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Proteini toplotnega šoka 90 (Hsp90) so šaperoni, katerih glavna naloga je pomagati proteinom zavzeti ustrezno nativno konformacijo, s čimer postanejo funkcionalni. Sodelujejo tudi v drugih celičnih procesih, kot so nadzor celičnega cikla, signalizacija, sposobnost preživetja celic, apoptoza in razgradnja proteinov. Pri fizioloških pogojih so v celicah prisotni v nizkih koncentracijah, pod stresnimi pogoji (kot so povišana temperatura, UV sevanje, izpostavljenost mrazu itd.) pa se njihova ekspresija poveča kot odziv na celični stres. Posledično so močno izraženi tudi v rakavih celicah, v katerih stabilizirajo konformacijsko nestabilne in napačno zvite proteine ter jim tako omogočajo preživetje, zato so zaviralci Hsp90 potencialne protirakave učinkovine. V sklopu magistrske naloge smo proučevali vezavo izbranih zaviralcev Hsp90 v alosterično vezavno mesto izoform Hsp90α in Hsp90β s pomočjo molekulskega sidranja, s katerim smo lahko napovedali orientacijo, položaj in konformacijo liganda vezanega v vezavnem mestu proteina ter ocenili njegovo aktivnost in selektivnost med izoformama. Kot izhodišče smo najprej pripravili knjižnico zaviralcev Hsp90 že sintetiziranih in ovrednotenih na Univerzi v Ljubljani, Fakulteti za farmacijo. Spojinam smo nato izračunali fizikalno-kemijske lastnosti. Na podlagi slednjih ter že znanih vrednosti antiproliferativnega delovanja zaviralcev v rakavih celičnih linijah (MCF-7, Sk-Br3 in SK-N-MC) smo izbrali spojine, ki bi se lahko pokazale kot optimalne za nadaljnje delo. Sledila je validacija programov za sidranje, s katero smo preverili, katerega od računalniških programov (Glide, Autodock 4.2 in AutodockVina) je najbolje uporabiti za sidranje spojin. Tako smo izbrane spojine TZZ-103, TZZ-122, TZZ-151, TMT-11, TJD-54, TMM-7b in TMM-17 sidrali z računalniškim programom Glide, za katerega smo ugotovili, da je najustreznejši in na osnovi dobljenih rezultatov ocenili vezavno konformacijo in selektivnost zaviralcev za posamezno izoformo. Kot selektivne so se izkazale spojine TZZ-151, TMT-11 ter TMM-7b, vendar bi to bilo potrebno potrditi tudi eksperimentalno. Rezultati magistrske naloge so pomemben korak k razumevanju vezave alosteričnih zaviralcev Hsp90 in njihove selektivnosti med izoformama Hsp90α in Hsp90β. Heat shock proteins 90 (Hsp90) are chaperones whose main function is to help proteins adopt the correct native conformation and thereby become functional. They are also involved in other cellular processes such as cell cycle control, signal transduction, cell viability, apoptosis and protein degradation. Under physiological conditions, they are present in cells at low levels, but under stress conditions (such as increased temperature, UV radiation, exposure to cold, etc.) their expression increases in response to cellular stress. As a result, they are also highly expressed in cancer cells, in which they stabilize conformationally unstable and misfolded proteins, helping them to survive, which is why Hsp90 inhibitors are potential anticancer drugs. In this Master's thesis, we investigated the binding of selected Hsp90 inhibitors to the allosteric binding site of Hsp90α and Hsp90β isoforms using molecular docking, which was used to predict the orientation, position, and conformation of the ligand bound in the protein binding site and to estimate its activity and selectivity between isoforms. As a starting point, we first prepared a library of Hsp90 inhibitors already synthesized and evaluated at the University of Ljubljana, Faculty of Pharmacy. Subsequently, the physicochemical properties of the compounds were calculated. Based on the latter and the already known antiproliferative activity of the inhibitors in cancer cell lines (MCF -7, Sk-Br3 and SK -N- MC), we selected compounds that could prove optimal for further work. This was followed by validation of docking programs, which we used to test which of the computer programs (Glide, Autodock 4.2, and AutodockVina) was best suited for docking compounds. Thus, the selected compounds TZZ-103, TZZ-122, TZZ-151, TMT-11, TJD-54, TMM-7b, and TMM-17 were docked using the computer program Glide, which we considered the most suitable, and based on the results obtained, the binding conformation and selectivity for each isoform was evaluated. Compounds TZZ-151, TMT-11 and TMM-7b were found to be selective, but this should be confirmed experimentally. The results provide important insight into the binding modes of allosteric Hsp90 inhibitors and their selectivity between the Hsp90α and Hsp90β isoforms. |
