Cellular Uptake and Ultrastructural Localization Underlie the Pro-apoptotic Activity of a Hydrocarbon-stapled BIM BH3 Peptide

Abstract

Hydrocarbon stapling has been applied to restore and stabilize the α-helical structure of bioactive peptides for biochemical, structural, cellular, and in vivo studies. The peptide sequence, in addition to the composition and location of the installed staple, can dramatically influence the properties of stapled peptides. As a result, constructs that appear similar can have distinct functions and utilities. Here, we perform a side-by-side comparison of stapled peptides modeled after the pro-apoptotic BIM BH3 helix to highlight these principles. We confirm that replacing a salt-bridge with an i, i + 4 hydrocarbon staple does not impair target binding affinity and instead can yield a biologically and pharmacologically enhanced α-helical peptide ligand. Importantly, we demonstrate by electron microscopy that the pro-apoptotic activity of a stapled BIM BH3 helix correlates with its capacity to achieve cellular uptake without membrane disruption and accumulate at the organellar site of mechanistic activity.

Selective Covalent Targeting of Anti-Apoptotic BFL-1 by Cysteine-Reactive Stapled Peptide Inhibitors

“…We first confirmed that BIM SAHB A1 and BIM SAHB A -3 have equivalent cellular uptake, as quantified by ImageXpress Micro (IXM) high content epifluorescence microscopy of treated A375P cells and mouse embryonic fibroblasts (MEFs), which we have used previously to benchmark the comparative cell penetrance of FITC-labeled stapled peptides(Bird et al, 2016) (Figure S3A–B). The mechanism of uptake for BIM SAHBs is consistent with macropinocytosis, as previously reported(Edwards et al, 2015; Walensky et al, 2004) and evidenced here by the epifluorescence microscopy pattern of treated A375P and MEF cells at 4 hours (Figure S3C–D). …”

Cell Penetration Profiling Using the Chloroalkane Penetration Assay

“…Overall, CAPA data supported that ZF5.3, BIM-SAHB A1 , and DP1 can access the cytosol more effectively than Tat, as suggested by previous literature. 28–30 …”

A critical assessment of the synthesis and biological activity of p53/human double minute 2‐stapled peptide inhibitors

“…The inhibition of the p53/human double minute 2 (Hdm2) interaction is of particular interest in this context, and a variety of studies on different variants of stapled peptides have been published (Walensky et al, 2004;Chang et al, 2013). Remarkably, despite their size, these stapled peptides have been shown to enter cells and reach their targets in the cytosol (Muppidi et al, 2014;Chu et al, 2015;Edwards et al, 2015). These properties have been linked to the constrained helical character of the peptides (Kim and Verdine, 2009).…”