We evaluated quantitatively the relative cell permeability of peptoids and peptides using a cell-based reporter gene-based assay. Generally, peptoids were much more cell permeable than the corresponding peptides, though the difference decreased with increasing length. These results suggest that peptoids may be useful reagents for manipulating the activities of intracellular proteins.There is currently considerable interest in developing chemical reagents with which to manipulate protein-protein interactions as tools for chemical biology or as potential drugs. Peptides have the type of protein-binding properties required for this type of application as they are capable of recognizing the relatively large, shallow surfaces typical of interaction domains. However, peptides have well-known limitations as pharmacological agents, the most serious being sensitivity to proteases and limited cell permeability. Therefore, several different types of peptidomimetics have been investigated, 1 with the hope of identifying a family of compounds that retain the favorable protein-binding properties of peptides but exhibit superior pharmacokinetic characteristics. In this endeavor, it would be useful to have a general method with which to compare the cell permeability of a given peptidomimetic compound with that of the corresponding peptide. We report here a method that allows such comparisons to be made conveniently and quantitatively using any transfectable cell type. Using peptoids as a model system, we show that these N-alkylated species are up to 20-fold more cell-permeable than analogous peptides. Along with their other favorable properties, 2-8 these data suggest that peptoids may be useful reagents for targeting intracellular proteins.Recently we developed a high-throughput assay for assessing the relative cell permeability of synthetic molecule-dexamethasone (Dex) derivative conjugates. 9 A schematic of the system is provided in the Supplementary Material (Fig. S1). Briefly, the molecule of interest is conjugated to a Dex derivative and incubated with HeLa cells that have been transfected with three plasmids. One encodes a fusion protein comprised of the Gal4 DNA-binding domain, the Glucocorticoid Receptor ligand binding domain and the VP16 transactivation domain (Gal4DBD-GRLBD-VP16). The other two carry a Gal4-responsive firefly luciferase reporter E-mail: thomas.kodadek@utsouthwestern.edu. (pG5B), and a constitutively expressed Renilla reniformis luciferase (pRL-SV40), respectively. Gal4DBD-GRLBD-VP16 is a potentially potent activator of pG5B expression, but is held in an inactive form by tight binding of Hsp90 to the GR LBD. However, binding of the steroid to this domain competes this interaction and allows the activator to drive firefly luciferase expression. Thus, the ratio of firefly luciferase to Renilla luciferase activity in these cells is a reflection of the amount of steroid-GR LBD complex formed, which, in turn, depends on the concentration of steroid conjugate that enters the cell. NIH Public Access...
Peptides are limited in their use as drugs due to low cell permeability and vulnerability to proteases. In contrast, peptoids are immune to enzymatic degradation and some peptoids have been shown to be relatively cell permeable. In order to facilitate future design of peptoid libraries for screening experiments, it would be useful to have a high-throughput method to estimate the cell permeability of peptoids containing different residues. In this paper, we report the strengths and limitations of a high-throughput cell-based permeability assay that registers the relative ability of steroid-conjugated peptides and peptoids to enter a cell. A comparative investigation of the physicochemical properties and side chain composition of peptoids and peptides is described to explain the observed higher cell permeability of peptoids over peptides. These data suggest that the conversion of the monomeric residues in peptides to an N-alkylglycine moiety in peptoids reduced the hydrogen-bonding potential of the molecules and is the main contributor to the observed permeability improvement.
Parasite glycosylphosphatidylinositol (GPI) is an important toxin in malaria disease, and people living in malaria-endemic regions often produce high levels of anti-GPI antibodies. The natural anti-GPI antibody response needs to be understood to aid the design of an efficient carbohydrate-based antitoxin vaccine. We present a versatile approach based on a synthetic GPI glycan array to correlate anti-GPI antibody levels and protection from severe malaria.
Cyclic peptides are of considerable interest as potential protein ligands. It has been postulated that cyclic molecules might be more cell permeable than their linear counterparts due to their reduced conformational flexibility. We report a study that tests this hypothesis by using a quantitative, reporter gene-based assay that measures the relative cell permeability of steroid conjugates of molecules of interest. We demonstrate that cyclic peptides are, in fact, not generally more permeable than their linear counterparts.
A highly convergent strategy for the synthesis of fully lipidated GPI anchors of malarial origin is reported. This strategy utilized three orthogonal protecting groups, which can be chemoselectively deprotected and functionalized in the late stage of the synthesis. Rapid access to the target GPIs in a highly efficient manner in sufficient quantities for the biological studies has been achieved.
Ap ractical chemoenzymatic method for the synthesis of 9-hydroxynonanoic acid and1 ,9-nonanedioic acid (i.e., azelaic acid) from oleic acid [(9Z)-octadec-9-enoic acid] was investigated. Biotransformation of oleic acid into 9-(nonanoyloxy)nonanoic acid via 10-hydroxyoctadecanoic acid and 10keto-octadecanoic acid was driven by aC -9 double bond hydratase from Stenotrophomonas maltophilia, an alcohol dehydrogenase from Micrococcus luteus, and aB aeyer-Villiger monooxygenase (BVMO) from Pseudomonas putida KT2440, which was expressedi nr ecombinant Escherichia coli.A fter productiono ft he ester (i.e. ,t he BVMO reactionp roduct), the compound wasc hemically hydrolyzed to nnonanoic acid and9 -hydroxynonanoic acid because n-nonanoic acid is toxic to E. coli.T he ester was also converted into 9-hydroxynonanoic acid and the n-nonanoic acid methyle ster, which can be oxygenated into the 9-hydroxynonanoic acid methyl ester by the AlkBGT from P. putida GPo1. Finally,9 -hydroxynonanoic acid was chemically oxidized to azelaic acid with ah ighy ield under fairly mild reactionc onditions.F or example,w hole-cell biotransformation at ah igh cell density (i.e., 10 gd ry cells/L) allowed the final ester product concentration and volumetric productivity to reach2 5mMa nd 2.8 mM h À1 ,r espectively.T he overall molar yield of azelaic acid from oleic acid was 58%, based on the biotransformation and chemical transformation conversion yields of 84% and 68%, respectively.Scheme 2. Chemical conversion of the ester intermediate (4)toa zelaic acid (7).
We report an efficient and convergent synthesis of a series of oligosaccharides comprised of the malaria GPI glycan (2a), a promising anti-malaria vaccine candidate currently in preclinical trials and several related oligosaccharide sequences (3-8) that are possible biosynthetic precursors of the malarial GPI. A flexible synthetic strategy is disclosed that relies on a late-stage coupling between oligomannosides of varying length and pseudo-disaccharide glycosyl acceptor 11 to readily access various malarial GPI structures. Phosphorylation was accomplished by mild and efficient H-phosphonate chemistry before the final deprotection was carried out by using sodium in ammonia. The direct connection of a thiol group via a phosphate diester linkage to the inositol moiety provides a handle for easy conjugation of the GPI glycan to carrier proteins, immobilization on carbohydrate microarrays and photo-affinity labels identification. These synthetic oligosaccharides will serve as molecular probes.
A practical chemoenzymatic synthetic method for 11-hydroxyundecanoic acid and 1,11-undecanedioic acid from ricinoleic acid (12-hydroxyoleic acid) was investigated.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.