liu.seSearch for publications in DiVA
Change search
ReferencesLink to record
Permanent link

Direct link
Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
Linköping University, Department of Physics, Chemistry and Biology, Biochemistry. Linköping University, The Institute of Technology.ORCID iD: 0000-0002-7642-9263
Childhood Cancer Research Unit, Department of Women and Child Health, Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm.
Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0002-2809-7591
Show others and affiliations
2013 (English)In: European Journal of Clinical Pharmacology, ISSN 0031-6970, E-ISSN 1432-1041, Vol. 69, no 9, 1641-1649 p.Article in journal (Refereed) Published
Abstract [en]


Important drugs in the treatment of childhood acute lymphoblastic leukaemia (ALL) are 6-mercaptopurine (6-MP) and methotrexate (MTX). Thiopurine methyltransferase (TPMT) is a polymorphic enzyme causing variability in 6-MP response and toxicity. The aim of this study was to investigate the fluctuation in TPMT enzyme activity over time and the effect of high-dose MTX infusions on TPMT enzyme activity and 6-MP metabolites in paediatric ALL patients.


Fifty-three children with ALL treated according to the NOPHO-ALL 2000 protocol were included in the study. TPMT enzyme activity was measured at six different times starting from diagnosis until after the end of maintenance treatment. TPMT and 6-MP metabolites were measured before the initiation of high-dose MTX (HD-MTX) infusions and at 66 h post-infusion. The interaction between MTX and TPMT was investigated in vitro using recombinant TPMT protein and a leukaemic cell line.


Forty percent of TPMT wild-type individuals had deceptively low TPMT enzyme activity according to genotype at the time of diagnosis. TPMT activity had decreased significantly 66 h after the start of HD-MTX infusions (−9.2 %; p = 0.013). MTX bound to recombinant TPMT protein severely inhibiting TPMT enzyme activity (remaining activity 16 %).


Our results show that TPMT genotyping should be performed in children with ALL, since 40 % of the children in our study who carried the wild-type TPMT gene were at risk of initial underdosing of 6-MP in cases where only TPMT enzyme activity was determined. MTX inhibits the TPMT enzyme activity after HD-MTX infusions due to protein binding.

Place, publisher, year, edition, pages
Springer Berlin/Heidelberg, 2013. Vol. 69, no 9, 1641-1649 p.
Keyword [en]
Leukaemia, 6-mercaptopurine, methotrexate, pharmacogenetics, thiopurine s-methyltransferase
National Category
Natural Sciences
URN: urn:nbn:se:liu:diva-80190DOI: 10.1007/s00228-013-1521-9ISI: 000323429900003OAI: diva2:546093
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2015-05-29Bibliographically approved
In thesis
1. Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
Open this publication in new window or tab >>Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In the treatment of leukemia and inflammatory bowel disease, thiopurines are commonly used drugs. Thiopurine S-methyltransferase (TPMT) is one of the drug metabolizing enzymes responsible of counteracting the formation of TGNs that will be incorporated into the DNA and RNA synthesis and thus induce apoptosis. TPMT is a polymorphic enzyme and to date about 30 different sequence variants have been identified. Individuals who are to be treated with thiopurines are genotyped and/ or phenotyped at the time of diagnosis in order to individualize the treatment, with thiopurine dosage adjusted to the TPMT activity. In the treatment of acute lymphoblastic leukemia (ALL) high-dose methotrexate (MTX) is administered intravenously during the consolidation phase of the therapy and used in lower doses in the other phases of the ALL therapy. In blood samples from 53 children with ALL, we found decreased TPMT enzyme activity after 66 hours infusion of high-dose MTX. TPMT was recombinantly expressed, and the potential binding of MTX to TPMT was investigated by a fluorescence method. This showed that MTX bound to TPMT at relevant plasma concentrations observed in patient samples. At the time of leukemia diagnosis, TPMT activity was not correlated with the genotype for TPMT wild-types, which demonstrates the importance of using genotyping as a golden standard for determination of TPMT status in individuals with haematological malignancies. The low enzyme activity of TPMT*2 and TPMT*5 protein was evaluated by expressing these sequence variants in Escherichia coli (E.coli), and then characterizing them biophysically. Our results showed that TPMT*2 and TPMT*5 in the native state did not bind the extrinsic probe anilinonaphthalene sulfonate (ANS), which shows that the three-dimensional structure is already affected and restructured in that state. Based on these findings, we concluded that ANS can be used to probe the status of the active site. In another study we investigated the characteristics of TPMT*6 and TPMT*8 and found that the cofactor, S-adenosylmethionine (SAM) had a stabilizing effect on those sequence variants and on TPMT wild-type. Analysis of the structure of the TPMT protein by nuclear magnetic resonance (NMR) spectroscopy, enabled partial assignment of the backbone residue, of 64% of the TPMT sequence. Forty residues in TPMT exhibited millisecond dynamics but only 15 of those residues could be assigned, which emphasizes the difficulties involved in determining the three-dimensional structure of TPMT by NMR spectroscopy. In conclusion the present studies contribute to the understanding of the molecular characteristics of TPMT.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. 71 p.
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1462
National Category
Natural Sciences
urn:nbn:se:liu:diva-80194 (URN)978-91-7519-851-4 (ISBN)
Public defence
2012-09-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (Swedish)
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2013-10-04Bibliographically approved

Open Access in DiVA

fulltext(355 kB)183 downloads
File information
File name FULLTEXT01.pdfFile size 355 kBChecksum SHA-512
Type fulltextMimetype application/pdf

Other links

Publisher's full text

Search in DiVA

By author/editor
Wennerstrand, PatriciaMårtensson, Lars-GöranLindqvist Appell, MalinZimdahl, Anna
By organisation
ChemistryFaculty of Science & EngineeringBiochemistryThe Institute of TechnologyClinical PharmacologyFaculty of Health SciencesDivision of Drug Research
In the same journal
European Journal of Clinical Pharmacology
Natural Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 183 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

Altmetric score

Total: 211 hits
ReferencesLink to record
Permanent link

Direct link