Click chemistry for the generation of cell permeable apoptotic peptides

Abstract

he use of proteins and peptides as drug molecules has been held back by their proteolytic instability and inability to cross-cellular membranes. Proteins and long peptides are often produced by expression in E. coli rather than by solid phase peptide synthesis. A drawback of in vivo protein and peptide synthesis is the difficulty to selectively modify the product peptide by the attachment of fluorescent dyes or ligation to other macromolecules like polysaccharides, lipids or peptides. Here we present a facile method to modify an expressed protein or peptide to create a C-terminal alkyne group. This functionality is then used inter alia for conjugation to the cell-penetrating peptide octa-arginine. This will provide a vector for delivery across the plasma membrane of cells. To demonstrate our method, we have produced in E. coli a peptide derived from the Bak protein; one of the key regulators of apoptosis in eukaryotic cells. In the cell it is usually found bound to Bcl-xL at the outer mitochondrial membrane. If this interaction is disrupted, Bak oligomerizes and forms pores which trigger mitochondria dependent apoptosis through cytochrome c release. Small peptides derived from the BH3 helix of Bak have been shown to induce apoptosis. We have expressed such a peptide in E. coli as a fusion protein. The ketosteroid isomerase fusion protein is insoluble and readily purified from cell extracts. The peptide is then cleaved from the fusion protein by reaction with cyanogen bromide at a strategically inserted methionine residue to generate a homoserine lactone at the C-terminus of the Bak peptide. This lactone is then used for direct amide formation with inexpensive propargylamine. The resulting alkynyl peptide serves as a reagent for highly efficient ‘click’ reactions to couple to a wide range of azides. Since the Bak peptide is not able to cross the cell membrane, the well-known octa-arginine cell penetrating peptide sequence was added as a delivery vector. Here we discuss the synthesis of this semi-synthetic peptide and its interaction with, and uptake into, cancer cell lines.