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Biophysical Characterization of Thiopurine S-Methyltransferase: A Key enzyme in the Effects of Thiopurine Drugs
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
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.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1462
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-80194ISBN: 978-91-7519-851-4 (print)OAI: oai:DiVA.org:liu-80194DiVA: diva2:546114
Public defence
2012-09-07, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 09:15 (Swedish)
Opponent
Supervisors
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2013-10-04Bibliographically approved
List of papers
1. Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene
Open this publication in new window or tab >>Characterization of a novel sequence variant, TPMT*28, in the human thiopurine methyltransferase gene
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2010 (English)In: Pharmacogenetics and genomics, ISSN 1744-6880, Vol. 20, no 11, 700-707 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: The activity of the human enzyme thiopurine methyltransferase (TPMT) varies greatly between individuals because of genetic polymorphism. TPMT is involved in the detoxification and activation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathioprine. These drugs are used in the treatment of acute lymphoblastic leukemia and inflammatory bowel disease. A total of 29 sequence variants have been identified so far in the TPMT gene. However, most of these variants are rare and not fully characterized. METHODS AND RESULTS: In this study, we describe the identification and characterization of a novel TPMT sequence variant, originally found in a Swedish man of Italian origin. Sequencing of the variable number tandem repeats region of the TPMT promoter and exons III-X revealed a T-to-C transition at nucleotide 611, causing an amino acid substitution from isoleucine to threonine at amino acid 204, positioned in an α-helix, approximately 16 Å from the active site. This new variant was found in the patient and in his son. Both had intermediate enzyme activity (8.1 U/ml packed red blood cells and 8.8 U/ml packed red blood cells, respectively) and neither carried other variants in the coding region of the gene. To be able to study this variant in more detail, the TPMT*28 variant was expressed in Escherichia coli, and an in-vitro characterization of the variant revealed that the protein was destabilized and showed a stronger tendency towards degradation at 37°C than the wild-type protein. The individuals carrying the TPMT*28 variant had less TPMT protein and lower TPMT activity in both red and white blood cells compared with a wild-type control. CONCLUSIONS: We present a detailed in-vivo and in-vitro characterization of a novel TPMT sequence variant (TPMT*28) causing decreased TPMT activity. Individuals carrying TPMT*28 might have an increased risk for developing severe side effects if treated with conventional doses of thiopurines.

Keyword
pharmacogenetics; protein stability; right-angle light scattering; single nucleotide polymorphism; thiopurine methyltransferase
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-60741 (URN)10.1097/FPC.0b013e3283402ee4 (DOI)20881512 (PubMedID)
Available from: 2010-10-25 Created: 2010-10-25 Last updated: 2015-05-29
2. Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*5
Open this publication in new window or tab >>Structural Characteristics Determine the Cause of the Low Enzyme Activity of Two Thiopurine S-Methyltransferase Allelic Variants: A Biophysical Characterization of TPMT*2 and TPMT*5
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2012 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 30, 5912-5920 p.Article in journal (Refereed) Published
Abstract [en]

The enzyme thiopurine S-methyltransferase (TPMT) is involved in the metabolism of thiopurine drugs used to treat acute lymphoblastic leukemia and inflammatory bowel disease. Thus far, at least 29 variants of the TPMT gene have been described, many of which encode proteins that have low enzyme activity and in some cases become more prone to aggregation and degradation. Here, the two naturally occurring variants, TPMT*2 (Ala80 → Pro) and TPMT*5 (Leu49 → Ser), were cloned and expressed in Escherichia coli. Far-UV circular dichroism spectroscopy showed that TPMT*2 was substantially destabilized whereas TPMT*5 showed much greater stability comparable to that of wild-type TPMT (TPMTwt). The extrinsic fluorescent molecule anilinonaphthalene sulfonate (ANS) was used to probe the tertiary structure during thermal denaturation. In contrast to TPMTwt, neither of the variants bound ANS to a large extent. To explore the morphology of the TPMT aggregates, we performed luminescent conjugated oligothiophene staining and showed fibril formation for TPMT*2 and TPMT*5. The differences in the flexibility of TPMTwt, TPMT*2, and TPMT*5 were evaluated in a limited proteolysis experiment to pinpoint stable regions. Even though there is only one amino acid difference between the analyzed TPMT variants, a clear disparity in the cleavage patterns was observed. TPMT*2 displays a protected region in the C-terminus, which differs from TPMTwt, whereas the protected regions in TPMT*5 are located mainly in the N-terminus close to the active site. In conclusion, this in vitro study, conducted to probe structural changes during unfolding of TPMT*2 and TPMT*5, demonstrates that the various causes of the low enzyme activity in vivo could be explained on a molecular level.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-80185 (URN)10.1021/bi300377d (DOI)000308262600005 ()
Note

funding agencies|Swedish Childhood Cancer Foundation||Swedish Research Council||EMBO||

Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2017-12-07Bibliographically approved
3. In vitro Protein Stability of Two Naturally Occurring Thiopurine S-methyltransferase Sequence Variants: Biophysical Characterization of TPMT*6 and TPMT*8
Open this publication in new window or tab >>In vitro Protein Stability of Two Naturally Occurring Thiopurine S-methyltransferase Sequence Variants: Biophysical Characterization of TPMT*6 and TPMT*8
2017 (English)In: ACS Omega, E-ISSN 2470-1343, Vol. 2, no 8, 4991-4999 p.Article in journal (Other academic) Published
Abstract [en]

Thiopurine S-methyltransferase (TPMT) is a polymorphic enzyme involved in the metabolism and inactivation of thiopurine substances administered as immunosuppressants in the treatment of malignancies and autoimmune diseases. In this study, the naturally occurring variants, TPMT*6 (Y180F) and TPMT*8 (R215H), have been biophysically characterized. Despite being classified as low and intermediate in vivo enzyme activity variants, respectively, our results demonstrate a discrepancy because both TPMT*6 and TPMT*8 were found to exhibit normal functionality in vitro. While TPMT*8 exhibited biophysical properties almost indistinguishable from those of TPMTwt, the TPMT*6 variant was found to be destabilized. Furthermore, the contributions of the cofactor S-adenosylmethionine (SAM) to the thermodynamic stability of TPMT were investigated, but only a modest stabilizing effect was observed. Also presented herein is a new method for studies of the biophysical characteristics of TPMT and its variants using the extrinsic fluorescent probe 8-anilinonaphthalene-1-sulfonic acid (ANS). ANS was found to bind strongly to all investigated TPMT variants with a Kd of approximately 0.2 μM and a 1:1 binding ratio as determined by isothermal titration calorimetry (ITC). Circular dichroism and fluorescence measurements showed that ANS binds exclusively to the native state of TPMT, and binding to the active site was confirmed by molecular modeling and simulated docking as well as ITC measurements. The strong binding of the probe to native TPMT and the conformity of the obtained results demonstrate the advantages of using ANS binding characteristics in studies of this protein and its variants.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keyword
Polymorphisms, protein stability, S-adenosylmethionine, thiopurine S-methyltransferase, anilinonaphtalene sulfonate
National Category
Biochemistry and Molecular Biology Medicinal Chemistry Biocatalysis and Enzyme Technology Biophysics
Identifiers
urn:nbn:se:liu:diva-80187 (URN)10.1021/acsomega.7b00801 (DOI)
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2017-08-29Bibliographically approved
4. Partially Assigned Chemical Shifts of Human Thiopurine S-methyltransferase Reveal Flexibility in Native Structure
Open this publication in new window or tab >>Partially Assigned Chemical Shifts of Human Thiopurine S-methyltransferase Reveal Flexibility in Native Structure
(English)Manuscript (preprint) (Other academic)
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-80188 (URN)
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2017-04-25Bibliographically approved
5. Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia
Open this publication in new window or tab >>Methotrexate binds to recombinant thiopurine S-methyltransferase and inhibits enzyme activity after high-dose infusions in childhood leukaemia
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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]

Purpose

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.

Methods

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.

Results

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 %).

Conclusions

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
Keyword
Leukaemia, 6-mercaptopurine, methotrexate, pharmacogenetics, thiopurine s-methyltransferase
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-80190 (URN)10.1007/s00228-013-1521-9 (DOI)000323429900003 ()
Available from: 2012-08-22 Created: 2012-08-22 Last updated: 2017-12-07Bibliographically approved

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