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Rak spada med najsmrtonosnejše bolezni in predstavlja enega izmed največjih izzivov v medicini. Trenutno se za boj s to boleznijo poslužujemo treh metod zdravljenja, in sicer kemoterapije, radioterapije in kirurških posegov. Čeprav je ob pravočasni diagnozi določene vrste raka možno uspešno zdraviti ali omejiti, zdravljenje običajno zaradi slabše selektivnosti zdravilnih učinkovin in invazivnosti posegov spremljajo hudi neželeni učinki, ki zmanjšajo kakovost pacientovega življenja. Rak se razvije kot plod številnih dejavnikov, tako genetskih kot dejavnikov okolja. Obstajajo tudi skupne točke različnih vrst raka, ena izmed katerih je opažena povišana koncentracija stresnih proteinov ali proteinov toplotnega šoka (Hsp), ki pripomorejo k preprečevanju apoptoze tumorskih celic in povečanem obsegu njihovega razmnoževanja. Zaviranje delovanja proteinov Hsp se je izkazalo kot učinkovit način zmanjšanja proliferacije tumorskih celic, večina dosedanjih odkritih zaviralcev Hsp pa deluje po mehanizmu kompetitivne inhibicije ATP-vezavne domene, kar je neselektiven način delovanja in lahko pripelje do škodljive indukcije odziva toplotnega šoka. Predmet novejših raziskav predstavlja šaperon Hsp90, ki se v kompleksih s košaperoni pojavlja kot pomemben člen pri razvoju številnih karcinomov. Tema magistrske naloge je načrtovanje novih zaviralcev protein-protein interakcije (PPI) med Hsp90 in košaperonom Cdc37, ki bi delovali selektivno in povzročali manj neželenih učinkov. Odkrivanja novih zaviralcev Hsp90 smo se lotili z uporabo računalniških metod virtualnega rešetanja in molekulskega sidranja v vezavno mesto na Hsp90. Sprva smo z vezavo že znanega zaviralca PPI Hsp90-Cdc37, DDO-5936, validirali uporabljeno programsko opremo. Nato smo se lotili sidranja treh spojin, odkritih na Fakulteti za farmacijo, ki so izkazovale antiproliferativni učinek na rakavih celicah. Sledeči korak je bil rešetanje knjižnic spojin in sidranje dobljenih zadetkov. V končni stopnji smo rešetali še knjižnico z več kot 3,2 milijoni komercialno dostopnih spojin s programom ROCS in nato še s programom EON ter primerjali prileganja dobljenih spojin s spojino 18h, ki je prav tako zaviralec preiskovane PPI z močnejšo afiniteto do vezavnega mesta kot DDO-5936. Rezultati so pokazali, da se nobena od sidranih spojin ne veže podobno kot spojina DDO-5936, prav tako zadetki virtualnih rešetanj niso zadostno strukturno in elektrostatsko podobni spojini 18h, da bi sklepali na podobno vezavo in učinek. Ugotovitve naše naloge predstavljajo podlago za optimizacijo spojin in generiranje novih knjižnic za nadaljnja rešetanja in raziskave. Cancer is one of the deadliest diseases and one of the biggest challenges for medicine. We are currently fighting the disease with chemotherapy, radiotherapy and surgery. With these methods, it is possible to control or cure many forms of cancer if diagnosed in time. The bad side are the many side effects associated with treatment because the drugs are not selective and the surgeries are too invasive, which negatively affects the patient's life. The development of cancer is the result of many factors, both genetic and environmental. However, there are some common features of cancers. One of these is the upregulation of so-called heat shock proteins (Hsp), which help prevent tumour cell apoptosis and increase cell proliferation. Inhibition of these proteins has been shown to be effective in reducing tumour cell proliferation. Most of the currently known inhibitors exert their effects by competitively inhibiting the ATP-binding domain of Hsps, which is not a selective mechanism of action and can lead to a toxic heat shock response. Recent research has focused on Hsp90-co-chaperone complexes, which play an important role in cancer development. The content of our master thesis is to develop new Hsp90-Cdc37 PPI inhibitors that act selectively and cause fewer side effects. We performed the design of new inhibitors using computational methods such as virtual screening and molecular docking to the Hsp90 binding site. First, we docked the already known Hsp90-Cdc37 PPI inhibitor DDO-5936 to validate the software we used. After successful validation, we proceeded to dock three compounds discovered at Faculty of Pharmacy that have been shown to have an antiproliferative effect on cancer cells. The next step was a ligand-based virtual screening of the compound library and subsequent docking of hits. In the final phase of our work, we used the software ROCS to screen a compound library consisting of more than 3.2 million commercially available compounds. Ten of the best hits were used to compare the overlay with compound 18h, which is also an inhibitor of the investigated PPI but has a greater affinity for the binding site and consequently better activity than DDO-5936. Ten thousand best hits were then further virtually screened using the software EON. Ten highest ranked hits were analysed and compared with 18h. The results showed that none of the docked compounds bind to the binding site, similar to DDO-5936. Moreover, the virtual screening hits showed insufficient structural and electrostatic similarity to 18h, from which we can conclude that they would not bind properly or show activity. Our results provide the basis for further optimization of the compounds and creation of new compound libraries that can be used in new research. |
