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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: 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.

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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EDA peptides are resistant to proteolytic degradation.Proteolytic stability was measured in the presence of (a) a cocktail of immobilized chymotrypsin and trypsin over a time range of 0–4 hours and (b) 50% mouse serum over a time range of 0–16 hours. (c, d) CD spectra of the non-modified and EDA2 peptides were obtained on a Jasco J-710 CD Spectrometer at 25°C in 10 mM sodium phosphate buffer at pH 6.5 and 7.4. EDA2 maintains its structure under both conditions.
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pone.0118796.g004: EDA peptides are resistant to proteolytic degradation.Proteolytic stability was measured in the presence of (a) a cocktail of immobilized chymotrypsin and trypsin over a time range of 0–4 hours and (b) 50% mouse serum over a time range of 0–16 hours. (c, d) CD spectra of the non-modified and EDA2 peptides were obtained on a Jasco J-710 CD Spectrometer at 25°C in 10 mM sodium phosphate buffer at pH 6.5 and 7.4. EDA2 maintains its structure under both conditions.

Mentions: To determine whether the addition of a covalent constraint promoted proteolytic stability, degradation of the EDA peptides was measured in the presence of purified proteases, serum, and culture media (Fig. 4 and S2 Fig.). The rate of peptide degradation was first measured using purified proteases (Fig. 4a). EDA peptides were incubated with a cocktail of immobilized trypsin and chymotrypsin over a time course of four hours. The amount of remaining peptide was quantified by LC/MS relative to an internal standard. While the non-modified control peptide was rapidly degraded with 50% lost within one hour, all of the triazolyl-linked peptides showed significantly enhanced proteolytic resistance with little to no degradation over the 4-hour time course, demonstrating that introduction of the linker appears to provide substantial resistance to proteolytic degradation.


Inhibiting EGFR dimerization using triazolyl-bridged dimerization arm mimics.

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

EDA peptides are resistant to proteolytic degradation.Proteolytic stability was measured in the presence of (a) a cocktail of immobilized chymotrypsin and trypsin over a time range of 0–4 hours and (b) 50% mouse serum over a time range of 0–16 hours. (c, d) CD spectra of the non-modified and EDA2 peptides were obtained on a Jasco J-710 CD Spectrometer at 25°C in 10 mM sodium phosphate buffer at pH 6.5 and 7.4. EDA2 maintains its structure under both conditions.
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Related In: Results  -  Collection

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

pone.0118796.g004: EDA peptides are resistant to proteolytic degradation.Proteolytic stability was measured in the presence of (a) a cocktail of immobilized chymotrypsin and trypsin over a time range of 0–4 hours and (b) 50% mouse serum over a time range of 0–16 hours. (c, d) CD spectra of the non-modified and EDA2 peptides were obtained on a Jasco J-710 CD Spectrometer at 25°C in 10 mM sodium phosphate buffer at pH 6.5 and 7.4. EDA2 maintains its structure under both conditions.
Mentions: To determine whether the addition of a covalent constraint promoted proteolytic stability, degradation of the EDA peptides was measured in the presence of purified proteases, serum, and culture media (Fig. 4 and S2 Fig.). The rate of peptide degradation was first measured using purified proteases (Fig. 4a). EDA peptides were incubated with a cocktail of immobilized trypsin and chymotrypsin over a time course of four hours. The amount of remaining peptide was quantified by LC/MS relative to an internal standard. While the non-modified control peptide was rapidly degraded with 50% lost within one hour, all of the triazolyl-linked peptides showed significantly enhanced proteolytic resistance with little to no degradation over the 4-hour time course, demonstrating that introduction of the linker appears to provide substantial resistance to proteolytic degradation.

Bottom Line: 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.

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