Cyclic peptide scaffold with ability to stabilize and deliver a helical cell-impermeable cargo across membranes of cultured cancer cells

Lawrence, Nicole; Philippe, Grégoire J.B.; Harvey, Peta J.; Condon, Nicholas D.; Benfield, Aurélie H.; Cheneval, Olivier; Craik, David J.; Henriques, Sónia Troeira

doi:10.1039/d0cb00099j

Cited by 14 publications

(17 citation statements)

References 51 publications

(90 reference statements)

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“…cPF4PD (GCQAPLYKKIIKKLLESGGXGGAPLYKKIIKKLCES; X is azidoalanine) is a helix–loop–helix cyclic peptide with antimalarial properties. It enters infected red blood cells and cancer cells and reaches their cytosol without disrupting the cell membrane , and was here included to investigate whether it can deliver cargo peptides inside cancer cells. The linear CPP p18 was also conjugated to KD3 to assess its effect on the activity and cellular delivery of KD3.…”

Section: Resultsmentioning

confidence: 99%

“…For example, Liu and collaborators found that conjugation of a CPP with PMI, a peptide that inhibits p53:MDM2/X interactions, induced cell membrane disruption at low concentrations, and therefore they employed liposome formulation as an alternative strategy to deliver PMI inside cells to reduce toxicity . In recent studies, we demonstrated the delivery of active p53 mimetics into the cytosol of cancer cells, followed by cell death without targeting p53:MDM2/X interactions; instead, cyclized CPP–cargo constructs induced toxicity via organelle/cell membrane disruption. , …”

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confidence: 99%

“…In the current study, we compared a set of cyclic CPPs for their ability to deliver a peptide inhibitor in the cytosol and induce cell death by modulating the targeted cancer pathway. We conjugated KD3, , a stapled peptide inhibitor of p53:MDM2/X interactions but unable to reach the cytosol of cells, onto the cyclic CPPs cTAT, cR10, CCPP12, and cPF4PD . We used copper catalyzed alkyne–azide cycloaddition (CuAAC) and introduced polyethylene glycol (PEG) linkers between CPP and KD3 to obtain active conjugates.…”

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confidence: 99%

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Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells

et al. 2021

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Peptides are being developed as targeted anticancer drugs to modulate cytosolic protein−protein interactions involved in cancer progression. However, their use as therapeutics is often limited by their low cell membrane permeation and/or inability to reach cytosolic targets. Conjugation to cell penetrating peptides has been successfully used to improve the cytosolic delivery of high affinity binder peptides, but cellular uptake does not always result in modulation of the targeted pathway. To overcome this limitation, we developed "angler peptides" by conjugating KD3, a noncell permeable but potent and specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions, with a set of cyclic cell-penetrating peptides. We examined their binding affinity for MDM2 and MDMX, the cell entry mechanism, and role in reactivation of the p53 pathway. We identified two angler peptides, cTAT−KD3 and cR10−KD3, able to activate the p53 pathway in cancer cells. cTAT−KD3 entered cells via endocytic pathways, escaped endosomes, and activated the p53 pathway in breast (MCF7), lung (A549), and colon (HCT116) cancer cell lines at concentrations in the range of 1−12 μM. cR10−KD3 reached the cytosol via direct membrane translocation and activated the p53 pathway at 1 μM in all the tested cell lines. Our work demonstrates that nonpermeable anticancer peptides can be delivered into the cytosol and inhibit intracellular cancer pathways when they are conjugated with stable cell penetrating peptides. The mechanistic studies suggest that direct translocation leads to less toxicity, higher cytosol delivery at lower concentrations, and lower dependencies on the membrane of the tested cell line than occurs for an endocytic pathway with endosomal escape. The angler strategy can rescue high affinity peptide binders identified from high throughput screening and convert them into targeted anticancer therapeutics, but investigation of their cellular uptake and cell death mechanisms is essential to confirming modulation of the targeted cancer pathways.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells

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“…To decrease the number of possible isomers and avoid changes in the overall charge of Lys-rich peptides after labeling, the dye can be conjugated using site-directed strategies. Some examples include conjugation of alkyne-derivatized dyes onto azide-containing peptides with copper-catalyzed azide-alkyne cycloaddition, and conjugation of dyes containing hydroxylamine with acetone-linked peptides using oxime ligation ( 77 ).…”

Section: Distinguishing Internalized and Membrane-bound Peptide Via Flow Cytometrymentioning

confidence: 99%

Mode-of-Action of Antimicrobial Peptides: Membrane Disruption vs. Intracellular Mechanisms

Benfield

Henriques

2020

Front. Med. Technol.

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Antimicrobial peptides are an attractive alternative to traditional antibiotics, due to their physicochemical properties, activity toward a broad spectrum of bacteria, and mode-of-actions distinct from those used by current antibiotics. In general, antimicrobial peptides kill bacteria by either disrupting their membrane, or by entering inside bacterial cells to interact with intracellular components. Characterization of their mode-of-action is essential to improve their activity, avoid resistance in bacterial pathogens, and accelerate their use as therapeutics. Here we review experimental biophysical tools that can be employed with model membranes and bacterial cells to characterize the mode-of-action of antimicrobial peptides.

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“… 13 Along those lines, very recently it was demonstrated that conjugation of cCPPs via triazole linkages to the peptide KD3, which was known to be a non-cell permeable stapled peptide inhibitor of p53/MDM2 and p53/MDMX interactions, 14 enables activation of apoptosis in cancer cells. 15 Alternative approaches for creating cell-permeable peptides to disrupt p53/MDMX interactions using cyclic peptide scaffolds, 16 polycationic tags or medchem optimization strategies have been extensively studied. 17,18 …”

Section: Introductionmentioning

confidence: 99%

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors

et al. 2021

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Cyclic peptide scaffold with ability to stabilize and deliver a helical cell-impermeable cargo across membranes of cultured cancer cells

Abstract: A new helix-loop-helix peptide scaffold with dual ability to transport cargo across cancer cell membranes and disrupt mitochondrial membrane function.

Cited by 14 publications

References 51 publications

Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells

Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells

Mode-of-Action of Antimicrobial Peptides: Membrane Disruption vs. Intracellular Mechanisms

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors

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