Article Figures & Data
Figures
- 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).
- 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.
- 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.
- 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.
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.
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.
- 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.
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.
Tables
- 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
ZINC13407541 13a 8n Residue H Residue H Residue H Gln156 0.417 Tyr93 0.293 Tyr93 0.159 Asn122 0.408 Gln156 0.278 Gln156 0.143 Arg76 0.242 Asn122 0.207 Thr86 0.096 Ser77 0.159 Thr86 0.120 Tyr154 0.091 Tyr93 0.134 Arg76 0.113 Ser137 0.074 Thr86 0.115 Ser77 0.101 Gly139 0.074 Met74 0.083 Met74 0.078 Thr46 0.063 Val84 0.083 Val84 0.078 Asn49 0.063 Leu146 0.027 Ala45 0.035 Ser77 0.063 Ile75 0.024 Gln67 0.031 Arg76 0.061 Gly38 0.012 Leu146 0.021 Ser155 0.052 Glu78 0.009 Asn49 0.015 Ile75 0.047 Ser159 0.009 Pro152 0.015 Leu146 0.045 Gln118 0.007 Tyr154 0.015 Met74 0.033 Thr119 0.007 Ser155 0.015 Val84 0.033 Ser34 0.024 Gly38 0.024 Pro152 0.024 Asn162 0.024 Ile164 0.024 Asp125 0.020 Tyr127 0.020 Ser159 0.020 Ala, Alanine; Arg, Arginine; Asn, Asparagine; Asp, Aspartic acid; Glu, Glutamic acid; Gly, Glycine; Ile, Isoleucine; Leu, Leucine; Met, Methionine; Pro, Proline; Ser, Serine; Thr, Threonine; Tyr, Tyrosine; Val, Valine.
- 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.
Compound ΔG(VinaMPI) [kcal/mol] ΔG(KDEEP) [kcal/mol] ZINC13407541 −7.0 ± 2.9 −6.4 ± 0.3 13a −7.3 ± 2.9 −6.9 ± 0.5 8n −5.8 ± 2.9 −4.9 ± 0.6 - 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.
Parameters Vehicle
(n = 7)ZINC13407541
(n = 8)8n
(n = 8)13a
(n = 5)p-Value FGF23 (pg/ml) 1413 ± 366 2359 ± 861* 1754 ± 445 1378 ± 289 0.0110 Phosphate (mg/dl) 4.6 ± 0.60 6.5 ± 1.02** 6.7 ± 0.58** 7.8 ± 1.08*** <0.0001 Calcium (mg/dl) 8.0 ± 1.23 8.9 ± 1.22 8.4 ± 1.77 8.3 ± 1.63 0.7208 PTH (pg/ml) 78 ± 12 122 ± 21** 118 ± 20** 138 ± 21*** 0.0002 1,25(OH)2D (pg/ml) 19 ± 13 65 ± 14*** 69 ± 19*** 116 ± 14*** <0.0001 Aldosterone (pg/ml) 278 ± 84 389 ± 68* 401 ± 113* 497 ± 71*** 0.0031 - 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.
Gene Vehicle
(n = 6)ZINC13407541
(n = 6)8n
(n = 7)13a
(n = 5)p-Value Osteoblast lineage Fgfr1 1.00 ± 0.35 0.51 ± 0.20** 0.56 ± 0.23** 0.21 ± 0.07*** 0.0001 Fgfr2 1.00 ± 0.12 0.98 ± 0.29 0.94 ± 0.32 0.44 ± 0.06** 0.0010 Fgfr3 1.00 ± 0.25 1.14 ± 0.24 1.16 ± 0.21 0.42 ± 0.17*** <0.0001 Fgfr4 1.00 ± 0.59 0.53 ± 0.25* 0.45 ± 0.19** 0.25 ± 0.06*** 0.0057 Fgf1 1.00 ± 0.25 1.14 ± 0.40 1.01 ± 0.39 0.38 ± 0.15* 0.0019 Fgf2 1.00 ± 0.32 1.04 ± 0.31 1.01 ± 0.27 0.52 ± 0.16* 0.0081 Fgf23 1.00 ± 0.33 0.88 ± 0.18 0.93 ± 0.41 0.46 ± 0.15* 0.0189 Dmp1 1.00 ± 0.48 0.77 ± 0.18 0.88 ± 0.16 0.38 ± 0.16*** 0.0002 Osteopontin 1.00 ± 0.25 0.55 ± 0.11** 0.56 ± 0.16** 0.64 ± 0.12*** <0.0001 Bsp 1.00 ± 0.34 0.87 ± 0.27 0.95 ± 0.29 0.90 ± 0.18 0.8607 Mepe 1.00 ± 0.16 1.68 ± 0.35* 1.86 ± 0.44* 5.42 ± 0.77*** <0.0001 Col1 1.00 ± 0.28 0.55 ± 0.16** 0.62 ± 0.23* 0.30 ± 0.10*** <0.0001 Alp 1.00 ± 0.26 0.66 ± 0.20* 0.60 ± 0.19** 0.36 ± 0.08*** 0.0001 Osteocalcin 1.00 ± 0.27 1.06 ± 0.32 1.25 ± 0.39 3.36 ± 0.97*** <0.0001 Runx2-II 1.00 ± 0.23 0.82 ± 0.41 0.91 ± 0.31 0.21 ± 0.11*** 0.0003 Opg 1.00 ± 0.20 1.02 ± 0.33 0.96 ± 0.29 0.92 ± 0.37 0.9488 RankL 1.00 ± 0.16 0.85 ± 0.18 0.97 ± 0.37 0.87 ± 0.11 0.6112 Fzd2 1.00 ± 0.38 0.51 ± 0.21** 0.62 ± 0.16* 0.17 ± 0.08*** <0.0001 Wnt10b 1.00 ± 0.27 0.53 ± 0.24** 0.64 ± 0.16* 0.12 ± 0.06*** <0.0001 Axin2 1.00 ± 0.25 0.57 ± 0.21** 0.67 ± 0.17* 0.11 ± 0.06*** <0.0001 Osteoclast Trap 1.00 ± 0.17 0.91 ± 0.24 1.08 ± 0.27 0.86 ± 0.22 0.3383 Mmp9 1.00 ± 0.21 0.81 ± 0.39 1.10 ± 0.34 1.11 ± 0.13 0.2358 Chondrocyte Collagen II 1.00 ± 0.45 3.72 ± 1.08* 3.43 ± 2.27* 14.4 ± 2.65*** <0.0001 VegfA 1.00 ± 0.38 0.83 ± 0.51 0.77 ± 0.48 0.81 ± 0.27 0.7852 Adipocyte PPARγ2 1.00 ± 0.32 0.43 ± 0.29** 0.48 ± 0.31* 0.10 ± 0.08*** 0.0001 aP2 1.00 ± 0.13 0.78 ± 0.15* 0.70 ± 0.16** 0.23 ± 0.11*** <0.0001 Lpl 1.00 ± 0.34 0.47 ± 0.23** 0.54 ± 0.24* 0.17 ± 0.07*** <0.0001 aP2, adipocyte fatty acid-binding protein 2; Bsp, bone sialoprotein; Col1, type I collagen; Fzd2, frizzled class receptor 2; Lpl, lipoprotein lipase; Mmp9, matrix metallopeptidase 9; Opg, osteoprotegerin; PPARg2, peroxisome proliferator-activated receptor gamma 2; RankL, receptor activator of nuclear factor kappa B ligand; Runx2-II, type II runt-related transcription factor-2; Trap, tartrate-resistant acid phosphatase; VegfA, vascular endothelial growth factor A.
- 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.
Gene Vehicle
(n = 6)ZINC13407541
(n = 6)8n
(n = 6)13a
(n = 5)p-Value Fgfr1 1.00 ± 0.19 0.98 ± 0.15 0.95 ± 0.13 0.90 ± 0.29 0.8270 Npt2a 1.00 ± 0.10 1.36 ± 0.15* 1.44 ± 0.19* 1.76 ± 0.44*** 0.0007 Npt2c 1.00 ± 0.13 1.37 ± 0.15* 1.41 ± 0.11** 1.98 ± 0.48*** <0.0001 Klotho 1.00 ± 0.12 1.32 ± 0.16* 1.34 ± 0.16* 1.66 ± 0.22*** <0.0001 Cyp24a1 1.00 ± 0.10 0.69 ± 0.15** 0.64 ± 0.11*** 0.66 ± 0.16*** 0.0004 Cyp27b1 1.00 ± 0.10 1.42 ± 0.14* 1.48 ± 0.14* 2.08 ± 0.61*** 0.0001 NCC 1.00 ± 0.15 0.64 ± 0.13*** 0.72 ± 0.17** 0.55 ± 0.12*** 0.0002
Data Supplement
- Supplemental Data -
Figure S1. The computationally predicted interaction of ZINC13407541 and its two analogs, 13a and 8n, with the N terminal domain of FGF23 (PDB code: 5W21) Q156A mutant shown in (A) 3D structure; in (B) (C) and (D) 2D residue-contacting map for ZINC13407541, 13a, and 8n, respectively.
Table S1. The raw results of hydrogen-bond analyses on binding poses of ZINC13407541 to the N-terminal domain of FGF23 in the molecular docking.
Table S2. The raw results of hydrogen-bond analyses on binding poses of 13a to the N-terminal domain of FGF23 in the molecular docking.
Table S3. The raw results of hydrogen-bond analyses on binding poses of 8n to the N-terminal domain of FGF23 in the molecular docking.
Table S4. The conservation scores in descending order of the residues shown in Table 1 in the main text.
Table S5. The computationally estimated free energies of binding (ΔG) with standard errors of ZINC13407541, 13a, and 8n to the N-terminal domain of FGF23 Q156A mutant using AutoDock VinaMPI and KDEEP.
- Supplemental PDB 1 -
Supplemental Auxiliary Production PDB 1. FGF23.pdb
- Supplemental PDB 2 -
Supplemental Auxiliary Production PDB 2. PDB_5W21.pdb
- Supplemental PDB 3 -
Supplemental Auxiliary Production PDB 3. ZINC13407541/ZINC13407541_Pose_1.pdb
- Supplemental PDB 4 -
Supplemental Auxiliary Production PDB 4. ZINC13407541/ZINC13407541_Pose_2.pdb
- Supplemental PDB 5 -
Supplemental Auxiliary Production PDB 5. ZINC13407541/ZINC13407541_Pose_3.pdb
- Supplemental PDB 6 -
Supplemental Auxiliary Production PDB 6. ZINC13407541/ZINC13407541_Pose_4.pdb
- Supplemental PDB 7 -
Supplemental Auxiliary Production PDB 7. ZINC13407541/ZINC13407541_Pose_5.pdb
- Supplemental PDB 8 -
Supplemental Auxiliary Production PDB 8. ZINC13407541/ZINC13407541_Pose_6.pdb
- Supplemental PDB 9 -
Supplemental Auxiliary Production PDB 9. ZINC13407541/ZINC13407541_Pose_7.pdb
- Supplemental PDB 10 -
Supplemental Auxiliary Production PDB 10. ZINC13407541/ZINC13407541_Pose_8.pdb
- Supplemental PDB 11 -
Supplemental Auxiliary Production PDB 11. ZINC13407541/ZINC13407541_Pose_9.pdb
- Supplemental PDB 12 -
Supplemental Auxiliary Production PDB 12. ZINC13407541/ZINC13407541_Pose_10.pdb
- Supplemental PDB 13 -
Supplemental Auxiliary Production PDB 13. ZINC13407541/ZINC13407541_Pose_11.pdb
- Supplemental PDB 14 -
Supplemental Auxiliary Production PDB 14. ZINC13407541/ZINC13407541_Pose_12.pdb
- Supplemental PDB 15 -
Supplemental Auxiliary Production PDB 15. ZINC13407541/ZINC13407541_Pose_13.pdb
- Supplemental PDB 16 -
Supplemental Auxiliary Production PDB 16. ZINC13407541/ZINC13407541_Pose_14.pdb
- Supplemental PDB 17 -
Supplemental Auxiliary Production PDB 17. ZINC13407541/ZINC13407541_Pose_15.pdb
- Supplemental PDB 18 -
Supplemental Auxiliary Production PDB 18. ZINC13407541/ZINC13407541_Pose_16.pdb
- Supplemental PDB 19 -
Supplemental Auxiliary Production PDB 19. ZINC13407541/ZINC13407541_Pose_17.pdb
- Supplemental PDB 20 -
Supplemental Auxiliary Production PDB 20. ZINC13407541/ZINC13407541_Pose_18.pdb
- Supplemental PDB 21 -
Supplemental Auxiliary Production PDB 21. ZINC13407541/ZINC13407541_Pose_19.pdb
- Supplemental PDB 22 -
Supplemental Auxiliary Production PDB 22. ZINC13407541/ZINC13407541_Pose_20.pdb
- Supplemental PDB 23 -
Supplemental Auxiliary Production PDB 23. 13a/13a_Pose_1.pdb
- Supplemental PDB 24 -
Supplemental Auxiliary Production PDB 24. 13a/13a_Pose_2.pdb
- Supplemental PDB 25 -
Supplemental Auxiliary Production PDB 25. 13a/13a_Pose_3.pdb
- Supplemental PDB 26 -
Supplemental Auxiliary Production PDB 26. 13a/13a_Pose_4.pdb
- Supplemental PDB 27 -
Supplemental Auxiliary Production PDB 27. 13a/13a_Pose_5.pdb
- Supplemental PDB 28 -
Supplemental Auxiliary Production PDB 28. 13a/13a_Pose_6.pdb
- Supplemental PDB 29 -
Supplemental Auxiliary Production PDB 29. 13a/13a_Pose_7.pdb
- Supplemental PDB 30 -
Supplemental Auxiliary Production PDB 30. 13a/13a_Pose_8.pdb
- Supplemental PDB 31 -
Supplemental Auxiliary Production PDB 31. 13a/13a_Pose_9.pdb
- Supplemental PDB 32 -
Supplemental Auxiliary Production PDB 32. 13a/13a_Pose_10.pdb
- Supplemental PDB 33 -
Supplemental Auxiliary Production PDB 33. 13a/13a_Pose_11.pdb
- Supplemental PDB 34 -
Supplemental Auxiliary Production PDB 34. 13a/13a_Pose_12.pdb
- Supplemental PDB 35 -
Supplemental Auxiliary Production PDB 35. 13a/13a_Pose_13.pdb
- Supplemental PDB 36 -
Supplemental Auxiliary Production PDB 36. 13a/13a_Pose_14.pdb
- Supplemental PDB 37 -
Supplemental Auxiliary Production PDB 37. 13a/13a_Pose_15.pdb
- Supplemental PDB 38 -
Supplemental Auxiliary Production PDB 38. 13a/13a_Pose_16.pdb
- Supplemental PDB 39 -
Supplemental Auxiliary Production PDB 39. 13a/13a_Pose_17.pdb
- Supplemental PDB 40 -
Supplemental Auxiliary Production PDB 40. 13a/13a_Pose_18.pdb
- Supplemental PDB 41 -
Supplemental Auxiliary Production PDB 41. 13a/13a_Pose_19.pdb
- Supplemental PDB 42 -
Supplemental Auxiliary Production PDB 42. 13a/13a_Pose_20.pdb
- Supplemental PDB 43 -
Supplemental Auxiliary Production PDB 43. 8n/8n_Pose_1.pdb
- Supplemental PDB 44 -
Supplemental Auxiliary Production PDB 44. 8n/8n_Pose_2.pdb
- Supplemental PDB 45 -
Supplemental Auxiliary Production PDB 45. 8n/8n_Pose_3.pdb
- Supplemental PDB 46 -
Supplemental Auxiliary Production PDB 46. 8n/8n_Pose_4.pdb
- Supplemental PDB 47 -
Supplemental Auxiliary Production PDB 47. 8n/8n_Pose_5.pdb
- Supplemental PDB 48 -
Supplemental Auxiliary Production PDB 48. 8n/8n_Pose_6.pdb
- Supplemental PDB 49 -
Supplemental Auxiliary Production PDB 49. 8n/8n_Pose_7.pdb
- Supplemental PDB 50 -
Supplemental Auxiliary Production PDB 50. 8n/8n_Pose_8.pdb
- Supplemental PDB 51 -
Supplemental Auxiliary Production PDB 51. 8n/8n_Pose_9.pdb
- Supplemental PDB 52 -
Supplemental Auxiliary Production PDB 52. 8n/8n_Pose_10.pdb
- Supplemental PDB 53 -
Supplemental Auxiliary Production PDB 53. 8n/8n_Pose_11.pdb
- Supplemental PDB 54 -
Supplemental Auxiliary Production PDB 54. 8n/8n_Pose_12.pdb
- Supplemental PDB 55 -
Supplemental Auxiliary Production PDB 55. 8n/8n_Pose_13.pdb
- Supplemental PDB 56 -
Supplemental Auxiliary Production PDB 56. 8n/8n_Pose_14.pdb
- Supplemental PDB 57 -
Supplemental Auxiliary Production PDB 57. 8n/8n_Pose_15.pdb
- Supplemental PDB 58 -
Supplemental Auxiliary Production PDB 58. 8n/8n_Pose_16.pdb
- Supplemental PDB 59 -
Supplemental Auxiliary Production PDB 59. 8n/8n_Pose_17.pdb
- Supplemental PDB 60 -
Supplemental Auxiliary Production PDB 60. 8n/8n_Pose_18.pdb
- Supplemental PDB 61 -
Supplemental Auxiliary Production PDB 61. 8n/8n_Pose_19.pdb
- Supplemental PDB 62 -
Supplemental Auxiliary Production PDB 62. 8n/8n_Pose_20.pdb
- Supplemental PDB 63 -
Supplemental Auxiliary Production PDB 63. Q156A/FGF23_Q156A.pdb
- Supplemental PDB 64 -
Supplemental Auxiliary Production PDB 64. Q156A/ZINC13407541_Pose_1.pdb
- Supplemental PDB 65 -
Supplemental Auxiliary Production PDB 65. Q156A/13a_Pose_1.pdb
- Supplemental PDB 66 -
Supplemental Auxiliary Production PDB 66. Q156A/8n_Pose_1.pdb
- Supplemental Data -
