Mechanism Matters: A Taxonomy of Cell Penetrating Peptides

Kauffman, W. Berkeley; Fuselier, Taylor; He, Jing; Wimley, William C.

doi:10.1016/j.tibs.2015.10.004

Cited by 293 publications

(347 citation statements)

References 98 publications

(169 reference statements)

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“…In addition, even a small molecule such as Sytox green (molecular mass, 600) showed only partial entrance in AS-48-treated parasites. The formation of a mixed phospholipid-peptide toroidal pore, predicted by molecular dynamics (44), will easily afford the translocation of AS-48 across the membrane, similar to some other cell-penetrating peptides (53).…”

Section: Discussionmentioning

confidence: 99%

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

Abengózar

Cebrián

Saugar

et al. 2017

Antimicrob Agents Chemother

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We report the feasibility of enterocin AS-48, a circular cationic peptide produced by Enterococcus faecalis, as a new leishmanicidal agent. AS-48 is lethal to Leishmania promastigotes as well as to axenic and intracellular amastigotes at low micromolar concentrations, with scarce cytotoxicity to macrophages. AS-48 induced a fast bioenergetic collapse of L. donovani promastigotes but only a partial permeation of their plasma membrane with limited entrance of vital dyes, even at concentrations beyond its full lethality. Fluoresceinated AS-48 was visualized inside parasites by confocal microscopy and seen to cause mitochondrial depolarization and reactive oxygen species production. Altogether, AS-48 appeared to have a mixed leishmanicidal mechanism that includes both plasma membrane permeabilization and additional intracellular targets, with mitochondrial dysfunctionality being of special relevance. This complex leishmanicidal mechanism of AS-48 persisted even for the killing of intracellular amastigotes, as evidenced by transmission electron microscopy. We demonstrated the potentiality of AS-48 as a new and safe leishmanicidal agent, expanding the growing repertoire of eukaryotic targets for bacteriocins, and our results provide a proof of mechanism for the search of new leishmanicidal bacteriocins, whose diversity constitutes an almost endless source for new structures at moderate production cost and whose safe use on food preservation is well established.

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Section: Discussionmentioning

confidence: 99%

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

Abengózar

Cebrián

Saugar

et al. 2017

Antimicrob Agents Chemother

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“…The ability of hydrocarbon staples to enhance cell penetration is perhaps their most important attribute. Several classes of cell penetration enhancement sequences have been identified and successfully employed (52). Enhancement by chemical stapling appears to be due to an increase in the overall hydrophobicity of the peptide as a result of the chemical clamp and is sequence-independent.…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Thomas

Cooper

Clayton

et al. 2016

Journal of Biological Chemistry

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Aberrant Ras signaling drives numerous cancers, and drugs to inhibit this are urgently required. This compelling clinical need combined with recent innovations in drug discovery including the advent of biologic therapeutic agents, has propelled Ras back to the forefront of targeting efforts. Activated Ras has proved extremely difficult to target directly, and the focus has moved to the main downstream Ras-signaling pathways. In particular, the Ras-Raf and Ras-PI3K pathways have provided conspicuous enzyme therapeutic targets that were more accessible to conventional drug-discovery strategies. The Ras-RalGEF-Ral pathway is a more difficult challenge for traditional medicinal development, and there have, therefore, been few inhibitors reported that disrupt this axis. We have used our structure of a Ral-effector complex as a basis for the design and characterization of ␣-helical-stapled peptides that bind selectively to active, GTPbound Ral proteins and that compete with downstream effector proteins. The peptides have been thoroughly characterized biophysically. Crucially, the lead peptide enters cells and is biologically active, inhibiting isoform-specific RalB-driven cellular processes. This, therefore, provides a starting point for therapeutic inhibition of the Ras-RalGEF-Ral pathway.Ras is well established as the most frequently mutated oncogene in human cancer. This small G protein cycles between an active GTP-bound state and an inactive GDP-bound state. Molecular switching between the "on" and "off" states is positively regulated by nucleotide guanine exchange factors (GEFs) 4 and negatively regulated by GTPase-activating proteins (GAPs). It is only the active, GTP-bound form of the protein that can bind to downstream effectors and facilitate signal transduction. Mutations in oncogenic Ras result in a constitutively active protein that remains fixed in the GTP-bound on-state, leading to unregulated activation of downstream pathways. These mutations are found in ϳ30% of all cancers, with a higher occurrence in specific cancer types such as pancreatic (71%) and colorectal (45%) (1). This makes Ras a crucial cancer therapeutic target; nevertheless, Ras has so far evaded direct attempts at inhibition, and many Ras-driven cancers are currently deemed undruggable. Ras signaling has proven difficult to disrupt by small, drug-like molecules because its activation of downstream cascades is accomplished through proteinprotein interactions, which have traditionally been avoided as drug targets due to the large, shallow surfaces involved in protein-protein interfaces (2-4). Likewise, there are no obvious clefts or small molecule binding pockets on Ras, and competitive inhibition with the nucleotide is unfeasible due to the extremely high affinity of GTP binding and its high concentration in the cellular environment (5, 6). Logical attempts to interfere with critical post-translational modifications of Ras, such as inhibition of farnesyltransferase, have also proved unsuccessful (for reviews, see Refs. 1, 6 and 7). More e...

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“…Most importantly, peptides can be designed to bind with high specificity to their in vivo targets and have relatively few off-target side effects. While the market for peptide-based drugs is currently estimated at more than 40 billion per year (57), a major limiting factor is the permeability barrier imposed by the plasma membrane (36)(37)(38). To overcome that limitation, over 100 peptide sequences have been identified capable of transporting bioactive molecules (38,58).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, for some conditions, the aforementioned global approaches may be unwarranted and a strategy that specifically targets the causal pathway or pathways more efficacious. In the current study, we do just that by using a cell-penetrating peptide (CPP) (36)(37)(38) consisting of the HIV TAT protein fused to an N-terminal 31-amino acid SNX9 fragment found to bind and specifically inhibit pSMAD3 nuclear import and biologic action. The results show that, while this TAT-SNX9 peptide had no effect on TGF-β signaling mediated via pSMAD2 or luciferase activity stimulated by bone morphogenetic protein 4 (BMP4), EGF, or PDGF, it inhibited (a) TGF-β induction of pSMAD3 target genes, soft agar TGF-β is considered a master switch in the pathogenesis of organ fibrosis.…”

Section: Introductionmentioning

confidence: 99%

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling

Kang¹,

Jung²,

Yin³

et al. 2017

Journal of Clinical Investigation

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ResultsA TAT-SNX9 peptide specifically blocks pSMAD3 nuclear import and profibrotic TGF-β signaling. We previously determined that SNX9 has an obligate role in mediating profibrotic TGF-β signaling dependent upon SMAD3 (24). In order to investigate colony formation, and cell migration; and (b) fibrotic changes associated with the bleomycin (BLM) and adenoviral models of lung fibrosis. Moreover, a 3-amino acid point mutant of the TAT-SNX9 peptide unable to bind pSMAD3 was ineffective in analogous in vitro and in vivo assays. Lysates from AKR-2B cells untreated (-) or stimulated (+) for 45 minutes with 5 ng/ml TGF-β were incubated with GST beads or the indicated fusion proteins immobilized on GST beads. Bound proteins were eluted and assessed by Western blot analysis for pSMAD3 or pSMAD2. Cell lysate reflects signal obtained from 10 μg total protein; the slower migrating band in the pSMAD3 lane is a nonspecific protein (right, representative of 3 separate experiments). (B) AKR-2B cells were transduced for 90 minutes with the indicated concentration of TAT peptide. Following washing and 1 hour TGF-β (5 ng/ml) treatment, nuclear fractions were isolated and assessed by Western blot analysis for pSMAD2, pSMAD3, or histone deacetylase 1 (HDAC) (left, representative of 3 separate experiments). Quantitation of nuclear pSMADs was performed with ImageJ software (NIH) and represents the mean ± SEM of 3 experiments (right). (C) Left panels: AKR-2B cells were incubated with vehicle (top panels) or transduced (bottom panels) as in B with TAT-SH3 or TAT-LC (1.8 μM). Following treatment with or without TGF-β (5 ng/ml) for 1 hour, immunofluorescence for SMAD3 or the HA-tagged TAT peptide was performed as described in Methods and nuclei were stained with DAPI. Upper and lower panels show 2 distinct microscopic fields for each condition. Original magnification in C: ×100. Right: quantitation of nuclear SMAD3 from 30 cells in each of 3 experiments. *P < 0.05; **P < 0.005; ***P < 0.0005, 1-way ANOVA followed by Dunnett's multiple comparisons test. The Journal of Clinical Investigation R E S E A R C H A R T I C L E2 5 4 3 jci.orgVolume 127 Number 7 July 2017 sion of this fragment could act in trans as a dominant inhibitor of pSMAD3 nuclear uptake. To address this issue, constructs were prepared expressing either the SNX9 SH3 or LC domains fused to the cell-penetrating TAT peptide from HIV (39). Subsequent to TAT peptide transduction, cultures were treated with TGF-β and nuclear accumulation of pSMAD2 or pSMAD3 determined. As shown in Figure 1B and Supplemental Figure 2, while the TAT-SH3 peptide inhibited nuclear import of pSMAD3 in a dose-dependent manner and increased cytoplasmic pSMAD3, it had no effect on pSMAD2. Furthermore, consistent with the inability of the LC domain to bind receptorregulated SMADs (R-SMADs) ( Figure 1A), it was similarly ineffective in modulating nuclear translocation ( Figure 1B). These biochemical findings were independently confirmed using immunofluorescence ( Figure 1C and Supplemental Figure 3). While t...

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Mechanism Matters: A Taxonomy of Cell Penetrating Peptides

Cited by 293 publications

References 98 publications

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

Enterocin AS-48 as Evidence for the Use of Bacteriocins as New Leishmanicidal Agents

Inhibition of Ral GTPases Using a Stapled Peptide Approach

Cell-penetrating peptides selectively targeting SMAD3 inhibit profibrotic TGF-β signaling

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