Novel FGF21 analogues through structure-based optimization for therapeutic development

Abstract

Fibroblast growth factor 21 (FGF21) plays a pivotal role in regulating metabolic processes and energy homeostasis, making it a promising therapeutic avenue for various obesity-related conditions. However, its therapeutic efficacy faces challenges due to its suboptimal pharmacokinetics and bioactivity. To overcome these limitations, we adapt a strategy in which key amino acid residues responsible for enhanced activity are pinpointed through sequence alignment and comparative analysis to develop long-acting FGF21 analogs. The mutant FGF21 analogs are fused with the Fc fragment. Here, we report the design, identification, and characterization of two distinct Fc-fused FGF21 analogs, Fc-FGF21(P119R) and Fc-FGF21(H125R), with significantly augmented potency. These findings hold promise for clinical applications, offering potential interventions for obesity-related metabolic disorders.

1. Introduction

The fibroblast growth factor (FGF) family is a large family of signaling polypeptides that play diverse roles in cell growth, proliferation, differentiation, angiogenesis and metabolism [1‒3]. Among these, fibroblast growth factor 21 (FGF21) is a member of the endocrine FGF19 subfamily [4‒6]. Structurally characterized by its distinctive β-trefoil fold, FGF21 binds to its receptor FGFR1c kinase through its N-terminal segment and its co-receptor β-Klotho (KLB) via its C-terminal tail [7‒9]. The formation of the FGF21-FGFR-KLB signaling complex(s) in a limited number of metabolic or endocrine tissues initiates intracellular signaling through the ERK1/2 cascade [7,10‒13]. The effects of FGF21 are pleotropic, maintaining metabolic and energy homeostasis by lowering blood glucose level and improving blood lipid profiles, with multiple potential metabolic benefits [12,14‒22].

Obesity, fatty liver diseases and diabetes mellitus are among the prevalent metabolic disorders, often complicating other organ-specific diseases [23]. The development of more effective pharmacotherapies is imperative. FGF21 is a competitive candidate. An FGF21 analogue has been planned to enter phase III clinical trials for patients with nonalcoholic steatohepatitis (NASH), liver fibrosis, and hypertriglyceridemia [24,25]. Notably, FGF21 does not induce hypoglycemia, cell proliferation or weight gain [12]. However, its limited pharmacokinetic property and biological potency remain significant challenges for its clinical application [26‒29].

Several strategies have been explored to increase the half-life and activity of FGF21. One such example is LY2405319, which incorporates an additional disulfide bond through the L118C and A134C mutations. This modification is aimed at bolstering its physical stability. Furthermore, LY2405319 has a deletion of four N-terminal amino acids at the N-terminus to mitigate proteolytic cleavage and an S167A mutation to eliminate O-linked glycosylation, facilitating large-scale, homogenous production in yeast [30]. Another innovative approach involves an Fc fusion with two strategic mutation sites in FGF21, effectively reducing aggregation and degradation [31]. Despite these promising advancements, FGF21 and its analogues continue to face challenges related to their relatively low efficacy. Furthermore, in the initial phases of clinical trials, they have not consistently met the primary endpoints for glycemic control [29], indicating that novel improvements are needed. For instance, the introduction of 5 mutation sites into FGF21 on the basis of its structure increases its potency [32]. Introduction of R185W and L166F mutations on the basis of the structure of β-Klotho in the complex of the FGF21 C-terminal peptide resulted in a tenfold increase in the potency of FGF21 [9]. Compared with wild-type FGF21, a novel FGF21-FGF19 chimera with G43C and A31C mutations and a β1-β2 loop from FGF19 exhibited improved anti-diabetic activities [33]. An FGF1ΔHBS-FGF21C-tail chimera, created by substituting the thermally labile and low-receptor-affinity core of FGF21 with an HS binding–deficient endocrinized core of FGF1, exhibited a fivefold longer half-life and a significantly improved efficacy in correcting hyperglycemia and ameliorating insulin resistance in diabetic mice and monkeys [34]. Despite these efforts, there remains untapped potential for further enhancements in the efficacy of FGF21.