Synthesis of new Hsp90β-selective inhibitors based on the N-phenylpyrrolamide scaffold

Proteini toplotnega šoka, katerih največji delež v celici predstavlja protein toplotnega šoka 90 (Hsp90), so del sistema za nadzor kakovosti celic, njihova naloga pa je zagotavljanje proteostaze. V prisotnosti celičnih stresorjev, povišane temperature, spremembe redoks potenciala in ob raznih bolezenskih stanjih se njihovo izražanje poveča. Poznane so štiri izoforme Hsp90, in sicer citoplazemski Hsp90β, stalno izražen v celici, in Hsp90α, ki se izrazi v citosolu kot odziv na celični stres. Za organele specifični izoformi se nahajata v endoplazemskem retikulumu (Grp94) in v mitohondrijih (TRAP-1). Stabilnost in funkcija mnogih onkoproteinov je odvisna prav od Hsp90, zato znanstveniki že več kot dve desetletji razvijajo zaviralce Hsp90 kot potencialne protitumorne učinkovine. Sprva so bili razviti neselektivni zaviralci N-končne domene Hsp90, vendar se je pri vseh kot neželeni učinek pojavil odziv toplotnega šoka (angl. heat shock response ali HSR), zato se je razvoj usmeril v selektivne zaviralce N-končne domene Hsp90β (Hsp90βN), ki HSR ne inducirajo. V sklopu magistrske naloge smo sintetizirali nove potencialne selektivne zaviralce Hsp90β z N-fenilpirolamidnim skeletom, ki se vežejo v N-končno domeno. Želeli smo pridobiti nove informacije o odnosu med strukturo in delovanjem spojin ter izboljšati njihovo afiniteto in selektivnost za Hsp90βN. Sintetizirali smo štiri različne analoge izhodiščne spojine IZS-274. Pri treh spojinah smo na fenilni obroč preko vmesnika -O-CH2- uvedli N-metilpiperidinski, piridinski ali 1-metoksfenilni fragment. Pri eni spojini pa smo na fenilni obroč neposredno preko kisikovega vmesnika uvedli 1-metoksifenilni fragment. Proučevali smo, kako uvedba distančnika -CH2- ter substituenti na fenilnem obroču vplivajo na vezavno afiniteto in selektivnost med izoformama Hsp90α in Hsp90β. Končnim spojinam smo določili afiniteto do N-končne domene človeških rekombinantnih proteinov Hsp90α in Hsp90β ter na podlagi rezultatov sklepali na njihovo selektivnost. Spojine 8, 14 in 26 niso izkazale afinitete do Hsp90α in Hsp90β pri najvišji testirani koncentraciji. Spojina 21 pa je v primerjavi z izhodiščno spojino sicer izkazala nižjo afiniteto do obeh izoform, vendar višjo selektivnost do izoforme Hsp90β. Na podlagi teh rezultatov lahko sklepamo, da je za zaviranje Hsp90 ključen bazični substituent, ki je analogen tistemu pri spojini IZS-274. Spojina 21 je bila poleg vrednotenja na rekombinantnem proteinu ovrednotena tudi na rakavi celični liniji Ewingovega sarkoma SK-N-MC, na kateri je delovala zaviralno z vrednostjo IC50 1,9 µM. Nova spoznanja nam bodo v pomoč pri nadaljnji optimizaciji in razvoju selektivnih zaviralcev Hsp90βN.Heat shock proteins (Hsp), the most abundant of which in a cell is heat shock protein 90 (Hsp90), serve as part of the cell\u27s quality control system and are responsible for proteostasis. Under certain stress conditions, such as elevated temperature, altered redox potential and various diseases, their expression levels increase in the cell. Four isoforms of Hsp90 have been identified. The Hsp90β isoform is constitutively expressed in the cytoplasm, whereas Hsp90α expression in the cytosol can be induced upon exposure to cellular stress. The organelle-specific isoforms Grp94 and TRAP-1 are found in the endoplasmic reticulum and mitochondria, respectively. Stability and function of many oncoproteins depend on Hsp90. Therefore, scientists have been working for more than two decades on the development of Hsp90 inhibitors as potential antitumor drugs. The first inhibitors developed were nonselectively targeting the N-terminal domain of all four isoforms. However, these inhibitors elicited a pro-survival heat shock response (HSR). Therefore, further research focused on the development of selective Hsp90β N-terminal domain inhibitors that do not induce the HSR. In this master\u27s thesis, we synthesized new selective Hsp90β isoform inhibitors based on the N-phenylpyrrolamide scaffold. We aimed to gain new information about the structure-activity relationship, improve affinity, and selectivity to Hsp90βN. We synthesized four analogs of the reference compound IZS-274. In three of these compounds, we attached a -O-CH2- spacer on the phenyl ring and then attached N-methylpiperidine, pyridine or 1-methoxyphenyl substituent. In one of the final compounds, we attached a -O- spacer on the phenyl group and then 1-methoxyphenyl substituent. We investigated how the introduction of -CH2- spacer and different substituents affect the binding affinity and isoform selectivity. We measured the affinity and selectivity for the N-terminal domain of the human recombinant Hsp90α and Hsp90β proteins. Results showed that compounds 8, 14 and 26 did not display affinity in the measured concentration range for any of these isoforms. Compound 21 showed lower affinity for both isoforms, but higher selectivity for the Hsp90β isoform compared with IZS-274, suggesting that a basic substituent analogous to that of IZS-274 is key to the inhibitory action of compounds. Compound 21 was also evaluated for its antiproliferative activity in the SK-N-MC Ewing sarcoma cancer cell line where it showed inhibitory activity with an IC50 value of 1.9 µM. The presented results will help in further optimization and development of Hsp90βN isoform-selective inhibitors