Simulacije milekularne dinamike i računarsko dizajniranje inhibitora protein lizin metiltransferaze EZH2
Molecular dynamics simulations and computational design of inhibitors of protein lysine methyltransferase EZH2
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Enhancer of Zeste Homolog 2 (EZH2) je epigenetski enzim koji vrši selektivno
metilovanje lizina 27 na histonu H3 (H3K27). Trimetilovani H3K27 je
represivan epigenetski signal, cime EZH2 utice na smanjenje transkripcije ciljnih
gena. Prekomerna aktivnost EZH2, izazvana povecanom ekspresijom ili mutacijama
njegovog katalitickog domena, dovedena je u vezu sa vecim brojem maligniteta kod
ljudi, a inhibicija ovog enzima smatra se perspektivnom strategijom u razvoju novih
antitumorskih lekova. Prvi selektivni inhibitori EZH2 otkriveni su 2012. godine i
njihova biološka karakterizacija potvrdila je terapijski potencijal inhibicije ovog epigenetskog
regulatora. Iako postoji potreba za otkricem novih inhibitora, znacajnu
prepreku u njihovom dizajnu do sada je predstavljao nedostatak trodimenzionalne
strukture kompleksa EZH2 i okarakterisanih liganada. Cilj istraživanja u okviru ove
doktorske disertacije bio je da se primenom racunarskih metoda uspostavi i validira
model vezivnog mesta EZH2, i d...a se, zatim, uspostavljeni model upotrebi u dizajnu
novih inhibitora kao potencijalnih antitumorskih lekova.
U prvoj fazi istraživanja izvršeno je homologo modeliranje katalitickog domena
EZH2 koji sadrži vezivno mesto za kofaktor, a za koje se kompetitivno vezuju
svi poznati inhibitori. Pocetni model usavršen je primenom simulacija konvencionalne
i ubrzane molekularne dinamike (MD), a zatim je daljim simulacijama istraživano
kako 13 inhibitora piridonske strukture stupa u interakciju sa vezivnim mestom
na enzimu. Korišcenjem prikupljenih trajektorija izvršena je procena slobodne
energije vezivanja inhibitora zasnovana na molekularnoj mehanici uz generalizovani
Bornov model rastvaraca (MM/GBSA). Izracunate energije upore ene su sa dostupnim
eksperimentalnim podacima i utvr eno je dobro slaganje izme u vrednosti koje
ukazuje na validnost uspostavljenog modela. Daljom analizom trajektorija i primenom
racunarske mutageneze u alanin identifikovane su intermolekulske interakcije
koje kljucno doprinose aktivnosti i selektivnosti proucavanih inhibitora. Na ovim
osnovama, definisan je farmakoforni model inhibitora EZH2 a njegovom validacijom
potvr ena je mogucnost uspešne identifikacije i inhibitora baziranih na drugim
osnovnim strukturama.
Efikasnost mnogih antitumorskih lekova ogranicava razvoj rezistencije tui
morskih celija, pri cemu je opisan veci broj mehanizama koji dovode do pojave rezistencije...
methylation of lysine 27 on histone H3. Trimethylated H3K27 constitutes
a repressive epigenetic mark, making EZH2 capable of transcriptional silencing of
target genes. Aberrant EZH2 activity, caused by either overexpression or point
mutations in the catalytic domain, has been related to a number of human malignancies,
making EZH2 inhibition a promising strategy in the development of novel
anticancer treatments. First disclosure of selective EZH2 inhibitors in 2012 and their
biological characterization has confirmed the therapeutic potential of inhibiting this
epigenetic regulator. While there is an outstanding need for the discovery of novel
EZH2 inhibitors, the lack of a three-dimensional structure of EZH2 complexed with
one of its ligands has been a significant obstacle to this end. The aim of research
conducted as part of this doctoral dissertation was to establish and validate a model
of EZH2 binding site using computational methods, and to subsequently utilize the
established mode...l as a foundation in the design of novel potential EZH2 inhibitors.
In the first stage of our research, homology modeling of the EZH2 catalytic
domain was conducted, which contains the cofactor binding site targeted
by all known inhibitors. The initial model was refined through conventional and
accelerated molecular dynamics (MD) simulations. Next, structures of EZH2 complexed
with 13 different pyridone inhibitors were simulated in order to study the key
interactions participating in ligand binding. An assessment of inhibitors’ binding
free energy was performed using the molecular mechanics/generalized Born surface
area (MM/GBSA) method. The computed energies were correlated to available
experimental data and good agreement was found supporting the validity of the
established model. Further analysis of MD trajectories and computational alanine
scanning facilitated the definition of key intermolecular interactions that contribute
to the observed potency and selectivity of the studied inhibitors. On this basis, a
pharmacophore model of EZH2 inhibitors was proposed and validated. It demonstrated
good capabilities in identifying truly active molecules, including those not
based on a pyridone scaffold, in presence of decoys.
The efficacy of anticancer drugs can become limited with the onset of resiiv
stance that can develop in cancer cells through various mechanisms...