Disulfide-rich Peptides Research Articles

Design, Synthesis, and Characterization of a Single-Chain Peptide Antagonist for the Relaxin-3 Receptor RXFP3

Relaxin-3 is a two-chain disulfide-rich peptide that is the ancestral member of the relaxin peptide family and, together with its G protein-coupled receptor RXFP3, is highly expressed in the brain. Strong evolutionary conservation of relaxin-3 suggests a critical biological function and recent studies have demonstrated modulation of sensory, neuroendocrine, metabolic, and cognitive systems. However, detailed studies of central relaxin-3-RXFP3 signaling have until now been severely hampered by the lack of a readily available high-affinity antagonist for RXFP3. Previous studies have utilized a complex two-chain chimeric relaxin peptide, R3(BΔ23-27)R/I5, in which a truncated relaxin-3 B-chain carrying an additional C-terminal Arg residue was combined with the insulin-like peptide 5 (INSL5) A-chain. In this study we demonstrate that, by replacing the native Cys in this truncated relaxin-3 B-chain with Ser, a single-chain linear peptide of 23 amino acids that retains high-affinity antagonism for RXFP3 can be achieved. In vivo studies demonstrate that this peptide, R3 B1-22R, antagonized relaxin-3/RXFP3 induced increases in feeding in rats after intracerebroventricular injection. Thus, R3 B1-22R represents an excellent tool for biological studies probing relaxin pharmacology and a lead molecule for the development of synthetically tractable, single-chain RXFP3 modulators for clinical use.

PredCSF: An Integrated Feature-Based Approach for Predicting Conotoxin Superfamily

Conotoxins are small disulfide-rich peptides that are invaluable channel-targeted peptides and target neuronal receptors. They show prospects for being potent pharmaceuticals in the treatment of Alzheimer's disease, Parkinson's disease, and epilepsy. Accurate and fast prediction of conotoxin superfamily is very helpful towards the understanding of its biological and pharmacological functions especially in the post-genomic era. In the present study, we have developed a novel approach called PredCSF for predicting the conotoxin superfamily from the amino acid sequence directly based on fusing different kinds of sequential features by using modified one-versus-rest SVMs. The input features to the PredCSF classifiers are composed of physicochemical properties, evolutionary information, predicted second structure and amino acid composition, where the most important features are further screened by random forest feature selection to improve the prediction performance. The prediction results show that PredCSF can obtain an overall accuracy of 90.65% based on a benchmark dataset constructed from the most recent database, which consists of 4 main conotoxin superfamilies and 1 class of non-conotoxin class. Systematic experiments also show that combing different features is helpful for enhancing the prediction power when dealing with complex biological problems. PredCSF is expected to be a powerful tool for in silico identification of novel conotonxins and is freely available for academic use at http://www.csbio.sjtu.edu.cn/bioinf/PredCSF.

NMR and protein structure in drug design: application to cyclotides and conotoxins

Nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for determining the structures, dynamics and interactions of molecules, and the derived information can be useful in drug design applications. This article gives a brief overview of the role of NMR in drug design and illustrates this role with examples studied in our laboratory in recent years on disulfide-rich peptides, including cyclotides and conotoxins. Cyclotides are head-to-tail cyclized proteins from plants that are exceptionally stable and hence make useful templates for the stabilization of bioactive peptide epitopes as well as potential leads for anti-HIV drugs. Natural cyclotides target cell membranes, so understanding cyclotide-membrane interactions is useful in applying cyclotides in drug design applications. NMR studies of these interactions are described in this article. Conotoxins are disulfide-rich peptides, from the venoms of marine cone snails, which are of pharmaceutical interest because they potently interact with a range of ion channels, transporters and other receptor sites implicated in disease states. Chemically re-engineering conotoxins to give them a cyclic backbone has been used to engender them with improved biopharmaceutical properties, such as are observed in cyclotides.

Spider-Venom Peptides as Therapeutics

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.

ChemInform Abstract: Native Chemical Ligation Applied to the Synthesis and Bioengineering of Circular Peptides and Proteins

AbstractReview: 69 refs.

Disulfide-Depleted Selenoconopeptides: Simplified Oxidative Folding of Cysteine-Rich Peptides

Despite the therapeutic promise of disulfide-rich, peptidic natural products, their discovery and structure/function studies have been hampered by inefficient oxidative folding methods for their synthesis. Here we report that converting the three disulfide-bridged mu-conopeptide KIIIA into a disulfide-depleted selenoconopeptide (by removal of a noncritical disulfide bridge and substitution of a disulfide- with a diselenide-bridge) dramatically simplified its oxidative folding while preserving the peptide's ability to block voltage-gated sodium channels. The simplicity of synthesizing disulfide-depleted selenopeptide analogs containing a single disulfide bridge allowed rapid positional scanning at Lys7 of mu-KIIIA, resulting in the identification of K7L as a mutation that improved the peptide's selectivity in blocking a neuronal (Na(v)1.2) over a muscle (Na(v)1.4) subtype of sodium channel. The disulfide-depleted selenopeptide strategy offers regioselective folding compatible with high throughput chemical synthesis and on-resin oxidation methods, and thus shows great promise to accelerate the use of disulfide-rich peptides as research tools and drugs.

Identification of Conus Peptidylprolyl Cis-Trans Isomerases (PPIases) and Assessment of Their Role in the Oxidative Folding of Conotoxins

Peptidylprolyl cis-trans isomerases (PPIases) are ubiquitous proteins that catalyze the cis-trans isomerization of prolines. A number of proteins, such as Drosophila rhodopsin and the human immunodeficiency viral protein HIV-1 Gag, have been identified as endogenous substrates for PPIases. However, very little is known about the interaction of PPIases with small, disulfide-rich peptides. Marine cone snails synthesize a wide array of cysteine-rich peptides, called conotoxins, many of which contain one or more prolines or hydroxyprolines. To identify whether PPIase-associated cis-trans isomerization of these residues affects the oxidative folding of conotoxins, we identified, sequenced, and expressed three functionally active isoforms of PPIase from the venom gland of Conus novaehollandiae, and we characterized their ability to facilitate oxidative folding of conotoxins in vitro. Three conotoxins, namely mu-GIIIA, mu-SIIIA, and omega-MVIIC, derived from two distinct toxin gene families were assayed. Conus PPIase significantly increased the rate of appearance of the native form of mu-GIIIA, a peptide containing three hydroxyprolines. In contrast, the presence of PPIase had no effect on the folding of mu-SIIIA and omega-MVIIC, peptides containing no or one proline residue, respectively. We further showed that an endoplasmic reticulum-resident PPIase isoform facilitated folding of mu-GIIIA more efficiently than two cytosolic isoforms. This is the first study to demonstrate PPIase-assisted folding of conotoxins, small disulfide-rich peptides with unique structural properties.

Site-Specific Effects of Diselenide Bridges on the Oxidative Folding of a Cystine Knot Peptide, ω-Selenoconotoxin GVIA

Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.

Invited reviewnative chemical ligation applied to the synthesis and bioengineering of circular peptides and proteins

Native chemical ligation methodology developed in the laboratory of Stephen Kent is a versatile approach to the linkage of peptide fragments using a native peptide bond. It is readily adaptable to the task of joining the N- and C-termini of peptides to produce cyclic molecules and we have used it for the cyclization of a range of disulfide-rich peptides. Specifically, it has been valuable for the synthesis of cyclotides, naturally occurring peptides characterized by a head-to-tail cyclized backbone and a knotted arrangement of three conserved disulfide bonds. Cyclotides have a diverse range of biological activities, including anti-HIV, antimicrobial, and insecticidal activities. They are ultrastable owing to their cyclic cystine knot motif, and native chemical ligation methodology has been invaluable in the synthesis of a range of native and modified cyclotides to explore their structure-activity relationships and applications in drug design. Similar studies have also been applied to a smaller cyclic peptide produced in sunflower seeds, sunflower trypsin inhibitor-1, which also shows promise as a template in drug design applications. We have also found native chemical ligation to be a valuable methodology for the cyclization of conotoxins, small disulfide-rich peptides from the venoms of marine cone snails. Conotoxins target a range of ions channels and receptors and are exciting leads in drug design applications. The synthetic cyclization of conotoxins with peptide linkers stabilizes them and improves their biopharmaceutical properties. In summary, this article illustrates the use of native chemical ligation technology in the cyclization of cyclotides, sunflower trypsin inhibitor-1, and conotoxins in our laboratory.

Cyclotides: macrocyclic peptides with applications in drug design and agriculture.

Cyclotides are disulfide-rich peptides from plants that are exceptionally stable as a result of their unique cyclic cystine knot structural motif. Their natural role is thought to be as plant defence agents, most notably against insect pests, but they also have potential applications in drug design and agriculture. This article identifies gaps in current knowledge on cyclotides and suggests future directions for research into this fascinating family of ultra-stable mini-proteins.

The M-superfamily of conotoxins: a review

The focus of this review is the M-superfamily of Conus venom peptides. Disulfide rich peptides belonging to the M-superfamily have three loop regions and the cysteine arrangement: CC-C-C-CC, where the dashes represent loops one, two, and three, respectively. Characterization of M-superfamily peptides has demonstrated that diversity in cystine connectivity occurs between different branches of peptides even though the cysteine pattern remains consistent. This superfamily is subdivided into five branches, M-1 through M-5, based on the number of residues in the third loop region, between the fourth and fifth cysteine residues. M-superfamily peptides appear to be ubiquitous in Conus venom. They are largely unexplained in indigenous biological function, and they represent an active area of research within the scientific community.

Scanning Mutagenesis of α-Conotoxin Vc1.1 Reveals Residues Crucial for Activity at the α9α10 Nicotinic Acetylcholine Receptor

Vc1.1 is a disulfide-rich peptide inhibitor of nicotinic acetylcholine receptors that has stimulated considerable interest in these receptors as potential therapeutic targets for the treatment of neuropathic pain. Here we present an extensive series of mutational studies in which all residues except the conserved cysteines were mutated separately to Ala, Asp, or Lys. The effect on acetylcholine (ACh)-evoked membrane currents at the alpha9alpha10 nicotinic acetylcholine receptor (nAChR), which has been implicated as a target in the alleviation of neuropathic pain, was then observed. The analogs were characterized by NMR spectroscopy to determine the effects of mutations on structure. The structural fold was found to be preserved in all peptides except where Pro was substituted. Electrophysiological studies showed that the key residues for functional activity are Asp(5)-Arg(7) and Asp(11)-Ile(15), because changes at these positions resulted in the loss of activity at the alpha9alpha10 nAChR. Interestingly, the S4K and N9A analogs were more potent than Vc1.1 itself. A second generation of mutants was synthesized, namely N9G, N9I, N9L, S4R, and S4K+N9A, all of which were more potent than Vc1.1 at both the rat alpha9alpha10 and the human alpha9/rat alpha10 hybrid receptor, providing a mechanistic insight into the key residues involved in eliciting the biological function of Vc1.1. The most potent analogs were also tested at the alpha3beta2, alpha3beta4, and alpha7 nAChR subtypes to determine their selectivity. All mutants tested were most selective for the alpha9alpha10 nAChR. These findings provide valuable insight into the interaction of Vc1.1 with the alpha9alpha10 nAChR subtype and will help in the further development of analogs of Vc1.1 as analgesic drugs.

CDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions

We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

Rapid sensitive analysis of cysteine rich peptide venom components

Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low, we combined ETD with a targeted chemical derivatization strategy to increase the charge state of cysteine-containing peptide toxins. Using this strategy, we obtained full sequences for 31 peptide toxins, using just 7% of the crude venom from the venom gland of a single cone snail (Conus textile).

Solution structure of Hyp10Pro variant of conomarphin, a cysteine-free and d-amino-acid containing conopeptide

Conotoxins are mainly disulfide-rich short peptides active on different ion channels, neurotransmitter receptors or transporters in nervous system, exhibiting highly diversified composition, structures and biological functions. Besides these kinds of conopeptides, some novel cysteine-free conopeptides have also been reported. Conomarphin, a cystine-free 15-residue conopeptide from Conus marmoreus, has been purified and classified into M-superfamily. In addition to its unique characteristic of a D-type phenylalanine at the third residue from the C-terminus, conomarphin has an unusual hydroxyproline residue at position 10. To make an effort to understand the role of hydroxyproline post-translational modification in conomarphin, 1H NMR solution structure of Hyp10Pro variant of conomarphin was resolved and compared with the native conomarphin in the present work. The Hyp10Pro conomarphin has a type II-β-turn near the C-terminus instead of a 3 10 helix in native conomarphin. The compact loop region in native conomarphin becomes more open when hydroxyproline is displaced by proline. This reveals that hydroxyproline residue is essential for the structure of conomarphin just like D-Phe13. The unusual post-translational modification of conomarphin implies a unique selectivity of hydroxylation in toxin sequence.

Detailed Atomistic Molecular Dynamics Simulations of α-Conotoxin AuIB in Water

We present results about the shape, size, structure, conformational stability, and hydrodynamics of alpha-conotoxin AuIB (a disulfide-rich peptide from the venom of Conus aulicus, recognized as a nicotinic acetylcholine antagonist with great pharmaceutical potential) from very long (0.5 mus) massively parallel molecular dynamics (MD) simulations in full atomistic detail. We extract coarse-grained descriptors of protein shape (ellipsoid), and of translational and rotational mobilities, i.e., the basic components at the lowest hierarchical level in a multiscale modeling strategy. Structural analysis reveals the folded conformation and asymmetric shape to be strongly favored for conotoxin. In accordance with experimental findings, conformational stability is observed and found to be linked to the presence of the alpha-helix along the 15 residues and to the existence of the two disulfide bonds. We find rotational (D(r)) and translational (D(t)) diffusivities to be suitable descriptors of coarse-grained dynamics, i.e., of the hydrodynamic behavior, and obtain D(r) = 3.62 (+/-0.17) x 10(8) s(- 1) and D(t) = 1.08 (+/-0.4) x 10(- 10) m(2) s(- 1). We further compare the MD-computed coarse-grained descriptors with first principles theoretical predictions based on the extended Hess-Doi Fokker-Planck approach which relates particle shape and dimensions to diffusion coefficients. An excellent agreement between simulation data and analytical predictions is observed for both dynamical descriptors. This comparison strongly suggests that diffusivities of rigid biomolecules much larger than the alpha-conotoxin AuIB studied here can be obtained from the coarse-grained shape descriptor (ellipsoid) derived from relatively short MD simulations.

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Structural studies of conotoxins

Conotoxins are small disulfide-rich peptides from the venoms of marine cone snails. They target a variety of ion channels, transporters, and receptors besides the interest in their natural functions in venoms and they are of much interest as drug leads. This short article gives an overview of the structural diversity of conotoxins, and illustrates this diversity with recent selected examples of conotoxin structures.

Conotoxins: natural product drug leads

Venomous marine cone snails harbour a variety of small disulfide-rich peptides called conotoxins, which target a broad range of ion channels, membrane receptors, and transporters. More than 700 species of Conus are thought to exist, each expressing a wide array of different peptides. Within this large repertoire of toxins, individual conotoxins are able to discriminate between different subtypes and isoforms of ion channels, making them valuable pharmacological probes or leads for drug design. This review gives a brief background to the discovery of conotoxins and describes their sequences, biological activities, and applications in drug design.

NMR-based mapping of disulfide bridges in cysteine-rich peptides: application to the mu-conotoxin SxIIIA.

Disulfide-rich peptides represent a megadiverse group of natural products with very promising therapeutic potential. To accelerate their functional characterization, high-throughput chemical synthesis and folding methods are required, including efficient mapping of multiple disulfide bridges. Here, we describe a novel approach for such mapping and apply it to a three-disulfide-bridged conotoxin, mu-SxIIIA (from the venom of Conus striolatus), whose discovery is also reported here for the first time. Mu-SxIIIA was chemically synthesized with three cysteine residues labeled 100% with (15)N/(13)C, while the remaining three cysteine residues were incorporated using a mixture of 70%/30% unlabeled/labeled Fmoc-protected residues. After oxidative folding, the major product was analyzed by NMR spectroscopy. Sequence-specific resonance assignments for the isotope-enriched Cys residues were determined with 2D versions of standard triple-resonance ((1)H, (13)C, (15)N) NMR experiments and 2D [(13)C, (1)H] HSQC. Disulfide patterns were directly determined with cross-disulfide NOEs confirming that the oxidation product had the disulfide connectivities characteristic of mu-conotoxins. Mu-SxIIIA was found to be a potent blocker of the sodium channel subtype Na(V)1.4 (IC50 = 7 nM). These results suggest that differential incorporation of isotope-labeled cysteine residues is an efficient strategy to map disulfides and should facilitate the discovery and structure-function studies of many bioactive peptides.