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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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A safety-catch cysteine protecting group, S-4,4'-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 nM (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Vicinal disulfide bridges, in which a disulfide bond is formed between adjacent cysteine residues, constitute an unusual but expanding class of potential allosteric disulfides. Although vicinal disulfide rings (VDRs) are relatively uncommon, they have proven to be functionally critical in almost all proteins in which they have been discovered. However, it has proved difficult to test whether these sterically constrained disulfides participate in functionally important redox transformations. We demonstrate that chemical replacement of VDRs with dicarba or diselenide bridges can be used to assess whether VDRs function as allosteric disulfides. Our approach leads to the hypothesis that not all VDRs participate in functionally important redox reactions.

Disulfide bond engineering is an important approach to improve the metabolic half-life of cysteine-containing peptides. Eleven analogues of oxytocin were synthesized including disulfide bond replacements by thioether, selenylsulfide, diselenide, and ditelluride bridges, and their stabilities in human plasma and activity at the human oxytocin receptor were assessed. The cystathionine (K(i) = 1.5 nM, and EC₅₀ = 32 nM), selenylsulfide (K(i) = 0.29/0.72 nM, and EC₅₀ = 2.6/154 nM), diselenide (K(i) = 11.8 nM, and EC₅₀ = 18 nM), and ditelluride analogues (K(i) = 7.6 nM, and EC₅₀ = 27.3 nM) retained considerable affinity and functional potency as compared to oxytocin (K(i) = 0.79 nM, and EC₅₀ = 15 nM), while shortening the disulfide bridge abolished binding and functional activity. The mimetics showed a 1.5-3-fold enhancement of plasma stability as compared to oxytocin (t(½) = 12 h). By contrast, the all-D-oxytocin and head to tail cyclic oxytocin analogues, while significantly more stable with half-lives greater than 48 h, had little or no detectable binding or functional activity.

alpha-Conotoxins are multiple disulfide bond containing peptides that are isolated from venomous marine cone snails. They display remarkable selectivity for different subtypes of nicotinic acetylcholine receptors (nAChRs). While alpha-conotoxins display poor resistance to in vivo degradation by proteases, which limits their use as drug leads, N-to-C cyclization via an oligopeptide spacer unit has been previously shown to improve stability. However, the effect of N-to-C cyclization on the formation of the disulfide bond framework is not fully understood. Four N-to-C cyclic analogs of alpha-conotoxin ImI; cImI-A, cImI-betaA, cImI-AG, and cImI-AGG were synthesized to evaluate the effect of oligopeptide spacer length on disulfide bond selectivity and stability to proteolysis. Different ratios of disulfide bond isomers were obtained for each analog using a nonselective random disulfide bond forming strategy, which was dependent on the length of the spacer. To identify each isomer obtained using the random strategy, and to gain access to disulfide bond isomers otherwise unattainable using the random strategy, both the native (globular) and ribbon isomers were synthesized in good yield and purity using a selective orthogonal cysteine protecting group strategy. As such, a random oxidation strategy showed a clear preference for the ribbon isomer in cImI-A. The cyclic globular isomers showed a high resistance to enzymatic degradation compared to the ribbon isomers, with the cImI-A and cImI-AG globular isomers demonstrating the highest stability. These results suggest that cyclization can improve the biochemical stability of conotoxins with potential applications in the development of drugs., ((c) 2010 Wiley Periodicals, Inc.)

Disulphide bonds play an important role in protein structure and function. Bovine kappa-casein (kappa-csn), an important glycoprotein in milk, contains two cysteines that can form disulphide bonds. On 2-D gels run under nonreducing conditions the kappa-csn in milk presented a complex pattern of monomers and disulphide-linked oligomers. Trains of spots corresponding to monomers to hexamers were observed as a result of the participation of different glycoforms and phosphoforms in oligomer formation. The dimers and trimers ran as doublets on the gel and analysis of the disulphide-linked peptides released from them after in-gel tryptic digestion showed they were the result of different disulphide linkages. The linkages were confirmed by MSMS. When milks with electrophoretically distinct genetic variants of kappa-csn were mixed and run on 2-D gels, they retained their distinct patterns indicating that disulphide exchange reactions or disulphide 'scrambling' was not occurring during 2-D analysis. The patterns observed represent the native distribution of kappa-csn in milk at harvest. The role and significance of the disulphide bonding of kappa-csn are discussed.

Background: Alpha-conotoxins have exciting therapeutic potential based on their high selectivity and affinity for nicotinic acetylcholine receptors. The spacing between the cysteine residues in alpha-conotoxins is variable, leading to the classification of sub-families. BuIA is the only alpha-conotoxin containing a 4/4 cysteine spacing and thus it is of significant interest to examine the structure of this conotoxin., Results: In the current study we show the native globular disulfide connectivity of BuIA displays multiple conformations in solution whereas the non-native ribbon isomer has a single well-defined conformation. Despite having multiple conformations in solution the globular form of BuIA displays activity at the nicotinic acetylcholine receptor, contrasting with the lack of activity of the structurally well-defined ribbon isomer., Conclusion: These findings are opposite to the general trends observed for alpha-conotoxins where the native isomers have well-defined structures and the ribbon isomers are generally disordered. This study thus highlights the influence of the disulfide connectivity of BuIA on the dynamics of the three-dimensional structure.

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NMR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-Hi:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-Hi:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing the presence of a triple-stranded antiparallel beta sheet consistent with the inhibitor cystine-knot (ICK) motif.

Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC50 17.85 μ m) while inhibiting apoptosis (EC50 2.2 μ m). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors. [ABSTRACT FROM AUTHOR]

Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer A chain and a 23-mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the individual synthetic chains through aerial oxidation remarkably resulted in the self-assembly of Mp1a as a homogenous product without the need for directed disulfide-bond formation. NMR analysis revealed a well-defined, unique structure containing an antiparallel α-helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non-specific Ca2+ influx in SH-SY5Y cells with a half maximal effective concentration (EC50) of 4.3 μ m. Mp1a also displayed broad-spectrum antimicrobial activity, with the highest potency against Gram-negative Acinetobacter baumannii (MIC 25 n m). Intraplantar injection (10 μ m) in mice elicited spontaneous pain and mechanical allodynia. Single- and two-chain mimetics of Mp1a revealed functional selectivity. [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]

A safety-catch cysteine protecting group, S-4,4′-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 n M (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells. [ABSTRACT FROM AUTHOR]

Background Most ant venoms consist predominantly of small linear peptides, although some contain disulfide-linked peptides as minor components. However, in striking contrast to other ant species, some Anochetus venoms are composed primarily of disulfide-rich peptides. In this study, we investigated the venom of the ant Anochetus emarginatus with the aim of exploring these novel disulfide-rich peptides. Methods The venom peptidome was initially investigated using a combination of reversed-phase HPLC and mass spectrometry, then the amino acid sequences of the major peptides were determined using a combination of Edman degradation and de novo MS/MS sequencing. We focused on one of these peptides, U 1 -PONTX-Ae1a (Ae1a), because of its novel sequence, which we predicted would form a novel 3D fold. Ae1a was chemically synthesized using Fmoc chemistry and its 3D structure was elucidated using NMR spectroscopy. The peptide was then tested for insecticidal activity and its effect on a range of human ion channels. Results Seven peptides named poneritoxins (PONTXs) were isolated and sequenced. The three-dimensional structure of synthetic Ae1a revealed a novel, compact scaffold in which a C-terminal β-hairpin is connected to the N-terminal region via two disulfide bonds. Synthetic Ae1a reversibly paralyzed blowflies and inhibited human L-type voltage-gated calcium channels (Ca V 1). Conclusions Poneritoxins from Anochetus emarginatus venom are a novel class of toxins that are structurally unique among animal venoms. General significance This study demonstrates that Anochetus ant venoms are a rich source of novel ion channel modulating peptides, some of which might be useful leads for the development of biopesticides. [ABSTRACT FROM AUTHOR]

In the past 100 years minimal venom research has focused on ticks despite several species possessing a highly paralytic and lethal venom cocktail of proteinaceous molecules. The saliva of the Australian paralysis tick, Ixodes holocyclus , has been responsible for 20 human fatalities from 1900 to 1945, and up to 100,000 domestic animal fatalities annually. In the last 50 years, research on this tick has focused on identifying the neurotoxins present in the saliva and in the last ten years the sequence of a potential neurotoxin, HT-1, has been determined. In this study we chemically synthesised HT-1 using Boc-chemistry in combination with native chemical ligation. Following successful oxidative folding, we determined the three-dimensional structure of HT-1 by NMR spectroscopy and found a novel structural fold with three of the four disulfide bonds comprising the inhibitory cystine knot (ICK) motif. The fourth disulfide bond connects the second loop to the N-terminal, which decreases the flexibility of the structure. [ABSTRACT FROM AUTHOR]