Ewing sarcoma (EWS) is a rare type of cancer that is usually diagnosed in children and adolescents. It occurs in the bones and soft tissue around the bones. EWS begins with a change in the DNA that leads to the gene for the fusion oncoprotein EWS::FLI1. EWS::FLI1 lacks the secondary and tertiary structure that is crucial for sufficient oncogenesis. However, we can use this as a weak point for EWS::FLI1inhibition, as this disordered structure has many binding sites for small molecules. EWS::FLI1 is a transcription factor that binds to the transcription modulator RNA helicase A (RHA). In addition, EWS::FLI1 also binds to the Hsp90 in cancer cells. Hsp90 is a heat shock protein whose main function is to protect proteins from different stress factors, e.g. UV light, hyperthermia, toxins and chemicals. In cancer cells, the expression of Hsp90 is increased so that they are more likely to survive.
In this master thesis we have synthesized the HEMTAC molecules. The structure contains an analog of the EWS::FLI1 inhibitor and an Hsp90 inhibitor, which are connected to each other via a linker. The EWS::FLI1 inhibitor inhibits the interaction between EWS::FLI1 and RHA. As Hsp90 inhibitors, we used the already known TZZ11 and PU-H71. TZZ11 is a C-terminal inhibitor and PU-H71 is an N-terminal inhibitor of Hsp90. PU-H71 has a high affinity for Hsp90 in cancer cells and a low affinity for Hsp90 in normal cells, which requires low-dose treatment to achieve selectivity for tumor cells. We used aliphatic chains and rings as linkers. By conjugating the above, we aimed to induce the degradation of EWS::FLI1 in Ewing sarcoma cells. We followed a similar approach for the synthesis of 24 and 27. We bound norbornane, a lipophilic molecule, to the EWS::FLI1 inhibitor. We wanted to investigate the influence of the lipophilic substituent on the inhibition of EWS::FLI1 and the induction of its degradation using hydrophobic tagging.
Compound 19, based on PU-H71 and piperidine linker, showed the most promising results among the synthesized compounds (IC50 0.92 ± 0.098 μM). For the synthesis of compound 22, we used an aliphatic chain linker, but it showed weaker inhibitory activity (IC50 13.0 ± 1.3 μM). Compounds 17 and 23, which are based on the inhibitor TZZ11, were not active. In addition, we tested compounds 11 and 18, which contain only the modified inhibitor of EWS::FLI1. The results showed that EWS::FLI1 inhibitor alone also contributes to antitumor activity. The results of this master thesis provide important insights for the further development of new HEMTAC molecules with stronger inhibitory activity.
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