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In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT6 and TPMT8.

ABSTRACT: 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 K_d 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.

SUBMITTER: Wennerstrand P

PROVIDER: S-EPMC6044926 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:OBJECTIVES:Bone marrow suppression is a common adverse effect of the immunosuppressive drug azathioprine. Polymorphisms in the gene encoding thiopurine S-methyltransferase (TPMT) can alter the metabolism of azathioprine, resulting in marrow toxicity and life-threatening infection. In a multicenter cohort of pediatric heart transplant (HT) recipients, we determined the frequency of TPMT genetic variation and assessed whether azathioprine-treated recipients with TPMT variants were at increased risk of infection. METHODS:We genotyped TPMT in 264 pediatric HT recipients for the presence of the TPMT*2, TPMT*3A, and TPMT*3C variant alleles. Data on infection episodes and azathioprine use were collected as part of each patient's participation in the Pediatric Heart Transplant Study. We performed unadjusted Kaplan-Meier analyses comparing infection outcomes between groups. RESULTS:TPMT variants were identified in 26 pediatric HT recipients (10%): *3A (n = 17), *3C (n = 8), and *2 (n = 1). Among those with a variant allele, *3C was most prevalent in black patients (4 of 5) and *3A most prevalent among white and Hispanic patients (16 of 20). Among 175 recipients (66%) who received azathioprine as part of the initial immunosuppressive regimen, we found no difference in the number of infections at 1 year after HT (0.7 ± 1.3; range, 0-6 versus 0.5 ± 0.9; range, 0-3; p = 0.60) or in freedom from infection and bacterial infection between non-variant and variant carriers. There was 1 infection-related death in each group. CONCLUSIONS:In this multicenter cohort of pediatric HT recipients, the prevalence of TPMT variants was similar across racial/ethnic groups to what has been previously reported in non-pediatric HT populations. We found no association between variant alleles and infection in the first year after HT. Because clinically detected cytopenia could have prompted dose adjustment or cessation, we recommend future studies assess the relationship of genotype to leukopenia/neutropenia in the pediatric transplantation population.

Project description:Pediatric Crohn's disease (CD) carries a higher genetic susceptibility and an increased risk of a more aggressive disease course than adult CD. Treatment of CD is based on immunomodulatory drugs, such as thiopurines. The enzyme mainly involved in drug metabolism is thiopurine S-methyltransferase (TPMT). An increased concentration of drug metabolites can cause adverse drug effects, such as myelosuppression and hepatotoxicity; therefore, assessing the activity of TPMT is essential both before and during treatment. TPMT genotyping result is not affected by previous thiopurine dose and currently is the primary component of TPMT activity and disease monitoring. Until now, more than 40 allelic variants of the TPMT gene have been reported, with most of them having an uncertain or no enzyme function. In this article, we report the first case of a novel TPMT allele, TPMT*45, that was identified in a Korean girl with CD whose findings suggested decreased TPMT activity. This newly observed variant is caused by a single nucleotide polymorphism resulting in nonsense mutation (c.676C>T, p.R226*) and the partial loss of amino acids in the TPMT protein. Initially, the patient began azathioprine at a standard dosage (1.5 mg/kg/day), and her laboratory results, including red blood cell (RBC) TPMT activity (6-methylmercaptopurine 2.68 nmol/mL/h and 6-methylmercaptopurine riboside 4.82 nmol/mL/h) along with thiopurine metabolite levels (6-thioguanine nucleotides 479.3 pmol/8×108 RBC), suggested an enzyme deficiency. The thiopurine dose was reduced to half (0.7 mg/kg/day), and the follow-up metabolite results as well as the associated inflammatory markers were continuously within reference ranges. Along with an improvement in the patient's subjective reports and clinical symptoms, the patient demonstrated a good treatment response to the adjusted dose. The results of our report illustrate the importance of TPMT genotyping and pharmacogenetic-based thiopurine dose adjustment. Further research should focus on the functional characterization and impact on this novel allele's treatment effect.

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In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT6 and TPMT8.

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Dataset Information

In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT*6 and TPMT*8.

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OmicsDI is part of the ELIXIR infrastructure

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In Vitro Protein Stability of Two Naturally Occurring Thiopurine S-Methyltransferase Variants: Biophysical Characterization of TPMT6 and TPMT8.