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New targets for an old drug

II. Hypoxanthine-guanine amidophosphoribosyltransferase as a new pharmacodynamic target of methotrexate

Abstract

Methotrexate has been a clinical agent used in cancer, immunosuppression, rheumatoid arthritis, and other highly proliferative diseases for many years, yet its underlying molecular mechanism of action in these therapeutic areas is still unclear. We have previously reported using a chemical proteomics technique on several other potential pharmacodynamic targets of methotrexate. Here, using a frontal affinity chromatography with mass spectrometry detection, we confirm one of these targets, hypoxanthine-guanine amidophosphoribosyltransferase, as a true binder of methotrexate with a Kd of 4.2 μM. These results complement and confirm our recent study, but more importantly, shed light into the mechanism of action of methotrexate in oncology and other highly proliferative diseases and may help explain some unaccounted for effects of this drug. For example, despite the fact that DNA salvage pathway enzymes are highly active, methotrexate can be effective if it only targets enzymes of the de novo pathway.

References

  1. 1.

    Arlington, S.A. (2001). Industrialization of R&D in the 21st Century. Pricewaters Coopers, ECPI, Barcelona, Spain.

    Google Scholar 

  2. 2.

    Banik, G.M. (2004, May). In silico ADME-Tox prediction: The more the merrier. Current Drug Discovery, pp. 31–34.

  3. 3.

    CHI. (2002, May). CHI's Third Annual Conference on Pharmacogenomics/Pharmacoproteomics Europe. Munich, Germany.

  4. 4.

    Leung, D., Hardouin, C., Boger, D.L., and Cravatt, B.F. (2003). Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 21: 687–691.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    Kim, E. and Park, J. M. (2003). Identification of novel target proteins of cyclic GMP signaling pathways using chemical proteomics. J. Biochem. Mol. Biol. 36:299–304.

    PubMed  CAS  Google Scholar 

  6. 6.

    Graves, P.R., Kwiek, J.J., Fadden, P., Ray, R., Hardeman, K., Coley, A.M., et al. (2002). Discovery of novel targets of quinoline drugs in the human purine binding proteome. Mol. Pharmacol. 62:1364–1372.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    Weber, G. and Prajda, N. (1994). Targeted and non-targeted actions of anti-cancer drugs. Adv. Enzyme Regul. 34:71–89.

    PubMed  Article  CAS  Google Scholar 

  8. 8.

    Allison, A.C. (2000). Immunosuppressive drugs: the first 50 years and a glance forward. Immunopharmacology 47:63–83.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Saravanan, V. and Hamilton, J. (2002). Advances in the treatment of rheumatoid arthritis: old versus new therapies. Expert Opin. Pharmacother. 3:845–856.

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Fairbanks, L.D., Ruckemann, K., Qiu, Y., Hawrylowicz, C.M., Richards, D.F., Swaminathan, R., et al. (1999). Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis? Biochem. J. 342(Pt. 1):143–152.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Cronstein, B.N. (1997). The mechanism of action of methotrexate. Rheum. Dis. Clin. North Am. 23:739–755.

    PubMed  Article  CAS  Google Scholar 

  12. 12.

    Costi, M.P. and Ferrari, S. (2001). Update on antifolate drugs targets. Curr. Drug Targets 2:135–166.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Allegra, C.J., Drake, J.C., Jolivet, J., and Chabner, B.A. (1985). Inhibition of phosphoribosylaminoimidazolecarboxamide transformylase by methotrexate and dihydrofolic acid polyglutamates. Proc. Natl. Acad. Sci. USA 82:4881–4885.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    Mauritz, R., Peters, G.J., Priest, D.G., Assaraf, Y.G., Drori, S., Kathmann, I., et al. (2002). Multiple mechanisms of resistance to methotrexate and novel antifolates in human CCRF-CEM leukemia cells and their implications for folate homeostasis. Biochem. Pharmacol. 63:105–115.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Toledo-Sherman, L.M., Desouza, L., Hosfield, C., Liao, L., Boutellier, K., Taylor, P., et al. (2004). New targets for an old drug: a chemical proteomics approach to unraveling the molecular mechanism of action of methotrexate. Clin. Proteomics 1:45–68.

    Article  CAS  Google Scholar 

  16. 16.

    Jones, G., Willett, P., Glen, R.C., Leach, A.R., and Taylor, R. (1997). Development and validation of a genetic algorithm for flexible docking. J. Mol. Biol. 267:727–748.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    Chen, Y.Z. and Zhi, D.G. (2001). Ligand-protein inverse docking and its potential use in the computer search of protein targets of a small molecule. Proteins 43:217–226.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Chan, N.W.C., Lewis, D.F., Hewko, S., Hindsgaul, O., and Schriemer, D.C. (2002). Frontal affinity chromatography for the screening of mixtures. Comb. Chem. High Throughput Screen. 5:395–406.

    PubMed  CAS  Google Scholar 

  19. 19.

    Weber, G., Nagai, M., Natsumeda, Y., Ichikawa, S., Nakamura, H., Eble, J.N., et al. (1991). Regulation of de novo and salvage pathways in chemotherapy. Adv. Enzyme Regul. 31:45–67.

    PubMed  CAS  Google Scholar 

  20. 20.

    Balendiran, G.K., Molina, J.A., Xu, Y., Torres-Martinez, J., Stevens, R., Focia, P.J., et al. (1999). Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding. Protein Sci. 8:1023–1031.

    PubMed  CAS  Article  Google Scholar 

  21. 21.

    Schoettle, S.L. and Christopherson, R.I. (1994). Inhibition of murine amido phosphoribosyl-transferase by folate derivatives. Adv. Exp. Med. Biol. 370:151–154.

    PubMed  CAS  Google Scholar 

  22. 22.

    Jones, R.J. and Twelves, C.J. (2002). Pemetrexed: a multitargeted antifolate (ALIMTA, LY-231514). Expert Rev. Anticancer Ther. 2:13–22.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Aherne, G.W., Hardcastle, A., Ward, E., Dobinson, D., Crompton, T., Valenti, M., et al. (2001). Pharmacokinetic/pharmacodynamic study of ZD9331, a nonpolyglutamatable inhibitor of thymidylate synthase, in a murine model following two curative administration schedules. Clin. Cancer Res. 7:2923–2930.

    PubMed  CAS  Google Scholar 

  24. 24.

    Romain, S., Martin, P.M., Klijn, J.G., van Putten, W.L., Look, M.P., Guirou, O., et al. (1997). DNA-synthesis enzyme activity: a biological tool useful for predicting antimetabolic drug sensitivity in breast cancer? Int. J. Cancer 74:156–161.

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Weber, G. and Prajda, N. (1994). Targeted and non-targeted actions of anti-cancer drugs. Adv. Enzyme Regul. 34:71–89.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Gordon, R.B., Keough, D.T., and Emmerson, B.T. (1987). HPRT-deficiency associated with normal PRPP concentration and APRT activity. J. Inherit. Metab. Dis. 10:82–88.

    PubMed  Article  CAS  Google Scholar 

  27. 27.

    Fung, K.P., Lam, W.P., Choy, Y.M., and Lee, C.Y. (1996). Effect of methotrexate on the intracellular phosphoribosyl pyrophosphate level and glucose transport of Ehrlich ascites tumor cells in vitro. Oncology 53:27–30.

    PubMed  CAS  Article  Google Scholar 

  28. 28.

    Slon-Usakiewicz, J.J., Ng, W., Foster, J.E., Dai, J.-R., Deretey, E., Toledo-Sherman, L., et al. (2004). Frontal affinity chromatography with MS detection of EphB2 tyrosine kinase receptor. 1. Comparison with conventional ELISA. J. Med. Chem. 47:5094–5100.

    PubMed  Article  CAS  Google Scholar 

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Correspondence to Jacek J. Slon-Usakiewicz.

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Slon-Usakiewicz, J.J., Pasternak, A., Reid, N. et al. New targets for an old drug. Clin Proteom 1, 227–234 (2004). https://doi.org/10.1385/CP:1:3-4:227

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Key Words

  • Methotrexate
  • HGPRT
  • chemical
  • proteomics
  • mechanism of action