Fibroblast growth factor 1 (FGF1), a well-characterized member of the FGF family, effectively lowers blood glucose levels in animal models of type 2 diabetes by stimulating glucose uptake. However, its significant mitogenic potential poses a major challenge for clinical application. Here, we present engineered variants of FGF1 designed to dissociate its potent glucose-lowering effects from its undesired proliferative activity, aiming for a future therapeutic agent for type 2 diabetes. Through a series of rational mutations focused on modulating receptor binding and heparan interactions, coupled with enhanced thermodynamic stability, we developed two lead FGF1 variants. Comprehensive in vitro studies confirmed that these variants exhibit significantly reduced mitogenic potential across various cell types compared to wild-type FGF1. Specifically, one variant showed profound loss of proliferation due to disrupted FGFR binding, while the other displayed attenuated mitogenicity linked to decreased heparin affinity. Critically, both fully maintained potent glucose-lowering properties in db/db mice without inducing hypoglycemia or changes in body weight. Furthermore, these engineered proteins demonstrate superior thermodynamic stability and markedly improved pharmacokinetic profile, critical attributes for drug development. Our findings highlight a successful strategy to uncouple the therapeutic benefits of FGF1 from its mitogenic side effects, offering promising, stable, and safe protein-based drug candidates for type 2 diabetes treatment.

Celon Pharma S A R and D Centre Marymoncka 15 Kazun Nowy 05 152 Poland

Department of Biology Faculty of Medicine Masaryk University Kamenice 753 5 Brno 62500 Czech Republic

Department of Medical Biotechnology Faculty of Biotechnology University of Wroclaw Joliot Curie 14a Wroclaw 50 383 Poland

Department of Protein Engineering Faculty of Biotechnology University of Wroclaw Joliot Curie 14a Wroclaw 50 383 Poland

Institute of Animal Physiology and Genetics of the CAS Rumburska 89 Libechov 27721 Czech Republic

International Clinical Research Center St Anne's University Hospital Pekarska 53 Brno 65691 Czech Republic

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