4.2 Article Proceedings Paper

Selective inhibition of vitamin D hydroxylases in human keratinocytes

Journal

STEROIDS
Volume 66, Issue 3-5, Pages 409-422

Publisher

ELSEVIER SCIENCE INC
DOI: 10.1016/S0039-128X(00)00159-8

Keywords

inhibitors; CYP24; 1 alpha-hydroxylase; 1 alpha,25(OH)(2)D-3; human keratinocytes; steroid

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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 vitro and in vivo. Levels and activity of 1 alpha ,25(OH)(2)D-3 are short-lived, 1 alpha ,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 1 alpha ,25(OH)(2)D-3 synthesis, catalyzed by 1 alpha -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 1 alpha -hydroxylase, H-3-25(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 1 alpha -hydroxylated products generated from H-3-25(OH)D-3 and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1 alpha ,25(OH)(2)D-3 to 1 alpha ,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 as 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-hydroxylation 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 differentiation, (C) 2001 Elsevier Science Inc. All rights reserved.

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