Prior models suggest that the terminal ends of the endocrine

Prior models suggest that the terminal ends of the endocrine FGFs individually determine FGFR and KLB recognition, with little or no cross-talk. Our results confirm that short C-terminal sequences are necessary and sufficient to bind KLB and, supportive of a common mechanism, the underlying structural determinants are highly conserved between FGF19 and FGF21. Nevertheless, Lys194 appeared to be a natural molecular break to curb FGF19 agonism (Figure 3C) in an FGFR isoform-specific manner since the FGF19,A194 mutant proved about tenfold more effective than FGF19 to signal via FGFR1 but not FGFR4 (Figure 4A,B). This novel finding was further corroborated in a binding assay in which the interaction of FGF19,A194 with FGFR1/KLB complex was enhanced but remained unchanged at FGFR4/KLB relative to native hormone (Table 2). Furthermore, the selective ability of the C-terminal lysine in FGF19 to suppress FGFR1-driven signaling relative to FGFR4 indicates that the C-terminal end of the protein operates in concert with the N-terminal region. How this is structurally accomplished remains to be studied, but it illustrates the cooperative action of KLB and FGFR to define high potency of these two endocrine FGFs. As such, the in vitro and in vivo activities of the engineered FGF21-19A hybrid far exceed those of either native protein (Figure 3, Figure 5D). In summary, we report here that short C-terminal peptides derived from two endocrine FGFs bind to the KLB co-receptor and can each fully antagonize FGF19 and FGF21 signaling. Despite only a modest degree of sequence identity in the C-terminal regions of FGF19 and FGF21, the critical amino acids involved in KLB recognition are highly conserved between these two proteins. Nonetheless, the differences in sequence can have biological purpose as evidenced by the impact of the FGF19 C-terminal amino A 804598 upon FGFR specificity. As C-terminal lysine residues are susceptible to selective proteolysis by Carboxypeptidase B-like exopeptidases [55], it is plausible that this amino acid in FGF19 might function physiologically to alter receptor selectively. Pragmatically, we believe that the antagonism of the intrinsically derived peptide being subsequently translated to super-agonism when the optimized sequence is integrated as a portion of a full-length protein is precedent setting, as this medicinal chemistry strategy has not been reported before. As well, the synthetic peptide-based KLB antagonist and the super-potent protein hybrid each constitute valuable reagents in further defining the biology of the endocrine FGFs. Collectively, our results deepen the understanding of the structural aspects in FGF19 and FGF21 ligand–receptor complex activation and provide experimental means for further advances in basic and translational research.
Author contributions AA, SP, PL, AMKH, and BA designed, performed and analyzed biochemical in vitro experiments. AA, JP, and PAM designed, performed and analyzed chemical peptide synthesis. BF and DPT designed, performed and analyzed the in vivo experiments. AK and RDD conceptualized, analyzed and supervised all studies. AA, AK, and RDD co-wrote the manuscript.
Declaration of interests
Acknowledgments We thank Dr. Fa Liu and Mr. Jay Levy for their assistance with peptide synthesis. We thank Emily Yates, Marita Rivir, Jenna Holland, Joyce Sorrell and Kathi Smith for their assistance with in vivo pharmacology studies.
Introduction Breast cancer is among the most common cancer worldwide, and has become as a big threaten to public health nowadays [1]. Though advanced technologies have been applied in diagnostics and treatment, the overall survival rate is still lower than 50%. Chemotherapy is still considered as primary or adjuvant option for most breast cancer patients [2]. However, though chemotherapy initially works well for 60–100% breast cancer patients, drug response rates reduce to 20–30% in the second-round chemotherapy, due to intrinsic or acquired drug resistance of cancer cells to chemo-drugs [3]. Therefore, new intervention methods to overcome drug resistance are still needed in treating with breast cancer patients.