Department 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.).
Department 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.)
Mol Pharmacol. 2021 Jun;101(6):408-421. doi: 10.1124/molpharm.121.000471. Epub 2022 Mar 26.
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/-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 mouse homolog of XLH a direct connect analog [()-2-(4-(-butyl)phenyl)cyclopent-1-ene-1-carbaldehyde oxime] (), which exhibited the greatest stability in microsomal assays, and [()-2-(()-4-methylstyryl)benzaldehyde oxime] (), 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 ( and ) 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)D] concentrations in mice. Long-term parenteral treatment with or also enhanced linear bone growth, increased mineralization of bone, and narrowed the growth plate in mice. The more potent compound had greater therapeutic effects in 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 mouse model.
过量的成纤维细胞生长因子 23（FGF23）会导致遗传性低磷血症佝偻病，如 X 连锁低磷血症（XLH）和肿瘤诱导性骨软化症（TIO）。一种能够特异性结合 FGF23 以防止成纤维细胞生长因子受体/-Klotho 复合物激活的小分子，相对于目前批准的全身性 FGF23 阻断抗体具有潜在优势。我们先前使用基于结构的药物设计方法，将 ZINC13407541（N-[[2-(2-苯乙烯基)环戊-1-基]亚甲基]羟胺）鉴定为 FGF23 的小分子拮抗剂。进一步的结构活性研究开发了一系列具有增强的药物样特性的 ZINC13407541 类似物。在这项研究中，我们在 XLH 的一种临床前小鼠同源物中测试了直接连接类似物 [()-2-(4-(-丁基)苯基)环戊-1-烯-1-甲酰基肟]（），其在微粒体测定中表现出最大的稳定性，和 [()-2-(()-4-甲基苯乙烯基)苯甲醛肟]（），其表现出体外效力的增加。通过冷冻电子显微镜结构和计算对接，我们确定了 FGF23 拮抗剂（ZINC13407541 及其类似物（和））在 FGF23 蛋白的 N 端结构域中的关键结合残基（Q156）。定点突变和双分子荧光互补-荧光共振能量转移测定证实了这三种拮抗剂的结合位点。我们发现，用这些化合物中的任何一种对 FGF23 进行药理抑制，均可阻断 FGF23 信号传导，并增加小鼠血清磷酸盐和 1,25-二羟基维生素 D [1,25(OH)D]的浓度。长期给予或也增强了小鼠的线性骨生长、骨矿化和生长板变窄。更有效的化合物在小鼠中具有更大的治疗效果。进一步优化这些 FGF23 抑制剂可能会产生多功能药物来治疗过量 FGF23 介导的疾病。意义：本研究使用基于结构的药物设计和药物化学方法来鉴定和优化具有不同稳定性和效力的小分子，这些小分子拮抗遗传性低磷血症佝偻病中过量的成纤维细胞生长因子 23（FGF23）的作用。研究结果证实，这些拮抗剂与 FGF23 的 N 端结合，抑制其与成纤维细胞生长因子受体/-Klotho 信号复合物的结合和激活。这些先导化合物的给药改善了临床前 XLH 小鼠模型中的磷酸盐稳态和骨骼表型异常。
