Designing Specific HSP70 Substrate Binding Domain Inhibitor for Perturbing Protein Folding Pathways to Inhibit Cancer
Kübra A Coşkun1, İrfan Koca2, Mehmet Gümüş3
1Department of Basic Pharmaceutical Sciences, Division of Biochemistry, Faculty of Pharmacy, Cumhuriyet University, Sivas, Turkey.
Background:
HSP70 is a survival factor for tumor cells in transformation and in tumor progression as well as in anti-apoptotic response.
Objective:
Several inhibitors targeting HSP70 ATPase function displayed off-target effects, but PES, which targets the HSP70 substrate binding domain, prevents tumor cell survival prominently. However, PES may not bind HSP70 in the absence of nucleotide. This research aimed to design a unique inhibitor molecule that works both in the presence and absence of nucleotides to amplify inhibition.
Methods:
A set of chimeric coumarine-pyrazole derivatives were determined by in silico techniques and synthesized to elucidate their inhibitory effects. Cell viability experiments displayed KBR1307 as the most efficient inhibitor. A set of characterization experiments were performed, and the results were compared to that of PES agent. Binding constant, ATP hydrolysis rate, and percent aggregation were determined in the presence and absence of inhibitors.
Results:
In silico docking experiments showed that only KBR1307 binds the HSP70 substrate binding domain and interacts with cochaperone interface. Binding experiments indicated that KBR1307 binds HSP70 both in the presence and absence of nucleotides, but PES does not. Both inhibitors significantly lower HSP70 ATPase activity and substrate protein disaggregation activity. However, KBR1307 displays a lower IC50 value at the MCF-7 cell line compared to PES. Both inhibitors do not alter HSP70 secondary structure composition and overall stability.
Conclusion:
KBR1307 effectively inhibits HSP70 compared to PES and provides a promising template for novel anticancer drug development.
Insights
A novel inhibitor, KBR1307, effectively targets Heat Shock Protein 70 (HSP70) in both nucleotide-present and absent states, offering a promising new avenue for cancer therapy development.
Area of Science:
- Molecular Biology
- Drug Discovery
- Oncology
Background:
- Heat Shock Protein 70 (HSP70) is crucial for tumor cell survival and progression.
- Existing HSP70 inhibitors targeting ATPase function have shown off-target effects.
- PES inhibitor targets the HSP70 substrate binding domain but may not bind without nucleotides.
Purpose of the Study:
- To design a novel inhibitor targeting HSP70 that functions in both nucleotide-bound and unbound states.
- To enhance the anti-cancer efficacy of HSP70 inhibition.
- To develop a new template for anticancer drug development.
Main Methods:
- In silico docking and synthesis of coumarine-pyrazole derivatives.
- In vitro characterization including binding assays and ATPase activity measurements.
- Cell viability assays (MCF-7 cell line) to determine IC50 values.
Main Results:
- KBR1307 binds the HSP70 substrate binding domain, including the cochaperone interface, irrespective of nucleotide presence.
- Unlike PES, KBR1307 demonstrates binding to HSP70 with or without nucleotides.
- KBR1307 exhibits superior inhibition of HSP70 ATPase and disaggregation activities, with a lower IC50 in MCF-7 cells compared to PES.
Conclusions:
- KBR1307 is a potent HSP70 inhibitor effective in both nucleotide-dependent and independent states.
- KBR1307 shows improved efficacy over PES and serves as a promising lead for novel anticancer drug development.
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