Abstract
20S-hydroxyvitamin D3 [20S(OH)D3] is anti-inflammatory and not hypercalcemic, suggesting its potential as a lead compound. In this study, side chain modified 20S(OH)D3 analogs (4, 13, 23 and 33) together with their 1α-OH derivatives were synthesized and their metabolism and biological activities tested. 4, 13 and 23 are good substrates for CYP27B1, enabling enzymatic synthesis of their 1α-OH derivatives 5, 14 and 24. However, 33 could not be hydroxylated by CYP27B1 and acts as an inhibitor. All analogs were poorer substrates for CYP24A1 than calcitriol, indicating improved catabolic stability. While the parent analogs showed minimal VDR stimulating activity, their 1α-OH derivatives were potent VDR agonists. 4, 5, 14 and 24 significantly upregulated the expression of CYP24A1 at the mRNA level, consistent with their VDR activation abilities and indicating that 1α-hydroxylation is required to produce analogs with strong activity. These analogs have anti-inflammatory activities that are influenced by side chain composition and by 1α-hydroxylation. To understand their molecular interactions with the VDR, 20S(OH)D3, 4 and 33 were co-crystalized with the VDR ligand binding domain, which revealed subtle differences to the calcitriol-bound receptor. This study demonstrates the potential of the 20S(OH)D3 scaffold for the development of novel anti-inflammatory agents.
Highlights
Vitamin D3 (D3) can be obtained from either dietary sources through intestinal absorption or endogenous production through dermal synthesis
A novel metabolic pathway (Fig. 1) has been reported by our group for activation and metabolism of D35,6 which starts with CYP11A1 acting on D3 to produce 20S-hydroxyvitamin D3 [20S(OH)D3] as the major product[5,6], and which operates in vivo7,8. 20S(OH)D3 displays many similar activities to that of 1,25(OH)2D3 including strong anti-proliferative, anti-leukemic, tumorostatic, anti-fibrotic and pro-differentiation activities[9,10,11,12,13,14] mediated through either vitamin D receptor (VDR) activation[9,15] or inhibition of RORα and RORγ16,17
This metabolite was later shown to cause translocation of the VDR into the nucleus acting as a biased VDR agonist[17,18], while other studies showed that it acts as an inverse agonist on RORα and RORγ16,17. 20S(OH)D3 exerts anti-inflammatory activities in vitro through inhibition of NFκB and decreasing production of proinflammatory cytokines by keratinocytes[10,15,19,20], melanoma cells[21], lymphocytes and macrophages[9,16], as well as in vivo as indicated by suppressive effects on collagen-induced arthritis, at a dose as low as 2 μg/kg in a mouse model[22]
Summary
Vitamin D3 (D3) can be obtained from either dietary sources through intestinal absorption or endogenous production through dermal synthesis. The modifications included replacing C24 with an oxygen (13), inserting a double bond between C24 and C25 (4), adding an amide group replacing carbons 23 and 24 (33) and adding two fluorine groups at C24 (23) Their abilities to be 1α-hydroxylated by the activation enzyme, CYP27B1, and metabolized by the catabolic enzyme, CYP24A1, were evaluated. The enzymatically-generated 1α-OH derivatives showed a much stronger ability to activate the VDR for the selected activities tested, including vitamin D response element (VDRE)-reporter (luciferase) activity, and CYP24A1 expression measured by real-time PCR, as compared to the parent compounds. Their abilities to inhibit the production of IFNγ by activated murine lymphocytes were determined
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