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Abstract Changing an oxygen atom of the phosphoester bond in phosphopeptides by a sulfur atom enables instantly targeting Golgi apparatus (GA) and selectively killing cancer cells by enzymatic self‐assembly. Specifically, conjugating cysteamine S‐phosphate to the C‐terminal of a self‐assembling peptide generates a thiophosphopeptide. Being a substrate of alkaline phosphatase (ALP), the thiophosphopeptide undergoes rapid ALP‐catalyzed dephosphorylation to form a thiopeptide that self‐assembles. The thiophosphopeptide enters cells via caveolin‐mediated endocytosis and macropinocytosis and instantly accumulates in GA because of dephosphorylation and formation of disulfide bonds in Golgi by themselves and with Golgi proteins. Moreover, the thiophosphopeptide potently and selectively inhibits cancer cells (HeLa) with the IC50(about 3 μM), which is an order of magnitude more potent than that of the parent phosphopeptide.
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Dynamic Continuum of Nanoscale Peptide Assemblies Facilitates Endocytosis and Endosomal Escape
Alkaline phosphatase (ALP) enables intracellular targeting by peptide assemblies, but how the ALP substrates enter cells remains elusive. Here we show that nanoscale phosphopeptide assemblies cluster ALP to enable caveolae-mediated endocytosis (CME) and endosomal escape. Specifically, fluorescent phosphopeptides undergo enzyme-catalyzed self-assembly to form nanofibers. Live cell imaging unveils that phosphopeptides nanoparticles, coincubated with HEK293 cells overexpressing red fluorescent protein-tagged tissue-nonspecific ALP (TNAP-RFP), cluster TNAP-RFP in lipid rafts to enable CME. Further dephosphorylation of the phosphopeptides produces peptidic nanofibers for endosomal escape. Inhibiting TNAP, cleaving the membrane anchored TNAP, or disrupting lipid rafts abolishes the endocytosis. Decreasing the transformation to nanofibers prevents the endosomal escape. As the first study establishing a dynamic continuum of nanoscale assemblies for cellular uptake, this work illustrates an effective design for enzyme-responsive supramolecular therapeutics and provides mechanism insights for understanding the dynamics of cellular uptake of proteins or exogenous peptide aggregates.
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- Award ID(s):
- 2011846
- PAR ID:
- 10506772
- Publisher / Repository:
- American Chemical Society
- Date Published:
- Journal Name:
- Nano Letters
- Volume:
- 21
- Issue:
- 9
- ISSN:
- 1530-6984
- Page Range / eLocation ID:
- 4078 to 4085
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acs.nanolett.1c01029
