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Rakava obolenja so drugi najpogostejši vzrok smrti v Sloveniji in ostalem razvitem svetu in s tem eden največjih javnozdravstvenih problemov. Tarčna terapija je pristop molekularne medicine za zdravljenje tovrstnih bolezni, ki interferira s specifičnimi celičnimi strukturami, udeleženimi v karcinogenezi. Učinkuje po različnih mehanizmih, med katerimi je tudi ustavitev proliferacije rakavih celic. Slednjo lahko dosežemo z blokado celičnih struktur, ki so udeležene pri delitvi rakavih celic. Mednje spada humani napetostno odvisni kalijev kanalček iz družine 'ether-à-go-go' (hEAG1), ki je odgovoren za regulacijo celičnega cikla. hEAG1 posredno omogoči tvorbo delitvenega vretena v delečih se celicah. Prav tako je v celicah udeležen pri vzdrževanju mirovnega membranskega potenciala. Ekspresija kanalčka je povečana v tumorskih celicah več kot 70% vseh vrst tumorjev. hEAG1 igra pomembno vlogo pri preživetju, rasti in delitvi tumorskih celic. Z njegovo inhibicijo je možno doseči smrt tumorskih celic, zato je pomembna tarča raziskav. Strukturno gledano je kanalček hEAG1 transmembranski tetramerni protein, ki ga fiziološko najdemo predvsem v nevronih. Po zgradbi je zelo soroden kanalčku hERG, ki je odgovoren za repolarizacijo srčnih celic. Selektivna inhibicija kanalčka hEAG1 še vedno predstavlja veliko težavo, saj se večina ligandov veže hkrati tudi na kanalček hERG, s čimer se poveča tveganje za nastanek aritmije. V okviru magistrske naloge smo z metodami računalniške kemije skušali poiskati nove selektivne zaviralce kanalčka hEAG1. Z vizualno preiskavo struktur kanalčkov hEAG1 in hERG ter analizo njunih aminokislinskih zaporedij smo primerjali oba kanalčka. V regijah kanalčkov, v katerih smo ugotovili strukturne razlike, smo ustvarili vezavne žepe in v dva najbolj specifična žepa, ki sta bila lastna kanalčku hEAG1, izvedli virtualno rešetanje s sidranjem. Oba vezavna žepa sta bila del senzorja napetosti v kanalčku. Za najboljših sto spojin zadetkov virtualnega rešetanja v vsakem žepu smo generirali farmakoforne modele. V prvem žepu so bile interakcije izključno hidrofobne, v drugem pa so modeli vsebovali še eno ionsko interakcijo, donor in akceptor vodikove vezi. Proučili smo še specifičnost aminokislin v okolici vezavnih žepov s primerjavo s kanalčkom hERG. Ugotovili smo, da so v prvem žepu specifične tri, v drugem pa dve aminokislini v kanalčku hEAG1. Na osnovi rezultatov sidranja smo zaključili, da spojin zadetkov ni smiselno testirati in vitro zaradi majhne verjetnosti, da bi se v izbrane vezavne žepe vezale z veliko afiniteto in močnimi interakcijami ter imele ustrezno selektivnost. Cancers are the second leading cause of death in Slovenia and other developed countries worldwide, making them one of the biggest public health concerns. Targeted therapy is a form of molecular medicine that interferes with specific cell structures involved in carcinogenesis. It functions through various mechanisms, including inhibition of tumour cell proliferation. The latter is achieved by inhibiting cell structures that are required for cell division. One of these is the human voltage-gated potassium channel of the 'ether-à-go-go' family (hEAG1), which is responsible for regulating the cell cycle. Indirectly, it forms the mitotic spindle. In addition, hEAG1 is involved in the regulation of resting membrane potential in cells. However, the channel is overexpressed in more than 70% of all cancers. The channel is responsible for cell survival, growth, and division, and thus its inhibition leads to cancer cell death. Structurally, the hEAG1 channel is a tetrameric transmembrane protein that is physiologically most abundant in neurons. Its structure is very similar to that of the hERG channel, which repolarizes cardiac cells. Selective inhibition of the hEAG1 channel remains a challenge because most of its ligands bind simultaneously to the hERG channel and additionally increase the risk of cardiac arrhythmias. In this master’s thesis, we used various computational methods to discover new selective hEAG1 channel inhibitors. By visually examining the structures of hEAG1 and hERG channels and analysing their amino acid sequences, we compared the two channels. The regions where we found structural differences were used to form binding pockets in the channels. In the hEAG1, two particularly specific pockets were used to perform virtual screening using molecular docking. Both binding pockets were part of the voltage sensor domain. We then generated pharmacophore models for the top hundred virtual hits. Pocket 1 solely exhibited hydrophobic interactions with the ligands, while pocket 2 offered the possibility of also forming an ionic interaction and hydrogen bonds through a donor and an acceptor. We then examined the specificity of amino acids surrounding the binding pocket by comparing them to the hERG channel. We concluded that there are three specific amino acids in pocket 1 and two specific amino acids in pocket 2 in the hEAG1 channel. Considering the result of molecular docking, we decided that the hit compounds are not worth testing in vitro because it is not likely possible to successfully bind into the pockets with high affinity or strong interactions and achieve a sufficient level of selectivity.

Kalijevi kanali se vpletajo v raznolike celične procese s primarno vlogo pri vzdrževanju ionske homeostaze. Ključno vlogo imajo tudi pri patofiziologiji napredovanja rakavih obolenj. Napetostno odvisni kalijev kanal hEAG1 je primer kanala, ki se vpleta v različne procese razvoja raka, saj se njegovo povečano izražanje pojavlja pri večini vrst raka pri človeku. Fiziološko se hEAG1 pojavlja skoraj izključno v centralnem živčnem sistemu in tako predstavlja skoraj popolno novo tarčo za razvoj novih protirakavih učinkovin. Težavo pri razvoju zaviralcev hEAG1 predstavlja kanal hERG, ki spada v isto družino ionskih kanalov kot hEAG1. Posledica zaviranja hERG je lahko podaljšanje srčnega intervala QT. Možnost pojava potencialno smrtno nevarnih aritmij zaradi zaviranja kanala hERG predstavlja velik izziv pri načrtovanju novih zaviralcev hEAG1. V sklopu doktorske disertacije smo začeli z iskanjem novega strukturnega tipa zaviralcev hEAG1. S pristopom na podlagi strukture znanih ligandov smo pripravili farmakoforni model, ki opisuje vezavo analogov naravne spojine purpurealidina I. Ti se vežejo na zunanjo stran napetostnega senzorja hEAG1, ki je odgovoren za prenos spremembe membranske napetosti v spremembo konformacije kanala. Poenostavljen farmakoforni model smo uporabili za virtualno rešetanje, pri katerem smo dobili 18 spojin zadetkov. Izbrane virtualne zadetke smo testirali z metodo vpete krpice membrane ter odkrili spojino z nizkim mikromolarnim zaviralnim delovanjem na hEAG1. Spojina zadetek virtualnega rešetanja, ki je izkazala dobro zaviralno delovanje hEAG1 in vitro, je služila kot osnova za sintezo novih analogov, s katerimi smo želeli izboljšati zaviralno delovanje na hEAG1 in selektivnost napram kanalu hERG. Selektivnost je bila dosežena na številne natrijeve in kalijeve ionske kanale z izjemo kanala hERG. Kljub temu, da nismo uspeli izboljšati selektivnosti na kanal hERG, pa nam je uspelo značilno povečati zaviranje hEAG1. Tako smo ugotovili, da je za zaviralno delovanje najpomembnejša pozitivno nabita aminska skupina, poleg tega pa je potrebno ohraniti tudi nitro in trifluorometilno skupino na aromatskem obroču. Z odstranitvijo hidroksilne skupine smo spojino zadetek pretvorili v nanomolarni zaviralec hEAG1 in tako petkrat povečali zaviralno delovanje v primerjavi z aktivno spojino iz virtualnega rešetanja. Glede na kinetiko vezave spojine zadetka, ki je značilno za spojine, ki se vežejo v osrednjo votlino kanala, smo natančneje preverili potencialno mesto, kamor se novoodkrite spojine vežejo. Pri tem smo uporabili pristope in silico in in vitro, s katerimi smo prišli do skupnega zaključka, da se nove spojine vežejo v še neovrednoteno mesto kanala hEAG1. Metoda in vitro je temeljila na izpodrivanju spojin, ki se vežejo na isto vezavno mesto. Preverili smo, ali spojina zadetek tekmuje s spojinama, katerih vezavno mesto je v osrednji votlini kanala oziroma na zunanji strani napetostnega senzorja (VSD). Nismo zaznali kompeticije niti z astemizolom, ki se veže v poro kanala, niti z mibefradilom, ki se veže v VSD. Na podlagi tega smo se odločili, da z metodo in silico še dodatno preverimo najpogosteje in najbolje raziskano mesto vezave v osrednji votlini. Za ta pristop smo pripravili homologni model hEAG1 v odprti konformaciji in vanj sidrali znane zaviralce hEAG1. Najboljše poze sidranja smo uporabili za simulacije molekulske dinamike. Na podlagi simulacij molekulske dinamike smo pripravili skupen farmakoforni model, ki opisuje vezavo zaviralcev v osrednjo votlino kanala. Ta model ni prepoznal novih zaviralcev hEAG1, kar je v skladu z rezultati in vitro potrdilo, da se kljub značilnemu pozitivno nabitemu centru v zaviralcih le-ti ne vežejo v osrednjo votlino. Velika podobnost tega skupnega farmakofornega modela s farmakofornim modelom za hERG pojasnjuje neselektivnost zaviralcev hEAG1, ki se vežejo v osrednjo votlino. Skupni farmakoforni model predstavlja nove možnosti za odkritje novih strukturnih razredov zaviralcev, ki se ne bodo vezali v osrednjo votlino in bodo izkazovali večjo selektivno delovanje na hEAG1 napram hERG. Antiproliferativno delovanje novih zaviralcev na različnih rakavih celičnih linijah smo preverili z 2D in 3D celičnimi modeli. Celično linijo MCF-7, z visokim izražanjem hEAG1, zavirajo novi zaviralci v mikromolarnem območju. Podobno jakost apoptotičnega delovanja smo opazili tudi na sferoidih celične linije Colo357. Zaradi prisotnosti skupine nitro, ki je nismo uspeli zamenjati z bioizosternimi zamenjavami, smo vrednotili mutagenost spojine zadetka z AMES-ovim testom. Ta ni izkazovala mutagenega delovanja pri necitotoksičnih koncentracijah, a je vseeno potrebna nadaljnja previdnost pri razvoju zaviralcev hEAG1 tega strukturnega razreda. Odkritje novega strukturnega tipa zaviralcev hEAG1 v sklopu doktorske disertacije predstavlja izvirni prispevek k znanosti na področju odkrivanja novih protirakavih učinkovin s tem novim mehanizmom delovanja. V delu smo uporabili inovativne pristope farmakofornega modeliranja v kombinaciji z molekulsko dinamiko za pripravo 3D farmakofornih modelov za odkrivanje in vrednotenje zaviralcev hEAG1. Rezultati doktorske disertacije predstavljajo novo izhodišče za nadaljnji razvoj zaviralcev hEAG1 s protirakavim delovanjem. Potassium channels are involved in a variety of cellular processes and play a primary role in maintaining ion homeostasis. They also play a critical role in the pathophysiology of cancer progression. The voltage-gated potassium channel hEAG1 is one such example involved in development of various cancers, as its increased expression occurs in most human cancers. Physiologically, it is found almost exclusively in the central nervous system and would therefore be an almost perfect target for cancer therapy. The problem is the hERG channel, which belongs to the same family of ion channels as hEAG1 and as an off-target can cause prolongation of the cardiac QT interval. The potential for potentially life-threatening arrhythmias presents a major challenge in the development of hEAG1 inhibitors. The primary goal of doctoral dissertation was to discover a new structural type of hEAG1 inhibitors. Using a computer-aided drug design approach, we created a pharmacophore model describing the binding of analogs of the natural compound purpurealidin I. These bind to the outside of the hEAG1 voltage sensor, which is responsible for transferring the change in membrane potential to the structural conformational change of the channel. A simplified pharmacophore model was used for virtual screening where 18 hit compounds were identified. Selected virtual hits were tested using the patch-clamp method and a compound with low micromolar inhibitory activity on hEAG1 was discovered. Hit compound from virtual screening with in vitro activity served as the basis for the synthesis of new analogs with which we aimed to improve inhibitory activity and selectivity. Selectivity was achieved for several sodium and potassium ion channels with the exception of the hERG channel. Although we were unable to improve selectivity at hERG, we were able to significantly increase activity at hEAG1. Thus, we found that the positively charged amino group is most important for the inhibitory effect, and in addition, it is necessary to retain the nitro and trifluoromethyl groups on the phenyl ring. Removal of the hydroxyl group transformed the hit compound into a nanomolar inhibitor, which increased the inhibitory activity fivefold compared to the virtual screening hit.Given the binding kinetics of the hit compound, which is characteristic of compounds that bind to the central cavity of the channel, we further investigated the potential binding site of the newly discovered compounds. We used both the in silico and in vitro approaches, with which we reached the common conclusion that the new compounds bind to the previously unevaluated site of the hEAG1 channel. The in vitro method was used to evaluate the potential displacement of compounds that bind to the same binding site. Thus, we checked whether the hit compound competes with compounds whose binding site is located in the central cavity or on the outside of the voltage sensor (VSD). We could not find any competition between astemizole that binds in central cavity or mibefradil that binds in VSD with the hit compound. Therefore, we decided to further verify the most common and best studied binding site in the central cavity using the in silico method. For this approach, we prepared a homology model of hEAG1 in the open conformation and docking of the known hEAG1 inhibitors into the binding site in the central cavity. The best-scored docking poses per ligand were used in molecular dynamics simulations. Based on the molecular dynamics simulations, we created a merged pharmacophore model describing the binding of the inhibitors to the central cavity. This model did not recognize our new hEAG1 inhibitors, confirming that they do not bind to the central cavity, as also observed in the in vitro asays, despite the characteristic positively charged center in the structure of the inhibitors. High similarity of the merged pharmacophore model with hERG pharmacophore model explains the non-selectivity of the hEAG1 inhibitors binding to the central cavity. The use of merged pharmacophore model will increase the chance of discovering new inhibitors that do not bind to the central cavity and thus have a higher probability of selective hEAG1 inhibition. The antiproliferative activity of the new hEAG1 inhibitors on various cancer cell lines was tested using 2D and 3D cell models. The MCF-7 cell line with high hEAG1 expression was inhibited by the addition of micromolar concentrations of the new inhibitors and similar apoptotic activity was also observed on the spheroids of the Colo357 cell line. Due to the presence of the nitro group, which we could not replace with bioisosteric substitutions, the mutagenicity of the hit compound was also evaluated using AMES assay. It showed no mutagenic effects at non-cytotoxic concentrations, but further caution is advised in development of hEAG1 inhibitors of this structural class. The discovery of a new structural type of hEAG1 inhibitors in this doctoral dissertation represents an original contribution to the science in the field of anticancer drug discovery. In this work, we used an innovative ligand-based pharmacophore modeling approach to create 3D pharmacophore models in combination with molecular dynamics to discover and evaluate hEAG1 inhibitors. The obtained results represent a good foundation for further development of hEAG1 inhibitors with antiproliferative activity.