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Inhibiting EGFR dimerization using triazolyl-bridged dimerization arm mimics.

Hanold LE, Oruganty K, Ton NT, Beedle AM, Kannan N, Kennedy EJ - PLoS ONE (2015)

Bottom Line: These peptides are modified to contain a triazolyl bridge between the peptide strands to constrain the EGFR dimerization arm β-loop.In this study, we demonstrate that these peptides have significantly improved proteolytic stability over the non-modified peptide sequence, and their inhibitory effects are dependent on the number of the methylene units and orientation of the introduced triazolyl bridge.We identified a peptide, EDA2, which downregulates receptor phosphorylation and dimerization and reduces cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
The epidermal growth factor receptor (EGFR) is overexpressed in multiple carcinomas and is the focus of a variety of targeted therapies. Here we report the design of peptide-based compounds that mimic the EGFR dimerization arm and inhibit allosteric activation of EGFR. These peptides are modified to contain a triazolyl bridge between the peptide strands to constrain the EGFR dimerization arm β-loop. In this study, we demonstrate that these peptides have significantly improved proteolytic stability over the non-modified peptide sequence, and their inhibitory effects are dependent on the number of the methylene units and orientation of the introduced triazolyl bridge. We identified a peptide, EDA2, which downregulates receptor phosphorylation and dimerization and reduces cell viability. This is the first example of a biologically active triazolyl-bridged peptide targeting the EGFR dimerization interface that effectively downregulates EGFR activation.

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EDA2 down-regulates activated EGFR and reduces cell viability.(a) Cells were stimulated with 50 ng/mL EGF for 5 min in the presence or absence of EDA peptides, followed by western blotting. An apparent decrease in phosphorylated EGFR was observed when treated with EDA2. Vertical lines indicate non-adjacent samples from the same western blot. (b) Quantification of EGFR phosphorylated at Tyr1068, normalized to tubulin, shows that EDA2 reduces phosphorylated EGFR by greater than 60%, while the non-modified and scrambled controls do not. Data is plotted as the average of at least three experiments, where error bars represent SEM. * p < 0.05, ** p < 0.01 relative to the EGF-stimulated control. All remaining means are not significant (p > 0.05) relative to the EGF-stimulated control. (c) A panel of cell lines was dosed daily with EDA2 or gefitinib for 5 days, and viability was quantified using the Cell Titer Blue assay. EDA2 reduced viability by 50%, 72%, and 34% in MDA-MB-231, PC-3, and Bx-PC-3 cells, respectively. Data is plotted as the average of quadruplicates, where error bars represent SEM.
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pone.0118796.g005: EDA2 down-regulates activated EGFR and reduces cell viability.(a) Cells were stimulated with 50 ng/mL EGF for 5 min in the presence or absence of EDA peptides, followed by western blotting. An apparent decrease in phosphorylated EGFR was observed when treated with EDA2. Vertical lines indicate non-adjacent samples from the same western blot. (b) Quantification of EGFR phosphorylated at Tyr1068, normalized to tubulin, shows that EDA2 reduces phosphorylated EGFR by greater than 60%, while the non-modified and scrambled controls do not. Data is plotted as the average of at least three experiments, where error bars represent SEM. * p < 0.05, ** p < 0.01 relative to the EGF-stimulated control. All remaining means are not significant (p > 0.05) relative to the EGF-stimulated control. (c) A panel of cell lines was dosed daily with EDA2 or gefitinib for 5 days, and viability was quantified using the Cell Titer Blue assay. EDA2 reduced viability by 50%, 72%, and 34% in MDA-MB-231, PC-3, and Bx-PC-3 cells, respectively. Data is plotted as the average of quadruplicates, where error bars represent SEM.

Mentions: The EDA peptides were first tested to see if they could downregulate EGFR activation. Receptor activation was monitored as a function of EGFR autophosphorylation on Tyr1068 in intact MDA-MB-231 cells (Fig. 5a and b and S3 Fig.). The receptor was stimulated with EGF for 5 min following a 30 min peptide pretreatment. As predicted, EDA2 effectively downregulated EGFR activation while all the EDA peptides (EDA1 and EDA3–6) showed little to no activity (S3 Fig.). EDA2 was found to reduce EGFR phosphorylation by greater than 60% as compared to untreated cells at a dose of 5 μM (Fig. 5b). In contrast, the non-modified and EDA2 scramble (EDA2-Scr) controls had no inhibitory effect on EGFR phosphorylation, indicating that this effect is dependent on both sequence and cyclization. To further demonstrate that this effect was not due to changes in EGFR expression, an independent assay showed that total EGFR levels were not affected (S4 Fig.).


Inhibiting EGFR dimerization using triazolyl-bridged dimerization arm mimics.

Hanold LE, Oruganty K, Ton NT, Beedle AM, Kannan N, Kennedy EJ - PLoS ONE (2015)

EDA2 down-regulates activated EGFR and reduces cell viability.(a) Cells were stimulated with 50 ng/mL EGF for 5 min in the presence or absence of EDA peptides, followed by western blotting. An apparent decrease in phosphorylated EGFR was observed when treated with EDA2. Vertical lines indicate non-adjacent samples from the same western blot. (b) Quantification of EGFR phosphorylated at Tyr1068, normalized to tubulin, shows that EDA2 reduces phosphorylated EGFR by greater than 60%, while the non-modified and scrambled controls do not. Data is plotted as the average of at least three experiments, where error bars represent SEM. * p < 0.05, ** p < 0.01 relative to the EGF-stimulated control. All remaining means are not significant (p > 0.05) relative to the EGF-stimulated control. (c) A panel of cell lines was dosed daily with EDA2 or gefitinib for 5 days, and viability was quantified using the Cell Titer Blue assay. EDA2 reduced viability by 50%, 72%, and 34% in MDA-MB-231, PC-3, and Bx-PC-3 cells, respectively. Data is plotted as the average of quadruplicates, where error bars represent SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4366150&req=5

pone.0118796.g005: EDA2 down-regulates activated EGFR and reduces cell viability.(a) Cells were stimulated with 50 ng/mL EGF for 5 min in the presence or absence of EDA peptides, followed by western blotting. An apparent decrease in phosphorylated EGFR was observed when treated with EDA2. Vertical lines indicate non-adjacent samples from the same western blot. (b) Quantification of EGFR phosphorylated at Tyr1068, normalized to tubulin, shows that EDA2 reduces phosphorylated EGFR by greater than 60%, while the non-modified and scrambled controls do not. Data is plotted as the average of at least three experiments, where error bars represent SEM. * p < 0.05, ** p < 0.01 relative to the EGF-stimulated control. All remaining means are not significant (p > 0.05) relative to the EGF-stimulated control. (c) A panel of cell lines was dosed daily with EDA2 or gefitinib for 5 days, and viability was quantified using the Cell Titer Blue assay. EDA2 reduced viability by 50%, 72%, and 34% in MDA-MB-231, PC-3, and Bx-PC-3 cells, respectively. Data is plotted as the average of quadruplicates, where error bars represent SEM.
Mentions: The EDA peptides were first tested to see if they could downregulate EGFR activation. Receptor activation was monitored as a function of EGFR autophosphorylation on Tyr1068 in intact MDA-MB-231 cells (Fig. 5a and b and S3 Fig.). The receptor was stimulated with EGF for 5 min following a 30 min peptide pretreatment. As predicted, EDA2 effectively downregulated EGFR activation while all the EDA peptides (EDA1 and EDA3–6) showed little to no activity (S3 Fig.). EDA2 was found to reduce EGFR phosphorylation by greater than 60% as compared to untreated cells at a dose of 5 μM (Fig. 5b). In contrast, the non-modified and EDA2 scramble (EDA2-Scr) controls had no inhibitory effect on EGFR phosphorylation, indicating that this effect is dependent on both sequence and cyclization. To further demonstrate that this effect was not due to changes in EGFR expression, an independent assay showed that total EGFR levels were not affected (S4 Fig.).

Bottom Line: These peptides are modified to contain a triazolyl bridge between the peptide strands to constrain the EGFR dimerization arm β-loop.In this study, we demonstrate that these peptides have significantly improved proteolytic stability over the non-modified peptide sequence, and their inhibitory effects are dependent on the number of the methylene units and orientation of the introduced triazolyl bridge.We identified a peptide, EDA2, which downregulates receptor phosphorylation and dimerization and reduces cell viability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, Georgia, United States of America.

ABSTRACT
The epidermal growth factor receptor (EGFR) is overexpressed in multiple carcinomas and is the focus of a variety of targeted therapies. Here we report the design of peptide-based compounds that mimic the EGFR dimerization arm and inhibit allosteric activation of EGFR. These peptides are modified to contain a triazolyl bridge between the peptide strands to constrain the EGFR dimerization arm β-loop. In this study, we demonstrate that these peptides have significantly improved proteolytic stability over the non-modified peptide sequence, and their inhibitory effects are dependent on the number of the methylene units and orientation of the introduced triazolyl bridge. We identified a peptide, EDA2, which downregulates receptor phosphorylation and dimerization and reduces cell viability. This is the first example of a biologically active triazolyl-bridged peptide targeting the EGFR dimerization interface that effectively downregulates EGFR activation.

Show MeSH
Related in: MedlinePlus