Combination of vitamin D metabolites with selective inhibitors of vitamin D metabolism.
1alpha,25(OH)2D3 exerts antiproliferative, differentiating effects on many cell types, including cancer tissues. In most of its target cells, levels of 1alpha,25(OH)2D3 are regulated by local synthesis via CYP27B and metabolism via CYP24. Rapidly induced by vitamin D, CYP24 repeatedly hydroxylates the vitamin D side chain and ultimately terminates hormonal activity. Aiming at increased hormone levels, lifetime and function, numerous vitamin D analogs have been synthesized with structural modifications, which impede oxidation of the vitamin D side chain. Our group followed a different strategy, namely, blocking 1,25(OH)2D3 metabolism with inhibitors of CYP24. As appropriate inhibitors, we exploited compounds termed azoles, which directly bind to the heme iron of the CYPs via an azole nitrogen and to other parts of the substrate site. We synthesized some 400 azoles and tested their potential to selectively inhibit CYP24, but not hormone synthesis by the related CYP27B. Using primary human keratinocyte cultures as the source of CYP24 and CYP27, we discovered some 50 inhibitors of CYP24 with IC50 values in the nanomole range and selectivities up to 60-fold. As the first representative of selective CYP24 inhibitors, VID400 underwent preclinical development. In human keratinocytes, VID400 stabilized levels of endogenously produced 1alpha,25(OH)2D3, and thereby strongly amplified and prolonged expression of CYP24, a surrogate marker of hormonal function. In parallel, antiproliferative activity showed up at 100-fold or more lower concentrations of 1alpha,25(OH)2D3. This data suggests that CYP24 inhibitors could become attractive drugs in antiproliferative therapy, used as single entities to increase or extend endogenous hormone function or in combination with low doses of potent analogs. Moreover, we used selective inhibitors as valuable tools to (a) elucidate regulatory mechanisms of vitamin D synthesis and metabolism, (b) determine intrinsic activities of the otherwise highly transient vitamin D metabolites and (c) model the active sites of CYP24 and CYP27B.
Schuster
Inge
I
Institute of Pharmaceutical Chemistry, University Vienna, Althanstrasse 15, 1090 Vienna, Austria. inge@tbi.univie.ac.at
Egger
Helmut
H
Reddy
G Satyanarayana
GS
Vorisek
Georg
G
D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000201:enzymology; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014807:Vitamin D / Q000378:metabolism*; D065668:Vitamin D3 24-Hydroxylase
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase