BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
권호 표지
DOI QR코드

DOI QR Code

Expression and purification of human mPGES-1 in E. coli and identification of inhibitory compounds from a drug-library

  • Published : 2008.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

Human microsomal prostaglandin E synthase-1 (mPGES-1) is a membrane associated protein that catalyzes the conversion of prostaglandin $H_2$ ($PGH_2$) into prostaglandin $E_2$ ($PGE_2$). In this study, the expression of human mPGES-1 in E. coli was significantly enhanced by modifying the utility of specific codons and the recombinant mPGES-1 was efficiently purified to homogeneity. The $K_m$ and $V_{max}$ of the purified enzyme were determined and the trimeric state characterized by chemical cross-linking with glutaraldehyde. The purified mPGES-1 was used for the screening of a chemical library of bioactive or drug compounds to identify novel inhibitors, and oxacillin and dyphylline were identified as moderately inhibiting mPGES-1 with $I_{C50}$ values of 100 and 200 ${\mu}M$, respectively. As these compounds competitively inhibited the catalysis of $PGH_2$, their binding sites appeared to be located near the $PGH_2$ binding pocket.

Keywords

References

  1. Smith, W.L., DeWitt, D.L. and Garavito, R.M. (2000) Cyclooxygenases: structural, cellular, and molecular biology. Annu. Rev. Biochem. 69, 145-182 https://doi.org/10.1146/annurev.biochem.69.1.145
  2. Garavito, R. M. and Dewitt, D.L. (1999) The cyclooxygenase isoforms: structural insights into the conversion of arachidonic acid to prostaglandins. Biochim. Biochpy. Act. 1441, 278-287 https://doi.org/10.1016/S1388-1981(99)00147-X
  3. Murakami, M., Naraba, H., Tanioka, T., Semmyo, N., Nakatami, Y., Kojima, F. Iked,a T., Fueki, M., Ueno, A, Oh S. and Kudo, I. (2000) Regulation of prostaglandin $E_{2}$ biosynthesis by inducible membrane associated prostaglandin $E_{2}$ synthase that acts in concert with cyclooxygenase-2. J. Biol. Chem. 275, 32783-32792 https://doi.org/10.1074/jbc.M003505200
  4. Mancini, J. A., Blood, K., Guay, J., Gordon, R., Claveau, D., Chan, C. C. and Riendeau, D. (2001) Cloning, expression, and up-regulation of inducible rat prostaglandin E synthase during lipopolysaccharide-induced pyresis and adjuvant-induced arthritis. J. Biol. Chem. 276, 4469-4475 https://doi.org/10.1074/jbc.M006865200
  5. Stichtenoth, D.O., Thoren, S., Bian, H., Peters-Golden, M., Jakobsson, P. J. and Crofford, L.J. (2001) Microsomal prostaglandin E synthase is regulated by proinflammatory cytokines and glucocorticoids in primary rheumatoid synovial cells. J. Immunol. 167, 469-574 https://doi.org/10.4049/jimmunol.167.1.469
  6. Filion, F., Bouchard, N., Goff, A. K., Lussier, J. G. and Sirois, J. (2001) Molecular cloning and induction of bovine prostaglandin E synthase by gonadotropins in ovarian follicles prior to ovulation in vivo. J. Biol. Chem. 276, 34323-34330 https://doi.org/10.1074/jbc.M103709200
  7. Kamei, D., Murakami, M., Nakatani, Y., Ishikawa, Y., Ishii, T. and Kudo, I. (2003) Potential role of microsomal prostaglandin E synthase-1 in tumorigenesis. J. Biol. Chem. 278, 19396-19405 https://doi.org/10.1074/jbc.M213290200
  8. Yoshimatsu, K., Altorki, N. K., Golijanin, D., Zhang, F., Jakobsson, P. J., Dannenberg, A. J. and Subbaramaiah, K. (2001) Inducible prostaglandin E synthase is overexpressed in non-small cell lung cancer. Clin. Cancer Res. 7, 2608-2610
  9. Jakobsson, P. J., Thoren, S., Morgenstern, R. and Samuelsson, B. (1999) Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc. Natl. Acsd. Aci. U.S.A. 96, 7220-7225
  10. Catley, M. C., Chivers, J. E., Cambridge, L. M., Holden, N., Slater, D. M., Staples, K. J., Bergmann, M. W., Loser, P., Barnes, P. J. and Newton, R. (2003) IL-1beta-dependent activation of NF-kappa B mediates $PGE_{2}$ release via the expression of cyclooxygenase-2 and microsomal prostaglandin E synthase. FEBS Lett. 547, 75-79 https://doi.org/10.1016/S0014-5793(03)00672-0
  11. Naraba, H., Murakami, M., Matsumoto, H., Shimbara, S., Ueno, A., Kudo, I. and Oh-ishi, S. (1998) Segregated coupling of phospholipases $A_{2}$, cyclooxygenases, and terminal prostanoid synthases in different phases of prostanoid biosynthesis in rat peritoneal macrophages. J. Immunol. 160, 2974-2982
  12. Fournier, T., Fadok, V. and Henson, P. M. (1997) Tumor necrosis factor-alpha inversely regulates prostaglandin $D_{2}$ and prostaglandin $E_{2}$ production in murine macrophages. Synergistic action of cyclic AMP on cyclooxygenase-2 expression and prostaglandin $E_{2}$ synthesis. J. Biol. Chem. 272, 31065-31072 https://doi.org/10.1074/jbc.272.49.31065
  13. Ushikubi, F., Segi, E., Sugimoto, Y., Murata, T., Matsuoka, T., Kobayashi, T., Hizaki, H., Tuboi, K., Katsuyama, M., Ichikawa, A., Tanaka, T., Yoshida, N. and Narumiya, S. (1998) Impaired febrile response in mice lacking the prostaglandin E receptor subtype $EP_{3}$. Nature 395, 281-284 https://doi.org/10.1038/26233
  14. Samad, T. A., Sapirstein A. and Woolf, C. J. (2002) Prostanoids and pain: unraveling mechanisms and revealing therapeutic targets. Trends Mol. Med. 8, 390-396 https://doi.org/10.1016/S1471-4914(02)02383-3
  15. Tanioka, T., Nakatani, Y., Semmyo, N., Murakami, M. and Kudo, I. (2000) Molecular identification of cytosolic prostaglandin $E_{2}$ synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin $E_{2}$ biosynthesis. J. Biol. Chem. 75, 32775-32782
  16. Tanikawa, N., Ohmiya, Y., Ohkubo, H., Hashimoto, K., Kangawa, K., Kojima, M., Ito, S. and Watanabe, K. (2002) Identification and Characterization of a Novel Type of Membrane-Associated Prostaglandin E Synthase. Biochem. Biophys. Res. Comm. 291, 884-889 https://doi.org/10.1006/bbrc.2002.6531
  17. Cheng, Y., Wang, M., Yu, Y., Lawson, J., Funk, C. D. and Fitzgerald, G. A. (2006) Cyclooxygenases, microsomal prostaglandin E synthase-1, and cardiovascular function. J. Clin. Invest. 116, 1391-1399 https://doi.org/10.1172/JCI27540
  18. Trebino, C. E., Stock, J. L., Gibbons, C. P., Naiman, B. M., Wachtmann, T. S., Umland, J. P., Pandher, K., Lapointe, J. M., Saha, S., Roach, M. L., Carter, D., Thomas, N. A., Durtschi, B. A., McNeish, J. D., Hambor, J. E., Jakobsson, P. J., Carty, T. J., Perez, J. R. and Audoly, L. P. (2003) Impaired inflammatory and pain responses in mice lacking an inducible prostaglandin E synthase. Proc. Natl. Acad. Sci. U. S. A. 100, 9044-9049
  19. Kudo, I. and Murakami, M. (2005) Prostaglandin E synthase, a terminal enzyme for prostaglandin $E_{2}$ biosynthesis. J. Biochem. Mol. Biol. 38, 633-638 https://doi.org/10.5483/BMBRep.2005.38.6.633
  20. Quraishi, O., Mancini, J. A. and Riendeau, D. (2002) Inhibition of inducible prostaglandin E(2) synthase by 15-deoxy- Delta(12,14)-prostaglandin J(2) and polyunsaturated fatty acids. Biochem Pharmacol. 63, 1183-1189 https://doi.org/10.1016/S0006-2952(02)00844-4
  21. Thoren, S. and Jakobsson, P. J. (2000) Coordinate up- and downregulation of glutathione-dependent prostaglandin E synthase and cyclooxygenase-2 in A549 cells. Inhibition by NS-398 and leukotriene $C_{4}$. Eur. J. Biochem. 267, 6428-6434 https://doi.org/10.1046/j.1432-1327.2000.01735.x
  22. Riendeau, D., Aspiotis, R., Ethier, D., Gareau, Y., Grimm, E. L., Guay, J., Guiral, S., Juteau, H., Mancini, J. A., Méthot, N., Rubin, J. and Friesen, R. W. (2005) Inhibitors of the inducible microsomal prostaglandin $E_{2}$ synthase (mPGES-1) derived from MK-886. Bioorg. Med. Chem. Lett. 15, 3352-3355 https://doi.org/10.1016/j.bmcl.2005.05.027
  23. Cote, B., Boulet, L., Brideau, C., Claveau, D., Ethier, D., Frenette, R., Gagnon, M., Giroux, A., Guay, J., Guiral, S., Mancini, J., Martins, E., Masse, F., Methot, N., Riendeau, D., Rubin, J., Xu, D., Yu, H., Ducharme, Y. and Friesen, R. W. (2007) Substituted phenanthrene imidazoles as potent, selective, and orally active mPGES-1 inhibitors. Bioorg. Med. Chem. Lett. 17, 6816-6820 https://doi.org/10.1016/j.bmcl.2007.10.033
  24. Huang, X., Yan, W., Gao, D., Tong, M., Tai, H. H. and Zhan, C. G. (2006) Structural and functional characterization of human microsomal prostaglandin E synthase-1 by computational modeling and site-directed mutagenesis. Bioorg. Med. Chem. 14, 3553-3562 https://doi.org/10.1016/j.bmc.2006.01.010
  25. Gierse, J. K., Hauser, S. D., Creely, D. P., Koboldt, C., Rangwala, S. H., Isakson, P. C. and Seibert, K. (1995) Expression and selective inhibition of the constitutive and inducible forms of human cyclooxygenase. Biochem J. 305, 479-484 https://doi.org/10.1042/bj3050479
  26. Wester, M. R., Stout, C. D. and Johnson, E. F. (2002) Purification and crystallization of N-terminally truncated forms of microsomal cytochrome P450 2C5. Methods Enzymol. 357, 73-79 https://doi.org/10.1016/S0076-6879(02)57667-9
  27. Thoren, S., Weinander, R., Saha, S., Jegerschold, C., Pettersson, P. L., Samuelsson, B., Hebert, H., Hamberg, M., Morgenstern, R. and Jakobsson, P. J. (2003) Human microsomal prostaglandin E synthase-1: purification, functional characterization, and projection structure determination. J. Biol. Chem. 278, 22199-22209 https://doi.org/10.1074/jbc.M303227200
  28. Ouellet, M., Falgueyret, J. P., Ear, P. H., Pen, A., Mancini, J. A., Riendeau, D. and Percival, M. D. (2002) Purification and characterization of recombinant microsomal prostaglandin E synthase-1. Protein Expr. Purif. 26, 489-495 https://doi.org/10.1016/S1046-5928(02)00566-1
  29. Masse, F., Guiral, S., Fortin, L. J., Cauchon, E., Ethier, D., Guay, J. and Brideau, C. (2005) An automated multistep high-throughput screening assay for the identification of lead inhibitors of the inducible enzyme mPGES-1. J. Biomol. Screen. 10, 599-605 https://doi.org/10.1177/1087057105276083
  30. Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275

Cited by

  1. Chemistry and biology of microsomal prostaglandin E2 synthase-1 (mPGES-1) inhibitors as novel anti-inflammatory agents: recent developments and current status vol.6, pp.34, 2016, https://doi.org/10.1039/C5RA25186A
  2. Microsomal prostaglandin E synthase-1 exhibits one-third-of-the-sites reactivity vol.440, pp.1, 2011, https://doi.org/10.1042/BJ20110977
  3. Prostaglandin synthases: Molecular characterization and involvement in prostaglandin biosynthesis vol.66, 2017, https://doi.org/10.1016/j.plipres.2017.04.003
  4. Inhibitors of Microsomal Prostaglandin E2Synthase-1 Enzyme as Emerging Anti-Inflammatory Candidates vol.34, pp.4, 2014, https://doi.org/10.1002/med.21306
  5. Targeting the eicosanoid pathway in non-small-cell lung cancer vol.13, pp.6, 2009, https://doi.org/10.1517/14728220902915567
  6. A Facilitated Approach to Evaluate the Inhibitor Mode and Potency of Compounds Targeting Microsomal Prostaglandin E Synthase-1 vol.9, pp.5, 2011, https://doi.org/10.1089/adt.2010.0350
  7. Identification of novel mPGES-1 inhibitors through screening of a chemical library vol.22, pp.24, 2012, https://doi.org/10.1016/j.bmcl.2012.10.085