Identification of Small-Molecule Inhibitors of Fibroblast Growth Factor 23 Signaling via In Silico Hot Spot Prediction and Molecular Docking to α-Klotho

Fibroblast growth factor 23 (FGF23) is a therapeutic target for treating hereditary and acquired hypophosphatemic disorders, such as X-linked hypophosphatemic (XLH) rickets and tumor-induced osteomalacia (TIO), respectively. FGF23-induced hypophosphatemia is mediated by signaling through a ternary complex formed by FGF23, the FGF receptor (FGFR), and alpha-Klotho. Currently, disorders of excess FGF23 are treated with an FGF23-blocking antibody, burosumab. Small-molecule drugs that disrupt protein/protein interactions necessary for the ternary complex formation offer an alternative to disrupting FGF23 signaling. In this study, the FGF23:alpha-Klotho interface was targeted to identify small-molecule protein/protein interaction inhibitors since it was computationally predicted to have a large fraction of hot spots and two druggable residues on alpha-Klotho. We further identified Tyr433 on the KL1 domain of alpha-Klotho as a promising hot spot and alpha-Klotho as an appropriate drug-binding target at this interface. Subsequently, we performed in silico docking of similar to 5.5 million compounds from the ZINC database to the interface region of alpha-Klotho from the ternary crystal structure. Following docking, 24 and 20 compounds were in the final list based on the lowest binding free energies to alpha-Klotho and the largest number of contacts with Tyr433, respectively. Five compounds were assessed experimentally by their FGF23-mediated extracellular signal-regulated kinase (ERK) activities in vitro, and two of these reduced activities significantly. Both these compounds were predicted to have favorable binding affinities to alpha-Klotho but not have a large number of contacts with the hot spot Tyr433. ZINC12409120 was found experimentally to disrupt FGF23:alpha-Klotho interaction to reduce FGF23-mediated ERK activities by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 +/- 0.23 mu M. Molecular dynamics (MD) simulations of the ZINC12409120:alpha-Klotho complex starting from in silico docking poses reveal that the ligand exhibits contacts with residues on the KL1 domain, the KL1-KL2 linker, and the KL2 domain of alpha-Klotho simultaneously, thereby possibly disrupting the regular function of alpha-Klotho and impeding FGF23:alpha-Klotho interaction. ZINC12409120 is a candidate for lead optimization.