Molecular design of inhibitors for RNA methylation regulating enzymes

The RNA m6A methylation plays crucial role in various physiological processes and therefore the development of chemical agents controlling this process has large biological and medical importance. In the present work, the results of the computational design of the inhibitory ligands for the enzymes regulating the RNA adenosine N6-methylation are presented. The structure of the enzyme-ligand complexes was established using the molecular docking and molecular dynamics approaches. The activity of the best predicted RNA m6A methyltransferase inhibitors was confirmed using enzyme inhibition and cell proliferation assays. These compounds are the first known RNA m6A methylation inhibitors and therefore of substantial interest for further biomedical studies

Small-Molecule Inhibitors of the RNA M6A Demethylases FTO Potently Support the Survival of Dopamine Neurons

The fat mass and obesity-associated protein (FTO), an RNA N6-methyladenosine (m6A) demethylase, is an important regulator of central nervous system development, neuronal signaling and disease. We present here the target-tailored development and biological characterization of small-molecule inhibitors of FTO. The active compounds were identified using high-throughput molecular docking and molecular dynamics screening of the ZINC compound library. In FTO binding and activity-inhibition assays the two best inhibitors demonstrated Kd = 185 nM; IC50 = 1.46 µM (compound 2) and Kd = 337 nM; IC50 = 28.9 µM (compound 3). Importantly, the treatment of mouse midbrain dopaminergic neurons with the compounds promoted cellular survival and rescued them from growth factor deprivation induced apoptosis already at nanomolar concentrations. Moreover, both the best inhibitors demonstrated good blood-brain-barrier penetration in the model system, 31.7% and 30.8%, respectively. The FTO inhibitors demonstrated increased potency as compared to our recently developed ALKBH5 m6A demethylase inhibitors in protecting dopamine neurons. Inhibition of m6A RNA demethylation by small-molecule drugs, as presented here, has therapeutic potential and provides tools for the identification of disease-modifying m6A RNAs in neurogenesis and neuroregeneration. Further refinement of the lead compounds identified in this study can also lead to unprecedented breakthroughs in the treatment of neurodegenerative diseases

Epitranskriptoomiliste protsesside madalmolekulaarsete regulaatorite arendus

Väitekirja elektrooniline versioon ei sisalda publikatsiooneViimase 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.  https://www.ester.ee/record=b545080

HIV Replication Is Increased by RNA Methylation METTL3/METTL14/WTAP Complex Activators

The N6-methyladenosine (m(6)A) modifications in both viral and host cell RNAs play an important role in HIV-1 virus genome transcription and virus replication. We demonstrate here that activators of the METTL3/METTL14/WTAP RNA methyltransferase complex enhance the production of virus particles in cells harboring HIV-1 provirus. In parallel, the amount of m(6)A residues in the host cell mRNA was increased in the presence of these activator compounds. Importantly, the m(6)A methylation of the HIV-1 RNA was also enhanced significantly (about 18%). The increase of virus replication by the small-molecule activators of the METTL3/METTL14/WTAP complex excludes them as potential anti-HIV-1 drug candidates. However, the compounds may be of large interest as activators for the latent HIV-1 provirus copies deposited in host cells' genome and the subsequent virus eradication by an antiviral compound.Peer reviewe

The structure and function of YTHDF epitranscriptomic m6A readers.

Specific RNA sequences modified by a methylated adenosine, N6-methyladenosine (m6A), contribute to the post-transcriptional regulation of gene expression. The quantity of m6A in RNA is orchestrated by enzymes that write and erase it, while its effects are mediated by proteins that bind to read this modification. Dysfunction of this post-transcriptional regulatory process has been linked to human disease. Although the initial focus has been on pharmacological targeting of the writer and eraser enzymes, interest in the reader proteins has been challenged by a lack of clear understanding of their functional roles and molecular mechanisms of action. Readers of m6A-modified RNA (m6A-RNA) – the YTH (YT521-B homology) domain-containing protein family paralogs 1–3 (YTHDF1–3, referred to here as DF1–DF3) – are emerging as therapeutic targets as their links to pathological processes such as cancer and inflammation and their roles in regulating m6A-RNA fate become clear. We provide an updated understanding of the modes of action of DF1–DF3 and review their structures to unlock insights into drug design approaches for DF paralog-selective inhibition.Peer reviewe