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Novel Small Molecule Fibroblast Growth Factor 23 Inhibitors Increase Serum Phosphate and Improve Skeletal Abnormalities in Hyp Mice

Zhousheng Xiao, Jiawang Liu, Shih-Hsien Liu, Loukas Petridis, Chun Cai, Li Cao, Guangwei Wang, Ai Lin Chin, Jacob W. Cleveland, Munachi O. Ikedionwu, Jesse D. Carrick, Jeremy C. Smith and Leigh Darryl Quarles
Molecular Pharmacology June 2022, 101 (6) 408-421; DOI: https://doi.org/10.1124/molpharm.121.000471

Abstract

Excess fibroblast growth factor (FGF) 23 causes hereditary hypophosphatemic rickets, such as X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO). A small molecule that specifically binds to FGF23 to prevent activation of the fibroblast growth factor receptor/α-Klotho complex has potential advantages over the currently approved systemically administered FGF23 blocking antibody. Using structure-based drug design, we previously identified ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine) as a small molecule antagonist for FGF23. Additional structure-activity studies developed a series of ZINC13407541 analogs with enhanced drug-like properties. In this study, we tested in a preclinical Hyp mouse homolog of XLH a direct connect analog [(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] (8n), which exhibited the greatest stability in microsomal assays, and [(E)-2-((E)-4-methylstyryl)benzaldehyde oxime] (13a), which exhibited increased in vitro potency. Using cryo-electron microscopy structure and computational docking, we identified a key binding residue (Q156) of the FGF23 antagonists, ZINC13407541, and its analogs (8n and 13a) in the N-terminal domain of FGF23 protein. Site-directed mutagenesis and bimolecular fluorescence complementation-fluorescence resonance energy transfer assay confirmed the binding site of these three antagonists. We found that pharmacological inhibition of FGF23 with either of these compounds blocked FGF23 signaling and increased serum phosphate and 1,25-dihydroxyvitamin D [1,25(OH)2D] concentrations in Hyp mice. Long-term parenteral treatment with 8n or 13a also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in Hyp mice. The more potent 13a compound had greater therapeutic effects in Hyp mice. Further optimization of these FGF23 inhibitors may lead to versatile drugs to treat excess FGF23-mediated disorders.

SIGNIFICANCE STATEMENT This study used structure-based drug design and medicinal chemistry approaches to identify and optimize small molecules with different stability and potency, which antagonize excessive actions of fibroblast growth factor 23 (FGF23) in hereditary hypophosphatemic rickets. The findings confirmed that these antagonists bind to the N-terminus of FGF23 to inhibit its binding to and activation of the fibroblast growth factor receptors/α-Klotho signaling complex. Administration of these lead compounds improved phosphate homeostasis and abnormal skeletal phenotypes in a preclinical Hyp mouse model.

Introduction

Fibroblast growth factor 23 (FGF23) is a hormone produced by osteoblasts/osteocytes in the bone, which activates the FGF receptor/α-Klotho (α-KL) binary receptor complex in renal proximal tubules to regulate phosphate reabsorption and 1,25(OH)2D metabolism and in distal tubules to adjust sodium and calcium reabsorption (Quarles, 2003, 2011). FGF23 plays a causal role in hereditary hypophosphatemic disorders, such as X-linked hypophosphatemia (XLH) and autosomal recessive hypophosphatemia rickets and acquired tumor-induced osteomalacia (TIO). Elevated FGF23 plays an adaptive role in maintaining phosphate homeostasis in chronic kidney disease but is also associated with left ventricular hypertrophy and increased cardiovascular mortality in this setting (Gutierrez et al., 2008, 2009; Hsu and Wu, 2009; Jean et al., 2009; Isakova et al., 2011).

Until recently, treatment of hereditary FGF23-mediated hypophosphatemic disorders consisted of 1,25(OH)2D and phosphate supplements. This approach does not cure the disease and is associated with toxicities related to excess phosphate and 1,25(OH)2D, including nephrocalcinosis (Colares Neto et al., 2019; Arango Sancho, 2020). TIO can be cured by resection of the tumor that is producing FGF23, but surgical removal of the tumor is not possible in approximately 50% of these patients (Fukumoto, 2014; Florenzano et al., 2017).

An FGF23 blocking antibody KRN23 (Burosumab, a human monoclonal IgG1 antibody targeting FGF23 and developed by Kyowa Hakko Kirin and Ultragenyx) has been recently approved for the treatment of XLH and TIO by the U.S. Food and Drug Administration (Carpenter et al., 2018; Lamb, 2018). KRN23 subcutaneously administered at an average dose of 0.98 mg/kg every 2 weeks improves rickets and increases serum phosphate levels in XLH (Carpenter et al., 2014), and was superior to conventional treatment with phosphate and 1,25(OH)2D supplements. To date, KRN23 has not been associated with toxicity at the doses used to treat hypophosphatemia, but the initial use of a high affinity FGF23 blocking antibody in preclinical rodent models resulted in excess mortality (Shalhoub et al., 2012), principally due to oversuppression of FGF23 and resulting hyperphosphatemia and 1,25(OH)2D toxicity (Stubbs et al., 2007; Yamazaki et al., 2008; Fukumoto, 2018). Additional disadvantages of KRN23 include the need for systemic administration, long half-life (elimination half-life of 18 days), and cost.

A small molecule drug to block FGF23 activation of the FGFRs/α−Klotho receptor would have several potential advantages over KRN23. We used homology modeling, molecular dynamics simulation, and virtual high-throughput screening to identify ZINC13407541 (N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine), which binds to FGF23 and inhibits FGF23 interaction with FGFRs (Xiao et al., 2016). The core ZINC13407541 structure contains two ring systems, bridged with an ethylene, and a carbaldehyde oxime functional group on the ortho- position of ring A that is essential for activity (Fig. 1). Subsequent medicinal chemistry investigation of structure-activity relationships based on ZINC13407541 produced 36 analogs with different stabilities and potencies (Downs et al., 2021). Of these, the 13a analog, created by replacing a five-membered aliphatic ring A with a six-membered ring and substituting a para-position hydrogen atom on the aryl ring B with electron-donating a methyl group (CH3), markedly increases potency of the FGF23 antagonists. The analog 8n, created by eliminating the ethylene bridge between the two ring systems of ZINC13407541 to create a direct connect derivative, decreased efficacy but increased metabolic stability (Downs et al., 2021).

Fig. 1.
Fig. 1.

Structure, in vitro efficacy, and metabolic stability of ZINC13407541 (A) and its analogs 8n (B) and 13a (C). IC50 values were extracted from three independent concentration-response experiments examining the compound-dependent inhibition of FGF23-induced ERK reporter activities in HEK293T cells expressing human α-KL after application of ZINC13407541 (n = 3 cells) and its analogs 8n (n = 3 cells) and 13a (n = 3 cells). Maximum percent inhibition values were calculated from the plateaus in inhibition at the highest inhibitor concentration (n = 3 cells for each compound). Metabolic stabilities were expressed as the half-life of the parent compound remaining after an incubation with human liver microsomes (n = 3 liver microsomes for each compound).

In this study, we refined the docking of these compounds to FGF23 three-dimensional (3D) structural data (Chen et al., 2018), performed mutagenesis and fluorescence resonance energy transfer (FRET) analysis to confirm binding of the antagonists to FGF23, and tested the in vivo efficacy of the most potent compound, 13a, in the preclinical Hyp mouse model of XLH.

Materials and Methods

Molecular Docking of Experimentally Verified Hits on the N-Terminal Domain of FGF23

To computationally analyze the binding conformation and affinity of ZINC13407541 and its two analogs, 13a and 8n, to FGF23, molecular docking of the three compounds to the crystal structure of FGF23 [Protein Data Bank (PDB) code: 5W21 (Chen et al., 2018)] and its Q156A mutant (Webb and Sali, 2014) was performed using AutoDock VinaMPI (Trott and Olson, 2010; Ellingson et al., 2013). A rectangular box of 40 × 30 × 40 Å (X × Y × Z directions) was geometrically centered at 88.37, 120.84, 60.25 Å to include the whole N-terminal domain of FGF23, and the entire box was used for searching ligand poses. An exhaustiveness of 30 was used for adequate sampling of ligand conformations within the box, and 20 binding poses were considered for each of the three compounds to wild-type FGF23. The files in PDB format of FGF23 and all poses in the docking are included in the “supplemental data files–PDB files” and their captions are included in the “supplemental file” as supporting information. After docking, each pose i of a compound was given a Boltzmann weight (Widom, 2002) Wi: Embedded Image

where ΔGi is the estimated free energy of binding for pose i, kB is Boltzmann constant, and T is human body temperature at 310 K. The hydrogen-bond analyses were performed on each pose i using LigPlot+ (Laskowski and Swindells, 2011) along with Wi to give each residue on FGF23 a weighted hydrogen-bond score H: Embedded Image

where Hi is the number of hydrogen bonds between pose i of the ligand and the residue. For hydrogen-bond calculation in LigPlot+ (Laskowski and Swindells, 2011), the maximum hydrogen-acceptor and maximum donor-acceptor distances were set at 3.0 and 4.0 Å, respectively. For residues whose H were not zero, each of them was given an estimated conservation score using The ConSurf Server (https://consurf.tau.ac.il/) (Ashkenazy et al., 2016) with default settings. Among the three compounds, the consensus residue with the highest H and conservation score was chosen as the potential binding site. Subsequently, the pose with hydrogen bonding to the potential binding site and with the lowest ΔG for each compound was plotted in three and two dimensions using visual molecular dynamics (VMD) (Humphrey et al., 1996) and LigPlot+ (Laskowski and Swindells, 2011), respectively. For nonbonded contact calculation in LigPlot+ (Laskowski and Swindells, 2011), the minimum and maximum contact distances were set at 2.0 and 4.0 Å, respectively. Dissociation constant (Kd) and machine deep learning (KDEEP) software (Jiménez et al., 2018), a protein-ligand absolute binding affinity predictor based on deep convolutional neural networks, was also used to obtain ΔG for the plotted pose of each compound as a comparison with AutoDock VinaMPI (Trott and Olson, 2010; Ellingson et al., 2013).

Chemicals and Reagents

Synthetic preparation of new ZINC13407541 analogs was conducted in the medicinal chemistry laboratory at University of Tennessee Health Science Center College of Pharmacy and Tennessee Technological University. All reagents used for organic synthesis were sourced from Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Fairfield, NJ), and stored in accordance with manufacturer recommendations. Each test compound was fully characterized by mass spectrometry and NMR (1H and 13C), with a >95% chemical purity which was determined with high-performance liquid chromatography (HPLC). The medicinal chemistry group also generated gram-scale quantitates for in vivo animal studies as we previously reported (Xiao et al., 2016). The compounds were stored at −20°C and tested both in vitro assays and in vivo in the laboratories. Recombinant human FGF23 protein (2604-FG-025) was purchased from R&D Systems (Minneapolis, MN).

Cell Culture and In Vitro Functional Assays

Human embryonic kidney 293 cells transformed with large T antigen (HEK293T) were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS and 1% penicillin and streptomycin.To test the effects of the novel compounds on FGF23-mediated activation of FGFR1/α-KL complex, HEK293T cells were transiently transfected with either empty expression vector or full-length human α-KL along with the extracellular signal-regulated kinase (ERK) luciferase reporter system (Urakawa et al., 2006) and Renilla luciferase-null as internal control plasmid. Transfections were performed by electroporation using Cell Line Nucleofector Kit R according to the manufacturer’s protocol (Amaxa, Inc., Gaithersburg, MD). Thirty-six hours after transfection, the transfected cells were treated with the test compound with a range of 10−9∼10−4 M in the presence or absence of 1 nM FGF23. After 5 hours, the cells were lysed and luciferase activities measured using a Synergy H4 Hybrid Multi-Mode Microplate Reader (Winooski, VT) and Promega Dual-Luciferase Reporter Assay System (Madison, WI).

In Vitro Absorption, Distribution, Metabolism, and Excretion Studies

We contracted the in vitro absorption, distribution, metabolism, and excretion screening with Eurofins Pharma Discovery Services. Microsomal stability of ZINC13407541 and ZINC13407541 analogs was determined in liver microsomes, including human liver microsomes and mouse liver microsomes to determine potential species differences in metabolism (Zeng et al., 2011). Aqueous solubility was assessed in PBS (pH 7.4), simulated intestinal fluid, and simulated gastric fluid. We also extended our assessment of compound in vitro absorption, distribution, metabolism, and excretion by assessing protein binding, permeability, and metabolism. We assessed protein binding using rapid equilibrium dialysis, as previously described (Song et al., 2008).

RNA Purification and Quantitative Real-Time Reverse-Transcription Polymerase Chain Reaction

For quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR), 1.0 μg total RNA isolated from heart, kidney, or long bone of 8-week-old mice was reverse transcribed as previously described (Qiu et al., 2012a, 2012b). Polymerase chain reactions contained 100 ηg template (cDNA or RNA), 300 ηM each forward and reverse primers, and 1XqPCR Supermix (Bio-Rad, Hercules, CA) in 50 μl. The threshold cycle (Ct) of tested-gene product from the indicated genotype was normalized to the Ct for cyclophilin A. Expression of total Klotho isoform transcripts was performed using the following Klotho-isoform-specific primers: Forward primer of mouse m-KL135 transcript: 5′- CAT TTC CCT GTG ACT TTG CTT G-3′, and reverse primer: 5′-ATG CAC ATC CCA CAG ATA GAC-3′. Forward primer of mouse s-KL70 transcript: 5′-GAG TCA GGA CAA GGA GCT GT-3′, and reverse primer: 5′-GGC CGA CAC TGG GTT TTG-3′. The sequences of primers for kidney and bone gene transcripts were previously reported (Qiu et al., 2012a, Qiu et al., 2012b). In addition, the fold change of tested gene transcripts was calculated from the relative levels of the normal gene transcripts in wild-type mice.

Bimolecular Fluorescence Complementation-Fluorescence Resonance Energy Transfer and Site-Directed Mutagenesis Study

We used a bimolecular fluorescence complementation (BiFC)-based FRET assay to measure the interaction of FGF-23/FGFR1/α-Klotho triple complexes in living cells (Shyu et al., 2008a, 2008b; Tian et al., 2007). For this, we used a FGFR1 and a α-Klotho cDNA construct fused to the N- and C-terminal nonfluorescent fragments of Venus [FGFR1-N-terminal fragment of Venus (residues 1–154, VN155) and α-Klotho-C-terminal fragment of Venus (residues 155–238, VC155)] and a FGF-23 cDNA construct fused to the full-length fluorescent protein Cerulean (Cerulean-FGF-23). The interaction between FGFR1-VN155 + α-Klotho-VC155 proteins reconstitutes an intact Venus (FGFR1-VN155/α-Klotho-VC155 dimer complexes), which serves as a FRET acceptor molecule. The FGF-23/FGFR1/α-Klotho triple complex formation brings Cerulean-FGF-23 (FRET donor) in proximity to the reconstituted Venus (FGFR1-VN155/α-Klotho-VC155 dimer complexes), allowing FRET to take place. For BiFC-based FRET assays, the HEK-293T cells will be cotransfected with Cerulean-FGF-23, FGFR1-VN155, and α-Klotho-VC155 constructs. The formation of FGFR1-VN155/α-Klotho-VC155 dimer complexes allows the FRET signal to be detected when a donor excitation (Cerulean-FGF-23) is applied. The HEK293 cells cultured in 96-well plates were treated with various compounds for 60 minutes. At the indicated time point, the emission signals of yellow fluorescent protein (YFP) (535 ± 8 nm) and cyan fluorescent protein (CFP) (486 ± 8 nm) were respectively measured by a Synergy H4 plate reader using an excitation light of 440 ± 10 nm. The background fluorescence was then measured from the wells containing only medium. After subtracting the background fluorescence from the recorded signal, net YFP and CFP readings were obtained. In this document, YFP/CFP emission ratio is used to represent the effect of FRET, which is equal to the net YFP reading divided by the net CFP reading from the same well. In addition, based on the identification of crucial contact residues that inhibit FGF-23 activity in the above computational modeling, we used a Q5 site-directed mutagenesis kit to generate amino-acid residue substitutions at the interaction sites (Q156A) in wild-type Cerulean-FGF-23WT cDNA to create mutant constructs (Cerulean-FGF-23Q156A) that disrupt the contact sites in small chemical/FGF-23 binding pocket of wild-type FGF-23. Then we used the same approach to cotransfect either Cerulean-FGF-23WT or Cerulean-FGF-23Q156A along with FGFR1-VN155 and α-Klotho-VC155 constructs into HEK293 cells to measure the changes of YFP/CFP emission ratio as well as the ERK luciferase reporter activities after treated with various compounds.

Animal Experiments

All animal research was conducted according to guidelines provided by National Institutes of Health and the Institute of Laboratory Animal Resources, National Research Council. The University of Tennessee Health Science Center’s Animal Care and Use Committee approved all animal studies (Protocol number: 18-111.0). All mice were maintained in our vivarium on a standard diet (7912; Harlan Teklad, Madison, WI). To generate hemizygous Hyp mice, we crossed male hemizygous with female wild-type to obtain both male and female hemizygous Hyp mice as previously described (Xiao et al., 2014). At 4 weeks of age, hemizygous Hyp mice were randomly assigned to the following experiments. We excluded the individual Hyp mice at extremes of the stated age range. For single-dose experiment, the 6-week-old Hyp mice received a single i.p. dose of ZINC13407541 (0, 50, 100, 200 mg/kg) or 13a (0, 10, 50, 100 mg/kg) at time 0 and were collected serum samples 0, 2, 4, 8, and 24 hours after administration. For short-term efficacy studies, the 6-week-old Hyp mice were divided into four different groups: (1) Vehicle control; (2) ZINC13407541 (100 mg/kg); (3) 8n (100 mg/kg); and (4) 13a (100 mg/kg). The mice were treated with intraperitoneal injection of either vehicle control (5% DMSO in corn oil), or ZINC13407541, or 8n, or 13a twice a day for 3 days. For long-term treatments, the Hyp mice were divided into four different groups: (1) Vehicle control; (2) ZINC13407541 (50 mg/kg); (3) 8n (50 mg/kg); and (4) 13a (50 mg/kg). The 4-week-old Hyp mice were treated with intraperitoneal injection of either vehicle control (5% DMSO in corn oil), or ZINC13407541, or 8n, or 13a twice a day for four weeks. We assessed the effects of the compounds on the skeletal phenotype at 8 weeks of age (after 4 weeks of treatment) using the methods previously described in studies that characterize the in vivo phenotypes of Hyp mice (Xiao et al., 2014). The serum samples were collected 4 hours after last dose administration. Serum FGF23 levels were measured using the FGF23 ELISA kit (Kainos Laboratories, Tokyo, Japan). Serum phosphorus levels were measured using a Phosphorus Liqui-UV kit (Stanbio Laboratories, Boerne, TX), and serum calcium levels were measured using a Calcium (CPC) Liquicolor kit (Stanbio Laboratories, Boerne, TX). Serum parathyroid hormone (PTH) levels were measured using the Mouse Intact PTH ELISA kit (Immutopics, Carlsbad, CA). Serum aldosterone levels were measured using aldosterone ELISA Kit (Cayman Chemical, MI). Serum 1,25(OH)2D levels were measured using the 1,25-dihydroxy-vitamin D EIA Kit (Immunodiagnostic Systems, Fountain Hills, AZ) as previously escribed (Xiao et al., 2014).

Bone Densitometry, Histomorphometric, and Micro-Computed Tomography Analysis

Bone mineral density (BMD) of femurs was assessed at 8 weeks of age using a small animal bone densitometer (Lunar Corp, Madison, WI). Calcein (Sigma-Aldrich, St. Louise, MO) double labeling of bone and histomorphometric analyses of periosteal mineral apposition rate in tibias were performed using the osteomeasure analysis system (Osteometrics, Inc., Decatur, GA). The distal femoral metaphyses were also scanned using a micro-computed tomography (CT) 40 scanner (Scanco Medical AG, Brüttisellen, Switzerland). A 3D images analysis was done to determine width of growth plate, bone volume, and cortical thickness (Ct.Th) as previously described (Xiao et al., 2008; Xiao et al., 2010).

Statistical Analysis

We evaluated differences between two groups by unpaired t test, multiple groups by one-way analysis of variance, and two groups over time by two-way analysis of variance with interactions. All values are expressed as means ± S.D. All computations were performed using a commercial biostatistics software (GraphPad Software Inc. La Jolla, CA).

Results

We first investigated the in vitro metabolic stability and IC50 for the representative compounds. Compound 8n and 13a were identified from structure activity relationship studies of 36 newly synthesized compounds with different stability and potency in the in vitro assays (Downs et al., 2021). Compound 8n, a direct connect analog of ZINC13407541showed the greatest stability and 13a the greatest potency of the 36 compounds generated. As shown in Fig. 1, compound 8n has eliminated the ethylene bridge to form a directly connected two-ring scaffold, which decreased maximum inhibition activity (% maximum inhibition 84%), but showed increased potency IC50 (2.8 µM) and metabolic stability (31 minutes) compared with ZINC13407541 (100%, 5.0 µM, and 8 minutes). In contrast, compound 13a has exchanged the cyclopentene with a phenyl group, showing no changes in metabolic stability (9 minutes) and similar maximum i3nhibition activity (% maximum inhibition 100%), but 36-fold higher potency for inhibiting FGF23 activity (13a, IC50 = 0.14 μM) compared with ZINC13407541 (IC50 = 5.0 μM).

We also performed the molecular docking of experimentally verified hits on the N-terminal domain of FGF23. Hydrogen-bond analyses after molecular docking (See Supplemental Tables 1–3 for results of the three compounds, ZINC13407541, and its two analogs, 13a and 8n) indicate that Gln156 is a consensus binding site in the N-terminal domain of FGF23 (Table 1). Moreover, Glutamine 156 (Gln156) is highly evolutionarily conserved (See Supp-lemental Table 4). Therefore, Gln156 may well be a binding site for the three compounds in experiments. Fig. 2A shows that ZINC13407541 and 13a adopt similar poses. Further, ZINC13407541 and 13a (See Fig. 2, B and C, respectively) each has two hydrogen bonds with Gln156 and Asparagine 122 (Asn122), and 8n (Fig. 2D) has one hydrogen bond with Gln156. shows the estimated free energies of binding (ΔG) of the three compounds to FGF23, calculated using two different methods: AutoDock VinaMPI (Trott and Olson, 2010; Ellingson et al., 2013) and KDEEP (Jiménez et al., 2018). The values ΔG (Trott and Olson, 2010; Jiménez et al., 2018) suggest that these three compounds bind to Gln156 on FGF23 with similar binding affinities. In addition, molecular docking of the three compounds to the N-terminal domain of FGF23 Q156A mutant shows that all the compounds no longer interact with Q156A (Supplemental Fig. 1) although ΔG of these compounds to the mutant (Supplemental Table 5) are similar to those to the wild-type (Table 2). This also suggests that Gln156 may be a key binding site.

Fig. 2.
Fig. 2.

The computationally predicted interaction of ZINC13407541 and its two analogs, 13a and 8n, with Gln156, the potential binding site, in the N-terminal domain of FGF23 (PDB code: 5W21) shown in (A) 3D structure; (B–D) two-dimensional residue-contacting map for ZINC13407541, 13a, and 8n, respectively. Hydrogen bonds are shown in red dash lines with donor-acceptor distances in Å. Hydrophobic interactions are shown in gray. The corresponding estimated free energies of binding (ΔG) for the three poses are shown in Table 2.

View this table:
TABLE 1

The weighted hydrogen-bond scores H (see eqs. 1 and 2 for details) in descending order for residues in the N-terminal domain of FGF23 that form hydrogen bonds with ZINC13407541, 13a, and 8n in the molecular docking

View this table:
TABLE 2

Computationally estimated free energies of binding (ΔG) with standard errors for ZINC13407541, 13a, and 8n to Gln156 in the N-terminal domain of FGF23 using AutoDock VinaMPI and KDEEP

The corresponding binding poses of ligands are shown in Fig. 2.

We next validated key binding residue in FGF-23 protein through the BiFC-based FRET assays, ERK reporter activity, and site-directed mutagenesis. We generated FGFR1-VN155, α-Klotho-VC155, and Cerulean (CFP)-FGF-23 wild-type constructs (Fig. 3A) to create a BiFC-based FRET assay for measuring FGF23/FGFR1/α-Klotho triple complex formation (Tian et al., 2007; Shyu et al., 2008a, 2008b). We also created a Cerulean (CFP)-FGF-23-Q156A mutant construct based on our molecular docking analysis above. We observed that ZINC13407541 reduced the YFP/CFP ratio at a concentration of 10 µM. Compound 8n (10 µM) had a similar effect as ZINC13407541. Compound 13a (5 µM) exhibited an even greater effect on YFP/CFP ratio, indicating more complete disruption of FGF23/FGFR1/α-Klotho triple complex formation (Fig. 3, B and D). In contrast, Q156A mutagenesis completely abolished the inhibition of YFP/CFP ratio by ZINC13407541 and its analogs (Fig. 3B). Mutagenesis alone had no effects on YFP/CFP ratio and FGF23/FGFR1/α-Klotho triple complex formation (Fig. 3B). Consistent with the BiFC-based FRET data, the ERK reporter activity was not affected in mutant FGF23Q156A when compared with wild-type FGF23WT, suggesting that the Q156A mutation does not change the formation and function of the FGF23/FGFR1/α-Klotho complex in an in vitro cell culture system. However, Q156A completely abolished the inhibition of the ERK reporter activity by ZINC13407541 (10 µM) and 8n (10 µM). Compound 13a (5 µM) thus inhibits FGF23Q156A-induced ERK reporter activity, indicating that 13a may have other effects, such as solvation free energies of the isolated molecules or bind other proteins in the ERK signaling cascade (Fig. 3, C and D). These results suggest that Q156 is key binding residue in FGF-23 protein for ZINC13407541 and its analogs, and the compounds binding to FGF-23 protein disrupts FGF23/FGFR1/α-Klotho triple complex conformation and its downstream signaling transduction (Fig. 3D).

Fig. 3.
Fig. 3.

Effects of ZINC13407541 and its analogs on CFP-FGF23WT or CFP-FGF23Q156A induced BiFC-based FRET ratio and ERK reporter activities. (A) Diagrams of fusion constructs for human CFP-FGF23WT, CFP-FGF23Q156A, α-KL-VN155, and FGFR1-VC155. (B) BiFC-based YFP/CFP ratio (n = 3 cells). (C) ERK reporter activities (n = 3 cells). (D) Computational model of FGF23 antagonist that targets FGF23/FGFR1/α-Klotho complex. Data are expressed as the mean ± S.D. from three independent experiments. *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by one-way ANOVA with Tukey’s multiple-comparisons test.

To examine the in vivo efficacy of both 8n and 13a analogs compared with the parent molecule ZINC13407541 in preclinical animal studies, we measured the mineral ion homeostasis changes in serum of Hyp mice treated with single-dose or short-term FGF23 inhibitors. In Hyp mice, a mouse homolog of XLH, administration of a single intraperitoneal injection of ZINC13407541(100 mg/kg) resulted in an increase in serum phosphate levels 2 hours after administration that peaked 4 hours after treatment and returned to original levels by 24 hours postinjection (Fig. 4, A and B). Also, the single IP injection of ZINC13407541 increased Hyp mice serum phosphate levels in a dose-dependent manner, the maximum effect achieved in the dose of 200 mg/kg ZINC13407541 (Fig. 4, A and B). In contrast, the ZINC13407541 treatment did not affect serum calcium levels (Fig. 4, C and D). Hyp mice treated with 100 mg/kg dose of ZINC13407541 after 24 hours or with 200 mg/kg dosing of ZINC13407541 after 4 hours exhibited a twofold increase in serum FGF23 levels (Fig. 4, E and F). Compound 13a showed the same time- and dose-dependent responses as observed with ZINC13407541, but the magnitude of the increase in serum phosphate levels in Hyp mice 4 hours after dosing was greater with 13a, consistent with its improved IC50 (Fig. 5, A and B). At 100 mg/kg, 13a almost completely corrected the phosphate levels of Hyp mice. No changes were observed in serum calcium in Hyp mice after its administration (Fig. 5, C and D). We observed a transient increase in FGF23 levels at 8 hours in response to single administration of compound 13a (100mg/kg) (Fig. 5E), but levels return to baseline by 24 hours. At the 4 hours’ time-point, when different doses of compound 13a were compared, there were no changes in serum FGF23 levels in Hyp mice (Fig. 5F). We also performed additional short-term treatments with ZINC13407541 (100 mg/kg), 8n (100 mg/kg), or 13a (100 mg/kg) i.p. injection twice a day for 3 days. We observed similar effects of these three compounds to increase serum phosphate levels in Hyp mice (Fig. 6, A and B). No changes in serum calcium or FGF23 levels were observed with this treatment regimen in Hyp mice (Fig. 6, C–F).

Fig. 4.
Fig. 4.

Time- and dose-dependent effects of ZINC13407541 on mineral ion homeostasis and FGF23 levels in Hyp mice. In left panels (A), (C), and (E), time-course assessments of serum phosphate, calcium, and FGF23 levels in Hyp mice that were given a single i.p. injection of ZINC13407541 (100mg/kg) during 24 hours. In right panels (B), (D), and (F), dose-response studies of serum phosphate, calcium, and FGF23 levels in Hyp mice that were given a single i.p. injection of ZINC13407541 (50, 100, and 200 mg/kg) after 4 hours. Data are expressed as the mean ± S.D. from serum samples of individual mice (n = 5). *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by 1-way ANOVA with Dunnett's test.

Fig. 5.
Fig. 5.

Time- and dose-dependent effects of 13a on mineral ion homeostasis and FGF23 levels in Hyp mice. In left panels (A), (C), and (E), time-course assessments of serum phosphate, calcium, and FGF23 levels in Hyp mice that were given a single i.p. injection of 13a (100mg/kg) during 24 hours. In right panels (B), (D), and (F), dose-response studies of serum phosphate, calcium, and FGF23 levels in Hyp mice that were given a single i.p. injection of 13a (10, 50, and 100 mg/kg) after 4 hours. Data are expressed as the mean ± S.D. from serum samples of individual mice (n = 5). *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by one-way ANOVA with Dunnett's test.

Fig. 6.
Fig. 6.

Short-term effects of ZINC13407541, 8n, and 13a on mineral ion homeostasis and FGF23 levels in Hyp mice. (A–C) Serum phosphate. (D–F) Serum calcium. (G–I) Serum FGF23. Data are expressed as the mean ± S.D. from serum samples of individual mice (n = 5). *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by two-way ANOVA with Bonferroni post hoc test.

Finally, to measure serum biochemistry and skeletal phenotype changes of Hyp mice treated with FGF23 inhibitors, we performed long-term exposure studies using half the effective dose (50 mg/kg) of ZINC13407541, 8n, and 13a as i.p. injection twice a day for 4 weeks in Hyp mice. Four- to 8-week-old Hyp mice were administered with vehicle as a control or treated with either ZINC13407541 (50mg/kg), 8n (50mg/kg), or 13a (50mg/kg) for 4 weeks followed by measurement of serum biochemistry and assessment of skeletal parameters. Serum biochemistry measurements showed that serum phosphate levels were elevated in all FGF23 antagonist treated groups compared with vehicle controls. The increase in serum phosphate was relevant greater in 13a, compared with ZINC13407541 and 8n treated mice. No statistically significant changes of FGF23 levels were observed in 8n and 13a treated groups, compared with vehicle treated mice (Table 3). There was a 1.5-fold increase of FGF23 in the ZINC13407541 treated group, similar to previous observations (Xiao et al., 2016). PTH and 1,25(OH)2D levels were increased in mice treated with ZINC13407541, 8n, or 13a (Xiao et al., 2018, 2019). FGF23 also has distal tubular effects to increase sodium reabsorption lead to suppression of aldosterone levels (Andrukhova et al., 2014; Han et al., 2016). ZINC13407541, 8n, and 13a resulted in stimulation in aldosterone levels compared with vehicle treated mice (Table 3), consistent with the inhibition of distal tubule effects of FGF23. With regard to skeletal effects, we observed that body weight, body length, femur length, and tail length were increased in Hyp mice treated with in ZINC13407541, 8n, or 13a compared with vehicle controls at the end of the treatment period (Fig. 7, A–D). Hyp mice treated with ZINC13407541, 8n, or 13a showed 15%, 18%, and 30% increments in femoral BMD, respectively, compared with vehicle controls (Fig. 8A). Micro-CT 3D images revealed that both ZINC13407541 and 8n treated groups had similar increases in trabecular bone volume, 51%, and Ct.Th, 30%. The Hyp mice treated with 13a had greater increases in both trabecular bone volume, 78%, and Ct.Th (44%) than either ZINC13407541 or 8n group (Fig. 8B). The width of the growth plate was reduced after treatment with ZINC13407541, 8n, or 13a. In summary, 13a had greater therapeutic effects on the healing of the growth plate in Hyp mice (Fig. 8B).

Fig. 7.
Fig. 7.

Long-term effects of ZINC13407541, 8n, and 13a on skeletal phenotypes in Hyp mice. (A) body length (n = 6–10). (B) body weight (n = 6–10). (C) tail length (n = 6–10). (D) femur length (n = 6). Data are expressed as the mean ± S.D. from serum samples of individual mice. *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by one-way ANOVA with Tukey’s multiple-comparisons test.

Fig. 8.
Fig. 8.

Long-term effects of ZINC13407541, 8n, and 13a on bone mineral density and bone structure in Hyp mice. (A) BMD (n = 6–10). (B) Micro-CT 3D images, including width of the growth plate (GP, double red arrow, n = 6), femoral bone volume (BV/TV, n = 6), and cortical thickness (Ct.Th, n = 6). Data are expressed as the mean ± S.D. from serum samples of individual mice (n = 5). *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by one-way ANOVA with Tukey’s multiple-comparisons test.

View this table:
TABLE 3

Serum biochemistry of Hyp mice treated with ZINC13407541 and its analogs

Data are expressed as the mean ± S.D. from serum samples of individual mice (n = 5–8). *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by one-way ANOVA with Tukey’s multiple-comparisons test.

To further explore the gene expression profiles changes in bone of Hyp mice, we performed a real-time RT-PCR analysis from long bone samples treated long term with FGF23 inhibitors. Hyp mice have a distinct gene expression profile in bone associated with the presence of rickets/osteomalacia (Liu et al., 2009; Martin et al., 2011; Xiao et al., 2014; Murali et al., 2016; Takashi et al., 2021). Real-time RT-PCR revealed that the antagonist-treated groups reduced FGFs/FGFRs-dependent signaling including Fgf1, Fgf2, Fgf23, Fgfr1, Fgfr2, Fgfr3, and Fgfr4 expressions in bone as well as attenuated wingless (Wnt)/β-catenin signaling, as evidence by reductions in Wnt10b and axis inhibition protein 2 (Axin 2) (Table 4). Reduction in expression of osteoblast message levels for Type 1 collagen, alkaline phosphatase (ALP), and dentin matrix protein 1 (Dmp1) were also observed in bone treated with ZINC13407541 and its analogs (Table 4). In contrast, mature osteoblast (Ob) markers, such as matrix extracellular phosphoglycoprotein (MEPE) and Osteocalcin and chondrocyte markers Type2 collagen but not vascular endothelial growth factor A (VegfA) were upregulated in femurs from the antagonists’ treated groups. In addition, adipocyte markers peroxisome proliferator-activated receptor gamma 2 (Pparγ2) adipocyte fatty acid-binding protein 2 (aP2), and lipoprotein lipase were downregulated in all antagonists’ treated groups (Table 4). The group treated with 13a had greater effects in all gene expressed markers than did either ZINC13407541 or the 8n group. However, there were no obvious changes in osteoclast markers, including osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RankL), matrix metallopeptidase 9 (Mmp9), and tartrate-resistant acid phosphatase (Trap) transcripts in the treated groups as compared with vehicle treated controls (Table 4).

View this table:
TABLE 4

Gene-expression profiles in bone of Hyp mice treated with ZINC13407541 and its analogs

Data are expressed as the fold changes relative to the housekeeping gene β-actin subsequently normalized to control mice (n = 5–7). *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by one-way ANOVA with Tukey’s multiple-comparisons test.

We also examined the renal FGF23 signaling changes in kidney of Hyp mice treated long-term with FGF23 inhibitors. We previously reported that Hyp mice have a characteristic gene expression profile in the kidney (Dai et al., 2012). Four weeks of treatment with FGF23 inhibitors altered FGF23 responsive gene expression in kidney of Hyp mice (Table 5). Compared with ZINC13407541 and 8n groups, 13a exhibited greater effects to increase type IIa sodium phosphate co-transporter (Npt2a) and Npt2c message expression, consistent with higher serum phosphate levels in the 13a treated groups. Consistent with FGF23 stimulation of cytochrome P450 family 27 subfamily b member 1 (Cyp27b1), and inhibition of Cyp24a1, leading to reductions in circulating 1,25(OH)2D, the administration of ZINC13407541 and its analogs to Hyp mice increased the serum concentration of 1,25(OH)2D (Table 3), in association with increased Cyp27b1 and decreased Cyp24a1 message expression (Table 5). FGF23 also stimulates sodium-chloride co-transporter (NCC) expression in the distal renal tubule cells leading to increased sodium (Na+) and potassium (K+) channel activity, sodium retention, and suppression of serum aldosterone. ZINC13407541, 8n, and 13a treatment suppressed NCC expression (Table 5), in association with increased circulating aldosterone levels in Hyp mice. In contrast, treatment with ZINC13407541, 8n, and 13a had no effects on Fgfr1 expression, but increased Klotho transcripts in kidney of Hyp mice (Table 3). These data indicate that ZINC13407541 and its analogs may have clinical utility in blocking the renal effects of excess FGF23.

View this table:
TABLE 5

Gene-expression profiles in kidney of Hyp mice treated with ZINC13407541 and its analogs (n = 5–6)

Data are expressed as the fold changes relative to the housekeeping gene β-actin subsequently normalized to control mice. *, **, and *** indicate statistically significant difference from vehicle control group. P values were determined by one-way ANOVA with Tukey’s multiple-comparisons test.

Discussion

In this study, we are pursuing the development of small molecules that bind to FGF23 and inhibit its activation of the FGFRs/α-Klotho binary receptor complex. Herein, we used a new 3D cryo-electron microscopy structure model to redock the FGF23 antagonists we identified in previous work (Xiao et al., 2016; Chen et al., 2018). We identified a key binding residue (Q156) of the FGF23 antagonists and developed a BiFC-based and ratiometric FRET assay to confirm that a series of small molecule FGF23 antagonists binding to Q156 based on the ZINC13407541 platform disrupt formation of the FGF23/α-KL/FGFR1 triple complex (Tian et al., 2007; Shyu et al., 2008a, 2008b). Furthermore, we report on the efficacy of ZINC13407541 derivatives that displayed either greater stability (8n) in in vitro microsomal assays or greater potency (13a) in in vitro functional assays in a preclinical mouse model. Indeed, two compound 8n and 13a (most potent) demonstrated short- and long-term efficacy in blocking FGF23 end-organ effects in Hyp mice, a human homolog of XLH. We found that pharmacological inhibition of FGF23 efficiently abrogates aberrant FGF23 signaling, corrects hypophosphatemia and aberrant 1,25(OH)2D levels, and improves both bone and renal gene expression profiles caused by elevated FGF23 levels in Hyp mice.

We observed that these small molecule FGF23 inhibitors antagonized both the proximal and distal tubular effects of FGF23 excess in Hyp mice in vivo. Increases in serum phosphate levels were observed as early as 3 to 5 hours after intraperitoneal injections of these compounds at a minimum effective dose was 50 mg/kg body weight, consistent with FGF23 regulation of phosphate metabolism in the kidneys. Long-term FGF23 inhibition with 8n and 13a treatment of Hyp mice for 4 weeks resulted in sustained increases in serum phosphate and 1,25(OH)2D levels, enhanced bone growth, increased bone mineralization, and improvement of the rachitic growth plate abnormalities, in accordance with FGF23 regulation of vitamin D metabolism in kidney and bone homeostasis in vivo. Indeed, the improvement in biochemistries in Hyp mice after treatment with these chemical FGF23 inhibitors was associated with reversal of FGF23’s renal effects on phosphate transporters and enzymes regulating vitamin D metabolism, including reversal of elevated FGF23 effects to suppresses the phosphate transporters Npt2a and Npt2c and Cyp27b1 the enzyme that 1-hydroxylates 25-hydroxyvitamin D [25-(OH)D], and to stimulate Cyp24a1, which degrades 1,25(OH)2D in the proximal tubule. The effects of excess FGF23 in Hyp mice stimulate the sodium chloride cotransporter in the distal tubule that leads to sodium retention and suppression of aldosterone (Xiao et al., 2019). Treatment with 8n and 13a reversed the excess FGF23 mediated renal tubule effects. In addition, treatment with 8n and 13a also increased α-Klotho transcript levels in Hyp mice. Although we did not test the effects of our compounds on blood pressure and cardiovascular effects of excess FGF23, these results suggest possible beneficial effects of these small molecule inhibitors. Compared with compound ZINC13407541and 8n, 13a had greater therapeutic effects on FGF23-induced abnormalities in Hyp mice, consistent with its lower IC50.

Excess FGF23 has both indirect effects on bone and cartilage mineralization mediated by the reductions in serum phosphate and 1,25(OH)2D (Liu et al., 2006, 2008), as well as possible direct effects due to FGF23 activation of FGFRs in osteoblasts (Shalhoub et al., 2011; Meng et al., 2020). We observed substantial improvement in bone abnormalities after only 4 weeks of treatment as well as alteration in gene expression profiles in bone. Treatment with 8n and 13a altered osteoblast function as evidence by decreased type II runt-related transcription factor-2 (Runx2-II), alkaline phosphatase (Alp), type I collagen (Col1), osteopontin (Opn) (Hoac et al., 2020), and Dmp1 and increased Mepe, bone sialoprotein (Bsp), and Osteocalcin expressions. FGF23 may also have effects on adipocytes to modulate adipogenesis (Xiao et al., 2019). This may explain the effects of FGF23 antagonists in reducing adipogenic markers in Hyp bone. Treatment with 8n and 13a reduced several Fgfs/Fgfrs, Wnt10b, frizzled class receptor 2 (Fzd2), and Axin2 gene transcripts, consistent with our previous findings that Hyp mice exhibited higher Fgfs/Fgfrs and Wnt signaling (Liu et al., 2009; Xiao et al., 2014; Murali et al., 2016; Takashi et al., 2021). Increased FGF23 levels were observed in response to single administration of ZINC13407541 and compound 13a, although the response of 13a was transient. Similarly, treatment with ZINC13407541 for 4 weeks resulted in statistically significant increase in serum FGF23, but treatment with 8n and 13a did not increase the FGF23 transcripts and serum FGF23 levels in Hyp mice. Changes in FGF23 may be compensating for the inhibition of FGF23 signaling, similar to the observed increase in serum FGF23 levels after treatment of patients with XLH with FGF23 blocking antibodies (https://www.ultragenyx.com/file.cfm/95/docs/Crysvita_Full_Prescribing_Information.pdf).

The short half-life of small molecules may be of considerable clinical benefit in titrating and reversing drug effects, which may be particularly important given the narrow therapeutic window for FGF23 suppression, particularly in chronic kidney disease (CKD). Preclinical CKD models show that inhibiting FGF23 with a blocking antibody increases mortality due to oversuppression of FGF23 (Shalhoub et al., 2012). Drawing analogies to treatment of secondary hyperparathyroidism with short acting calcimimetics in CKD to lower PTH levels and pointing to data that even partial reductions of FGF23 may be sufficient to reduce cardiovascular effects, a short-acting, titratable FGF23 antagonist might reduce FGF23 dose-dependent cardiotoxicity with acceptable side effects in CKD (Quarles, 2003, 2012; Weber et al., 2003). Further studies are needed to test the effects of these small molecules in models of CKD.

In conclusion, these studies further advance the premise that the development of FGF23 small molecule inhibitors is feasible for treatment of FGF23-mediated hypophosphatemic diseases and possibly other adverse effects of excess FGF23. Small molecules are generally more cost-effective, have a longer shelf life, and are more easily manufactured. In addition, the potential to modify our leads to develop an oral anti-FGF23 therapy has the potential to simplify the administration of FGF23 antagonists. Further lead optimization of these compounds is warranted to increase their drug-like properties and reduce their potential off-target effects.

Authorship Contributions

Participated in research design: Xiao, Carrick, Smith, Quarles.

Conducted experiments: Xiao, J.W. Liu, Cai, Cao, Wang, Chin, Cleveland, Ikedionwu.

Contributed new reagents or analytic tools: Xiao, J.W. Liu, Carrick.

Performed data analysis: Xiao, S.H. Liu, Petridis, Smith, Quarles.

Wrote or contributed to the writing of the manuscript: Xiao, S.H. Liu, Petridis, Carrick, Smith, Quarles.

Footnotes

    • Received December 8, 2021.
    • Accepted March 20, 2022.

  • This work was supported by National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant RO1-DK121132] (to L.D.Q.) and the Compute and Data Environment for Science at the Oak Ridge National Laboratory, which is supported by the Office of Science of the U.S. Department of Energy [Contract DE-AC05-00OR22725].

  • No author has an actual or perceived conflict of interest with the contents of this article.

  • A preprint of this article was deposited in bioRxiv [https://doi.org/10.1101/2020.08.04.236877].

  • https://doi.org/10.1124/molpharm.121.000471.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

13a
[(E)-2-((E)-4-methylstyryl)benzaldehyde oxime]
ALP
alkaline phosphatase
BiFC
bimolecular fluorescence complementation
BMD
bone mineral density
CFP
cyan fluorescent protein
CKD
chronic kidney disease
Ct
threshold cycle
Ct.Th
cortical thickness
Cyp
cytochrome P450
3D
three-dimensional
Dmp1
Dentin matrix protein 1
ERK
extracellular signal-regulated kinase
FGF
fibroblast growth factor
FGFR
fibroblast growth factor receptor
FRET
fluorescence resonance energy transfer
Gln
glutamine
HEK293T
human embryonic kidney 293 cells transformed with large T antigen
α-KL
α-Klotho
MEPE
matrix extracellular phosphoglycoprotein
8n
[(E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime]
NCC
sodium-chloride co-transporter
Npt
sodium phosphate co-transporter
1,25(OH)2D
1,25-dihydroxyvitamin D
25(OH)D
25-hydroxyvitamin D
PDB
protein data bank
PTH
parathyroid hormone
RT-PCR
reverse-transcription polymerase chain reaction
TIO
tumor-induced factor osteomalacia
VC155
C-terminal fragment of Venus (residues 155–238)
VN155
N-terminal fragment of Venus (residues 1–154)
Wnt
wingless
XLH
X-linked hypophosphatemia
YFP
yellow fluorescent protein
ZINC13407541
N-[[2-(2-phenylethenyl)cyclopenten-1-yl]methylidene]hydroxylamine

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Therapeutic Effects of FGF23 Antagonists in Hyp Mice

Zhousheng Xiao, Jiawang Liu, Shih-Hsien Liu, Loukas Petridis, Chun Cai, Li Cao, Guangwei Wang, Ai Lin Chin, Jacob W. Cleveland, Munachi O. Ikedionwu, Jesse D. Carrick, Jeremy C. Smith and Leigh Darryl Quarles
Molecular Pharmacology June 1, 2022, 101 (6) 408-421; DOI: https://doi.org/10.1124/molpharm.121.000471
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