrosis. According to the tissue distribution study using [¹⁴C]-labeled 11c, the compound was determined to be a peripheral 5HT_2A antagonist. Collectively, first-in-class compound 11c shows promise as a therapeutic agent for the treatment of MASLD and MASH.

Fig. 1. Strategy and history of 5HT_2A antagonists.

Library and rational design for the development of compound 11c as a potent 5HT_2A antagonist. Previous research efforts were discontinued due to limited oral absorption and safety.

Fig. 2. Molecular docking of compounds to Human 5HT_2A.

a The predicted binding model of compound 2 binding to 5HT_2A (left panel) and interactions with 5HT_2A side chains (right panel). b The predicted binding model of compound 11c binding to 5HT_2A (left panel) and interactions with 5HT_2A side chains. The three-dimensional structure of human 5HT_2A is shown with a ribbon expression. 5HT_2A side chains and compounds 2 and 11c are represented as sticks. PHD phenylalanine, ILE Isoleucine, PRO Proline, VAL valine, SER Serine, TRP Tryptophan.

Fig. 3. 5HT_2A antagonist, compound 11c, attenuates HFD-induced obesity, MASLD, and metabolic syndrome.

a–h Twelve-week-old mice were treated with vehicle or compound 11c (5 and 10 mpk) daily oral gavage with a high-fat diet for eight weeks, n = 6 for vehicle, n = 7 for compound 11c 5mpk, and n = 6 for 10mpk. a body weight trend of vehicle- or compound 11c-treated mice, two-way ANOVA with post hoc Dunnett’s test. b Percent fat and lean mass (body composition) of vehicle- or compound 11c-treated mice, two-way ANOVA with post hoc Dunnett’s test. c Intraperitoneal glucose tolerance test (IPGTT) after 16 h fasting, two-way ANOVA with post hoc Dunnett’s test. d Tissue weight of vehicle-treated or compound 11c-treated mice, two-way ANOVA with post hoc Dunnett’s test. e Representative liver histology by H&E staining in vehicle- or compound 11c-treated mice. Scale bars, 100 μm. f Total NAFLD activity score (NAS) of vehicle- or compound 11c-treated mice. Scale bars, 100 μm, 2-sided Mann-Whitney’s U-test. g Serum cholesterol (CHO), Serum triglyceride (TG), and hepatic TG levels of vehicle- or compound 11c-treated mice, one-way ANOVA with post hoc Dunnett’s test. h Relative mRNA expression of lipogenesis-related genes in vehicle- or compound 11c-treated mice, two-way ANOVA with post hoc Tukey’s Honestly Significant Difference (HSD) test. Data are expressed as the means Data are expressed as the means ± SEM.

Fig. 4. In vivo efficacy of Compound 11c for CDAHFD-induced liver fibrosis and inflammation C57BL/6 J mice (6 weeks old) underwent 12 weeks of CDAHFD feeding and for the same period oral injections of compound 11c.

a Body weight of CDAHFD-fed control and compound 11c-treated mice, one-way ANOVA with post hoc Tukey’s Honestly Significant Difference (HSD) test. The data are presented as mean ± SD (n = 5 per group). b, c Relative liver Col1a1 and α-SMA RNA expressions in mice, measured using quantitative RT-PCR, one-way ANOVA with post hoc Tukey’s Honestly Significant Difference (HSD) test. The data are presented as mean ± SEM (n = 5 per group). d Representative sirius red and immunohistochemical staining images (α-SMA, TNF-α, and IL-1β) from CDAHFD-subjected vehicle and compound 11c-treated mice (scale bars, 20 μm, arrows: positive areas). e–h Percentage of the sections positive for fibrotic area (sirius red), α-SMA, TNF-α, and IL-1β, 2-sided Mann-Whitney’s U-test. The data are presented as mean ± SD (n = 4–5 per group).