A comparative protease stability study of synthetic macrocyclic peptides that mimic two endocrine hormones
Department of Chemistry, The College of New Jersey, P.O. Box 7718, Ewing, NJ 08628, USA.Bioorganic & medicinal chemistry letters (Impact Factor: 2.42). 12/2012; 23(4). DOI: 10.1016/j.bmcl.2012.12.041
Peptide therapeutics have traditionally faced many challenges including low bioavailability, poor proteolytic stability and difficult cellular uptake. Conformationally constraining the backbone of a peptide into a macrocyclic ring often ameliorates these problems and allows for the development of a variety of new drugs. Such peptide-based pharmaceuticals can enhance the multi-faceted functionality of peptide side chains, permitting the peptides to bind cellular targets and receptors necessary to impart their role, while protecting them from degrading cellular influences. In the work described here, we developed three cyclic peptides, VP mimic1, VP mimic2 and OT mimic1, which mimic endocrine hormones vasopressin and oxytocin. Making notable changes to the overall structure and composition of the parent hormones, we synthesized the mimics and tested their durability against treatment with three proteases chosen for their specificity: pepsin, alpha-chymotrypsin, and pronase. Vasopressin and oxytocin contain a disulfide linkage leaving them particularly vulnerable to deactivation from the reducing environment inside the cell. Thus, we increased the complexity of our assays by adding reducing agent glutathione to each mixture. Subsequently, we discovered each of our mimics withstood protease treatment with less degradation and/or a slower rate of degradation as compared to both parent hormones and a linear control peptide.
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ABSTRACT: α-helices are the most common form of secondary structure found in proteins. In order to study controlled protein folding, as well as manipulate the interface of helical peptides with targets in protein-protein interactions, many techniques have been developed to induce and stabilize α-helical structure in short synthetic peptides. Furthermore, short, non-natural β-peptides have been established that fold into predictable 14-helices that mimic α-helical structure. We created a panel of short 6-8 residue α- and β-peptides that used confirmed primary sequence design features which influence helical control and directly compared the helicity across peptides with the most minimal epitopes. Using CD spectroscopy, we found that both α- and β-peptides abided by their respective design principles, with no significant "cross-helicity" inducing an α- or a β-peptide to fold into the oppositely controlled helix. Generally, the β-peptide of the most optimal sequence displayed the largest percent of 14-helicity, whereas the two α-peptides of most favorable design showed some α-helicity and a marked 310-helical contribution. Overall, the results can inform future peptidomimetic designs, especially in the development of short, structured peptides with biological function.Journal of biomolecular Structure & Dynamics 03/2014; 33(3). DOI:10.1080/07391102.2014.897260 · 2.92 Impact Factor
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ABSTRACT: Protease stability is a key consideration in the development of peptide-based drugs. A major approach to increase the bioavailability of pharmacologically active peptides is the incorporation of non-natural amino acids. Due to the unique properties of fluorine, fluorinated organic molecules have proven useful in the development of therapeutically active small molecules as well as in materials and crop science. This study presents data on the ability of fluorinated amino acids to influence proteolytic stability when present in peptide sequences that are based on ideal protease substrates. Different model peptides containing fluorinated amino acids or ethylglycine in the P2, P1'or P2' positions were designed according to the specificities of the serine protease, α-chymotrypsin (EC 220.127.116.11) or the aspartic protease, pepsin (EC 18.104.22.168). The proteolytic stability of the peptides toward these enzymes was determined by an analytical RP-HPLC assay with fluorescence detection and compared to a control sequence. Molecular modeling was used to support the interpretation of the structure-activity relationship based on the analysis of potential ligand-enzyme interactions. Surprisingly, an increase in proteolytic stability was observed only in a few cases. Thus, this systematic study shows that the proteolytic stability of fluorinated peptides is not predictable, but rather is a very complex phenomenon that depends on the particular enzyme, the position of the substitution relative to the cleavage site and the fluorine content of the side chain.Amino Acids 09/2014; 46(12). DOI:10.1007/s00726-014-1819-7 · 3.29 Impact Factor
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