Selective inhibition of vitamin D hydroxylases in human keratinocytes

Human keratinocytes convert 25(OH)D-3 to hormonally active 1 alpha ,25(OH)( 2)D-3 and respond to its antiproliferative/prodifferentiating action in vit ro and in vivo. Levels and activity of 1 alpha ,25(OH)(2)D-3 are short-live d, 1 alpha ,25(OH)(2)D-3 induces 24-hydroxylase (CYP24) that rapidly metabo lizes the hormone, yielding a cascade of side-chain oxidized products and t his eventually results in the loss of activity. Aiming at stabilizing the l evels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1 alpha ,25(OH)(2)D-3 synthesis, catalyzed by 1 alpha -hydroxylase - a rela ted member of cytochrome P-450 (CYP) superfamily. We describe here the test ing protocol, using primary human keratinocyte cultures as an appropriate s ource of CYP24 and 1 alpha -hydroxylase, H-3-25(OH)D-3 (at physiological co ncentrations) as substrate and sensitive HPLC techniques to analyze the com plex metabolite profiles. Four hundred potential inhibitors were screened b y this method; most of them were synthesized in our laboratory. These compo unds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, w e present VID400 and SDZ 89-443, as first examples of powerful selective CY P24 inhibitors, As anticipated, these compounds increased the levels of 1 a lpha -hydroxylated products generated from H-3-25(OH)D-3 and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in met abolism, namely to preferential conversion of 1 alpha ,25(OH)(2)D-3 to 1 al pha ,25(OH)(2)-3epi-D-3. As spin-off from our program, selective inhibitors of la-hydroxylase were also found (e.g. SDZ 88-357). Using H-3-25(OH)D-3 a s substrate in the absence of SDZ 88-357, CYP24 showed high preference for freshly generated 1 alpha -hydroxylated metabolites over abundant 25(OH)D-3 . In the presence of SDZ 88-357, we noticed a great increase in 24-hydroxyl ation of H-3-25(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 different iation, (C) 2001 Elsevier Science Inc. All rights reserved.