Evaluation of Cyclic Peptide Inhibitors of the Grb7 Breast Cancer Target: Small Change in Cargo Results in Large Change in Cellular Activity

Jianrong Sang,Gabrielle M Watson,Ketav Kulkarni,Xiuquan Ma,Aaron G Poth,Jacqueline A Wilce,Aurélie H Benfield,David J Craik,Sónia T Henriques

doi:10.3390/molecules24203739

Abstract

Grb7 is an adapter protein, overexpressed in HER2+ve breast and other cancers, and identified as a therapeutic target. Grb7 promotes both proliferative and migratory cellular pathways through interaction of its SH2 domain with upstream binding partners including HER2, SHC, and FAK. Here we present the evaluation of a series of monocyclic and bicyclic peptide inhibitors that have been developed to specifically and potently target the Grb7 SH2-domain. All peptides tested were found to inhibit signaling in both ERK and AKT pathways in SKBR-3 and MDA-MB-231 cell lines. Proliferation, migration, and invasion assays revealed, however, that the second-generation bicyclic peptides were not more bioactive than the first generation G7-18NATE peptide, despite their higher in vitro affinity for the target. This was found not to be due to steric hindrance by the cell-permeability tag, as ascertained by ITC, but to differences in the ability of the bicyclic peptides to interact with and penetrate cellular membranes, as determined using SPR and mass spectrometry. These studies reveal that just small differences to amino acid composition can greatly impact the effectiveness of peptide inhibitors to their intracellular target and demonstrate that G7-18NATE remains the most effective peptide inhibitor of Grb7 developed to date.

Highlights

Cancer progression occurs with the dysregulation of critical signaling pathways controlling cell proliferation, migration, and survival
We discovered that greater activity of G7-18NATE-Pen correlated with its superior cellular permeability over the bicyclic peptides, highlighting that even a small change to a cargo can affect its successful delivery by cell penetrating peptide (CPP) and its effective activity
The G7-B7M2 peptide combines both of these modifications, overall enhancing the in vitro higher affinity for the growth receptor bound protein 7 (Grb7)-SH2 domain in vitro

Summary

Introduction

Cancer progression occurs with the dysregulation of critical signaling pathways controlling cell proliferation, migration, and survival. These often involve receptor and non-receptor tyrosine kinases that initiate and propagate these signaling cascades. Tyrosine kinases and their downstream interaction partners constitute targets for therapeutics development aimed to halt the aberrant behavior of the cancer cell [1,2]. Molecules 2019, 24, 3739 in the overexpression of the growth receptor bound protein 7 (Grb7) [4] This adapter protein normally functions through interactions with FAK in the formation of focal adhesions that are important for cell migration [5,6]. Grb overexpression has been determined as an independent indication of poor prognostic cancer progression [8,9,10,11]

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Results

Conclusion