Selective inhibition of vitamin D hydroxylases in human keratinocytes.
Human keratinocytes convert 25(OH)D(3) to hormonally active 1alpha,25(OH)(2)D(3) and respond to its antiproliferative/prodifferentiating action in vitro and in vivo. Levels and activity of 1alpha,25(OH)(2)D(3) are short-lived. 1alpha,25(OH)(2)D(3) induces 24-hydroxylase (CYP24) that rapidly metabolizes the hormone, yielding a cascade of side-chain oxidized products and this eventually results in the loss of activity. Aiming at stabilizing the levels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1alpha,25(OH)(2)D(3) synthesis, catalyzed by 1alpha-hydroxylase - a related member of cytochrome P-450 (CYP) superfamily. We describe here the testing protocol, using primary human keratinocyte cultures as an appropriate source of CYP24 and 1alpha-hydroxylase, (3)H-25(OH)D(3) (at physiological concentrations) as substrate and sensitive HPLC techniques to analyze the complex metabolite profiles. Four hundred potential inhibitors were screened by this method; most of them were synthesized in our laboratory. These compounds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, we present VID400 and SDZ 89-443, as first examples of powerful selective CYP24 inhibitors. As anticipated, these compounds increased the levels of 1alpha-hydroxylated products generated from (3)H-25(OH)D(3) and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1alpha,25(OH)(2)D(3) to 1alpha,25(OH)(2)-3epi-D(3). As spin-off from our program, selective inhibitors of 1alpha-hydroxylase were also found (e.g. SDZ 88-357). Using (3)H-25(OH)D(3) as substrate in the absence of SDZ 88-357, CYP24 showed high preference for freshly generated 1alpha-hydroxylated metabolites over abundant 25(OH)D(3). In the presence of SDZ 88-357, we noticed a great increase in 24-hydroxylation of (3)H-25(OH)D(3). Besides their use as valuable tools in elucidating regulatory mechanisms, inhibitors of VD hydroxylases may give rise to novel therapeutic strategies, especially in defects of cell growth and differentiation.
Schuster
I
I
Novartis Research Institute, Vienna, Austria. inge@tbi.univie.ac.at
Egger
H
H
Bikle
D
D
Herzig
G
G
Reddy
G S
GS
Stuetz
A
A
Stuetz
P
P
Vorisek
G
G
D001393:Azoles / Q000138:chemical synthesis / Q000494:pharmacology; D002478:Cells, Cultured; D002762:Cholecalciferol / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014316:Tritium; D065668:Vitamin D3 24-Hydroxylase
D001393:Azoles; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D014316:Tritium; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D065668:Vitamin D3 24-Hydroxylase