Despite great therapeutic advances in the field of biological agents, small synthetic molecules still represent an important therapeutic pillar in management of cancer as well as autoimmune disorders. Among such drugs, thiopurines have been commonly used in the treatment of acute lymphoblastic leukaemia and inflammatory bowel disease. Due to their narrow therapeutic index, individualized approach in application of these drugs is crucial, which represents a complex challenge of the current medicine. One of the most important factors influencing thiopurine therapy is thiopurine S-methyltransferase (TPMT), as interindividual differences in response to thiopurines often result from genetic polymorphisms in its gene, metabolic factors stabilizing TPMT, or drug interactions accompanying concomitant treatment. The overall goal of the research presented in this thesis was to contribute to efficacy and safety of thiopurine therapy. Therefore, we aim to elucidate the function and endogenous role of TPMT, further, to identify novel genetic factors of the response to thiopurines, and to define molecular mechanisms of the drug interactions in thiopurine therapy. To unravel the endogenous substrate of TPMT, we focused on selenium compounds, since esides thiopurines, selenopurines are also known TPMT substrates. One of the most important selenium compounds natively present in biological systems is selenocysteine - a key building block of selenoproteins, which are involved in numerous important biological processes in maintaining homeostasis of cells. By STD NMR spectroscopy and fluorescence measurement of tryptophan residues, we demonstrated binding of selenocysteine (Sec) to human recombinant TPMT. The molecular docking analysis positioned Sec into the active site of TPMT with orientation suited for the methylation reaction. With 77Se NMR we finally detected Se-methylselenocysteine (MeSec), produced in the enzymatically catalysed reactionin the presence of the TPMT and the methyl donor S-adenozylmethionine, suggesting the previously unknown role of the enzyme as selenocisteine Se-methyltransferaze. In the pursuit of novel biological markers of response to thiopurines, we first characterized the gene encoding TPMT. We recognised a crucial importance of the exact DNA sequence in the TPMT promoter containing variable number of tandem repeats (VNTR). Motifs ABnC are in complete linkage disequilibrium with clinically established genetic polymorphisms TPMT*3C and TPMT*3A. The motif AB2C is in statistically significant complete linkage disequilibrium with the TPMT*3C allele, and the motifs with three or more B repeats, including a previously unreported VNTR allele AB7C, appear together with TPMT*3A alleles. Since TPMT*3 alleles are clinically relevant, the motifs ABnC represent a viable indirect pharmacogenomic marker in future thiopurine treatment. In our further search for biomarkers predicting the response to thiopurines, we tested the susceptibility to 6-mercaptopurine and 6-thioguanine on the established in vitro model, the lymphoblastoid cell lines (LCL). While the activity of TPMT expectedly correlated with the common genetic polymorphisms, lower TPMT activity did not correlate with enhanced cytotoxicity to 6-mercaptopurine (6MP), which is in contrast to the established correlation in vivo. This finding indicates the importance of other unknown factors that mask the influence of TPMT. In fact, higher levels of ABCB1 expression in LCL predicted their lower sensitivity to 6MP, suggesting this transporter as a potential biological marker for the resistance to 6-mercaptopurine. We further examined mechanisms of the interaction between thiopurines and other drugs often applied concomitantly in the therapy. Their mutual influence remains unexplained, therefore, we first reviewed the biochemical properties of the binding to TPMT, kinetic characteristics of substrate methylation and the clinical outcome of concomitant therapy, and complemented by in silico analysis. The molecular docking and chemical structures of known TPMT inhibitors elucidated the molecular basis for the binding to TPMT. In this context, we specifically focused on the interaction of TPMT by sulfasalazine, a derivative of 5-aminosalicylic acid, which is used as the first line treatment of intestinal bowel disease and has been recognised as a potent TPMT inhibitor. By measuring the change in fluorescence of tryptophan residues in human recombinant TPMT, we confirmed the binding of sulfasalazine and TPMT. Our in silico analysis proposed the primary binding of sulfasalazine to the co-factor binding site, but also indicated possible binding to the substrate binding site. We experimentally proved this by determining the mixed inhibition of the enzyme.
The results of this doctoral thesis provide important findings on TPMT and thiopurines from the biochemical and pharmacological point of view. The evidence of involvement of TPMT in the metabolism of selenium compounds represents a good starting point for further delineation of the endogenous role of TPMT and the inclusion of TPMT in pathological processes through selenium metabolism. By discovering the relationship between the genetic polymorphisms TPMT*3 and the ABnC motifs in the TPMT promoter region, and by identifying the impact of ABCB1 expression on the 6-mercaptopurine resistance, we expanded the range of potential markers of susceptibility to thiopurins, which will contribute to future improvements of the outcome of thiopurine treatment.
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