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Abstract Rapid cellular uptake of synthetic molecules remains a challenge, and the motif frequently employed to generate prodrugs, succinic ester, unfortunately lowers the efficacy of the desired drugs due to their slow ester hydrolysis and low cell entry. Here we show that succinic ester‐containing diglycine drastically boosts the cellular uptake of supramolecular assemblies or prodrugs. Specifically, autohydrolysis of the diglycine‐activated succinic esters turns the nanofibers of the conjugates of succinic ester and self‐assembling motif into nanoparticles for fast cellular uptake. The autohydrolysis of diglycine‐activated succinic esters and drug conjugates also restores the efficacy of the drugs. 2D nuclear magnetic resonance (NMR) suggests that a “U‐turn” of diglycine favors intramolecular hydrolysis of diglycine‐activated succinic esters to promote autohydrolysis. As an example of rapid autohydrolysis of diglycine‐activated succinic esters for instant cellular uptake, this work illustrates a nonenzymatic bond cleavage approach to develop effective therapeutics for intracellular targeting. 

Enzyme-instructed self-assembly (EISA) and hydrogelation is a versatile approach for generating soft materials. Most of the substrates for alkaline phosphatase catalysed EISA utilize phosphotyrosine ( p Tyr) as the enzymatic trigger for EISA and hydrogelation. Here we show the first example of phosphonaphthyl ( p NP) and phosphobiphenyl ( p BP) motifs acting as faster enzymatic triggers than phosphotyrosine for EISA and hydrogelation. This work illustrates novel enzyme triggers for rapid enzymatic self-assembly and hydrogelation. 

Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of alpha-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an alpha-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes. 

Although peptide assemblies have been explored extensively, the self-assembly of negatively charged peptides (NCPs) received little attention. Stimulated by the fact that acidic stretch is a common feature in the intrinsically disordered regions of histone chaperones, we explored the use of the assemblies of NCPs for trafficking histone proteins. Our results show that the peptides that contain glutamic acid (E)-repeat, at neutral or basic pH, self-assemble to form micelles in solution. Circular dichroism indicates that increasing pH favored the peptides to populate more in disordered and alpha helix conformations. Being innocuous to cells, the assemblies of these NCPs traffic histone 2B (H2B) to mitochondria. Structure-activity study indicates that self-assembly, proper stereochemistry, and acidic repeats are necessary for trafficking H2B. This work, as the first example of peptide assemblies for protein trafficking, illustrates a supramolecular approach for controlling cellular processes and provides insights for mimicking chaperones and controlling protein-protein interactions. 

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. 

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. 

Since mitochondria contribute to tumorigenesis and drug resistance in cancer, mitochondrial genetic engineering promises a new direction for cancer therapy. Here, we report the use of the perimitochondrial enzymatic noncovalent synthesis (ENS) of peptides for delivering genes selectively into the mitochondria of cancer cells for mitochondrial genetic engineering. Specifically, the micelles of peptides bind to the voltage-dependent anion channel (VDAC) on mitochondria for the proteolysis by enterokinase (ENTK), generating perimitochondrial nanofibers in cancer cells. This process, facilitating selective delivery of nucleic acid or gene vectors into mitochondria of cancer cells, enables the mitochondrial transgene expression of CRISPR/Cas9, FUNDC1, p53, and fluorescent proteins. Mechanistic investigation indicates that the interaction of the peptide assemblies with the VDAC and mitochondrial membrane potential are necessary for mitochondria targeting. This local enzymatic control of intermolecular noncovalent interactions enables selective mitochondrial genetic engineering, thus providing a strategy for targeting cancer cells.