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Sterol 14alpha-demethylases (CYP51) serve as primary targets for antifungal drugs, and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands that demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors.

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BackgroundThe 1α,25-dihydroxy-3-epi-vitamin-D3 (1α,25(OH)2-3-epi-D3), a natural metabolite of the seco-steroid vitamin D3, exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. In vivo action of 1α,25(OH)2-3-epi-D3 is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1α,25(OH)2D3. To further unveil the structural mechanism and structure-activity relationships of 1α,25(OH)2-3-epi-D3 and its receptor complex, we characterized some of its in vitro biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD).Methodology/Principal FindingsIn the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1α,25(OH)2D3. We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1α,25(OH)2-3-epi-D3 in primary human keratinocytes and biochemical properties are comparable to 1α,25(OH)2D3.Conclusions/SignificanceThe physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)2D3 lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)2D3.