Discovery and optimization of Grb7 antagonists as potential antitumor therapeutics
thesisposted on 03.02.2017, 03:47 by Ambaye, Nigus Dessalew
The growth factor receptor bound protein 7 (Grb7) is a non‐catalytic adaptor protein that was originally identified as a binding partner of the growth factor receptor (ErbB‐2). Amplification and overexpression of Grb7 is frequently implicated in the development of aggressive, recalcitrant and advanced tumor phenotypes making it a promising target in human cancers. Apart from several peptide‐based experimental antagonists, to date there exist no potent drug that is developed or known to act against Grb7. Thus, the overall objective of the research conducted in this thesis was to design novel, potent, selective and cell permeable Grb7 antagonists that may have potential as antitumor therapeutics. The first part of the thesis deals with optimization studies around a known lead polypeptide designed to bind to the Grb7 SH2 domain. A total of 11 polypeptides were synthesized with a yield varying from 2 to 17%. The peptide antagonists were found to have moderate binding affinity ranging from 0.6 – 60 μM as determined by isothermal titration calorimetry. Moreover, a short 7 residue cell permeablising sequence was incorporated into the inhibitor peptide construct and its ability to translocate peptides across membranes tested in in vitro and cell based assays. The second part of the thesis focused on small molecule antagonist discovery and optimization. Starting from known peptide leads and an experimental structure of the Grb7 SH2 domain, a series of ligand design techniques comprising shape based similarity searches, molecular docking and 2D‐similarity searching was effected to identify non‐peptide antagonists. In total, three structurally distinct classes of small molecular antagonists with moderate in vitro affinity and in vivo activity were identified from the study. The last part of the project reports the crystal structure determination of two Grb7 SH2 domain constructs. High‐resolution crystal structures of the apo‐ and peptide-bound forms of the Grb7 SH2 domain were determined. All together, extensive biophysical, computational and structural analyses were conducted on the Grb7 SH2 domain and ligands that will form the foundation for future Grb7 inhibitor development.