Development of Zalfermin, a Long-Acting Proteolytically Stabilized FGF21 Analog

• Published in: Journal of medicinal chemistry Vol. 67; no. 14; pp. 11769 - 11788
• Main Authors: Sass-Ørum, Kristian, Tagmose, Tina Møller, Olsen, Jørgen, Sjölander, Annika, Wahlund, Per-Olof, Han, Dan, Vegge, Andreas, Reedtz-Runge, Steffen, Wang, Zhe, Gao, Xiang, Wieczorek, Birgit, Lamberth, Kasper, Lykkegaard, Kirsten, Nielsen, Peter Kresten, Thøgersen, Henning, Yu, Mingrui, Wang, Jianhua, Drustrup, Jørn, Zhang, Xujia, Garibay, Patrick, Hansen, Kristian, Hansen, Ann Maria Kruse, Andersen, Birgitte
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 25.07.2024; Amer Chemical Soc
• Subjects: Animals; Chemistry, Medicinal; Fibroblast Growth Factors - metabolism; Humans; Life Sciences & Biomedicine; Macaca fascicularis; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity - drug therapy; Obesity - metabolism; Pharmacology & Pharmacy; Proteolysis - drug effects; Science & Technology; BETA-KLOTHO; GLYCOPEGYLATED FGF21; PHASE 1B/2A; ROBUST REDUCTION; LIVER FAT VOLUME; METABOLIC-ACTIVITY; NONALCOHOLIC STEATOHEPATITIS; DIABETIC MONKEYS; PROOF-OF-CONCEPT; CIRCULATING FGF21
• ISSN: 0022-2623; 1520-4804; 1520-4804
• DOI: 10.1021/acs.jmedchem.4c00391

Here, we describe the development of the FGF21 analog zalfermin (NNC0194-0499, 15), intended for once-weekly sc dosing. Protein engineering was needed to address inherent druggability issues of the natural FGF21 hormone. Thus, deamidation of Asp121 was solved by mutation to glutamine, and oxidation of Met168 was solved by mutation to leucine. N-terminal region degradation by dipeptidyl peptidase IV was prevented by alanine residue elongation. To prevent inactivating metabolism by fibroblast activation protein and carboxypeptidase-like activity in the C-terminal region, and to achieve t 1/2 extension (53 h in cynomolgus monkeys), we introduced a C18 fatty diacid at the penultimate position 180. The fatty diacid binds albumin in a reversible manner, such that the free fraction of zalfermin potently activates the FGF-receptor complex and retains receptor selectivity compared with FGF21, providing strong efficacy on body weight loss in diet-induced obese mice. Zalfermin is currently being clinically evaluated for the treatment of metabolic dysfunction-associated steatohepatitis.