Heat shock proteins (Hsp), the most abundant of which in a cell is heat shock protein 90 (Hsp90), serve as part of the cell's 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's 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.
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