Functional analysis of Hsp70 inhibitors

Area of Science:

• Molecular biology
• Cellular biology
• Biochemistry

Background:

• Heat shock proteins (Hsp70) are crucial molecular chaperones and recognized anti-cancer targets.
• Multiple Hsp70 paralogs reside in different cellular compartments, necessitating isoform-specific investigation for therapeutic targeting.
• Small molecule inhibitors VER-155008 and 2-phenylethynesulfonamide (PES) were developed to target Hsp70s.

Purpose of the Study:

• To identify which Hsp70 isoform(s) must be downregulated to decrease cancer cell viability.
• To elucidate the molecular mechanism of action of Hsp70 inhibitors VER-155008 and PES in vitro.
• To determine the isoform specificity of these small molecule inhibitors.

Main Methods:

• Cancer cell viability assays.
• Simultaneous knockdown of Hsp70 isoforms (HSPA1 and HSPA8) using RNA interference.
• In vitro biochemical assays to analyze inhibitor binding and mechanism of action.
• Analysis of inhibitor interaction with Hsp70 nucleotide-binding domain (NBD) and substrate-binding domain (SBD).

Main Results:

• Significant reduction in cancer cell viability necessitates the simultaneous knockdown of heat-inducible Hsp70 (HSPA1) and constitutive Hsc70 (HSPA8).
• VER-155008 acts as an ATP-competitive inhibitor by arresting the Hsp70 nucleotide-binding domain (NBD) in a half-open conformation, disrupting allosteric regulation.
• Under tested conditions, PES exhibited non-specific, detergent-like interactions with the Hsp70 substrate-binding domain (SBD).
• Neither VER-155008 nor PES demonstrated isoform specificity.

Conclusions:

• Targeting both HSPA1 and HSPA8 concurrently is essential for reducing cancer cell proliferation.
• VER-155008's ATP-competitive inhibition mechanism offers a potential therapeutic strategy, though lacking isoform specificity.
• The non-specific action of PES limits its utility as a targeted anti-cancer therapeutic.
• Further research is needed to develop isoform-specific Hsp70 inhibitors for effective cancer therapy.