Racionalni dizajn, sinteza i in vitro ispitivanja selektivnih inhibitora histon deacetilaze 6

Abstract

Histon deacetilaze su metaloenzimi koji hidrolizuju acetilovane bočne amino grupe lizina nahistonskim i nehistonskim proteinima. Uklanjanjem acetil grupe sa lizina generiše se pozitivnonaelektrisanje na histonima, koje gradi jonske veze sa negativno naelektrisanim DNK molekulima.Kao posledica građenja jonskih veza, hromatin postaje gusto pakovan, teže dostupantranskripcionim faktorima, koji posledično dovodi do represije genske transkripcije. Uepigenetičkim istraživanjima, derivati hidroksamskih kiselina se koriste u dizajniranju inhibitorahiston deacetilaza. Posledica inhibicije histon deacetilaza je indukcija apoptoze, zastoj u ćelijskomciklusu, smanjenje rasta tumora, inhibicija migracije i invazije malignih ćelija. U zavisnosti odhomologije u primarnoj sekvenci i ćelijske lokalizacije, histon deacetilaze su podeljene u četiriklase. Klasi I pripadaju HDAC1, HDAC2, HDAC3 i HDAC8 izoforme. Klasa II se deli na dvepodklase – klasu IIa (HDAC4, HDAC5, HDAC7 i HDAC9), dok klasi IIb pripadaju HDAC6 iHDAC10. Posebno se izdvaja izoforma HDAC11 u okviru klase IV humanih histon deacetilaza.Prva faza istraživanja u ovoj doktorskoj disertaciji je kompjutersko dizajniranje selektivnihinhibitora histon deacetilaze 6. Dizajniranje pomenutih inhibitora je omogućeno kombinovanjemstudija kvantitativnog odnosa strukture i aktivnosti (3D-QSAR), pretrage novih fragmenata, kao istudija molekulskog dokinga na trodimenzionalnim strukturama histon deacetilaza 1 i 6. Druga fazaistraživanja bila je sinteza odabranih jedinjenja na osnovu zaključaka in silico studija racionalnogdizajna selektivnih HDAC6 inhibitora. Treća faza istraživanja u ovoj doktorskoj disertaciji jeodređivanje vrednosti inhibitornih koncentracija (IC50) novosintetisanih jedinjenja na humanimHDAC1, HDAC3, HDAC6 i HDAC8 izoenzimima u in vitro enzimskim testovima. Ispitivanjeantikancerskih 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 sevalidni i prediktivni 3D-QSAR modeli. Tehnikom virtuelnog skrininga odabrani su odgovarajućifragmenti za dizajniranje selektivnih HDAC6 inhibitora. Molekulskim dokingom je predviđen načinvezivanja dizajniranih jedinjenja za humane HDAC1 i HDAC6 izoforme. Sintetisane su dve klasederivata hidroksamskih kiselina, derivati 1-benzhidrilnog piperazina i klasa 5,5-difenil-2,4-imidazolidindiona i okarakterisani metodama strukturne analize. Na osnovu rezultata in vitroenzimskih testiranja, identifikovano je sedam nanomolarnih inhibitora histon deacetilaze 6, od kojihsu dva inhibitora: BDR-6, IC50 = 186 nM i BDR-9, IC50 = 31 nM selektivna za HDAC6 izoformu ijedan inhibitor je izdvojen kao dualni HDAC6/8 inhibitor. Ispitivanjima uticaja sintetisanih inhibitorana ćelijsko preživljavanje, apoptozu, promene u ćelijskom ciklusu, migraciju i invaziju ćelija kanceradojke (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 najboljimantikancerskim osobinama. Najpotentniji sintetisani selektivni HDAC6 inhibitor, BDR-9 predstavljanecitotoksično jedinjenje sa antimigratornim osobinama na MCF-7 ćelijama kancera dojke. U ovojdoktorskoj disertaciji prikazan je sistematičan protokol za preklinički razvoj novih selektivnihinhibitora histon deacetilaze 6, koji može da bude iskorišćen u budućim istraživanjima u oblastiotkrića lekova koji deluju na kancerski epigenom.

Histone deacetylases are metalloenzymes that hydrolyze acetylated lysine side chains on histoneand non-histone proteins. Removal of the acetyl group from lysine generates a positive charge onhistones that forms ionic bonds with negatively charged DNA molecules. As a consequence offorming ionic bonds, chromatin becomes densely packed, the access of the transcription factors isprevented, which in turn leads to the repression of gene transcription. In epigenetic drugdiscovery, 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, tumorgrowth inhibition, inhibition of migration and invasion of malignant cells. Depending on thehomology in the primary sequence and cell localization, histone deacetylases are classified intofour classes. Class I is consisted of HDAC1, HDAC2, HDAC3 and HDAC8 isoforms. Class II isdivided 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 selectivehistone deacetylase 6 inhibitors. The second goal of the research was the synthesis of selectedcompounds based on the predictions of in silico studies. The third research goal in this doctoraldissertation is to determine the inhibitory concentrations (IC50) of newly synthesized compoundsagainst 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 weregenerated. The most promising fragments for the design of selective HDAC6 inhibitors wereselected by virtual screening technique. Molecular docking study predicted the binding modes ofdesigned compounds toward the human HDAC1 and HDAC6 isoforms. Two classes ofhydroxamic 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 inhibitorswere identified, of which two inhibitors: BDR-6, IC50 = 186 nM and BDR-9, IC50 = 31 nM areselective 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 potentselective HDAC6 inhibitor, BDR-9 is a non-cytotoxic compound with anti-migratory properties onMCF-7 breast cancer cells. In this doctoral dissertation, a systematic protocol for the preclinicaldrug discovery of novel selective histone deacetylase 6 inhibitors is presented, that can be used infuture research of drug candidates that target the cancer epigenome.