Biological evaluation and synthesis of calcitroic acid.

We describe the synthesis and broad profiling of calcitroic acid (CTA) as vitamin D receptor (VDR) ligand. The x-ray co-crystal structure of the Danio Rerio VDR ligand binding domain in complex with CTA and peptide MED1 confirmed an agonistic conformation of the receptor. CTA adopted a similar conformation as 1,25(OH)2D3 in the binding pocket. A hydrogen bond with His333 and a water molecule were observed in the binding pocket, which was accommodated due to the shorter CTA side chain. In contrast, 1,25(OH)2D3 interacted with His423 and His333 due to its longer side chain. In vitro, the EC50 values of CTA and CTA-ME for VDR-mediated transcription were 2.89 µM and 0.66 µM, respectively, confirming both compounds as VDR agonists. CTA was further evaluated for interaction with fourteen nuclear receptors demonstrating selective activation of VDR. VDR mediated gene regulation by CTA in intestinal cells was observed for the VDR target gene CYP24A1. CTA at 10 µM upregulated CYP24A1 with similar efficacy as 1,25(OH)2D3 at 20 nM and 100-fold stronger compared to lithocholic acid at 10 µM. CTA reduced the transcription of iNOS and IL-1ß in interferon γ and lipopolysaccharide stimulated mouse macrophages resulting in a reduction of nitric oxide production and secretion of IL-1ß. These observed anti-inflammatory properties of 20 µM CTA were similar to 20 nM 1,25(OH)2D3.

Synthesis and biological evaluation of calcioic acid.

Herein, we describe the synthesis of calcioic acid following a recently developed synthetic strategy for calcitroic acid. Several improvements to reaction conditions were made, which resulted in higher yields. The improved workup and isolation procedures are described. Furthermore, we investigated the interaction between the vitamin D receptor (VDR) and calcioic acid. Calcioic acid was able to bind VDR with a binding constant of 71 µM. In cells, calcioic acid reduced the transcription of VDR target gene CYP24A1 in the presence 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) but did not induce the transcription of CYP24A1. Therefore, calcioic acid is a very weak VDR antagonist. With the generation of gram quantities, further studies are expected to reveal if calcioic acid is solely a water-soluble metabolite of vitamin D or if it mediates other biological functions through biomolecules other than VDR.

Alternative binding sites at the vitamin D receptor and their ligands.

In recent decades, the majority of ligands developed for the vitamin D receptor (VDR) bind at its deeply buried genomic ligand binding pocket. Theses ligands can be categorized into agonists and partial agonists/antagonists. A limited number of ligands, most of them peptides, bind the VDR‒coactivator binding site that is formed in the presence of an agonist and inhibit coactivator recruitment, and therefore transcription. Another solvent exposed VDR‒ligand binding pocket was identified for lithocholic acid, improving the overall stability of the VDR complex. Additional proposed interactions with VDR are discussed herein that include the alternative VDR‒ligand binding pocket that may mediate both non-genomic cellular responses and binding function 3 that was identified for the androgen receptor. Many VDR ligands increase blood calcium levels at therapeutic concentrations in vivo, thus the identification of alternative VDR‒ligand binding pockets might be crucial to develop non-calcemic and potent ligands for VDR to treat cancer and inflammatory disease.