Download files
Abstract
Protein-protein interactions (PPIs) regulate the cell’s protein-folding machinery, which relies on a multiprotein complex formed with heat shock protein 70 (Hsp70). Hsp70 is a molecular chaperone responsible for partially folding nascent peptides and refolding misfolded proteins, whereupon it transfers the partially folded proteins to Hsp90. The transfer process is mediated by the co-chaperone heat shock organising protein (HOP). HOP binds to Hsp70 via its tetratricopeptide repeat 1 (TPR1) domain. Hence, the PPI between HOP and Hsp70 is critical for the Hsp70’s function. This interaction is particularly important in cancer cells, where Hsp70 is overexpressed to fold the rapidly produced proteins and facilitate cancer growth.
This thesis describes the de-novo design, synthesis and biological evaluation of molecules aimed to regulate the interaction between Hsp70 and HOP. Peptides were designed based on the sequence HOP’s TPR1 domain. The aim was to mimics the HOP interaction with Hsp70 and inhibits Hsp70’s function by modulating Hsp70-HOP interaction. The author synthesized seven molecules in this series. Using solid phase peptide synthesis, the seven peptides were purified by HPLC and verified by LCMS, 1H NMR and 2D NMR. These compounds were then tested in a binding assay and a functional luciferase refolding assay. A structurally unique Hsp70 inhibitor, C1, was identified as lead molecule, which is the first molecule to directly regulate PPI and inhibit protein folding events.
The author then synthesized tagged version of C1 and tested in a protein pulldown assay against Hsp70 to assess its ability to bind to Hsp70. To identify the domain binding site, a pulldown assay was run using the C1-Tag against Hsp70’s substrate binding domain (SBD). The author demonstrated that C1 bound to Hsp70 at the SBD. Finally, a structure-activity relationship study on C1 was carried out by producing molecules to perform alanine. C1 is a pentapeptide, and each amino acid residue was substituted for alanine in the backbone. Of the five derivatives produced, the author synthesized three.
This project is demonstrating a proof of a successful approach for designing new small molecules that will modulate protein-protein interactions and interfere in large dynamic protein complexes.