Development of cyclopeptide inhibitors specifically disrupting FXR–coactivator interaction in the intestine as a novel therapeutic strategy for MASH

Abstract

Intestinal farnesoid X receptor (FXR) antagonists have been proven to be efficacious in ameliorating metabolic diseases, particularly for the treatment of metabolic dysfunction-associated steatohepatitis (MASH). All the reported FXR antagonists target to the ligand-binding pocket (LBP) of the receptor, whereas antagonist acting on the non-LBP site of nuclear receptor (NR) is conceived as a promising strategy to discover novel FXR antagonist. Here, we have postulated the hypothesis of antagonizing FXR by disrupting the interaction between FXR and coactivators, and have successfully developed a series of macrocyclic peptides as FXR antagonists based on this premise. The cyclopeptide DC646 not only exhibits potent inhibitory activity of FXR, but also demonstrates a high degree of selectivity towards other NRs. Moreover, cyclopeptide DC646 has high potential therapeutic benefit for the treatment of MASH in an intestinal FXR-dependent manner, along with a commendable safety profile. Mechanistically, distinct from other known FXR antagonists, cyclopeptide DC646 specifically binds to the coactivator binding site of FXR, which can block the coactivator recruitment, reducing the circulation of intestine-derived ceramides to the liver, and promoting the release of glucagon-like peptide-1 (GLP-1). Overall, we identify a novel cyclopeptide that targets FXR-coactivator interaction, paving the way for a new approach to treating MASH with FXR antagonists.

Keywords

intestine-targeted drugs / FXR antagonists / cyclopeptide / metabolic dysfunction-associated steatohepatitis / protein–protein interactions

References

[1] Li F , Jiang C , Krausz KW et al. Microbiome remodelling leads to inhibition of intestinal farnesoid X receptor signalling and decreased obesity. Nat Commun 2013; 4: 2384.

[2] Sun L , Xie C , Wang G et al. Gut microbiota and intestinal FXR mediate the clinical benefits of metformin. Nat Med 2018; 24: 1919- 29.

[3] Wu Q , Sun L , Hu X et al. Suppressing the intestinal farnesoid X receptor/sphingomyelin phosphodiesterase 3 axis decreases atherosclerosis. J Clin Invest 2021; 131: e142865.

[4] Zheng X , Chen T , Jiang R et al. Hyocholic acid species improve glucose homeostasis through a distinct TGR5 and FXR signaling mechanism. Cell Metab 2021; 33: 791- 803.e7.

[5] Jiang C , Xie C , Lv Y et al. Intestine-selective farnesoid X receptor inhibition improves obesity-related metabolic dysfunction. Nat Commun 2015; 6: 10166.

[6] Jiang C , Xie C , Li F et al. Intestinal farnesoid X receptor signaling promotes nonalcoholic fatty liver disease. J Clin Invest 2015; 125: 386- 402.

[7] Jiang J , Ma Y , Liu Y et al. Glycine-β-muricholic acid antagonizes the intestinal farnesoid X receptor-ceramide axis and ameliorates NASH in mice. Hepatol Commun 2022; 6: 3363- 78.

[8] Takahashi S , Luo Y , Ranjit S et al. Bile acid sequestration reverses liver injury and prevents progression of nonalcoholic steatohepatitis in Western diet-fed mice. J Biol Chem 2020; 295: 4733- 47.

[9] Xie C , Jiang C , Shi J et al. An intestinal farnesoid X receptorceramide signaling axis modulates hepatic gluconeogenesis in mice. Diabetes 2017; 66: 613- 26.

[10] Huang F , Zheng X , Ma X et al. Theabrownin from Pu-erh tea attenuates hypercholesterolemia via modulation of gut microbiota and bile acid metabolism. Nat Commun 2019; 10: 4971.

[11] Zhang C , Liu Y , Wang Y et al. Discovery of betulinic acid derivatives as potent intestinal farnesoid X receptor antagonists to ameliorate nonalcoholic steatohepatitis. J Med Chem 2022; 65: 13452- 72.

[12] Ravindranathan P , Lee TK , Yang L et al. Peptidomimetic targeting of critical androgen receptor-coregulator interactions in prostate cancer. Nat Commun 2013; 4: 1923.

[13] Estebanez-Perpina E , Arnold LA , Nguyen P et al. A surface on the androgen receptor that allosterically regulates coactivator binding. Proc Natl Acad Sci USA 2007; 104: 16074- 9.

[14] Axerio-Cilies P , Lack NA , Nayana MRS et al. Inhibitors of androgen receptor activation function-2 (AF2) site identified through virtual screening. J Med Chem 2011; 54: 6197- 205.

[15] Caboni L , Kinsella GK , Blanco F et al. “True” antiandrogensselective non-ligand-binding pocket disruptors of androgen receptor-coactivator interactions: novel tools for prostatecancer. J Med Chem 2012; 55: 1635- 44.

[16] Chai X , Sun H , Zhou W et al. Discovery of N-(4-(benzyloxy)-phenyl)-sulfonamide derivatives as novel antagonists of the human androgen receptor targeting the activation function 2. J Med Chem 2022; 65: 2507- 21.

[17] Xu X , Xu X , Liu P et al. Structural basis for small molecule NDB (N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-(dimethylamino) benzamide) as a selective antagonist of farnesoid X receptor α (FXRα) in stabilizing the homodimerization of the receptor. J Biol Chem 2015; 290: 19888- 99.

[18] Teno N , Iguchi Y , Oda K et al. Discovery of orally active and nonsteroidal farnesoid X receptor (FXR) antagonist with propensity for accumulation and responsiveness in ileum. ACS Med Chem Lett 2021; 12: 420- 5.

[19] Fosgerau K , Hoffmann T . Peptide therapeutics: current status and future directions. Drug Discov Today 2015; 20: 122- 8.

[20] Sharma K , Sharma KK , Sharma A et al. Peptide-based drug discovery: current status and recent advances. Drug Discov Today 2022; 28: 103464.

[21] Thakur R , Suri CR , Kaur IP et al. Peptides as diagnostic, therapeutic, and theranostic tools: progress and future challenges. Crit Rev Ther Drug Carrier Syst 2023; 40: 49- 100.

[22] Li Z , Zhang B , Wang N et al. A novel peptide protects against diet-induced obesity by suppressing appetite and modulating the gut microbiota. Gut 2022; 72: 686- 98.

[23] Peng J , Li C , Khamrakulov M et al. Rhodium(III)-catalyzed C-H alkenylation: access to maleimide-decorated tryptophan and tryptophan-containing peptides. Org Lett 2020; 22: 1535- 41.

[24] Zhang Q , Xie X , Peng J et al. Direct C4-acetoxylation of tryptophan and tryptophan-containing peptides via palladium(II)-catalyzed C-H activation. Org Lett 2021; 23: 4699- 704.

[25] Li J , Sun J , Zhang X et al. Synthesis of maleimide-braced peptide macrocycles and their potential anti-SARS-CoV-2 mechanisms. Chem Commun 2023; 59: 868- 71.

[26] Zhang Y , Hu S , Li Y et al. Macrocyclization of maleimidedecorated peptides via late-stage Rh(III)-catalyzed Trp(C7) alkenylation. Org Lett 2023; 25: 2456- 60.

[27] Hu S , Zhang Y , Xie X et al. Rh(III)-catalyzed late-stage C-H alkenylation and macrolactamization for the synthesis of cyclic peptides with unique Trp(C7)-alkene crosslinks. Chin Chem Lett 2024; 35: 109408.

[28] Jin L , Feng X , Rong H et al. The antiparasitic drug ivermectin is a novel FXR ligand that regulates metabolism. Nat Commun 2013; 4: 1937.

[29] Merk D , Sreeramulu S , Kudlinzki D et al. Molecular tuning of farnesoid X receptor partial agonism. Nat Commun 2019; 10: 2915.

[30] Kajani S , Laker RC , Ratkova E et al. Hepatic glucagon action: beyond glucose mobilization. Physiol Rev 2024; 104: 1021- 60.

[31] Adorini L , Trauner M . FXR agonists in NASH treatment. J Hepatol 2023; 79: 1317- 31.

[32] Clifford BL , Sedgeman LR , Williams KJ et al. FXR activation protects against NAFLD via bile-acid-dependent reductions in lipid absorption. Cell Metab 2021; 33: 1671- 84.e4.

[33] Tang Y , Fan Y , Wang Y et al. A current understanding of FXR in NAFLD: the multifaceted regulatory role of FXR and novel lead discovery for drug development. Biomed Pharmacother 2024; 175: 116658.

[34] Sun L , Cai J , Gonzalez F . The role of farnesoid X receptor in metabolic diseases, and gastrointestinal and liver cancer. Nat Rev Gastroenterol Hepatol 2021; 18: 335- 47.

[35] Harrison SA , Bedossa P , Guy CD et al. A phase 3, randomized, controlled trial of resmetirom in NASH with liver fibrosis. N Engl J Med 2024; 390: 497- 509.

[36] Gonzalez FJ , Jiang C , Patterson AD . An intestinal microbiotafarnesoid X receptor axis modulates metabolic disease. Gastroenterology 2016; 151: 845- 59.

[37] Jiao TY , Ma YD , Guo XZ et al. Bile acid and receptors: biology and drug discovery for nonalcoholic fatty liver disease. Acta Pharmacol Sin 2022; 43: 1103- 19.

[38] Yan Y , Niu Z , Sun C et al. Hepatic thyroid hormone signalling modulates glucose homeostasis through the regulation of GLP-1 production via bile acid-mediated FXR antagonism. Nat Commun 2022; 13: 6408.

[39] Trabelsi MS , Daoudi M , Prawitt J et al. Farnesoid X receptor inhibits glucagon-like peptide-1 production by enteroendocrine L cells. Nat Commun 2015; 6: 7629.

[40] Nevola R , Epifani R , Imbriani S et al. GLP-1 receptor agonists in non-alcoholic fatty liver disease: current evidence and future perspectives. Int J Mol Sci 2023; 24: 1703.

[41] Montandon SA , Somm E , Loizides-Mangold U et al. Multitechnique comparison of atherogenic and MCD NASH models highlights changes in sphingolipid metabolism. Sci Rep 2019; 9: 16810.

[42] Yun C , Yan S , Liao B et al. The microbial metabolite agmatine acts as an FXR agonist to promote polycystic ovary syndrome in female mice. Nat Metab 2024; 6: 947- 62.

[43] Zhong X , Liu Y , Gao X et al. Caffeic acid phenethyl ester suppresses intestinal FXR signaling and ameliorates nonalcoholic fatty liver disease by inhibiting bacterial bile salt hydrolase activity. Acta Pharmacol Sin 2023; 44: 145- 56.

[44] Pathak P , Liu H , Boehme S et al. Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism. J Biol Chem 2017; 292: 11055- 69.

[45] Pathak P , Xie C , Nichols RG et al. Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid receptor-1 signaling to improve metabolism. Hepatology 2018; 68: 1574- 88.

[46] Chen L , Jiao T , Liu W et al. Hepatic cytochrome P450 8B1 and cholic acid potentiate intestinal epithelial injury in colitis by suppressing intestinal stem cell renewal. Cell Stem Cell 2022; 29: 1366- 81.e9.

[47] Kleiner DE , Brunt EM , Van Natta M et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005; 41: 1313- 21.

[48] Soisson SM , Parthasarathy G , Adams AD et al. Identification of a potent synthetic FXR agonist with an unexpected mode of binding and activation. Proc Natl Acad Sci USA 2008; 105: 5337- 42.

[49] Akwabi-Ameyaw A , Caravella JA , Chen L et al. Conformationally constrained farnesoid X receptor (FXR) agonists: naphthoic acid-based analogs of GW 4064. Bioorg Med Chem Lett 2008; 18: 4339- 43.

[50] Akwabi-Ameyaw A , Caravella JA , Chen L et al. Conformationally constrained farnesoid X receptor (FXR) agonists: alternative replacements of the stilbene. Bioorg Med Chem Lett 2011; 21: 6154- 60.

[51] Zheng W , Lu Y , Lin S et al. A novel class of natural FXR modulators with a unique mode of selective co-regulator assembly. ChemBioChem 2017; 18: 721- 5.

[52] Flatt B , Martin R , Wang TL et al. Discovery of XL335 (WAY-362450), a highly potent, selective, and orally active agonist of the farnesoid X receptor (FXR). J Med Chem 2009; 52: 904- 7.

[53] Downes M , Verdecia MA , Roecker AJ et al. A chemical, genetic, and structural analysis of the nuclear bile acid receptor FXR. Mol Cell 2003; 11: 1079- 92.

[54] Lundquist JT , Harnish DC , Kim CY et al. Improvement of physiochemical properties of the tetrahydroazepinoindole series of farnesoid X receptor (FXR) agonists: beneficial modulation of lipids in primates. J Med Chem 2010; 53: 1774- 87.

[55] Zheng W , Lu Y , Tian S et al. Structural insights into the heterodimeric complex of the nuclear receptors FXR and RXR. J Biol Chem 2018; 293: 12535- 41.

[56] Lu Y , Zheng W , Lin S et al. Identification of an oleanane-type triterpene hedragonic acid as a novel farnesoid X receptor ligand with liver protective effects and anti-inflammatory activity. Mol Pharmacol 2018; 93: 63- 72.

[57] Jiang L , Xiao D , Li Y et al. Structural basis of tropifexor as a potent and selective agonist of farnesoid X receptor. Biochem Biophys Res Commun 2021; 534: 1047- 52.

[58] Jiang L , Liu X , Wei H et al. Structural insight into the molecular mechanism of cilofexor binding to the farnesoid X receptor. Biochem Biophys Res Commun 2022; 595: 1- 6.

[59] Schmidtke P , Le Guilloux V , Maupetit J et al. Fpocket: online tools for protein ensemble pocket detection and tracking. Nucleic Acids Res 2010; 38: W582- 9.

RIGHTS & PERMISSIONS

The Author(s). Published by Oxford University Press on behalf of Higher Education Press.