Therapeutic Potential of a Novel Vitamin D3 Oxime Analogue, VD1-6, with CYP24A1 Enzyme Inhibitory Activity and Negligible Vitamin D Receptor Binding

Abstract

The regulation of vitamin D₃ actions in humans occurs mainly through the Cytochrome P450 24-hydroxylase (CYP24A1) enzyme activity. CYP24A1 hydroxylates both 25-hydroxycholecalciferol (25(OH)D₃) and 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃), which is the first step of vitamin D catabolism. An abnormal status of the upregulation of CYP24A1 occurs in many diseases, including chronic kidney disease (CKD). CYP24A1 upregulation in CKD and diminished activation of vitamin D₃ contribute to secondary hyperparathyroidism (SHPT), progressive bone deterioration, and soft tissue and cardiovascular calcification. Previous studies have indicated that CYP24A1 inhibition may be an effective strategy to increase endogenous vitamin D activity and decrease SHPT. This study has designed and synthesized a novel C-24 O-methyloxime analogue of vitamin D₃ (VD1-6) to have specific CYP24A1 inhibitory properties. VD1-6 did not bind to the vitamin D receptor (VDR) in concentrations up to 10^-7 M, assessed by a VDR binding assay. The absence of VDR binding by VD1-6 was confirmed in human embryonic kidney HEK293T cultures through the lack of CYP24A1 induction. However, in silico docking experiments demonstrated that VD1-6 was predicted to have superior binding to CYP24A1, when compared to that of 1,25(OH)₂D₃. The inhibition of CYP24A1 by VD1-6 was also evident by the synergistic potentiation of 1,25(OH)₂D₃-mediated transcription and reduced 1,25(OH)₂D₃ catabolism over 24 h. A further indication of CYP24A1 inhibition by VD1-6 was the reduced accumulation of the 24,25(OH)D₃, the first metabolite of 25(OH)D catabolism by CYP24A1. Our findings suggest the potent CYP24A1 inhibitory properties of VD1-6 and its potential for testing as an alternative therapeutic candidate for treating SHPT.

Keywords: CYP24A1; HEK293T; catabolism inhibition; in silico docking; vitamin D3.

Scheme 1

Synthesis of the 24-O-methyloxime derivative VD1-6.

Figure 1

Mean fluorescence polarization (n = 3) measurements in millipolarization (mP) for the bound 1 nM Fluormone™/3.62 nM VDR full-length protein interacting with each of the 16 tested concentrations (0.0007 to 1 × 10⁴ nM, represented at Log scale in the figure) for each of the three tested compounds. Error bars represent the standard deviation from the mean. Solid lines connecting the points represent the curve fitting, using sigmoidal dose–response modelling with varying slopes.

Figure 2

OEDocking simulated orientation of 1,25(OH)₂D₃ (left) and VD1-6 (right) in the CYP24Al active site. Depicted as a 3D binding pocket view, using VIDA (upper panels), and ligand-interaction diagram (lower panels), using the OEChem toolkit.

Figure 3

Effect of VD1-6 (10⁻¹¹–10⁻⁷ M), 1,25(OH)₂D₃ (10⁻⁹ M), and in combination on the mRNA levels of CYP24A1, CYP27B1, and VDR, CALB1, and SLC34A3 in HEK293T cell culture at 24 h. * p < 0.05 vs. vehicle control; ^# p < 0.05 vs. 1,25(OH)₂D₃ (10⁻⁷ M) treatment. Values are represented as mean ± SD.

Figure 4

Levels of 1,25(OH)₂D₃ in HEK293T cell culture at 0, 8, and 24 h in the absence and presence of VD1-6 at 10⁻⁸ M and 10⁻⁷ M. Mean ± S.D, n = 4/group; ^a p < 0.05 vs 0 h; ^b p < 0.05 vs. 1,25(OH)₂D₃ alone at 8 h; ^c p < 0.05 vs. 1,25(OH)₂D₃ alone at 24 h.

Figure 5

24,25(OH)₂D₃ levels (A) and relative CYP24A1 mRNA levels (B) in HEK293T cell culture at 72 h in the absence and presence of VD1-6 (10⁻¹¹–10⁻⁷ M). Mean ± S.D, n = 4/group; ^a p < 0.05 vs. 25(OH)D₃ only treatment; ^b p < 0.05 vs. 25(OH)D₃ + 10⁻¹¹ M VD1-6 treatment; ^c p < 0.05 vs. 25(OH)D₃ + 10⁻¹⁰ M VD1-6 treatment; ^d p < 0.05 vs. 25(OH)D₃ + 10⁻⁹ M VD1-6 treatment; ^e p < 0.05 vs. 25(OH)D₃ + 10⁻⁸ M VD1-6 treatment.