The role of olefin geometry in the activity of hydrocarbon stapled peptides targeting eukaryotic translation initiation factor 4E (eIF4E).

Hydrocarbon stapled (HCS) peptides are a class of cross-linked α-helix mimetics. The technology relies on the use of α,α'-disubstituted alkenyl amino acids, which fully contrain the helical region to typically yield peptides with enhanced structural ordering and biological activity. Recently, monosubstituted alkenyl amino acids were disclosed for peptide stapling; however, the impact that this tether has on HCS peptide structure and activity has not yet been fully explored. By applying this HCS to the disordered peptide eIF4E-binding protein 1 (4E-BP1), we discovered that this type of tethering has a dramatic effect on olefin geometry and activity of the resultant stapled peptides, where the putative trans isomer was found to exhibit enhanced in vitro and cellular inhibitory activity against eIF4E protein-protein interactions. We further demonstrated that the metathesis catalyst used for ring-closing metathesis can influence monosubstituted HCS peptide activity, presumably through alteration of the cis/trans olefin ratio. This study represents one of the first in-depth analyses of olefin isomers of a stapled peptide and highlights an additional feature for medicinal chemistry optimization of this class of peptide-based probes.

Consideration of Binding Kinetics in the Design of Stapled Peptide Mimics of the Disordered Proteins Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 and Eukaryotic Translation Initiation Factor 4G.

Protein disorder plays a crucial role in signal transduction and is key for many cellular processes including transcription, translation, and cell cycle. Within the intrinsically disordered protein interactome, the α-helix is commonly used for binding, which is induced via a disorder-to-order transition. Because the targeting of protein-protein interactions (PPIs) remains an important challenge in medicinal chemistry, efforts have been made to mimic this secondary structure for rational inhibitor design through the use of stapled peptides. Cap-dependent mRNA translation is regulated by two disordered proteins, 4E-BP1 and eIF4G, that inhibit or stimulate the activity of the m7G cap-binding translation initiation factor, eIF4E, respectively. Both use an α-helical motif for eIF4E binding, warranting the investigation of stapled peptide mimics for manipulating eIF4E PPIs. Herein, we describe our efforts toward this goal, resulting in the synthesis of a cell-active stapled peptide for further development in manipulating aberrant cap-dependent translation in human diseases.

Chemoenzymatic reversible immobilization and labeling of proteins without prior purification.

Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label, or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this approach for polypeptide modification could be particularly useful for large-scale production of protein conjugates for therapeutic or industrial applications.