Delivery of Antibody Mimics into Mammalian Cells via Anthrax Toxin Protective Antigen

Abstract

Antibody mimics have significant scientific and therapeutic utility for the disruption of protein–protein interactions inside cells; however, their delivery to the cell cytosol remains a major challenge. Here we show that protective antigen (PA), a component of anthrax toxin, efficiently transports commonly used antibody mimics to the cytosol of mammalian cells when conjugated to the N-terminal domain of LF (LFN). In contrast, a cell-penetrating peptide (CPP) was not able to deliver any of these antibody mimics into the cell cytosol. The refolding and binding of a transported tandem monobody to Bcr-Abl (its protein target) in chronic myeloid leukemia cells were confirmed by co-immunoprecipitation. We also observed inhibition of Bcr-Abl kinase activity and induction of apoptosis caused by the monobody. In a separate case, we show disruption of key interactions in the MAPK signaling pathway after PA-mediated delivery of an affibody binder that targets hRaf-1. We show for the first time that PA can deliver bioactive antibody mimics to disrupt intracellular protein–protein interactions. This technology adds a useful tool to expand the applications of these modern agents to the intracellular milieu.

Peptide- and proton-driven allosteric clamps catalyze anthrax toxin translocation across membranes

“…Based upon the stereochemically altered peptide series tested, isotactic αL and αD peptides may cyclically populate sections of helical structure in the channel. This allosteric and stereochemistry-centered model is consistent with a recent functional analysis of synthetic anthrax toxin substrates (40) and a crystal structure revealing the LF N -helix interactions with the α-clamp (9). An extended-chain model (17), by contrast, would not be selective of stereochemistry, as all three stereochemical configurations tested could easily make extended chain (32).…”

Anticancer RuII and RhIII Piano‐Stool Complexes that are Histone Deacetylase Inhibitors

“…[3] This new class of targeted drugs exhibit ab road range of therapeutic mechanisms,i ncludingi nhibition of extracellularg rowth receptors, [4] activation of cell death pathways, [5] retardation of cell motility, [6] kinase inhibition, [7] and toxin delivery, [8] to name af ew.S ubsequently,i nhibition of enzymes associated with key regulatory pathways in cancer is an attractive alternative to targetingD NA. [9] In principle, "molecularly targeted" agents are highly selective agents againstt he growth and survival of tumour cells, whilst sparing normalcells.…”

Emerging Strategies for Developing Next-Generation Protein Therapeutics for Cancer Treatment

“…This leaves many promising untapped intracellular targets, catalyzing significant efforts to develop proteins capable of accessing and binding targets inside the cell. Major strategies for cellular internalization include peptide stapling [37], protein supercharging [38], toxin-based delivery [39,40], and fusion of proteins to cell-penetrating peptides [41,42]. Despite these efforts, the field of intracellular targeting remains inchoate, imposing restrictions on the targets currently tractable to protein therapeutics.…”