Strained Cyclic Disulfides Enable Cellular Uptake by Reacting with the Transferrin Receptor

Abstract

In this study, we demonstrate that appendage of a single asparagusic acid residue (AspA tag) is sufficient to ensure efficient cellular uptake and intracellular distribution of fully unprotected peptides. We apply this new delivery method to induce apoptotic response in cancer cells using long (up to 20mer) BH3 domain peptides. Moreover, to understand the molecular mechanism of the cellular uptake, we perform chemical proteomics experiments and identify the direct molecular targets of the asparagusic acid tag. Our findings document covalent bond formation between the asparagusic acid moiety and the cysteines 556 and 558 on the surface of the transferrin receptor resulting in subsequent endocytic uptake of the payload. We believe that the small size, low cellular toxicity and the efficient transferrin receptor-mediated uptake render the AspA tag highly attractive for various life science applications.

Epidithiodiketopiperazines: Strain-Promoted Thiol-Mediated Cellular Uptake at the Highest Tension

“…Also unlike AspA controls, 12 preincubation of the cells with DTT or TCEP did not strongly increase the activity of ETPs (Figure 4C). Most importantly, the knockdown of the transferrin receptor (TFRC) with siRNA inhibited the uptake of AspA controls 14 but failed to inhibit ETP-mediated uptake. The observed partial inactivation by TFRC knockdown down to ∼65% was most revealing (Figures 4C and S7).…”

“…11−14 Disulfides in general are increasingly recognized to enter cells by thiol-mediated uptake, i.e., covalent attachment by disulfide exchange with exofacial thiols followed by efficient uptake via diverse, to a good part unknown mechanisms. 11−23 The emergence of thiol-mediated uptake called for the application of ring tension.…”

“…11 Uptake efficiencies were found to increase with ring tension from relaxed disulfides 3 with θ ≈ 90° to lipoic acid derivatives 4 with θ = 35° and asparagusic acid derivatives 5 with θ = 27°. 12,13 The most efficient “AspA tag” as in 5 allowed the delivery of functional peptides, 14 liposomes and polymersomes 13 into cells, and the transferrin receptor (TFRC) has been identified as one of the targets. 14 The power and promise of strain-promoted thiol-mediated uptake at θ = 27° provided a compelling incentive to drive disulfide ring tension to the extreme.…”

Disulfide‐Unit Conjugation Enables Ultrafast Cytosolic Internalization of Antisense DNA and siRNA

“…[9] Conjugates of hydrophobic molecules,s uch as cholesterol and lipids,a nd ONs have also been reported, [10] but because they form micelles,these conjugates do not exist as single molecules.ON conjugates with ligands for receptors on the cell surface are efficiently taken up by the target cells. [13,14] To our knowledge,t here have been no reports of the use of disulfide units for the intracellular delivery of ONs.T herefore,wesynthesized series of ONs modified with disulfide groups and evaluated their cellular uptake.Then, we found that the modification of ONs with repeated linear disulfide units enables the rapid cytosolic internalization of ONs without the endosomal trap.T he internalization of disulfide-modified ONs is triggered by the formation of ad isulfide bond with at hiol group of ap rotein on the cell surface and affords potent gene silencing effects ( Figure 1). [11] This strategy is useful for targeting specific cells that express ap articular receptor.F inally,m ost of the methods described above have problems in endosomal escape of ONs.…”

Glycosylated Cell‐Penetrating Peptides (GCPPs)

“…acids were optimal. [41][42][43][44] After these structural and functional discoveries, the potential of CPPs has led to the development of myriadp enetrating peptides equences fort he cellulari nternalization of different payloads. [14,16] Similaru ptake properties were found for the enantiomeric TAT [49][50][51][52][53][54][55][56][57] peptidea nd in penetrating peptoidsw ith guanidiniums ide chains anchored at the nitrogen of the amide group.…”