Abstract
1α,25(OH)2D3exerts antiproliferative, differentiating effects on many cell types, including cancer tissues. In most of its target cells, levels of 1α,25(OH)2D3are 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)2D3metabolism 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 1α,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 1α,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.
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References
Akiyoshi-Shibata M, Sakaki T, Ohyama Y, Noshiro M, Okuda K, Yabusaki Y (1994) Further oxidation of hydroxycalcidiol by calcidiol-24-hydroxylase: a study with the mature enzyme expressed in Escherichia coli. Eur J Biochem 224:335–343
Astecker N, Reddy GS, Herzig G, Vorisek G, Schuster I (2000) 1α,25-Dihydroxy-3-epi-vitamin D3a physiological metabolite of 1α,25-dihydroxyvitamin D3: its production and metabolism in primary human keratinocytes. Mol Cell Endocrinol 170:91–101
Beaune P, Dansette PM, Mansuy D, Kiffel L, Finck AC, Leroux JP, Homberg JC (1987) Human anti-endoplasmic reticulum autoantibodies appearing in a drug-induced hepatitis are directed against a human cytochrome P450 that hydroxylates the drug. Proc Natl Acad Sci 84:551–555
Beckman M, Tadikonda P, Werner E, Prahl JM, Yamada S, DeLuca HF (1996) Human 25-hydroxyvitamin D3–24-hydroxylase, a multicatalytic enzyme. Biochemistry 35:8465–8472
Bikle DD, Pillai S (1993) Vitamin D, calcium and epidermal differentiation. Endocr Rev 14:3–19
Bikle DD, Nemanic MK, Whitney JO, Elias PM (1986) Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3. Biochemistry 25:1545–1548
Bouillon R, Okamura WH, Norman AW (1996) Structure-function relationships in the vitamin D endocrine system. Endocr Rev 16:200–257
Brodie A (2002) Aromatase inhibitors in breast cancer. Trends Endocrinol Metab 13:61–65
Brown AJ, Dusso A, Slatopolsky E (1999) Vtamin D. Am J Physiol 277:157–175
Bury Y, Herdick M, Uskokovic MR, Carlberg C (2001) Gene regulatory potential of 1αlpha, 25-dihydroxyvitamin D3analogues with two side chains. J Cell Biochem Suppl 36:179–190
Chen ML, Heinrich G, Ohyama YL, Okuda K, Omdahl JL, Chen TC, Holick MF (1994) Expression of 25-hydroxyvitamin D-24 hydroxylase mRNA in cultured human keratinocytes. Proc Soc Exp Biol Med 207:57–61
Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159
Colston KW, Colston MJ, Feldman D (1981) 1,25-dihydroxyvitamin D3and melanoma: the presence of receptors and inhibition of cell growth in culture. Endocrinology 108:1083–1086
Dusso A, Brown A, Slatopolsky E (1994) Extrarenal production of calcitriol. Semin Nephrol 14:144–155
Falorni A, Laureti S, Candeloro P, Perrino S, Coronella C, Bizzaro A, Bellastella A, Santeusanio F, DeBellis A (2002) Steroid-cell autoantibodies are preferentially expressed in women with premature ovarian failure who have adrenal autoimmunity. Fertility Sterility 78:270–279
Flanagan JN, Whitlatch LW, Chen TC, Zhu XH, Holick MT, Kong X-F, Holick MF (2001) Enhancing 1α-hydroxylase activity with the 25-hydroxyvitamin D-1α-hydroxylase gene in cultured human keratinocytes and mouse skin. J Invest Dermatol 116:910–914
Garcion E, Wion-Barbot N, Montero-Menei CN, Berger F, Wion D (2002) New clues about vitamin D functions in the nervous system. Trends Endocrinol Metab 13:100–105
Hashimoto K, Matsumoto K, Higashiyama M, Nishida Y, Yoshikawa K (1990) Growth-inhibitory effects of 1,25-dihydroxyvitamin D3on normal and psoriatic keratinocytes. Brit J Dermatol 123:93–98
Itin PH, Pittelkow MR, Kumar R (1994) Effects of vitamin D-metabolites on proliferation and differentiation of cultured epidermal keratinocytes grown in serum-free or defined culture medium. Endocrinology 135:1793–1798
Jones G, Strugnell SA, DeLuca HF (1998) Current understanding of the molecular actions of vitamin D. Physiol Rev 78:1193–1231
Kan PB, Hirst MA, Feldman D (1985) Inhibition of steroidogenic cytochrome P450 enzymes in rat testis by ketoconazole and related imidazole antifungal drugs. J Steroid Biochem 23:1023–1029
Kang S, Li X-Y, Duell EA, Voorhees JJ (1997) The retinoid X receptor agonist 9-cis-retinoic acid and the 24-hydroxylase inhibitor ketoconazole increase activity of 1,25-dihydroxyvitamin D3in human skin in vivo. J Invest Dermatol 108:513–581
Lehmann B, Tiebel O, Meurer M (1999) Expression of vitamin D325-hydroxylase (CYP27) mRNA after induction by vitamin D3or UVB radiation in keratinocytes of human skin equivalents -a preliminary study. Arch Dermatol Res 291:507–510
Lehmann B, Knuschke P, Meurer M (2000) A novel pathway for hormonally active calcitriol. Horm Res 54:312–315
Ly LH, Zhao XY, Holloway L, Feldman D (1999) Liarozole acts synergistically with 1α,25-dihydroxyvitamin D3to inhibit growth of DU145 human prostate cancer cells by blocking 24-hydroxylase activity. Endocrinology 140:2071–2076
Matsumoto K, Azumi Y, Kiyoki M, Okumura H, Hashimoto K, Yoshikawa K (1991) Involvement of endogenously produced 1,25-dihydroxyvitamin D3in the growth and differentiation of human keratinocytes. Biochim Biophys Acta 1092:311–318
Ohiyama Y, Noshiro M, Eggertsen G, Gotoh O, Kato Y, Bjorkhem I, Okuda K (1993) Structural characterization of the gene encoding 25-hydroxyvitamin D324-hydroxylase. Biochemistry 32:76–82
Omdahl JL, Bobrovnikova EA, Choe S, Dwivedi PP, May BK (2001) Overview of regulatory cytochrome P450 enzymes of the vitamin D pathway. Steroids 66:381–389
Ortiz de Montellano PR, Reich NO (1986) Inhibition of cytochrome P450 enzymes. In: Ortiz de Montellano PR (ed) Cytochrome P450, structure, mechanism and biochemistry. Plenum Press, New York, p 273
Raag R, Poulos TL (1989) Crystal structure of the carbon monoxide-substrate-cytochrome P-450 CAM ternary complex. Biochemistry 28:7586–7592
Reddy GS, Siu-Caldera M-L, Schuster I, Astecker N, Tserng KY, Mulralidharan WH, Okamura WH, McLane JA, Uskokovic MR (1997) Target tissue-specific metabolism of 1,25(OH)2D3through A-ring modification. In: Norman AW, Bouillon R, Thomasset M (eds) Vitamin D. Chemistry, biology and clinical applications of the steroid hormone, University of California, Riverside, pp 139–146
Reddy GS, Muralidharan KR, Okamura WH, Tserng K-Y, McLane JA (2001) Metabolism of 1α,25-dihydroxyvitamin D3and its C-3 epimer 1α,25-dihydroxy-3-epi-vitamin D3in neonatal human keratinocytes. Steroids 66:441–450
Schssler M, Astecker N, Herzig G, Vorisek G, Schuster I (2001) Skin is an autonomous organ in synthesis, two-step activation and degradation of vitamin D3: CYP27 in epidermis completes the set of essential vitamin D3-hydroxylases. Steroids 66:399–408
Schuster I (1985) The interaction of representative members from two classes of antifungals the azoles and the allylamines -with cytochromes P450 in steroidogenic tissues and liver. Xenobiotica 15:529–546
Schuster I, Herzig G, Schmid J, Vorisek G, Woolaway K (1998) Formation of vitamin D metabolites in keratinocytes and fibroblasts from psoriatic patients. J Dermatol Sci 16:S139
Schuster I, Egger H, Herzig G, Reddy GS, Schssler M, Vorisek G (2000) Constitutive activity of 1α-hydroxylase in primary human keratinocytes, revealed by selective inhibition of CYP24. In: Norman AW, Bouillon R, Thomasset M (eds) Vitamin D endocrine system. structural, biological, genetic and clinical aspects. University of California, Riverside, pp 182–185
Schuster I, Egger H, Astecker N, Herzig G, Schssler M, Vorisek G (2001a) Selective inhibitors of CYP24: mechanistic tools to explore vitamin D metabolism in human keratinocytes. Steroids 66:451–462
Schuster I, Egger H, Bikle D, Herzig G, Reddy GS, Stuetz A, Stuetz P, Vorisek G (2001b) Selective inhibition of vitamin D hydroxylases in human keratinocytes. Steroids 66:409–422
Schuster I, Egger H, Nussbaumer P, Kroemer R (2003) Inhibitors of vitamin D hydroxylases: structure—activity relationships J Cell Biochem 88:372–380
Smith EL, Walworth NC, Holick MF (1986) Effect of 1α,25-dihydroxyvitamin D3on the morphologic and biochemical differentiation of cultured human epidermal keratinocytes grown in serum-free conditions. J Invest Dermatol 86:709–714
Uskokovic M, Norman AW, Manchand PS, Studzinski GP, Campbell MJ, Koeffler H, Takeuchi A, Siu-Caldera M-L, Rao DS, Reddy GS (2001) Highly active analogs of 1α,25-dihydroxyvitamin D3that resist metabolism through C-24 oxidation and C-3 epimerization pathways. Steroids 66:463–471
White RE, Coon MJ (1980) Oxygen activation by cytochrome P450. Annu Rev Biochem 49:315–356
Zehnder D, Bland R, Chana RS, Wheeler DC, Howie AJ, Williams MC, Stewart PM, Hewison M (2002) Synthesis of 1,25-dihydroxyvitamin D3by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion. J Am Soc Nephrol 13:621–629
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Schuster, I., Egger, H., Herzig, G., Reddy, G.S., Vorisek, G. (2003). Combination of Vitamin D Metabolites with Selective Inhibitors of Vitamin D Metabolism. In: Reichrath, J., Tilgen, W., Friedrich, M. (eds) Vitamin D Analogs in Cancer Prevention and Therapy. Recent Results in Cancer Research, vol 164. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-55580-0_13
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DOI: https://doi.org/10.1007/978-3-642-55580-0_13
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