Heat shock proteins (Hsp) are a group of functionally related proteins that are responsible for stabilizing and for proper folding proteins. Their main roles are to inhibit the denaturation and aggregation of proteins in the cell and to stabilize the developed polypeptide chain upon exposure to cellular stress. Because of this, they are very important in the growth and survival of cancer cells, where protein synthesis and cellular stress are increased. Therefore, Hsp inhibitors are potential anticancer agents.
Hsp90 is a 90 kDa large, most abundant, and ubiquitous heat shock protein. It is a molecular chaperone responsible for the proper folding of more than two hundred proteins, among others many oncoproteins, including kinases and transcription factors, which are crucial in malignant transformation. Novobiocin, a coumarin antibiotic, was the first discovered Hsp90 C-terminal domain inhibitor. Unfortunately, novobiocin and other coumarin antibiotics have shown to have relatively low cell activity and therefore limited therapeutic potential. However, novobiocin and its analogues provide a good basis for the development of analogues with improved inhibitory activity.
The aim of the master’s thesis was to synthesize and evaluate the physicochemical and biological properties of substituted N-phenylindolamides as Hsp90 inhibitors. We started from the known Hsp90 inhibitors and tried to improve their activity with structural changes. We preserved the basic scaffold, which is crucial for the activity. The pyrrole ring on the left side of the molecule was replaced with various substituted indoles, and the chain on the right side of the molecule was extended. By changing the structure, we synthesized three new compounds that differ from known Hsp90 inhibitors in size, polarity, and the possibility of making interactions with the binding site on the target.
The final synthesized compounds were biologically tested to determine whether they bind to the N-terminal domain of the two isoforms of the Hsp90 protein, Hsp90N alpha and Hsp90N beta. Compounds’ binding to Hsp90 was determined by the fluorescence-based thermal shift assay (FTSA), which determines the thermal stability of the free and ligand-bound protein. Our final compounds (15, 16 and 18) do not bind to the N-terminal domain of the isoform alpha and beta Hsp90 at a concentration of 200 µM, therefore they are not active. Based on the results in Table I, we can conclude that our compounds are too large to bind to the N-terminal domain of Hsp90.
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