Development of small-molecule regulators of epitranscriptomic processes
Date
2021-07-12
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Abstract
Viimase kümnendi jooksul on kiiresti kasvanud huvi RNA erinevate modifikatsioonide vastu, mis reguleerivad geeniekspressiooni organismides. Sellist teadusharu, mis tegeleb RNA modifikatsioonide ning nende bioloogilise toimega, nimetatakse epitranskriptoomikaks. Kõige levinum RNA modifikatsioon on ühe nukleotiidi, adenosiini, metüleerimine kuuendas asendis ehk N6-metüüladenosiin (m6A). RNA m6A modifikatsioonid mängivad olulist rolli rakkude diferentseerumise, immuuntaluvuse, närvisignaalide ülekande, vähihaiguste ning paljude muude füsioloogiliste ning patoloogiliste seisundite reguleerimise juures. RNA m6A metüleerimist või demetüleerimist on võimalik reguleerida neis protsessides osalevate ensüümide inhibeerimise ja/või aktiveerimise teel.
Käesolevas väitekirjas rakendati arvutipõhiseid molekulaarmodelleerimise meetodeid (molekulaarsildamine, virtuaalne sõelumine ning molekulaardünaamika simulatsioonid) inhibiitorite leidmiseks RNA m6A metüleerimise ning demetüleerimisega tegelevatele ensüümidele. Töös käigus avastati unikaalsed RNA m6A metüültransferaasi kompleksi METTL3/METTL14/WTAP aktivaatorid ning leiti inhibiitorid kahele m6A demetülaasile, FTO ning ALKBH5. Arvutipõhiselt leitud ühenditele mõõdeti inhibeerimise või aktiveerimise aktiivsused ning sidumise afiinsused kõigi uuritud ensüümvalkude korral. Töö oluliseks osaks oli leitud ühendite toime uurimine erinevate haiguste katseklaasi (in vitro) mudelites. Leiti, et METTL3/METTL14/WTAP ensüümkompleksi aktivaatorid suurendavad HIV viiruse paljunemist. ALKBH5 ensüümi inhibiitorid seevastu pidurdasid vähirakkudele paljunemist. Eriti huvipakkuv on leitud FTO ensüümi inhibiitorite närvirakke kaitsev toime.
Töö oluliseks praktiliseks väärtuseks on see, et kõik leitud aktiivsed ühendid võivad olla lähtepunktiks uute ravimikandidaatide väljatöötamisel.
Over the last decade, there has been a rapidly growing interest in various chemical modifications of RNA because of their high importance in gene expression. The field of science that deals with various modifications of RNA has been called epitranscriptomics. The most common RNA modification is the methylation at the sixth position of adenosine nucleotide, or N6-methyladenosine (m6A). RNA m6A modifications play critical role in regulating cell differentiation, immune tolerance, neuronal signaling, carcinogenesis and other, both physiological and pathological, conditions. The methylation and demethylation of RNA m6A can be regulated by inhibiting and/or activating enzymes involved in these processes. In this dissertation, computer-based molecular modeling techniques such as molecular docking, virtual screening, and molecular dynamics simulations were used to develop inhibitors and activators for enzymes involved in the methylation and demethylation of RNA m6A. This research led to the discovery of activators of the RNA m6A methyltransferase METTL3/METTL14/WTAP complex and identification of new inhibitors for two demethylases, FTO and ALKBH5. The inhibitory or activating activities of the compounds as well as their binding affinities were measured experimentally for each enzyme protein studied. An essential part of this dissertation involves the study of the effects of the compounds developed in different in vitro disease models. Thus it was found that the METTL3/METTL14/WTAP enzyme complex activators enhance the HIV replication. On the contrary, the ALKBH5 enzyme inhibitors suppress the growth of the cancer cell cultures. The most interesting observation was the detection of the neuroprotective effect of the developed new FTO enzyme inhibitors. An important practical value of the current work is that all the active compounds developed can be used for the further development of novel medical drug candidates.
Over the last decade, there has been a rapidly growing interest in various chemical modifications of RNA because of their high importance in gene expression. The field of science that deals with various modifications of RNA has been called epitranscriptomics. The most common RNA modification is the methylation at the sixth position of adenosine nucleotide, or N6-methyladenosine (m6A). RNA m6A modifications play critical role in regulating cell differentiation, immune tolerance, neuronal signaling, carcinogenesis and other, both physiological and pathological, conditions. The methylation and demethylation of RNA m6A can be regulated by inhibiting and/or activating enzymes involved in these processes. In this dissertation, computer-based molecular modeling techniques such as molecular docking, virtual screening, and molecular dynamics simulations were used to develop inhibitors and activators for enzymes involved in the methylation and demethylation of RNA m6A. This research led to the discovery of activators of the RNA m6A methyltransferase METTL3/METTL14/WTAP complex and identification of new inhibitors for two demethylases, FTO and ALKBH5. The inhibitory or activating activities of the compounds as well as their binding affinities were measured experimentally for each enzyme protein studied. An essential part of this dissertation involves the study of the effects of the compounds developed in different in vitro disease models. Thus it was found that the METTL3/METTL14/WTAP enzyme complex activators enhance the HIV replication. On the contrary, the ALKBH5 enzyme inhibitors suppress the growth of the cancer cell cultures. The most interesting observation was the detection of the neuroprotective effect of the developed new FTO enzyme inhibitors. An important practical value of the current work is that all the active compounds developed can be used for the further development of novel medical drug candidates.
Description
Väitekirja elektrooniline versioon ei sisalda publikatsioone
Keywords
RNA, modifications, adenosine, methylation, enzymes, ligands, molecular modelling