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 α-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:α-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 α-Klotho. We further
identified Tyr433 on the KL1 domain of α-Klotho as a promising
hot spot and α-Klotho as an appropriate drug-binding target
at this interface. Subsequently, we performed in silico docking of
∼5.5 million compounds from the ZINC database to the interface
region of α-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 α-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 α-Klotho but not have a large number of contacts with the
hot spot Tyr433. ZINC12409120 was found experimentally to disrupt
FGF23:α-Klotho interaction to reduce FGF23-mediated ERK activities
by 70% and have a half maximal inhibitory concentration (IC50) of 5.0 ± 0.23 μM. Molecular dynamics (MD) simulations
of the ZINC12409120:α-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
α-Klotho simultaneously, thereby possibly disrupting the regular
function of α-Klotho and impeding FGF23:α-Klotho interaction.
ZINC12409120 is a candidate for lead optimization.