NMEGylation: A novel modification to enhance the bioavailability of therapeutic peptides

Abstract

We have evaluated “NMEGylation”—the covalent attachment of a oligo-N-methoxyethylglycine (NMEG) chain—as a new form of peptide/protein modification to enhance the bioavailability of short peptides. OligoNMEGs are hydrophilic PEG-like molecules made by solid-phase synthesis, typically up to 40 monomers in length. They have been studied as non-fouling surface coatings, and as monodisperse mobility modifiers for free-solution conjugate capillary electrophoresis. However, polyNMEGs have not been demonstrated prior to this work as modifiers of therapeutic proteins. In prior published work, we identified a short peptide, “C20”, as a potential extracellular inhibitor of the fusion of human respiratory syncytial virus (hRSV) with mammalian cells. The present study was aimed at improving the C20 peptide's stability and solubility. To this end we synthesized and studied a series of NMEGylated C20 peptide-peptoid bioconjugates comprising different numbers of NMEGs at either the N- or C- terminus of C20. NMEGylation was found to greatly improve this peptide's solubility and serum stability; however, longer polyNMEGs (n > 3) deleteriously affected peptide binding to the target protein. By incorporating just one NMEG monomer, along with a glycine monomer as a flexible spacer, at C20's N-terminus (NMEG-Gly-C20), we increased both solubility and serum stability greatly, while recovering a binding affinity comparable to that of unmodified C20 peptide. Our results suggest that NMEGylation with an optimized number of NMEG monomers and a proper linker could be useful, more broadly, as a novel modification to enhance bioavailability and efficacy of therapeutic peptides.

Polypeptoids: A perfect match for molecular definition and macromolecular engineering?

“…In a similar approach, Barron et al suggested attachment of short N‐methoxyethylglycine oligomers to therapeutic proteins . Taking advantage of the facile synthesis of large libraries of structurally diverse peptoids, Zuckermann and coworkers have also studied the use of cationic peptoids as nonviral vectors for gene delivery …”

Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability

“…However, the stability gained through introduction of D-amino acids at the same key positions rendered the peptide without activity, emphasizing the importance of not hindering helical propensity. Other examples of strategies that has been tested in order to increase peptide stability for other AMPs include using all-D-amino acids (Wade et al, 1990), β-peptides and α-peptoids (Godballe et al, 2011) or by conjugating methoxyethylglycine chains to the terminals (Park et al, 2011).…”

Library Construction, Selection and Modification Strategies to Generate Therapeutic Peptide-Based Modulators of Protein–Protein Interactions

“…Peptides can be conjugated to specialized protein transduction domains [135] which include basic cellpenetrating peptides, α-helical peptides and viral fusion proteins [136], that are capable of crossing biological membranes by various mechanisms [137]. Conjugation to carrier molecules such as polyethylene glycol or the novel peptoid N-methoxyethyl glycine, or human serum proteins such as human serum albumin, slows renal clearance and thus improves tissue uptake, improves proteolytic stability and lowers immunogenicity [138,139]. Conjugation of peptides to proteinogenic moieties can be achieved simply by expression of genetically fused species, while conjugation to nonnatural molecules can be achieved either post synthesis using activated molecules reactive with amino acid side chains [140], or using synthesized amino acid derivatives during SPPS [141].…”