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Ziconotide (ω‐conotoxin MVIIA) is an approved analgesic for the treatment of chronic pain. However, the need for intrathecal administration and adverse effects have limited its widespread application. Backbone cyclization is one way to improve the pharmaceutical properties of conopeptides, but so far chemical synthesis alone has been unable to produce correctly folded and backbone cyclic analogues of MVIIA. In this study, an asparaginyl endopeptidase (AEP)‐mediated cyclization was used to generate backbone cyclic analogues of MVIIA for the first time. Cyclization using six‐ to nine‐residue linkers did not perturb the overall structure of MVIIA, and the cyclic analogues of MVIIA showed inhibition of voltage‐gated calcium channels (CaV2.2) and substantially improved stability in human serum and stimulated intestinal fluid. Our study reveals that AEP transpeptidases are capable of cyclizing structurally complex peptides that chemical synthesis cannot achieve and paves the way for further improving the therapeutic value of conotoxins. [ABSTRACT FROM AUTHOR]

In contrast to Möbius and trypsin inhibitor cyclotides, members of the bracelet subfamily are typically intractable to chemical synthesis and folding. In a significant advance in the field, the bracelet cyclotides ribe 33 and Cter 27 were successfully produced synthetically in moderate yield in a recent study. That synthetic method was a breakthrough as members of the bracelet subfamily of cyclotides had hitherto eluded attempts to be synthetically produced, apart from one report of cyO2 production in which a complicated folding strategy was used. In the current study the successful in vitro folding of three mutants of bracelet cyclotide Cter 27 is reported. This study broadens our understanding of the folding of bracelet cyclotides and elucidates the three dimensional structure of synthetic Cter 27, providing a new class of cyclotide molecular grafting scaffold for drug design applications. [ABSTRACT FROM AUTHOR]

Ruthenium‐catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5‐disubstituted 1,2,3‐triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross‐linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor‐1. NMR and X‐ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole‐bridged peptides also displayed superior half‐lives in liver S9 stability assays compared to disulfide‐bridged peptides. This work establishes a foundation for the application of 1,5‐disubstituted 1,2,3‐triazoles as disulfide mimetics. [ABSTRACT FROM AUTHOR]

The design of disulfide bond mimetics is an important strategy for optimising cysteine-rich peptides in drug development. Mimetics of the drug lead conotoxin MrIA, in which one disulfide bond is selectively replaced of by a 1,4-disubstituted-1,2,3-triazole bridge, are described. Sequential copper-catalyzed azide-alkyne cycloaddition (CuAAC; click reaction) followed by disulfide formation resulted in the regioselective syntheses of triazole-disulfide hybrid MrIA analogues. Mimetics with a triazole replacing the Cys4-Cys13 disulfide bond retained tertiary structure and full in vitro and in vivo activity as norepinephrine reuptake inhibitors. Importantly, these mimetics are resistant to reduction in the presence of glutathione, thus resulting in improved plasma stability and increased suitability for drug development. [ABSTRACT FROM AUTHOR]

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ABSTRACT The cyclotides are a family of small head-to-tail cyclic plant defense proteins. In addition to their cyclic backbone, cyclotides comprise three disulfide bonds in a knotted arrangement, resulting in a highly cross-braced structure that provides exceptional chemical and proteolytic stability. A number of bioactivities have been associated with cyclotides, including insecticidal, antimicrobial, anti-viral and cytotoxic, and these activities are related to an ability to target and disrupt biological membranes. Kalata B2 and to a lesser extent kalata B1, isolated from Oldenlandia affinis, self-associate to tetramers and octamers in aqueous buffers, and this oligomerization has been suggested to be relevant for their ability to form pores in membranes. Here we demonstrate by solution NMR spectroscopy analysis that the oligomerization of kalata B2 is concentration dependent and that it involves the packing of hydrophobic residues normally exposed on the surface of kalata B2 into a multimeric hydrophobic core. Interestingly, the hydrophobic surface that is 'quenched' has previously been shown to be responsible for the ability of kalata B2 to insert into membranes. Thus, it seems unlikely that the oligomers observed in aqueous solution are related to any multimeric state present in a membrane environment, and responsible for the formation of pores. The ability to self-associate might alternatively provide a mechanism for preventing self-toxicity when stored at high concentrations in intracellular compartments. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 453-460, 2013. [ABSTRACT FROM AUTHOR]

1. Excitation–contraction coupling in skeletal muscle is dependent on a physical interaction between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR). 2. A number of peptides derived from the II–III loop region of the DHPR have been shown to be functionally active in stimulating the release of calcium via RyR channels. Their function has been found to correlate with the presence of a basic helical region located at the N-terminus of the II–III loop. 3. The entire recombinant skeletal DHPR II–III loop is an efficient activator of RyR1 and RyR2. 4. The skeletal DHPR II–III loop is comprised of a series of a-helices, but its tertiary structure has been determined to be unstructured and flexible. 5. Fluorescence quenching experiments have been used to identify and measure the binding affinity of the II–III loop with fragments of the RyR. [ABSTRACT FROM AUTHOR]

Our aim was to determine whether lipoamino acid conjugation of peptides that are high-affinity activators of ryanodine receptor (RyR) channels would (a) render the peptides membrane permeable, (b) alter their structure or (a) reduce their activity. The peptides correspond to the A region of the II-III loop of the skeletal dihydropyridine receptor.The lipoamino acid conjugation increased the apparent permeability of the peptide across the Caco-2 cell monolayer by up to~20-fold.Nuclear magnetic resonance showed that thea-helical structure of critical basic residues, required for optimal activation of RyRs, was retained after conjugation.The conjugated peptides were more effective in enhancing resting Ca2+ release, Ca2+-induced Ca2+ release and caffeine-induced Ca2+ release from isolated sarcoplasmic reticulum (SR) than their unconjugated counterparts, and significantly enhanced caffeine-induced Ca2+ release from mechanically skinned extensor digitorum longus (EDL) fibres.The effect of both conjugated and unconjugated peptides on Ca2+ release from skeletal SR was 30-fold greater than their effect on either cardiac Ca2+ release or on the Ca2+ Mg2+ ATPase.A small and very low affinity effect of the peptide in slowing Ca2+ uptake by the Ca2+, Mg2+ ATPase was exacerbated by lipoamino acid conjugation in both isolated SR and in skinned EDL fibres.The results show that lipoamino acid conjugation of A region peptides increases their membrane permeability without impairing their structure or efficacy in activating skeletal and cardiac RyRs.British Journal of Pharmacology (2005) 144, 743-754. doi:10.1038/sj.bjp.0705981 [ABSTRACT FROM AUTHOR]

Innentitelbild: Application and Structural Analysis of Triazole-Bridged Disulfide Mimetics in Cyclic Peptides (Angew. Keywords: disulfide mimetics; inhibitors; peptides; peptidomimetics; triazole bridges EN disulfide mimetics inhibitors peptides peptidomimetics triazole bridges 11258 11258 1 07/03/20 20200706 NES 200706 B 1,5-Disubstituierte 1,2,3-Triazole b können als Mimetika von Disulfidbindungen genutzt werden. Disulfide mimetics, inhibitors, peptides, peptidomimetics, triazole bridges. [Extracted from the article]

The Oxygen Diffusion Barrier Hypothesis states that aging in the liver is associated with restricted oxygen uptake that explains the age-related impairment of phase I drug clearance observed in vivo with preservation of in vitro phase I enzyme activity and in vivo phase II drug clearance. Aging in the liver may be secondary to oxidative stress. Therefore we examined the effects of oxidative injury on oxygen uptake, and phase I and phase II drug metabolism in the liver. Oxidative stress was induced in the perfused rat liver with hydrogen peroxide. The intrinsic clearances of propranolol and morphine were used as markers of phase I and phase II activity, respectively. Oxidative injury was associated with a 14$pL9% (P=0.03) reduction in oxygen uptake. The decrease in the intrinsic clearance of propranolol was greater than that of morphine (57$pL14% vs 34$pL7% P<0.005). This result supports the concept of a restriction of oxygen supply constraining hepatic drug metabolism following oxidative stress. This has implications for aging and hepatic drug metabolism. [ABSTRACT FROM AUTHOR]