FGF21 is an endocrine signaling protein belonging to the family of fibroblast growth factors (FGFs). It has emerged as a molecule of interest for treating various metabolic diseases due to its role in regulating glucogenesis and ketogenesis in the liver. However, FGF21 is prone to heat, proteolytic, and acid-mediated degradation, and its low molecular weight makes it susceptible to kidney clearance, significantly reducing its therapeutic potential. Protein engineering studies addressing these challenges have generally shown that increasing the thermostability of FGF21 led to improved pharmacokinetics. Here, we describe the computer-aided design and experimental characterization of FGF21 variants with enhanced melting temperature up to 15 °C, uncompromised efficacy at activation of MAPK/ERK signaling in Hep G2 cell culture, and ability to stimulate proliferation of Hep G2 and NIH 3T3 fibroblasts cells comparable with FGF21-WT. We propose that stabilizing the FGF21 molecule by rational design should be combined with other reported stabilization strategies to maximize the pharmaceutical potential of FGF21.

Department of Biology Faculty of Medicine Masaryk University Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Enantis Ltd Biotechnology Incubator INBIT Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

Loschmidt Laboratories Centre for Toxic Compounds in the Environment RECETOX Faculty of Science Masaryk University Brno Czech Republic

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Ornitz D.M., Itoh N. The fibroblast growth factor signaling pathway. Wiley Inter Rev Dev Biol. 2015;4:215–266. doi: 10.1002/wdev.176. PubMed DOI PMC

Yun Y.-R., Won J.E., Jeon E., Lee S., Kang W., Jo H., et al. Fibroblast growth factors: biology, function, and application for tissue regeneration. J Tissue Eng. 2010;2010 doi: 10.4061/2010/218142. PubMed DOI PMC

Eswarakumar V.P., Lax I., Schlessinger J. Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev. 2005;16:139–149. doi: 10.1016/j.cytogfr.2005.01.001. PubMed DOI

Faham S., Hileman R.E., Fromm J.R., Linhardt R.J., Rees D.C. Heparin structure and interactions with basic fibroblast growth factor. Science. 1996;271:1116–1120. doi: 10.1126/science.271.5252.1116. PubMed DOI

Kan M., Wang F., Xu J., Crabb J.W., Hou J., McKeehan W.L. An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science. 1993;259:1918–1921. doi: 10.1126/science.8456318. PubMed DOI

Ding X., Boney-Montoya J., Owen B.M., Bookout A.L., Coate K.C., Mangelsdorf D.J., et al. βKlotho is required for fibroblast growth factor 21 effects on growth and metabolism. Cell Metab. 2012;16:387–393. doi: 10.1016/j.cmet.2012.08.002. PubMed DOI PMC

Goetz R., Mohammadi M. Exploring mechanisms of FGF signalling through the lens of structural biology. Nat Rev Mol Cell Biol. 2013;14:166–180. doi: 10.1038/nrm3528. PubMed DOI PMC

Jones S.A. Physiology of FGF15/19. Adv Exp Med Biol. 2012;728:171–182. doi: 10.1007/978-1-4614-0887-1_11. PubMed DOI

Ornitz D.M. FGFs, heparan sulfate and FGFRs: complex interactions essential for development. BioEssays News Rev Mol Cell Dev Biol. 2000;22:108–112. doi: 10.1002/(SICI)1521-1878(200002)22:2<108::AID-BIES2>3.0.CO;2-M. PubMed DOI

Nishimura T., Nakatake Y., Konishi M., Itoh N. Identification of a novel FGF, FGF-21, preferentially expressed in the liver. Biochim Biophys Acta (BBA) - Gene Struct Expr. 2000;1492:203–206. doi: 10.1016/S0167-4781(00)00067-1. PubMed DOI

Kharitonenkov A., Shiyanova T.L., Koester A., Ford A.M., Micanovic R., Galbreath E.J., et al. FGF-21 as a novel metabolic regulator. J Clin Invest. 2005;115:1627–1635. doi: 10.1172/JCI23606. PubMed DOI PMC

Lee S., Choi J., Mohanty J., Sousa L.P., Tome F., Pardon E., et al. Structures of β-klotho reveal a ’zip code’-like mechanism for endocrine FGF signalling. Nature. 2018;553:501–505. doi: 10.1038/nature25010. PubMed DOI PMC

Zhang X., Ibrahimi O.A., Olsen S.K., Umemori H., Mohammadi M., Ornitz D.M. Receptor specificity of the fibroblast growth factor family. Complete mammalian FGF family. J Biol Chem. 2006;281:15694–15700. doi: 10.1074/jbc.M601252200. PubMed DOI PMC

Agrawal A., Parlee S., Perez-Tilve D., Li P., Pan J., Mroz P.A., et al. Molecular elements in FGF19 and FGF21 defining KLB/FGFR activity and specificity. Mol Metab. 2018;13:45–55. doi: 10.1016/j.molmet.2018.05.003. PubMed DOI PMC

Shao W., Jin T. Hepatic hormone FGF21 and its analogues in clinical trials. Chronic Dis Transl Med. 2021 doi: 10.1016/j.cdtm.2021.08.005. PubMed DOI PMC

Buchtova M., Chaloupkova R., Zakrzewska M., Vesela I., Cela P., Barathova J., et al. Instability restricts signaling of multiple fibroblast growth factors. Cell Mol Life Sci. 2015;72:2445–2459. doi: 10.1007/s00018-015-1856-8. PubMed DOI PMC

Chen G., Gulbranson D.R., Yu P., Hou Z., Thomson J.A. Thermal stability of fibroblast growth factor protein is a determinant factor in regulating self-renewal, differentiation, and reprogramming in human pluripotent stem cells. Stem Cells. 2012;30:623–630. doi: 10.1002/stem.1021. PubMed DOI PMC

Ren X., Zhao M., Lash B., Martino M.M., Julier Z. Growth factor engineering strategies for regenerative medicine applications. Front Bioeng Biotechnol. 2020;7:469. doi: 10.3389/fbioe.2019.00469. PubMed DOI PMC

Mitchell A.C., Briquez P.S., Hubbell J.A., Cochran J.R. Engineering growth factors for regenerative medicine applications. Acta Biomater. 2016;30:1–12. doi: 10.1016/j.actbio.2015.11.007. PubMed DOI PMC

Paluck S.J., Nguyen T.H., Lee J.P., Maynard H.D. A heparin-mimicking block copolymer both stabilizes and increases the activity of fibroblast growth factor 2 (FGF2) Biomacromolecules. 2016;17:3386–3395. doi: 10.1021/acs.biomac.6b01182. PubMed DOI PMC

Wu J., Mao Z., Hong Y., Han L., Gao C. Conjugation of basic fibroblast growth factor on a heparin gradient for regulating the migration of different types of cells. Bioconjug Chem. 2013;24:1302–1313. doi: 10.1021/bc300670t. PubMed DOI

Kajio T., Kawahara K., Kato K. Stabilization of basic fibroblast growth factor with dextran sulfate. FEBS Lett. 1992;306:243–246. doi: 10.1016/0014-5793(92)81009-b. PubMed DOI

Anitua E., Sánchez M., Orive G., Andia I. Delivering growth factors for therapeutics. Trends Pharmacol Sci. 2008;29:37–41. doi: 10.1016/j.tips.2007.10.010. PubMed DOI

Feito M.J., Lozano R.M., Alcaide M., Ramírez-Santillán C., Arcos D., Vallet-Regí M., et al. Immobilization and bioactivity evaluation of FGF-1 and FGF-2 on powdered silicon-doped hydroxyapatite and their scaffolds for bone tissue engineering. J Mater Sci Mater Med. 2011;22:405–416. doi: 10.1007/s10856-010-4193-3. PubMed DOI

Yoneda A., Asada M., Oda Y., Suzuki M., Imamura T. Engineering of an FGF–proteoglycan fusion protein with heparin-independent, mitogenic activity. Nat Biotechnol. 2000;18:641–644. doi: 10.1038/76487. PubMed DOI

Dvorak P., Bednar D., Vanacek P., Balek L., Eiselleova L., Stepankova V., et al. Computer-assisted engineering of hyperstable fibroblast growth factor 2. Biotechnol Bioeng. 2018;115:850–862. doi: 10.1002/bit.26531. PubMed DOI

Onuma Y., Higuchi K., Aiki Y., Shu Y., Asada M., Asashima M., et al. A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture. PloS One. 2015;10 doi: 10.1371/journal.pone.0118931. PubMed DOI PMC

An Y.J., Lee K.W., Jung Y.-E., Jeong Y.E., Kim S.-J., Woo J., et al. Improvement of FGF7 thermal stability by introduction of mutations in close vicinity to disulfide bond and surface salt bridge. Int J Pept Res Ther. 2022;28:85. doi: 10.1007/s10989-022-10394-1. DOI

Kharitonenkov A., Beals J.M., Micanovic R., Strifler B.A., Rathnachalam R., Wroblewski V.J., et al. Rational design of a fibroblast growth factor 21-based clinical candidate, LY2405319. PLoS One. 2013;8 doi: 10.1371/journal.pone.0058575. PubMed DOI PMC

Weng Y., Ishino T., Sievers A., Talukdar S., Chabot J.R., Tam A., et al. Glyco-engineered long acting FGF21 variant with optimal pharmaceutical and pharmacokinetic properties to enable weekly to twice monthly subcutaneous dosing. Sci Rep. 2018;8:4241. doi: 10.1038/s41598-018-22456-w. PubMed DOI PMC

Zakrzewska M., Krowarsch D., Wiedlocha A., Olsnes S., Otlewski J. Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. J Mol Biol. 2005;352:860–875. doi: 10.1016/j.jmb.2005.07.066. PubMed DOI

Hecht R., Li Y.-S., Sun J., Belouski E., Hall M., Hager T., et al. Rationale-based engineering of a potent long-acting FGF21 analog for the treatment of type 2 diabetes. PLoS One. 2012;7 doi: 10.1371/journal.pone.0049345. PubMed DOI PMC

Mu J., Pinkstaff J., Li Z., Skidmore L., Li N., Myler H., et al. FGF21 analogs of sustained action enabled by orthogonal biosynthesis demonstrate enhanced antidiabetic pharmacology in rodents. Diabetes. 2012;61:505–512. doi: 10.2337/db11-0838. PubMed DOI PMC

Camacho R.C., Zafian P.T., Achanfuo-Yeboah J., Manibusan A., Berger J.P. Pegylated Fgf21 rapidly normalizes insulin-stimulated glucose utilization in diet-induced insulin resistant mice. Eur J Pharmacol. 2013;715:41–45. doi: 10.1016/j.ejphar.2013.06.023. PubMed DOI

Verzijl C.R.C., Van De Peppel I.P., Struik D., Jonker J.W. Pegbelfermin (BMS-986036): an investigational PEGylated fibroblast growth factor 21 analogue for the treatment of nonalcoholic steatohepatitis. Expert Opin Investig Drugs. 2020;29:125–133. doi: 10.1080/13543784.2020.1708898. PubMed DOI

Sanyal A., Charles E.D., Neuschwander-Tetri B.A., Loomba R., Harrison S.A., Abdelmalek M.F., et al. Pegbelfermin (BMS-986036), a PEGylated fibroblast growth factor 21 analogue, in patients with non-alcoholic steatohepatitis: a randomised, double-blind, placebo-controlled, phase 2a trial. Lancet. 2019;392:2705–2717. doi: 10.1016/S0140-6736(18)31785-9. PubMed DOI

Stanislaus S., Hecht R., Yie J., Hager T., Hall M., Spahr C., et al. A novel Fc-FGF21 with improved resistance to proteolysis, increased affinity toward β-Klotho, and enhanced efficacy in mice and cynomolgus monkeys. Endocrinology. 2017;158:1314–1327. doi: 10.1210/en.2016-1917. PubMed DOI

Tillman E.J., Brock W.J., Rolph T. Efruxifermin, a long‐acting Fc‐fusion FGF21 analogue, reduces body weight gain but does not increase sympathetic tone or urine volume in Sprague Dawley rats. Br J Pharmacol. 2022;179:1384–1394. doi: 10.1111/bph.15725. PubMed DOI PMC

Dunshee D.R., Bainbridge T.W., Kljavin N.M., Zavala-Solorio J., Schroeder A.C., Chan R., et al. Fibroblast activation protein cleaves and inactivates fibroblast growth factor 21. J Biol Chem. 2016;291:5986–5996. doi: 10.1074/jbc.M115.710582. PubMed DOI PMC

Coppage A.L., Heard K.R., DiMare M.T., Liu Y., Wu W., Lai J.H., et al. Human FGF-21 is a substrate of fibroblast activation protein. PLoS One. 2016;11 doi: 10.1371/journal.pone.0151269. PubMed DOI PMC

Yan J., Nie Y., Cao J., Luo M., Yan M., Chen Z., et al. The roles and pharmacological effects of FGF21 in preventing aging-associated metabolic diseases. Front Cardiovasc Med. 2021;8 doi: 10.3389/fcvm.2021.655575. PubMed DOI PMC

Talukdar S., Zhou Y., Li D., Rossulek M., Dong J., Somayaji V., et al. A long-acting FGF21 molecule, PF-05231023, decreases body weight and improves lipid profile in non-human primates and type 2 diabetic subjects. Cell Metab. 2016;23:427–440. doi: 10.1016/j.cmet.2016.02.001. PubMed DOI

Charles E.D., Neuschwander-Tetri B.A., Pablo Frias J., Kundu S., Luo Y., Tirucherai G.S., et al. Pegbelfermin (BMS-986036), PEGylated FGF21, in patients with obesity and type 2 diabetes: results from a randomized phase 2 study. Obesity (Silver Spring) 2019;27:41–49. doi: 10.1002/oby.22344. PubMed DOI PMC

Bednar D., Beerens K., Sebestova E., Bendl J., Khare S., Chaloupkova R., et al. FireProt: energy- and evolution-based computational design of thermostable multiple-point mutants. PLoS Comput Biol. 2015;11 doi: 10.1371/journal.pcbi.1004556. PubMed DOI PMC

Beerens K., Mazurenko S., Kunka A., Marques S.M., Hansen N., Musil M., et al. Evolutionary analysis as a powerful complement to energy calculations for protein stabilization. ACS Catal. 2018;8:9420–9428. doi: 10.1021/acscatal.8b01677. DOI

Chang J., Zhang C., Cheng H., Tan Y.-W. Rational design of adenylate kinase thermostability through coevolution and sequence divergence analysis. Int J Mol Sci. 2021;22:2768. doi: 10.3390/ijms22052768. PubMed DOI PMC

Howell S.C., Inampudi K.K., Bean D.P., Wilson C.J. Understanding thermal adaptation of enzymes through the multistate rational design and stability prediction of 100 adenylate kinases. Structure. 2014;22:218–229. doi: 10.1016/j.str.2013.10.019. PubMed DOI

Vilim J., Ghazalova T., Petulova E., Horackova A., Stepankova V., Chaloupkova R., et al. Computer-assisted stabilization of fibroblast growth factor FGF-18. Comput Struct Biotechnol J. 2023;21:5144–5152. doi: 10.1016/j.csbj.2023.10.009. PubMed DOI PMC

Zhu L., Zhao H., Liu J., Cai H., Wu B., Liu Z., et al. Dynamic folding modulation generates FGF21 variant against diabetes. EMBO Rep. 2021;22 doi: 10.15252/embr.202051352. PubMed DOI PMC

Goetz R., Beenken A., Ibrahimi O.A., Kalinina J., Olsen S.K., Eliseenkova A.V., et al. Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members. Mol Cell Biol. 2007;27:3417–3428. doi: 10.1128/MCB.02249-06. PubMed DOI PMC

Studer G., Rempfer C., Waterhouse A.M., Gumienny R., Haas J., Schwede T. QMEANDisCo-distance constraints applied on model quality estimation. Bioinformatics. 2020;36:1765–1771. doi: 10.1093/bioinformatics/btz828. PubMed DOI PMC

Studer G., Biasini M., Schwede T. Assessing the local structural quality of transmembrane protein models using statistical potentials (QMEANBrane) Bioinformatics. 2014;30:i505–i511. doi: 10.1093/bioinformatics/btu457. PubMed DOI PMC

Benkert P., Biasini M., Schwede T. Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics. 2011;27:343–350. doi: 10.1093/bioinformatics/btq662. PubMed DOI PMC

Anandakrishnan R., Aguilar B., Onufriev A.V. H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res. 2012;40:W537–W541. doi: 10.1093/nar/gks375. PubMed DOI PMC

Gordon J.C., Myers J.B., Folta T., Shoja V., Heath L.S., Onufriev A. H++: a server for estimating pKas and adding missing hydrogens to macromolecules. Nucleic Acids Res. 2005;33:W368–W371. doi: 10.1093/nar/gki464. PubMed DOI PMC

Myers J., Grothaus G., Narayanan S., Onufriev A. A simple clustering algorithm can be accurate enough for use in calculations of pKs in macromolecules. Proteins. 2006;63:928–938. doi: 10.1002/prot.20922. PubMed DOI

Case D.A., Babin V., Berryman J.T., Betz R.M., Cai Q., Cerutti D.S., et al. AMBER 14. San Francisco: University of California, 2014.

The PyMOL molecular graphics system, Version 2.0 Schrödinger, LLC, 2015.

Waterhouse A., Bertoni M., Bienert S., Studer G., Tauriello G., Gumienny R., et al. SWISS-MODEL: homology modeling of protein structures and complexes. Nucleic Acids Res. 2018;46:W296–W303. doi: 10.1093/nar/gky427. PubMed DOI PMC

Schymkowitz J., Borg J., Stricher F., Nys R., Rousseau F., Serrano L. The FoldX web server: an online force field. Nucleic Acids Res. 2005;33:W382–W388. doi: 10.1093/nar/gki387. PubMed DOI PMC

Kellogg E.H., Leaver-Fay A., Baker D. Role of conformational sampling in computing mutation-induced changes in protein structure and stability. Proteins. 2011;79:830–838. doi: 10.1002/prot.22921. PubMed DOI PMC

Ashkenazy H., Erez E., Martz E., Pupko T., Ben-Tal N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 2010;38:W529–W533. doi: 10.1093/nar/gkq399. PubMed DOI PMC

Tina K.G., Bhadra R., Srinivasan N. PIC: protein interactions calculator. Nucleic Acids Res. 2007;35:W473–W476. doi: 10.1093/nar/gkm423. PubMed DOI PMC

Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. doi: 10.1093/nar/25.17.3389. PubMed DOI PMC

Huang Y., Niu B., Gao Y., Fu L., Li W. CD-HIT Suite: a web server for clustering and comparing biological sequences. Bioinformatics. 2010;26:680–682. doi: 10.1093/bioinformatics/btq003. PubMed DOI PMC

Frickey T., Lupas A. CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics. 2004;20:3702–3704. doi: 10.1093/bioinformatics/bth444. PubMed DOI

Katoh K., Rozewicki J., Yamada K.D. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019;20:1160–1166. doi: 10.1093/bib/bbx108. PubMed DOI PMC

Larsson A. AliView: a fast and lightweight alignment viewer and editor for large datasets. Bioinformatics. 2014;30:3276–3278. doi: 10.1093/bioinformatics/btu531. PubMed DOI PMC

Mika S., Rost B. UniqueProt: creating representative protein sequence sets. Nucleic Acids Res. 2003;31:3789–3791. doi: 10.1093/nar/gkg620. PubMed DOI PMC

Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. PubMed DOI

Donato M.T., Tolosa L., Gómez-Lechón M.J. Culture and functional characterization of human hepatoma HepG2 cells. Methods Mol Biol. 2015;1250:77–93. doi: 10.1007/978-1-4939-2074-7_5. PubMed DOI

Lin X., Li G., He X., Ma X., Zhang K., Zhang H., et al. FGF21 inhibits apolipoprotein(a) expression in HepG2 cells via the FGFR1-ERK1/2-Elk-1 pathway. Mol Cell Biochem. 2014;393:33–42. doi: 10.1007/s11010-014-2044-0. PubMed DOI

Quest GraphTM ED50 Calculator." AAT Bioquest Inc. 11 Feb. 2024.https://www.aatbio.com/tools/ed50-calculator.

Alford R.F., Leaver-Fay A., Jeliazkov J.R., O’Meara M.J., DiMaio F.P., Park H., et al. The Rosetta all-atom energy function for macromolecular modeling and design. J Chem Theory Comput. 2017;13:3031–3048. doi: 10.1021/acs.jctc.7b00125. PubMed DOI PMC

Guerois R., Nielsen J.E., Serrano L. Predicting changes in the stability of proteins and protein complexes: a study of more than 1000 mutations. J Mol Biol. 2002;320:369–387. doi: 10.1016/S0022-2836(02)00442-4. PubMed DOI

Gao K., Oerlemans R., Groves M.R. Theory and applications of differential scanning fluorimetry in early-stage drug discovery. Biophys Rev. 2020;12:85–104. doi: 10.1007/s12551-020-00619-2. PubMed DOI PMC

Kumar S., Tsai C.-J., Nussinov R. Factors enhancing protein thermostability. Protein Eng. 2000;13:179–191. doi: 10.1093/protein/13.3.179. PubMed DOI

Miotto M., Olimpieri P.P., Di Rienzo L., Ambrosetti F., Corsi P., Lepore R., et al. Insights on protein thermal stability: a graph representation of molecular interactions. Bioinformatics. 2019;35:2569–2577. doi: 10.1093/bioinformatics/bty1011. PubMed DOI PMC

Tsukada Y., Miyazawa K., Kitamura N. High intensity ERK signal mediates hepatocyte growth factor-induced proliferation inhibition of the human hepatocellular carcinoma cell line HepG2. J Biol Chem. 2001;276:40968–40976. doi: 10.1074/jbc.M010890200. PubMed DOI

Jung Y.-E., Lee K.W., Cho J.H., Bae D.-W., Jeong B.-G., Jung Y.-J., et al. Heating-mediated purification of active FGF21 and structure-based design of its variant with enhanced potency. Sci Rep. 2023;13:1005. doi: 10.1038/s41598-023-27717-x. PubMed DOI PMC

Baker D., Agard D.A. Kinetics versus thermodynamics in protein folding. Biochemistry. 1994;33:7505–7509. doi: 10.1021/bi00190a002. PubMed DOI

Quantitative and qualitative differences in the activation of a fibroblast growth factor receptor by different FGF ligands