Distinct BimBH3 (BimSAHB) stapled peptides for structural and cellular studies

Abstract

Hydrocarbon stapling is a chemical approach to restoring and fortifying the natural α-helical structure of peptides that otherwise unfold when taken out of context from the host protein. By iterating the peptide sequence, staple type, and sites of insertion, discrete compositions can be generated to suit a diversity of biochemical, structural, proteomic, cellular, and drug development applications. Here, we reinforce key design considerations to avoid pitfalls and maximize progress when applying stapled peptides in chemistry and biology research.

Figure 1. A tale of two BIM SAHBs

In order to accomplish a challenging NMR analysis of the hit-and-run interaction between BIM BH3 and BAX (left), we adjusted the sequence of our prototype high α-helicity, high affinity, and cell permeable BIM SAHB_A (aa 146–166) peptide (right) to enhance its solubility and weaken (i.e. slow down) its BAX-activating capability (left). Whereas Okamoto et al. successfully applied this refashioned BIM SAHB_A (145–164) peptide for structural determination (left), their application of this weakened-by-design BIM SAHB_A (145–164) in binding and cellular studies (red arrow) led to the conclusion that stapling BIM BH3 does not enhance binding affinity or biological activity. In agreement with these results, we never used BIM SAHB_A (145–164) in cellular studies because of its relatively low α-helicity and weak binding activity, and instead applied our original highly α-helical, potent, and cell permeable BIM SAHB_A (aa 146–166) construct in cellular and in vivo analyses(5, 6, 10). Indeed, the versatility of peptide chemistry in general and hydrocarbon stapling in particular allows for the production of a diverse spectrum of stapled peptides that can be tailored to suit a host of research and therapeutic goals.

Figure 2. Design and synthesis of stapled peptides for protein interaction research and therapeutic targeting

Single peptide α-helices embedded within proteins can serve as key binding determinants for protein interaction, reflecting Nature's solution to high fidelity protein targeting. Although potentially well-suited as prototype therapeutics for disrupting pathologic protein interactions, when taken out of context from the full-length protein, such structured peptides can unfold, limiting their biological activity and rendering them susceptible to rapid proteolysis in vivo. Hydrocarbon stapling can restore bioactive secondary structure, and combined with facile iteration of peptide sequence composition (varying template length and charge), staple type, staple placement, and a host of derivatizations, a library of stapled peptides can be generated to suit a diversity of research and therapeutic applications.