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Investigating Apoptozole as a Chemical Probe for HSP70 Inhibition.

Evans LE, Cheeseman MD, Yahya N, Jones K - PLoS ONE (2015)

Bottom Line: Apoptozole is a recently identified small molecule, which has been reported to possess strong affinity for the HSP70 isoforms HSP72 and HSC70.Unfortunately, using both biochemical and biophysical techniques, we were unable to find any experimental evidence that apoptozole binds to HSP70 in a specific and developable way.Instead, we provide experimental evidence that apoptozole forms aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London SW7 3RP, United Kingdom.

ABSTRACT
The use of chemical tools to validate clinical targets has gained in popularity over recent years and the importance of understanding the activity, selectivity and mechanism of action of these compounds is well recognized. Dysregulation of the HSP70 protein family has been linked to multiple cancer types and drug resistance, highlighting their importance as popular targets for anti-cancer drug development. Apoptozole is a recently identified small molecule, which has been reported to possess strong affinity for the HSP70 isoforms HSP72 and HSC70. We investigated apoptozole as a potential chemical tool for HSP70 inhibition. Unfortunately, using both biochemical and biophysical techniques, we were unable to find any experimental evidence that apoptozole binds to HSP70 in a specific and developable way. Instead, we provide experimental evidence that apoptozole forms aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner.

No MeSH data available.


Related in: MedlinePlus

Competitive displacement curves for ATP-FAM and HSP72 with ATP, ADP, VER-155008 and apoptozole.IC50 values are the geometric mean and pIC50 values are the geometric mean ± SEM from 3 independent measurements.
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pone.0140006.g002: Competitive displacement curves for ATP-FAM and HSP72 with ATP, ADP, VER-155008 and apoptozole.IC50 values are the geometric mean and pIC50 values are the geometric mean ± SEM from 3 independent measurements.

Mentions: We first addressed the binding site of apoptozole. Using reported evidence from ligand-directed protein labelling and molecular modelling, it has been proposed that apoptozole binds in the ATP-binding site of HSP70.[27] This would likely require apoptozole to be competitive with ATP for binding and for the binding of the ligands to be mutually exclusive. Using an ATP-derived fluorescent probe (ATP-FAM)[21] a fluorescence polarisation (FP) assay was developed to enable the detection of ATP-competitive inhibitors of human HSP72 (S1 and S2 Figs). IC50 determination was performed at the same time for the known ATP-competitive HSP70 inhibitor VER-155008 (S3 Fig)[21], and the endogenous ligands ATP and ADP. IC50 values of 490 nM, 1200 nM and 670 nM were determined for VER-155008, ATP and ADP, respectively. In contrast, apoptozole failed to show any measureable ATP-competitive binding to HSP72, with no displacement of the ATP-FAM probe observed at concentrations up to 80 μM (Fig 2). The lack of ATP-competitive binding strongly suggested that apoptozole was not binding in the ATP-binding site of HSP72.


Investigating Apoptozole as a Chemical Probe for HSP70 Inhibition.

Evans LE, Cheeseman MD, Yahya N, Jones K - PLoS ONE (2015)

Competitive displacement curves for ATP-FAM and HSP72 with ATP, ADP, VER-155008 and apoptozole.IC50 values are the geometric mean and pIC50 values are the geometric mean ± SEM from 3 independent measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4601772&req=5

pone.0140006.g002: Competitive displacement curves for ATP-FAM and HSP72 with ATP, ADP, VER-155008 and apoptozole.IC50 values are the geometric mean and pIC50 values are the geometric mean ± SEM from 3 independent measurements.
Mentions: We first addressed the binding site of apoptozole. Using reported evidence from ligand-directed protein labelling and molecular modelling, it has been proposed that apoptozole binds in the ATP-binding site of HSP70.[27] This would likely require apoptozole to be competitive with ATP for binding and for the binding of the ligands to be mutually exclusive. Using an ATP-derived fluorescent probe (ATP-FAM)[21] a fluorescence polarisation (FP) assay was developed to enable the detection of ATP-competitive inhibitors of human HSP72 (S1 and S2 Figs). IC50 determination was performed at the same time for the known ATP-competitive HSP70 inhibitor VER-155008 (S3 Fig)[21], and the endogenous ligands ATP and ADP. IC50 values of 490 nM, 1200 nM and 670 nM were determined for VER-155008, ATP and ADP, respectively. In contrast, apoptozole failed to show any measureable ATP-competitive binding to HSP72, with no displacement of the ATP-FAM probe observed at concentrations up to 80 μM (Fig 2). The lack of ATP-competitive binding strongly suggested that apoptozole was not binding in the ATP-binding site of HSP72.

Bottom Line: Apoptozole is a recently identified small molecule, which has been reported to possess strong affinity for the HSP70 isoforms HSP72 and HSC70.Unfortunately, using both biochemical and biophysical techniques, we were unable to find any experimental evidence that apoptozole binds to HSP70 in a specific and developable way.Instead, we provide experimental evidence that apoptozole forms aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Cancer Therapeutics Unit at The Institute of Cancer Research, London SW7 3RP, United Kingdom.

ABSTRACT
The use of chemical tools to validate clinical targets has gained in popularity over recent years and the importance of understanding the activity, selectivity and mechanism of action of these compounds is well recognized. Dysregulation of the HSP70 protein family has been linked to multiple cancer types and drug resistance, highlighting their importance as popular targets for anti-cancer drug development. Apoptozole is a recently identified small molecule, which has been reported to possess strong affinity for the HSP70 isoforms HSP72 and HSC70. We investigated apoptozole as a potential chemical tool for HSP70 inhibition. Unfortunately, using both biochemical and biophysical techniques, we were unable to find any experimental evidence that apoptozole binds to HSP70 in a specific and developable way. Instead, we provide experimental evidence that apoptozole forms aggregates under aqueous conditions that could interact with HSP70 proteins in a non-specific manner.

No MeSH data available.


Related in: MedlinePlus