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Novel Small Molecule Fibroblast Growth Factor 23 Inhibitors Increase Serum Phosphate and Improve Skeletal
Zhousheng Xiao1, Jiawang Liu1, Shih-Hsien Liu1
1Department of Medicine, College of Medicine (Z.X., C.C., L.C., G.W.W., L.D.Q.) and Department of Pharmaceutical Sciences, College of Pharmacy (J.L.), University of Tennessee Health Science Center, Memphis, Tennessee; University of Tennessee (UT)/Oak Ridge National Laboratory (ORNL) Center for Molecular Biophysics, Oak Ridge National Laboratory, Oak Ridge, Tennessee (S.H.L., L.P., J.C.S.); Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee (L.P., J.C.S.); and Department of Chemistry, Tennessee Technological University, Cookeville, Tennessee (A.L.C., J.W.C., M.O.I., J.D.C.).
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.
Insights
Researchers developed small molecule drugs that block excess fibroblast growth factor 23 (FGF23). These FGF23 inhibitors improved bone growth and mineralization in a mouse model of hereditary hypophosphatemic rickets.
Area of Science:
- Medicinal Chemistry
- Pharmacology
- Molecular Biology
Background:
- Excess fibroblast growth factor 23 (FGF23) causes hereditary hypophosphatemic rickets (HHR), including X-linked hypophosphatemia (XLH) and tumor-induced osteomalacia (TIO).
- Current FGF23-blocking therapies involve systemically administered antibodies, highlighting a need for alternative small molecule antagonists.
- Small molecules offer potential advantages over antibodies for FGF23 inhibition.
Purpose of the Study:
- To identify and optimize small molecule antagonists targeting FGF23 for treating HHR.
- To investigate the binding mechanism and therapeutic efficacy of novel FGF23 inhibitors in a preclinical model.
Main Methods:
- Structure-based drug design and medicinal chemistry were employed to develop FGF23 antagonists.
- Cryo-electron microscopy, computational docking, site-directed mutagenesis, and FRET assays were used to determine the binding site.
- Preclinical studies in *Hyp* mice (a model for XLH) assessed the efficacy of the compounds *in vivo*.
Main Results:
- Two analogs, (E)-2-(4-(tert-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime (8n) and (E)-2-((E)-4-methylstyryl)benzaldehyde oxime (13a), were identified with improved drug-like properties.
- Compounds 8n and 13a bind to the N-terminus of FGF23, inhibiting its interaction with the FGF receptor/α-Klotho complex.
- Treatment with 8n and 13a normalized serum phosphate and 1,25-dihydroxyvitamin D levels, enhanced bone growth, and improved mineralization in *Hyp* mice, with 13a showing greater potency.
Conclusions:
- Small molecule inhibitors of FGF23, particularly compound 13a, demonstrate significant therapeutic potential for HHR.
- These compounds effectively block FGF23 signaling by binding to its N-terminus.
- Further optimization of these FGF23 inhibitors could lead to novel treatments for FGF23-mediated disorders.
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