Evaluation of the cell permeability of bicyclic peptoids and bicyclic peptide-peptoid hybrids

Wang, Hee Myeong; Seo, Chang Deok; Lee, KangJu; Park, Jun Hyung; Lim, Hyun‐Suk

doi:10.1016/j.bioorg.2022.105976

Cited by 9 publications

(6 citation statements)

References 59 publications

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“…The chloroalkane penetration assay has been successfully applied to measure cytosolic and nuclear delivery for a large range of drug modalities including small molecules, peptides, natural products, and biotherapeutics. − In this work, we report the first-ever application of CAPA to measuring protein delivery by LNPs. CAPA allowed us to compare cytosolic delivery to more common measurements of total cellular uptake and functional delivery.…”

Section: Discussionmentioning

confidence: 99%

“…Several new assays have been developed for more direct measurement of cytosolic delivery without interference from the material trapped at the cell surface or in endosomes . Recently, we developed an assay called the chloroalkane penetration assay (CAPA). − Our initial work applied CAPA to measure the cytosolic delivery of cell-penetrant peptides, , and since then, we and others have adapted CAPA for measuring cytosolic delivery of DNA-encoded libraries, − peptoids, , small molecules including bivalent chemical degraders and protein inhibitors, , antisense oligonucleotides, , siRNAs, oligochalcogenides, , and cell-penetrant streptavidins . CAPA has also been applied in 3D deep tissue models and in bacteria. , To the best of our knowledge, CAPA has not been used to measure cytosolic penetration of proteins delivered using LNPs.…”

Section: Introductionmentioning

confidence: 99%

“…28 Recently, we developed an assay called the chloroalkane penetration assay (CAPA). 29−31 Our initial work applied CAPA to measure the cytosolic delivery of cell-penetrant peptides, 29,30 and since then, we and others have adapted CAPA for measuring cytosolic delivery of DNA-encoded libraries, 32−34 peptoids, 35,36 small molecules including bivalent chemical degraders and protein inhibitors, 37,38 antisense oligonucleotides, 39,40 siRNAs, 41 oligochalcogenides, 42,43 and cell-penetrant streptavidins. 44 CAPA has also been applied in 3D deep tissue models and in bacteria.…”

Section: ■ Introductionmentioning

confidence: 99%

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Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay

Wang,

Zhang,

et al. 2024

Biochemistry

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Protein therapeutics are an expanding area for research and drug development, and lipid nanoparticles (LNPs) are the most prominent nonviral vehicles for protein delivery. The most common methods for assessing protein delivery by LNPs include assays that measure the total amount of protein taken up by cells and assays that measure the phenotypic changes associated with protein delivery. However, assays for total cellular uptake include large amounts of protein that are trapped in endosomes or are otherwise nonfunctional. Assays for functional delivery are important, but the readouts are indirect and amplified, limiting the quantitative interpretation. Here, we apply an assay for cytosolic delivery, the chloroalkane penetration assay (CAPA), to measure the cytosolic delivery of a (−30) green fluorescent protein (GFP) fused to Cre recombinase (Cre(−30)GFP) fusion protein by LNPs. We compare these data to the data from total cellular uptake and functional delivery assays to provide a richer analysis of uptake and endosomal escape for LNP-mediated protein delivery. We also use CAPA for a screen of a small library of lipidoids, identifying those with a promising ability to deliver Cre(−30)GFP to the cytosol of mammalian cells. With careful controls and optimized conditions, we expect that CAPA will be a useful tool for investigating the rate, efficiency, and mechanisms of LNPmediated delivery of therapeutic proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay

Wang,

Zhang,

et al. 2024

Biochemistry

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“…In this paper, two amphiphilic compounds ( LP-4DNJ-3C and LP-4DNJ-6C ) based on 1-deoxynojirimycin and α-peptoid conjugates with the octadecyl chain as the lipophilic group were designed and synthesized. Peptoids are the mimetics of the peptides, which exhibit some advantages compared with the peptides, such as chain flexibility, cell permeability, biocompatibility, and excellent stability with proteolytic enzymes in vivo . − 1-Deoxynojirimycin (DNJ) was isolated from white mulberry root barks, which was a potent inhibitor of α-glucosidases and has been widely used to construct multivalent glycosidase inhibitors . Additionally, the self-assembly behaviors, α-glucosidase inhibition activity, and hypoglycemic effect of LP-4DNJ-3C and LP-4DNJ-6C were studied.…”

Section: Introductionmentioning

confidence: 99%

Amphiphilic α-Peptoid-deoxynojirimycin Conjugate-based Multivalent Glycosidase Inhibitor for Hypoglycemic Effect and Fluorescence Imaging

Wang,

Yan,

Rong

et al. 2024

J. Med. Chem.

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Multivalent glycosidase inhibitors based on 1-deoxynojirimycin derivatives against α-glucosidases have been rapidly developed. Nonetheless, the mechanism based on self-assembled multivalent glucosidase inhibitors in living systems needs to be further studied. It remains to be determined whether the self-assembly possesses sufficient stability to endure transit through the small intestine and subsequently bind to the glycosidases located therein. In this paper, two amphiphilic compounds, 1-deoxynojirimycin and α-peptoid conjugates (LP-4DNJ-3C and LP-4DNJ-6C), were designed. Their self-assembling behaviors, multivalent α-glucosidase inhibition effect, and fluorescence imaging on living organs were studied. LP-4DNJ-6C exhibited better multivalent α-glucosidase inhibition activities in vitro. Moreover, the self-assembly of LP-4DNJ-6C could effectively form a complex with Nile red. The complex showed fluorescence quenching effect upon binding with α-glucosidases and exhibited potent fluorescence imaging in the small intestine. This result suggests that a multivalent hypoglycemic effect achieved through self-assembly in the intestine is a viable approach, enabling the rational design of multivalent hypoglycemic drugs.

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“…The smaller the CP 50 , the more cellpenetrant the ct-compound. CAPA has been broadly adopted in industry and academia, and it has been successfully applied to peptides, 15,[47][48][49][50] antisense oligonucleotides and siRNAs with different lengths and chemical modifications, 45,51,52 cyclotides, 53 peptidomimetic macrocycles, [54][55][56] PROTACs, 57 and lipid nanoparticle-mediated delivery of proteins. 58 It has even been adapted for 3D tissue models to measure deep penetration of biotherapeutics, 59 and for bacterial cells to evaluate the bacterial penetration of small molecules.…”

Section: Introductionmentioning

confidence: 99%

Comparing cell penetration of biotherapeutics across human cell lines

Batistatou,

Kritzer

2024

Preprint

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A major obstacle in biotherapeutics development is maximizing cell penetration. Ideally, assays would allow for optimization of cell penetration in the cell type of interest early in the drug development process. However, few assays exist to compare cell penetration across different cell types, independent of drug function. In this work, we applied the chloroalkane penetration assay (CAPA) in seven mammalian cell lines as well as primary cells. Careful controls were used to ensure data could be compared across cell lines. We compared the penetration of several peptides and drug-like oligonucleotides and saw significant differences among the cell lines. To help explain these differences, we quantified the relative activities of endocytosis pathways in these cell lines and correlated them with the penetration data. Based on these results, we knocked down clathrin in a cell line with an efficient permeability profile and observed reduced penetration of peptides, but not oligonucleotides. Finally, we used small-molecule endosomal escape enhancers and observed enhancement of cell penetration of some oligonucleotides, but only in some of the cell lines tested. CAPA data provide valuable points of comparison among different cell lines, including primary cells, for evaluating the cell penetration of various classes of peptides and oligonucleotides.

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Evaluation of the cell permeability of bicyclic peptoids and bicyclic peptide-peptoid hybrids

Cited by 9 publications

References 59 publications

Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay

Investigating the Cytosolic Delivery of Proteins by Lipid Nanoparticles Using the Chloroalkane Penetration Assay

Amphiphilic α-Peptoid-deoxynojirimycin Conjugate-based Multivalent Glycosidase Inhibitor for Hypoglycemic Effect and Fluorescence Imaging

Comparing cell penetration of biotherapeutics across human cell lines

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