Hiding in plain sight: Optimizing topoisomerase IIα inhibitors into Hsp90β selective binders

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• Heat shock protein 90 (Hsp90) chaperones are crucial for stabilizing oncogenic proteins, making them a target for cancer therapy.
• Existing Hsp90 inhibitors often fail due to inducing a heat shock response (HSR), limiting their clinical utility.
• Developing isoform-selective Hsp90 inhibitors is a strategy to overcome HSR and improve therapeutic efficacy.

Purpose of the Study:

• To repurpose ATP-competitive inhibitors of bacterial DNA gyrase B and human topoisomerase IIα for Hsp90 inhibition.
• To identify novel Hsp90 inhibitors with selectivity for specific isoforms, particularly Hsp90β over Hsp90α.
• To evaluate the anticancer potential and safety profile of selective Hsp90 inhibitors.

Main Methods:

• Virtual screening of in-house inhibitor libraries against Hsp90.
• Biochemical assays to evaluate Hsp90 binding affinity and isoform selectivity.
• In vitro cancer cell line growth inhibition assays.
• Analysis of Hsp90 client protein levels and cell cycle progression.
• Toxicology studies in zebrafish larvae.
• Molecular modeling and STD NMR studies to elucidate selectivity mechanisms.

Main Results:

• Compound 11 showed low micromolar affinity for Hsp90 with 12-fold selectivity for Hsp90β over Hsp90α.
• 16 out of 29 analogs exhibited Hsp90β preference.
• Eleven compounds inhibited cancer cell growth in vitro.
• Compound 24e demonstrated Hsp90β selectivity (≥27-fold), reduced Hsp90 client proteins, induced G0/G1 cell cycle arrest without HSR, and showed selectivity against topoisomerase IIα and kinases.
• Compound 24e was non-toxic in zebrafish larvae.
• Molecular modeling and STD NMR confirmed Serine to Alanine switch at S52A in Hsp90α drives isoform selectivity.

Conclusions:

• Repurposed inhibitors targeting bacterial DNA gyrase B and human topoisomerase IIα can be effective Hsp90 inhibitors.
• Compound 24e is a potent and selective Hsp90β inhibitor with promising anticancer activity and a favorable safety profile.
• The S52A mutation is key to achieving selectivity between Hsp90α and Hsp90β isoforms, offering a rational design strategy for future drug development.