Farmakogenomsko vrednotenje dovzetnosti na tiopurine

doktorska disertacija

Dunja Urbančič (Avtor), Irena Mlinarič-Raščan (Mentor)

Povzetek

Kljub velikemu napredku zdravljenja, ki ga je prinesel pojav bioloških zdravil, ostajajo enostavne sintezne zdravilne učinkovine steber zdravljenja tako rakavih kot avtoimunskih bolezni. V to skupino sodijo tiopurini, ki se kot predzdravila pogosto uporabljajo pri zdravljenju akutne limfoblastne levkemije ter vnetne črevesne bolezni. Zaradi ozkega terapevtskega okna je pomembno, da je odmerjanje tiopurinov prilagojeno posamezniku, kar predstavlja kompleksen izziv sodobne terapije. Velik vpliv na izid zdravljenja s tiopurini ima encim tiopurin-S-metiltransferaza (TPMT). Interindividualna raznolikost odziva na tiopurine je namreč lahko posledica prisotnosti genskih polimorfizmov, metabolnih dejavnikov, ki stabilizirajo TPMT, prav tako pa je lahko rezultat interakcij z zdravilnimi učinkovinami v sočasni terapiji. Poglaviten namen raziskovalnega dela v sklopu doktorske disertacije je bil doprinesti k učinkoviti in varni terapiji s tiopurini, zato smo si zastavili sledeče cilje: poglobiti razumevanje delovanja encima TPMT in njegove endogene vloge, najti nove genetske dejavnike odziva na tiopurine ter opredeliti molekularne mehanizme interakcij med zdravilnimi učinkovinami, ki se pojavljajo ob sočasnem jemanju s tiopurini. V sklopu raziskovanja endogene vloge smo se osredotočili na selenove spojine, saj je znano, da TPMT poleg tiopuinov katalizira tudi reakcijo metilacije njihovih selenovih analogov. Med najpomembnejše selenove spojine, ki jih najdemo v organizmu, sodi selenocistein, ki je kot gradnik selenoproteinov udeležen pri ključnih procesih za vzdrževanje homeostaze v celicah. Z uporabo spektroskopije STD NMR in merjenjem fluorescence triptofanov v TPMT smo pokazali, da se selenocistein veže na humani rekombinantni TPMT. Analiza molekulskega sidranja je predpostavila lego selenocisteina v aktivnem mestu TPMT, ki bi bila relevantna za reakcijo metilacije. Ob prisotnosti encima in donorja metilne skupine, S-adenozilmetionina (SAM), smo potrdili encimsko katalizirano nastajanje metiliranega produkta Se-metilselenocisteina. To dokazuje doslej neznano vlogo encima kot selenocistein-Se-metiltransferaze. Pri iskanju novih bioloških označevalcev odziva na tiopurine smo se najprej osredotočili na raziskovanje lastnosti gena za TPMT. Ugotovili smo, da ima natančna določitev sekvence DNA v promotorski regiji, ki vsebuje variabilna števila tandemskih ponovitev (VNTR), velik pomen. Motivi z zaporedjem ABnC so namreč v t.i. vezavnem nesorazmerju s klinično pomembnimi polimorfizmi TPMT*3C in TPMT*3A. Motiv AB2C se pojavlja skupaj z alelom TPMT*3C, medtem ko se motivi z večjim številom ponovitev B (AB3C, AB4C, AB5C ter celo AB7C) pojavljajo skupaj z alelom TPMT*3A. Motiv AB7C je prvič dokazan na naši populaciji in opisan v pričujoči publikaciji. Omenjena polimorfizma sta klinično relevantna, zato bi bilo lahko v prihodnosti določanje motivov VNTR enako dober biološki označevalec pri uravnavanju odmerka tiopurinov, kot so polimorfizmi posameznih nukleotidov (SNP). Pri nadaljnjem iskanju bioloških označevalcev za napovedovanje odziva smo uporabili model limfoblastoidnih celičnih linij (LCL), na katerih smo določali občutljivost na 6-merkaptopurin in 6-tiogvanin. Ugotovili smo, da je bila aktivnost TPMT pričakovano odvisna od najpogostejših SNP v genu, a ni korelirala z dovzetnostjo celic LCL na 6-merkaptopurin, kar bi sicer pričakovali na osnovi in vivo delovanja. To spoznanje kaže na obstoj drugih nepoznanih pomembnih dejavnikov, ki celicam v prisotnosti citotoksičnih spojin omogočajo preživetje. Kot tovrstni kazalec rezistence oz. preobčutljivosti celic LCL na tiopurine se je izkazal nivo izražanja gena za prenašalni P-glikoprotein, ABCB1. V klinični praksi se tiopurini pogosto uporabljajo hkrati z drugimi zdravilnimi učinkovinami, način medsebojnega vplivanja pa pogosto ni znan, zato smo s pomočjo sodobnih in silico metod raziskovali mehanizme interakcij s sicer znanimi substrati in inhibitorji TPMT. Povzeli smo biokemijske značilnosti vezave na TPMT in kinetične karakteristike pretvorbe substratov ter klinične posledice terapije tiopurinov sočasno z inhibitorji TPMT. Z molekulskim sidranjem inhibitorjev v vezavna mesta TPMT smo dopolnili vpogled v molekularne osnove vezave. Še posebno smo se osredotočili na interakcijo TPMT s sulfasalazinom, derivatom aminosalicilne kisline, ki se uporablja pri zdravljenju vnetne črevesne bolezni in se je v in vitro študijah izkazal kot močan inhibitor TPMT. Z merjenjem spremembe fluorescence triptofanov v humanem rekombinantnem TPMT smo dokazali vezavo sulfasalazina na encim. Naša in silico analiza je pokazala dobro sidranje sulfasalazina v vezavno mesto za ko-faktor SAM, a tudi potencialno vezavo na vezavno mesto za substrat. To smo eksperimentalno potrdili tudi z encimsko kinetiko, ki je pokazala inhibicijo mešanega tipa. Rezultati tega doktorskega dela podajajo pomembne nove izsledke o TPMT in tiopurinih z biokemijskega in farmakološkega vidika. Dokaz vpletenosti TPMT v metabolizem selenovih spojin predstavlja dobro izhodišče za natančnejše določanje endogene vloge TPMT ter vpetosti TPMT v patološke procese preko selenovega metabolizma. Z odkritjem povezave med genskimi polimorfizmi TPMT*3 in motivi ABnC v promotoju TPMT ter z identifikacijo vpliva izražanja ABCB1 na rezistenco 6-merkaptopurina smo razširili nabor potencialnih označevalcev dovzetnosti na tiopurine, kar bo v klinični praksi doprineslo k izboljšanju napovedi izida zdravljenja s tiopurini.

Ključne besede

farmakogenomika;tiopurini;tiopurin-S-metiltransferaza;farmakogenomika tiopurinov;citotoksičnost interakcij;zdravilne učinkovine;odziv na tiopurine;disertacije;

Podatki

Jezik:	Slovenski jezik
Leto izida:	2018
Tipologija:	2.08 - Doktorska disertacija
Organizacija:	UL FFA - Fakulteta za farmacijo
Založnik:	[D. Urbančič]
UDK:	615:543.544.5(043.3)
COBISS:	4633969
Št. ogledov:	5
Št. prenosov:	0
Ocena:	0 (0 glasov)
Metapodatki:

Ostali podatki

Sekundarni jezik:	Angleški jezik
Sekundarni naslov:	Pharmacogenomic evaluation of susceptibility to thiopurines
Sekundarni povzetek:	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 TPMT3C and TPMT3A. The motif AB2C is in statistically significant complete linkage disequilibrium with the TPMT3C allele, and the motifs with three or more B repeats, including a previously unreported VNTR allele AB7C, appear together with TPMT3A alleles. Since TPMT3 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 TPMT3 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.
Sekundarne ključne besede:	Biološka zdravila;
Vrsta dela (COBISS):	Doktorska disertacija
Komentar na gradivo:	Univ. v Ljubljani, Fak. za farmacijo
Strani:	X, 178 str.
ID:	15645127