The stress protein Hsp90 plays a pivotal role in maintaining cellular homeostasis and facilitating the folding of many – including oncogenic – proteins, identifying it as an important factor in cancer progression and therefore a promising therapeutic target. The numerous adverse effects of N-terminal ATP-binding domain (NTD) inhibitors, resulting from nonselective inhibition of all four Hsp90 isoforms, have led to the development of inhibitors with alternative mechanisms of action. Thus, selective inhibitors of individual Hsp90 isoforms, inhibitors of the allosteric binding site on the C-terminal domain (CTD) and inhibitors of protein-protein interactions between Hsp90 and its co-chaperone Cdc37 were investigated.
In the experimental phase, libraries of selective inhibitors of individual Hsp90 isoforms were prepared based on literature data. Utilizing the structures of selective Hsp90β inhibitors, pharmacophore models for high-throughput virtual screening were developed and obtained virtual hits were assessed based on pharmacophore matching.
Further on, eleven CTD inhibitors with the 4,5,6,7-tetrahydrobenzothiazole scaffold were designed and synthesized, exploring the impact of the aromatic substituents, spacers, and substitution at the basic center on the inhibitory activity. To achieve selective inhibition of the mitochondrial Hsp90 isoform (TRAP1), triphenylphosphonium ion (TPP+) was attached to two known Hsp90 CTD inhibitors on different spacers and the change in activity before and after the introduction of this mitochondria-targeting moiety was observed. In the end, sixteen inhibitors of protein-protein interactions between Hsp90 and Cdc37 were synthesized to obtain information on the structure-activity relationship (SAR). The anticancer activity of the inhibitors was evaluated in breast cancer and Ewing sarcoma cell lines.
The results of cell-based assays indicated the importance of the para-substitution for the orientation of other structural elements in Hsp90-Cdc37 interaction inhibitors. As predicted, the potency of TRAP1 inhibitors was enhanced by TPP+ attachment. In addition, in Hsp90 CTD inhibitors, the importance of the aromatic ring at position 6 (compounds 8a-d) and the corresponding spacer to the basic center (compounds 11a-d and 12-14) were confirmed by cell-based assays. The most promising compound 11b thus inhibited the growth of the breast cancer cell line MCF-7 with an IC50 value of 0.43 ± 0.10 µM.
The new findings of the master's thesis significantly contribute to a better understanding of the allosteric binding site on Hsp90 CTD and represent a starting point for the further design of both C-terminal inhibitors and selective inhibitors of individual Hsp90 isoforms and inhibitors of Hsp90 protein-protein interactions with Cdc37.
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