Therapeutic applications of the cell-penetrating HIV-1 Tat peptide

Rizzuti, Mafalda; Nizzardo, Monica; Zanetta, Chiara; Ramirez, Agnese; Corti, Stefania

doi:10.1016/j.drudis.2014.09.017

Cited by 173 publications

(124 citation statements)

References 95 publications

(113 reference statements)

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“…One of the most promising delivery vehicles is the TAT protein transduction domain of HIV-1. It has been used to deliver a wide variety of cargo into cells and has shown efficacy in preclinical models in vivo (38,59,60). In addition, several clinical trials are currently underway using TAT-mediated delivery (57,(60)(61)(62)(63)(64), although no therapy as of yet has received FDA approval, as issues associated with efficacy, bioavailablilty, and targeting remain primary challenges.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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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“…For instance, CPPs (TAT, penetratin, etc.) are currently being tested in several preclinical and clinical trials (7,8). However, CPPs exposed to cells or serum are rapidly degraded, and this can consequently render these compounds less effective in vivo or in vitro (9 -13).…”

Section: Cell-penetrating Peptides (Cpps)mentioning

confidence: 99%

An l- to d-Amino Acid Conversion in an Endosomolytic Analog of the Cell-penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape

Najjar

Erazo-Oliveras

Brock

et al. 2017

Journal of Biological Chemistry

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Edited by F. Peter GuengerichCell-penetrating peptides (CPPs) are well established as delivery agents for otherwise cell-impermeable cargos. CPPs can also theoretically be used to modulate intracellular processes. However, their susceptibility to proteolytic degradation often limits their utility in these applications. Previous studies have explored the consequences for cellular uptake of converting the residues in CPPs from L-to D-stereochemistry, but conflicting results have been reported and specific steps en route to intracellular activity have not been explored. Here we use dimeric fluorescence TAT as a model CPP to explore the broader consequences of L-to D-stereochemical conversion. We show that inversion of chirality provides protease resistance without altering the overall mode of cellular entry, a process involving endocytic uptake followed by endosomal escape and cytosolic access. However, whereas inversion of chirality reduces endocytic uptake, the D-peptide, once in the endosome, is significantly more prone to escape than its L-counterpart. Moreover, the D-peptide is retained in the cytosol of cells for several days, whereas the L-peptide is degraded within hours. Notably, while the L-peptide is relatively innocuous to cells, the D-peptide exerts a prolonged antiproliferative activity. Together, our results establish connections between chirality, protease resistance, cellular penetration, and intracellular activity that may be useful for the development of future delivery agents with improved properties. Cell-penetrating peptides (CPPs)2 have become important tools for the delivery of macromolecular cargoes inside cells (1-3). These delivery agents show promise in therapeutic applications and are useful reagents for cell biology assays (4 -6). For instance, CPPs (TAT, penetratin, etc.) are currently being tested in several preclinical and clinical trials (7,8). However, CPPs exposed to cells or serum are rapidly degraded, and this can consequently render these compounds less effective in vivo or in vitro (9 -13). To protect CPPs from degradation, a common strategy has been to employ D-amino acids instead of their L-amino acid counterparts. D-Peptides are protease-resistant, and this approach has been applied to CPPs such as TAT, R9, penetratin, hLF, pVEC, and sweet arrow peptide (10, 14 -17). In addition, the extended in vivo half-lives of D-peptides over L-peptides have contributed to the successful development of D-polyarginine CPPs as cancer contrast agents (18,19). How inversion of chirality impacts the multifaceted functions of CPPs, however, remains unclear.Several reports have indicated that cellular uptake of CPPs is independent of peptide backbone chirality (16,20). Uptake of the CPPs studied was thought to involve direct plasma membrane translocation. This is because uptake persisted at 4°C, a condition that typically abolishes endocytosis (16). In many cases, however, the penetration of CPPs into cells involves different routes of cellular entry (21). Instead of crossing the plasma membr...

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“…Tat can increase cellular drug uptake by activating different types of endocytosis pathways, as well as direct translocation. 77 The ability of Tat to deliver macromolecular cargo to the brain will greatly facilitate the development of anticerebral ischemia drugs. However, this Tat-based drug delivery approach is potentially fraught with several scientific and technical problems, which include a lack of cell selectivity, instability, complicated influences by peptide cargo, uncertainty in guaranteeing an effective concentration in its target and immediate degradation after oral administration.…”

Section: Perspectivesmentioning

confidence: 99%

Therapeutic Targets for Cerebral Ischemia Based on the Signaling Pathways of the GluN2B C Terminus

Sun

Zhang

You

et al. 2015

Stroke

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Therapeutic applications of the cell-penetrating HIV-1 Tat peptide

Cited by 173 publications

References 95 publications

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

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

An l- to d-Amino Acid Conversion in an Endosomolytic Analog of the Cell-penetrating Peptide TAT Influences Proteolytic Stability, Endocytic Uptake, and Endosomal Escape

Therapeutic Targets for Cerebral Ischemia Based on the Signaling Pathways of the GluN2B C Terminus

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