Histon deacetilaze su metaloenzimi koji hidrolizuju acetilovane bočne amino grupe lizina na
histonskim i nehistonskim proteinima. Uklanjanjem acetil grupe sa lizina generiše se pozitivno
naelektrisanje na histonima, koje gradi jonske veze sa negativno naelektrisanim DNK molekulima.
Kao posledica građenja jonskih veza, hromatin postaje gusto pakovan, teže dostupan
transkripcionim faktorima, koji posledično dovodi do represije genske transkripcije. U
epigenetičkim istraživanjima, derivati hidroksamskih kiselina se koriste u dizajniranju inhibitora
histon deacetilaza. Posledica inhibicije histon deacetilaza je indukcija apoptoze, zastoj u ćelijskom
ciklusu, smanjenje rasta tumora, inhibicija migracije i invazije malignih ćelija. U zavisnosti od
homologije u primarnoj sekvenci i ćelijske lokalizacije, histon deacetilaze su podeljene u četiri
klase. Klasi I pripadaju HDAC1, HDAC2, HDAC3 i HDAC8 izoforme. Klasa II se deli na dve
podklase – klasu IIa (HDAC4, HDAC5, HDAC7 i HDAC9), dok klasi IIb pripadaju HDAC6 i
HDAC10. Posebno se izdvaja izoforma HDAC11 u okviru klase IV humanih histon deacetilaza.
Prva faza istraživanja u ovoj doktorskoj disertaciji je kompjutersko dizajniranje selektivnih
inhibitora histon deacetilaze 6. Dizajniranje pomenutih inhibitora je omogućeno kombinovanjem
studija kvantitativnog odnosa strukture i aktivnosti (3D-QSAR), pretrage novih fragmenata, kao i
studija molekulskog dokinga na trodimenzionalnim strukturama histon deacetilaza 1 i 6. Druga faza
istraživanja bila je sinteza odabranih jedinjenja na osnovu zaključaka in silico studija racionalnog
dizajna selektivnih HDAC6 inhibitora. Treća faza istraživanja u ovoj doktorskoj disertaciji je
određivanje vrednosti inhibitornih koncentracija (IC50) novosintetisanih jedinjenja na humanim
HDAC1, HDAC3, HDAC6 i HDAC8 izoenzimima u in vitro enzimskim testovima. Ispitivanje
antikancerskih osobina sintetisanih jedinjenja na ćelijama tumora dojke (MDA-MB-231 i MCF-7)
predstavlja četvrtu fazu istraživanja u ovoj doktorskoj disertaciji.
Kao rezultat kompjuterskog dizajniranja selektivnih inhibitora histon deacetilaze 6 izdvojeni se
validni i prediktivni 3D-QSAR modeli. Tehnikom virtuelnog skrininga odabrani su odgovarajući
fragmenti za dizajniranje selektivnih HDAC6 inhibitora. Molekulskim dokingom je predviđen način
vezivanja dizajniranih jedinjenja za humane HDAC1 i HDAC6 izoforme. Sintetisane su dve klase
derivata hidroksamskih kiselina, derivati 1-benzhidrilnog piperazina i klasa 5,5-difenil-2,4-
imidazolidindiona i okarakterisani metodama strukturne analize. Na osnovu rezultata in vitro
enzimskih testiranja, identifikovano je sedam nanomolarnih inhibitora histon deacetilaze 6, od kojih
su dva inhibitora: BDR-6, IC50 = 186 nM i BDR-9, IC50 = 31 nM selektivna za HDAC6 izoformu i
jedan inhibitor je izdvojen kao dualni HDAC6/8 inhibitor. Ispitivanjima uticaja sintetisanih inhibitora
na ćelijsko preživljavanje, apoptozu, promene u ćelijskom ciklusu, migraciju i invaziju ćelija kancera
dojke (MDA-MB-231 i MCF-7) izdvojen je neselektivni HDAC inhibitor, BDR-8 (HDAC1, IC50 =
408 nM; HDAC3 IC50 = 207 nM; HDAC6, IC50 = 124 nM i HDAC8, IC50 = 245 nM) sa najboljim
antikancerskim osobinama. Najpotentniji sintetisani selektivni HDAC6 inhibitor, BDR-9 predstavlja
necitotoksično jedinjenje sa antimigratornim osobinama na MCF-7 ćelijama kancera dojke. U ovoj
doktorskoj disertaciji prikazan je sistematičan protokol za preklinički razvoj novih selektivnih
inhibitora histon deacetilaze 6, koji može da bude iskorišćen u budućim istraživanjima u oblasti
otkrića lekova koji deluju na kancerski epigenom.Histone deacetylases are metalloenzymes that hydrolyze acetylated lysine side chains on histone
and non-histone proteins. Removal of the acetyl group from lysine generates a positive charge on
histones that forms ionic bonds with negatively charged DNA molecules. As a consequence of
forming ionic bonds, chromatin becomes densely packed, the access of the transcription factors is
prevented, which in turn leads to the repression of gene transcription. In epigenetic drug
discovery, hydroxamic acid derivatives are used in the design of histone deacetylase inhibitors.
Inhibition of human histone deacetylases leads to induction of apoptosis, cell cycle arrest, tumor
growth inhibition, inhibition of migration and invasion of malignant cells. Depending on the
homology in the primary sequence and cell localization, histone deacetylases are classified into
four classes. Class I is consisted of HDAC1, HDAC2, HDAC3 and HDAC8 isoforms. Class II is
divided into two subclasses - class IIa (HDAC4, HDAC5, HDAC7 and HDAC9), and class IIb
(HDAC6 and HDAC10). The HDAC11 is the unique isoform within the class IV HDACs.
The first goal of research in this doctoral dissertation was computational drug design of selective
histone deacetylase 6 inhibitors. The second goal of the research was the synthesis of selected
compounds based on the predictions of in silico studies. The third research goal in this doctoral
dissertation is to determine the inhibitory concentrations (IC50) of newly synthesized compounds
against human HDAC1, HDAC3, HDAC6 and HDAC8 isoenzymes in in vitro enzyme assays.
Evaluation of anticancer properties of synthesized compounds on breast tumor cells (MDA-MB-
231 and MCF-7) is the fourth goal set in this doctoral dissertation.
As a result of computational drug design study, valid and predictive 3D-QSAR models were
generated. The most promising fragments for the design of selective HDAC6 inhibitors were
selected by virtual screening technique. Molecular docking study predicted the binding modes of
designed compounds toward the human HDAC1 and HDAC6 isoforms. Two classes of
hydroxamic acid derivatives, 1-benzhydryl piperazine derivatives and 5,5-diphenyl-imidazolidine-
2,4-dione derivatives were synthesized and characterized by NMR analysis and mass spectrometry.
Considering the results of in vitro enzyme assays, seven nanomolar histone deacetylase 6 inhibitors
were identified, of which two inhibitors: BDR-6, IC50 = 186 nM and BDR-9, IC50 = 31 nM are
selective for HDAC6 isoform and one inhibitor was disclosed as dual HDAC6 / 8 inhibitor.
Evaluation of the effects of synthesized inhibitors on cell survival, apoptosis, changes in cell cycle,
migration and invasion of breast cancer cells (MDA-MB-231 and MCF-7) singled out a non-
selective HDAC inhibitor, BDR-8 (HDAC1, IC50 = 408 nM; HDAC3 IC50 = 207 nM, HDAC6, IC50
= 124 nM and HDAC8, IC50 = 245 nM) with the best anticancer properties. The most potent
selective HDAC6 inhibitor, BDR-9 is a non-cytotoxic compound with anti-migratory properties on
MCF-7 breast cancer cells. In this doctoral dissertation, a systematic protocol for the preclinical
drug discovery of novel selective histone deacetylase 6 inhibitors is presented, that can be used in
future research of drug candidates that target the cancer epigenome
