Construction of evaluation system for vitamin D analogs

2005 Fiscal Year Final Research Report Summary

• Project/Area Number: 16580074
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied biochemistry
• Research Institution: Toyama Prefectural University
• Principal Investigator: SAKAKI Toshiyuki Toyama Prefectural Univ, Dept.Biotechnology, Professor, 工学部, 教授 (70293909)
• Co-Investigator(Kenkyū-buntansha): OKANO Toshio Kobe Pharmaceutical Univ, Dept.Hygienic Sciences, Professor, 衛生化学研究室, 教授 (20131542) ; TAKITA Teisuke Kyoto Univ, Dept.Enzyme Chemistry, Assistant Professor, 大学院・農学研究科, 助手 (70263126)
• Project Period (FY): 2004 – 2005
• Keywords: vitamin D / cytochrome P450 / metabolism of vitamin D analog / CYP24A1 / CYP27B1 / site-directed mutagenesis / osteoporosis / cancer

Research Abstract

Mouse CYP27B1 was successfully overexpressed by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1α-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D_3. In addition, we revealed that Arg458 of mouse CYP27B1 is responsible for the interaction with adrenodoxin.
We revealed the species-based difference of CYP24A-dependent vitamin D metabolism, and tried to identify amino-acid residues that cause the species-based difference by site-directed mutagenesis. It was demonstrated that the amino-acid residues at positions 416 and 500 play a crucial role in substrate-binding, and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1α,25(OH)_2D_3 could be located close to amino acid residues at positions 416 and 500, respectively.
We examined CYP24A1-dependent metabolism of such vitamin D analogs as A-ring diastereomers and 20-epimer of 1α,25(OH)_2D_3, 2α-propoxy- 1α,25(OH)_2D_3 2α-(3-hydroxypropoxy)- 1α,25(OH)_2D_3, and 26,26,26,27,27,27- F_6-1α,25(OH)_2D_3. Good correlation was observed between increasing effect on serum calcium level in rats and k_<cat>/K_m value of CYP24A1 for each of the vitamin D analogs. In addition, species-based difference was also observed in CYP24A1-dependent metabolism of vitamin D analogs between humans and rats. These results strongly suggest the usefulness of the recombinant systems harboring CYP24A1 for predicting the metabolism and efficacy of vitamin D analogs before clinical trials.

Research Products

• [Journal Article] Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1α,25-dihydroxyvitamin D_3 : Implications for drug-induced osteomalacia. (2006)
  • Author(s): Xu, Y., Hashizume, T., Shuhart, M., Davis, C.L., Sakaki, T.他
  • Journal Title: Molecular Pharmacology 69 Pages: 56-65
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Synthesis of 25-hydroxy-19-norvitamin D_3 analo (2006)
  • Author(s): Arai, M.A., Tsutsumi, R., Chen, T.C., Sakaki, T., Urushino, N., 他
  • Journal Title: Heterocycles 66 Pages: 469-479
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Interaction between Mitochondrial CYP27B1 and Adrenodoxin : Role of Arginine 458 of Mouse CYP27B1 (2006)
  • Author(s): Urushino, N., Yamamoto, K., Kagawa, N., Ikushiro, S., Sakaki, T.他
  • Journal Title: Biochemistry 45 Pages: 4405-4412
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Structure-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis : amino acid resides responsible for species-based difference of CYP24A1 between humans and rats. (2006)
  • Author(s): Hamamoto, H., Kusudo, T., Urushino, N., Masuno, H., Sakaki, T.他
  • Journal Title: Molecular Pharmacology (印刷中)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Intestinal and hepatic CYP3A4 catalyze hydroxylation of 1α,25-dihydroxyvitamin D3: Implications for drug-induced osteomalacia. (2006)
  • Author(s): Xu, Y., Hashizume, T., Shuhart, M., Davis, C.L., Nelson, W.L., Sakaki, T., Kalhorn, T.F., Watkins, P.B., Schuetz, E.G.
  • Journal Title: Mol.Pharmacol. 69 Pages: 56-65
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Synthesis of 25-hydroxy-19-norvitamin D_3 analogs and their antiproliferative activities on prostate cells (2006)
  • Author(s): Arai, M.A., Tsutsumi, R., Chen, T.C., Sakaki, T., Urushino, N., Inouye, K., Kittaka, A.
  • Journal Title: Heterocycles 66 Pages: 469-479
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Interaction between Mitochondrial CYP27B1 and Adrenodoxin : Role of Arginine 458 of Mouse CYP27B1. (2006)
  • Author(s): Urushino, N., Yamamoto, K., Kagawa, N., Ikushiro, S., Kamakura, M., Yamada, S., Kato, S., Inouye, K., Sakaki, T.
  • Journal Title: Biochemistry 45 Pages: 4405-4412
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Structure-function analysis of vitamin D24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid resides responsible for species-based difference of CYP24A1 between humans and rats. (2006)
  • Author(s): Hamamoto, H., Kusudo, T., Urushino, N., Masuno, H., Yamamoto, K., Yamada, S., Kamakura, M., Ohta, M., Inouye, K., Sakaki, T.
  • Journal Title: Mol.Pharmacol. (in press)
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Metabolism of 26,26,26,27,27,27-F_G-1α,23S,25-trihydroxy vitamin D_3 by human UDP-glucuronosyltransferase 1A3 (2005)
  • Author(s): Kasai, N., Sakaki, T., Shinkyo, R., Ikushiro, S., Iyanagi, T., Ohta, M.他
  • Journal Title: Drug Metabolism and Disposition 33 Pages: 102-107
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] 2α-propoxy-1α,25-dihydroxyvitamin D_3 and 2α(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D_3 by human CYP27A1 and CYP24A1 (2005)
  • Author(s): Abe, D., Sakaki, T., Kusudo, T., Kittaka, A., Saito, N., Suhara, Y., Fujishima, T., 他
  • Journal Title: Drug Metabolism and Disposition 33 Pages: 778-784
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Identification of the amino acid residue of CYP27B1 responsible for binding of 25hydroxyvitamin D_3 whose mutation causes vitamin D-dependent rickets type 1. (2005)
  • Author(s): Yamamoto, K., Uchida, E., Urushino, N., Sakaki, T., Kagawa, N.Sawada, N 他
  • Journal Title: Journal of Biological Chemistry 280 Pages: 30511-30516
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Metabolism of vitamin D_3 by cytochromes P450. (2005)
  • Author(s): Sakaki, T., Kagawa, N., Yamamoto, K., Inouye, K.
  • Journal Title: Frontiesr in Bioscience 10 Pages: 119-134
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Metabolism of 26,26,26,27,27,27-F_6- 1α, 23S, 25-trihydroxyvitamin D_3 by human UDP-glucuronosyltransferase 1A3. (2005)
  • Author(s): Kasai, N., Sakaki, T., Shinkyo, R., Ikushiro, S., Iyanagi, T., Ohta, M., Inouye, K.
  • Journal Title: Drug Metab.Disp. 33 Pages: 102-107
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Measurement and characterization of C-3 epimerization activity toward vitamin D_3. (2005)
  • Author(s): Kamao, M., Hatakeyama, S., Sakaki, T., Sawada, N., Inouye, K., Kubodera, N., Reddy, S., Okano, T.
  • Journal Title: Arch.Biochem.Biophys. 436 Pages: 196-205
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Metabolism of 2α-propoxy-1α,25-dihydroxyvitamin D_3 and 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D3 by human CYP27A1 and CYP24A1 (2005)
  • Author(s): Abe, D., Sakaki, T., Kusudo, T., Kittaka, A., Saito, N., Suhara, Y., Fujishima, T., Takayama, H., Hamamoto, H., Kamakura, M., Ohta, M., Inouye, K.
  • Journal Title: Drug Metab.Disp. 33 Pages: 778-784
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D_3 whose mutation causes vitamin D-dependent rickets type 1. (2005)
  • Author(s): Yamamoto, K., Uchida, E., Urushino, N., Sakaki, T., Kagawa, N., Sawada, N., Kamakura, M., Kato, S., Inouye, K., Yamada, S.
  • Journal Title: J Biol.Chem. 280 Pages: 30511-30516
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Novel metabolism of 1α,25-dihydroxyvitamin D_3 with C_<24>- C_<25> bond cleavage catalyzed by human CYP24A1 (2004)
  • Author(s): Sawada N., Kusudo T., Sakaki T., Hatakeyama S., Okano T.他
  • Journal Title: Biochemistry 43 Pages: 4530-4537
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] C-3 Epimerization of vitamin D_3 metabolites and further metabolism of C-3 epimers : 25-Hydroxyvitamin D_3 is metabolized to 3-epi-25-hydroxyvitamin D_3 and subsequently (2004)
  • Author(s): Kamao M., Tatematsu S., Hatakeyana S., Sakaki T., Okano T.他
  • Journal Title: Journal of Biological Chemistry 279 Pages: 15897-15907
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Homology modeling of human 25-hydroxyvitamin D_31α-hydroxylase (CYP27B1) based on the crystal structure of rabbit CYP2C5 (2004)
  • Author(s): Yamamoto K., Masuno H., Sawada N., Sakaki T.他
  • Journal Title: Journal of Steroid Biochemistry and Molecular Biology 89-90 Pages: 167-171
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Conversion of vitamin D_3 to 1a,25-dihydroxyvitamin D_3 by Streptomyces griseolus cytochrome P450SU-1(CYP105A1) (2004)
  • Author(s): Sawada N., Sakaki T., Yoneda S Kusudo T., Shinkyo R., Ohta M.他
  • Journal Title: Biochemical and Biophysical Research Communications 320 Pages: 156-164
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES (2004)
  • Author(s): Uchida E., Kagawa N., Sakaki T., Urushino N., Sawada N.他
  • Journal Title: Biochemical and Biophysical Research Communications 323 Pages: 505-511
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Metabolism of A-ring diastereomers of 1α,25-dihydroxyvitamin D_3 by CYP24A1 (2004)
  • Author(s): Kusudo T., Sakaki T., Abe D., Fujishima T., Kittaka A.他
  • Journal Title: Biochemical and Biophysical Research Communications 321 Pages: 774-782
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Metabolism of vitamin D by human microsomal CYP2R1 (2004)
  • Author(s): Shinkyo R., Sakaki T., Kamakura M., Ohta M.Inouye K.
  • Journal Title: Biochemical and Biophysical Research Communications 324 Pages: 451-457
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Conversion of vitamin D_3 to 1α,25-dihydroxyvitamin D_3 by Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) (2004)
  • Author(s): Sawada, N., Sakaki.T., Yoneda, S., Kusudo, T., Shinkyo, R., Ohta, M., Inouye, K.
  • Journal Title: Biochem.Biophys.Res.Commun. 320 Pages: 156-164
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Metabolism of A-ring diastereomers of 1α,25-dihydroxyvitamin D_3 by CYP24A1. (2004)
  • Author(s): Kusudo, T., Sakaki, T., Abe, D., Fujishima, T., Kittaka, A., Takayama, H., Hatakeyama, S., Ohta, M., Inouye, K.
  • Journal Title: Biochem.Biophys.Res.Commun. 321 Pages: 774-782
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Purification and characterization of mouse CYP27B1 overproduced by an Escherichia coli system coexpressing molecular chaperonins GroEL/ES. (2004)
  • Author(s): Uchida, E., Kagawa, N., Sakaki, T., Urushino, N., Sawada, N., Kamakura, M., Ohta, M., Kato, S., Inouye, K.
  • Journal Title: Biochem.Biophys.Res.Commun. 323 Pages: 505-511
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Metabolism of vitamin D by human microsomal CYP2R1. (2004)
  • Author(s): Shinkyo, R., Sakaki, T., Kamakura, M., Ohta, M., Inouye, K.
  • Journal Title: Biochem.Biophys.Res.Commun. 324 Pages: 451-457
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Enzymatic properties of human CYP2W1 expressed in Escherichia coli.
  • Author(s): Yoshioka, H., Kasai, N., Ikushiro, S., Shinkyo, R., Kamakura, M., Ohta, M., Inouye, K., Sakaki, T.
  • Journal Title: Biochem.Biophys.Res.Commun. 345 Pages: 169-174
  • Description: 「研究成果報告書概要(欧文)」より