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Find video protocols related to scientific articles indexed in Pubmed.
The role of disulfide bonds in structure and activity of chlorotoxin.
Future Med Chem
PUBLISHED: 11-19-2014
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Background: Chlorotoxin is a small scorpion peptide that inhibits glioma cell migration. We investigated the importance of a major component of chlorotoxin's chemical structure - four disulfide bonds - to its tertiary structure and biological function. Results: Five disulfide bond analogs of chlorotoxin were synthesized, with l-?-aminobutyric acid residues replacing each or all of the disulfide bonds. Chemical oxidation and circular dichroism experiments revealed that Cys III-VII and Cys V-VIII were essential for native structure formation. Cys I-IV and Cys II-VI were important for stability of enzymatic proteolysis but not for the inhibition of human umbilical vein endothelial cell migration. Conclusion: The disulfide bonds of chlorotoxin are important for its structure and stability and have a minor role in its activity against cell migration.
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The Evolution of Momordica Cyclic Peptides.
Mol. Biol. Evol.
PUBLISHED: 11-08-2014
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Cyclic proteins have evolved for millions of years across kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here we show that cyclic mini-proteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 million years ago. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired via a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases.
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Insights Into the Molecular Flexibility of Theta-Defensins by NMR Relaxation Analysis.
J Phys Chem B
PUBLISHED: 11-07-2014
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?-defensins are mammalian cyclic peptides that have antimicrobial activities and show potential as stable scaffolds for peptide-based drug design. The cyclic cystine ladder structural motif of ?-defensins has been characterized using NMR spectroscopy and is important for their structure and stability. However, the effect of the pronounced elongated topology of ?-defensins on their molecular motion is not yet understood. Studies of molecular motion by NMR relaxation measurements have been facilitated by the recent development of a semi-recombinant method for producing cyclic peptides that allows for isotopic labeling. Here we undertook a multi-field 15N NMR relaxation analysis of the anti-HIV ?-defensin, HTD-2, and interpreted the experimental data using various models of overall and internal molecular motion. We found that it was necessary to apply a model that includes internal motion to account for the variations in the experimental T1 and NOE data at different backbone amide sites in the peptide. Although an isotropic model with internal motion was the simplest model that provided a satisfactory fit with the experimental data, we cannot exclude the possibility that overall motion is anisotropic, especially considering the strikingly elongated topology of ?-defensins. The presence of flexible side chains, self-association, interactions with solvent, and internal motions are all potential contributors to the observed relaxation data. Internal motions consistent with the constraints imposed by the cyclic cystine ladder were observed in that the order parameters, S2, show that residues in the turns are more flexible than those in the ?-sheet. This study provides insights into the dynamics of ?-defensins and information that might be useful in their applications as scaffolds in drug design.
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Cyclic Penta- and Hexaleucine Peptides without N-Methylation Are Orally Absorbed.
ACS Med Chem Lett
PUBLISHED: 10-09-2014
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Development of peptide-based drugs has been severely limited by lack of oral bioavailability with less than a handful of peptides being truly orally bioavailable, mainly cyclic peptides with N-methyl amino acids and few hydrogen bond donors. Here we report that cyclic penta- and hexa-leucine peptides, with no N-methylation and five or six amide NH protons, exhibit some degree of oral bioavailability (4-17%) approaching that of the heavily N-methylated drug cyclosporine (22%) under the same conditions. These simple cyclic peptides demonstrate that oral bioavailability is achievable for peptides that fall outside of rule-of-five guidelines without the need for N-methylation or modified amino acids.
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Translational diffusion of cyclic peptides measured using pulsed-field gradient NMR.
J Phys Chem B
PUBLISHED: 09-15-2014
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Cyclic peptides are increasingly being recognized as valuable templates for drug discovery or design. To facilitate efforts in the structural characterization of cyclic peptides, we explore the use of pulse-field gradient experiments as a convenient and noninvasive approach for characterizing their diffusion properties in solution. We present diffusion coefficient measurements of five cyclic peptides, including dichC, SFTI-1, cVc1.1, kB1, and kB2. These peptides range in size from six to 29 amino acids and have various therapeutically interesting activities. We explore the use of internal standards, such as dioxane and acetonitrile, to evaluate the hydrodynamic radius from the diffusion coefficient, and show that 2,2-dimethyl-2-silapentane-5-sulfonic acid, a commonly used chemical shift reference, can be used as an internal standard to avoid spectral overlap issues and simplify data analysis. The experimentally measured hydrodynamic radii correlate with increasing molecular weight and in silico predictions. We further applied diffusion measurements to characterize the self-association of kB2 and showed that it forms oligomers in a concentration-dependent manner, which may be relevant to its mechanism of action. Diffusion coefficient measurements appear to have broad utility in cyclic peptide structural biology, allowing for the rapid characterization of their molecular shape in solution.
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Racemic and Quasi-Racemic X-ray Structures of Cyclic Disulfide-Rich Peptide Drug Scaffolds.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 08-28-2014
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Cyclic disulfide-rich peptides have exceptional stability and are promising frameworks for drug design. We were interested in obtaining X-ray structures of these peptides to assist in drug design applications, but disulfide-rich peptides can be notoriously difficult to crystallize. To overcome this limitation, we chemically synthesized the L- and D-forms of three prototypic cyclic disulfide-rich peptides: SFTI-1 (14-mer with one disulfide bond), cVc1.1 (22-mer with two disulfide bonds), and kB1 (29-mer with three disulfide bonds) for racemic crystallization studies. Facile crystal formation occurred from a racemic mixture of each peptide, giving structures solved at resolutions from 1.25?Å to 1.9?Å. Additionally, we obtained the quasi-racemic structures of two mutants of kB1, [G6A]kB1, and [V25A]kB1, which were solved at a resolution of 1.25?Å and 2.3?Å, respectively. The racemic crystallography approach appears to have broad utility in the structural biology of cyclic peptides.
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Holocyclotoxin-1, a cystine knot toxin from Ixodes holocyclus.
Toxicon
PUBLISHED: 08-27-2014
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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.
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The plant decapeptide OSIP108 can alleviate mitochondrial dysfunction induced by cisplatin in human cells.
Molecules
PUBLISHED: 07-11-2014
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We investigated the effect of the Arabidopsis thaliana-derived decapeptide OSIP108 on human cell tolerance to the chemotherapeutic agent cisplatin (Cp), which induces apoptosis and mitochondrial dysfunction. We found that OSIP108 increases the tolerance of HepG2 cells to Cp and prevents Cp-induced changes in basic cellular metabolism. More specifically, we demonstrate that OSIP108 reduces Cp-induced inhibition of respiration, decreases glycolysis and prevents Cp-uptake in HepG2 cells. Apart from its protective action against Cp in human cells, OSIP108 also increases the yeast Saccharomyces cerevisiae tolerance to Cp. A limited yeast-based study of OSIP108 analogs showed that cyclization does not severely affect its activity, which was further confirmed in HepG2 cells. Furthermore, the similarity in the activity of the D-stereoisomer (mirror image) form of OSIP108 with the L-stereoisomer suggests that its mode of action does not involve binding to a stereospecific receptor. In addition, as OSIP108 decreases Cp uptake in HepG2 cells and the anti-Cp activity of OSIP108 analogs without free cysteine is reduced, OSIP108 seems to protect against Cp-induced toxicity only partly via complexation. Taken together, our data indicate that OSIP108 and its cyclic derivatives can protect against Cp-induced toxicity and, thus, show potential as treatment options for mitochondrial dysfunction- and apoptosis-related conditions.
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Fmoc-based synthesis of disulfide-rich cyclic peptides.
J. Org. Chem.
PUBLISHED: 06-11-2014
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Disulfide-rich cyclic peptides have exciting potential as leads or frameworks in drug discovery; however, their use is faced with some synthetic challenges, mainly associated with construction of the circular backbone and formation of the correct disulfides. Here we describe a simple and efficient Fmoc solid-phase peptide synthesis (SPPS)-based method for synthesizing disulfide-rich cyclic peptides. This approach involves SPPS on 2-chlorotrityl resin, cyclization of the partially protected peptide in solution, cleavage of the side-chain protecting groups, and oxidization of cysteines to yield the desired product. We illustrate this method with the synthesis of peptides from three different classes of cyclic cystine knot motif-containing cyclotides: Möbius (M), trypsin inhibitor (T), and bracelet (B). We show that the method is broadly applicable to peptide engineering, illustrated by the synthesis of two mutants and three grafted analogues of kalata B1. The method reduces the use of highly caustic and toxic reagents and is better suited for high-throughput synthesis than previously reported methods for producing disulfide-rich cyclic peptides, thus offering great potential to facilitate pharmaceutical optimization of these scaffolds.
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Structure-activity relationship study of the plant-derived decapeptide OSIP108 inhibiting Candida albicans biofilm formation.
Antimicrob. Agents Chemother.
PUBLISHED: 06-09-2014
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We performed a structure-activity relationship study of the antibiofilm plant-derived decapeptide OSIP108. Introduction of positively charged amino acids R, H, and K resulted in an up-to-5-fold-increased antibiofilm activity against Candida albicans compared to native OSIP108, whereas replacement of R9 resulted in complete abolishment of its antibiofilm activity. By combining the most promising amino acid substitutions, we found that the double-substituted OSIP108 analogue Q6R/G7K had an 8-fold-increased antibiofilm activity.
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Improving on nature: making a cyclic heptapeptide orally bioavailable.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 05-16-2014
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The use of peptides in medicine is limited by low membrane permeability, metabolic instability, high clearance, and negligible oral bioavailability. The prediction of oral bioavailability of drugs relies on physicochemical properties that favor passive permeability and oxidative metabolic stability, but these may not be useful for peptides. Here we investigate effects of heterocyclic constraints, intramolecular hydrogen bonds, and side chains on the oral bioavailability of cyclic heptapeptides. NMR-derived structures, amide H-D exchange rates, and temperature-dependent chemical shifts showed that the combination of rigidification, stronger hydrogen bonds, and solvent shielding by branched side chains enhances the oral bioavailability of cyclic heptapeptides in rats without the need for N-methylation.
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Structural parameters modulating the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-1.
Eur J Med Chem
PUBLISHED: 05-15-2014
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Peptides are emerging as a new class of therapeutics due to their high potency and specificity for a range of targets, including the inhibition of protein-protein interactions. Disulfide-rich cyclic peptides, in particular, have attracted much attention in drug design due to their ultra-stable structure. Moreover, some of them have been shown to internalize into cells, which makes them potential scaffolds to deliver pharmaceutically bioactive sequences to intracellular targets. Here we examined the effects of structural modifications on the cell-penetrating properties of two disulfide-rich cyclic cell-penetrating peptides, Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1). We found that the cellular uptake of MCoTI-II can be improved by increasing the overall positive charge of the native sequence. On the other hand, mutations to SFTI-1 did not significantly influence its cellular uptake, suggesting a non-specific endocytosis-dependent mechanism of cellular uptake. This study provides an understanding of the structural features affecting the internalization of MCoTI-II and SFTI-1, and hence provides a guide for the development of these disulfide-rich cyclic scaffolds into potential drug leads.
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Anticancer and toxic properties of cyclotides are dependent on phosphatidylethanolamine phospholipid targeting.
Chembiochem
PUBLISHED: 03-31-2014
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Cyclotides, ultrastable disulfide-rich cyclic peptides, can be engineered to bind and inhibit specific cancer targets. In addition, some cyclotides are toxic to cancer cells, though not much is known about their mechanisms of action. Here we delineated the potential mode of action of cyclotides towards cancer cells. A novel set of analogues of kalata B1 (the prototypic cyclotide) and kalata B2 and cycloviolacin O2 were examined for their membrane-binding affinity and selectivity towards cancer cells. By using solution-state NMR, surface plasmon resonance, flow cytometry and bioassays we show that cyclotides are toxic against cancer and non-cancerous cells and their toxicity correlates with their ability to target and disrupt lipid bilayers that contain phosphatidylethanolamine phospholipids. Our results suggest that the potential of cyclotides as anticancer therapeutics might best be realised by combining their amenability to epitope engineering with their ability to bind cancer cell membranes.
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Evolutionary origins of a bioactive peptide buried within Preproalbumin.
Plant Cell
PUBLISHED: 03-28-2014
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The de novo evolution of proteins is now considered a frequented route for biological innovation, but the genetic and biochemical processes that lead to each newly created protein are often poorly documented. The common sunflower (Helianthus annuus) contains the unusual gene PawS1 (Preproalbumin with SFTI-1) that encodes a precursor for seed storage albumin; however, in a region usually discarded during albumin maturation, its sequence is matured into SFTI-1, a protease-inhibiting cyclic peptide with a motif homologous to unrelated inhibitors from legumes, cereals, and frogs. To understand how PawS1 acquired this additional peptide with novel biochemical functionality, we cloned PawS1 genes and showed that this dual destiny is over 18 million years old. This new family of mostly backbone-cyclic peptides is structurally diverse, but the protease-inhibitory motif was restricted to peptides from sunflower and close relatives from its subtribe. We describe a widely distributed, potential evolutionary intermediate PawS-Like1 (PawL1), which is matured into storage albumin, but makes no stable peptide despite possessing residues essential for processing and cyclization from within PawS1. Using sequences we cloned, we retrodict the likely stepwise creation of PawS1's additional destiny within a simple albumin precursor. We propose that relaxed selection enabled SFTI-1 to evolve its inhibitor function by converging upon a successful sequence and structure.
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Plant-derived decapeptide OSIP108 interferes with Candida albicans biofilm formation without affecting cell viability.
Antimicrob. Agents Chemother.
PUBLISHED: 02-24-2014
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We previously identified a decapeptide from the model plant Arabidopsis thaliana, OSIP108, which is induced upon fungal pathogen infection. In this study, we demonstrated that OSIP108 interferes with biofilm formation of the fungal pathogen Candida albicans without affecting the viability or growth of C. albicans cells. OSIP108 displayed no cytotoxicity against various human cell lines. Furthermore, OSIP108 enhanced the activity of the antifungal agents amphotericin B and caspofungin in vitro and in vivo in a Caenorhabditis elegans-C. albicans biofilm infection model. These data point to the potential use of OSIP108 in combination therapy with conventional antifungal agents. In a first attempt to unravel its mode of action, we screened a library of 137 homozygous C. albicans mutants, affected in genes encoding cell wall proteins or transcription factors important for biofilm formation, for altered OSIP108 sensitivity. We identified 9 OSIP108-tolerant C. albicans mutants that were defective in either components important for cell wall integrity or the yeast-to-hypha transition. In line with these findings, we demonstrated that OSIP108 activates the C. albicans cell wall integrity pathway and that its antibiofilm activity can be blocked by compounds inhibiting the yeast-to-hypha transition. Furthermore, we found that OSIP108 is predominantly localized at the C. albicans cell surface. These data point to interference of OSIP108 with cell wall-related processes of C. albicans, resulting in impaired biofilm formation.
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A comparative study of extraction methods reveals preferred solvents for cystine knot peptide isolation from Momordica cochinchinensis seeds.
Fitoterapia
PUBLISHED: 02-21-2014
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MCoTI-I and MCoTI-II (short for Momordica cochinchinensis Trypsin Inhibitor-I and -II, respectively) are attractive candidates for developing novel intracellular-targeting drugs because both are exceptionally stable and can internalize into cells. These seed-derived cystine knot peptides are examples of how natural product discovery efforts can lead to biomedical applications. However, discovery efforts are sometimes hampered by the limited availability of seed materials, highlighting the need for efficient extraction methods. In this study, we assessed five extraction methods using M. cochinchinensis seeds, a source of well-characterized cystine knot peptides. The most efficient extraction of nine known cystine knot peptides was achieved by a method based on acetonitrile/water/formic acid (25:24:1), followed by methods based on sodium acetate (20 mM, pH 5.0), ammonium bicarbonate (5 mM, pH 8.0), and boiling water. On average, the yields obtained by these four methods were more than 250-fold higher than that obtained using dichloromethane/methanol (1:1) extraction, a previously applied standard method. Extraction using acetonitrile/water/formic acid (25:24:1) yielded the highest number of reconstructed masses within the majority of plant-derived cystine knot peptide mass range but only accounted for around 50% of the total number of masses, indicating that any single method may result in under-sampling. Applying acetonitrile/water/formic acid (25:24:1), boiling water, and ammonium bicarbonate (5 mM, pH 8.0) extractions either successively or discretely significantly increased the sampling number. Overall, acetonitrile/water/formic acid (25:24:1) can facilitate efficient extraction of cystine-knot peptides from M. cochinchinensis seeds but for discovery purposes the use of a combination of extraction methods is recommended where practical.
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The chemistry and biology of theta defensins.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 02-06-2014
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Cyclic peptides are found in a diverse range of organisms and are characterized by their stability and role in defense. Why is only one class of cyclic peptides found in mammals? Possibly we have not looked hard enough for them, or the technologies needed to identify them are not fully developed. We also do not yet understand their intriguing biosynthesis from two separate gene products. Addressing these challenges will require the application of chemical tools and insights from other classes of cyclic peptides. Herein, we highlight recent developments in the characterization of theta defensins and describe the important role that chemistry has played in delineating their modes of action. Furthermore, we emphasize the potential of theta defensins as antimicrobial agents and scaffolds for peptide drug design.
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The C-terminal propeptide of a plant defensin confers cytoprotective and subcellular targeting functions.
BMC Plant Biol.
PUBLISHED: 01-28-2014
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Plant defensins are small (45-54 amino acids), basic, cysteine-rich proteins that have a major role in innate immunity in plants. Many defensins are potent antifungal molecules and are being evaluated for their potential to create crop plants with sustainable disease resistance. Defensins are produced as precursor molecules which are directed into the secretory pathway and are divided into two classes based on the absence (class I) or presence (class II) of an acidic C-terminal propeptide (CTPP) of about 33 amino acids. The function of this CTPP had not been defined.
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Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro.
Chem. Biol.
PUBLISHED: 01-16-2014
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Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 ?M) compared with wild-type EGF-A (KD = 1.2 ?M), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.
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Solution structure, membrane interactions, and protein binding partners of the tetraspanin Sm-TSP-2, a vaccine antigen from the human blood fluke Schistosoma mansoni.
J. Biol. Chem.
PUBLISHED: 01-15-2014
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The tetraspanins (TSPs) are a family of integral membrane proteins that are ubiquitously expressed at the surface of eukaryotic cells. TSPs mediate a range of processes at the surface of the plasma membrane by providing a scaffold for the assembly of protein complexes known as tetraspanin-enriched microdomains (TEMs). We report here the structure of the surface-exposed EC2 domain from Sm-TSP-2, a TSP from Schistosoma mansoni and one of the better prospects for the development of a vaccine against schistosomiasis. This is the first solution structure of this domain, and our investigations of its interactions with lipid micelles provide a general model for interactions between TSPs, membranes, and other proteins. Using chemical cross-linking, eight potential protein constituents of Sm-TSP-2-mediated TEMs were also identified. These include proteins important for membrane maintenance and repair, providing further evidence for the functional role of Sm-TSP-2- and Sm-TSP-2-mediated TEMs. The identification of calpain, Sm29, and fructose-bisphosphate aldolase, themselves potential vaccine antigens, suggests that the Sm-TSP-2-mediated TEMs could be disrupted via multiple targets. The identification of further Sm-TSP-2-mediated TEM proteins increases the available candidates for multiplex vaccines and/or novel drugs targeting TEMs in the schistosome tegument.
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Semienzymatic cyclization of disulfide-rich peptides using Sortase A.
J. Biol. Chem.
PUBLISHED: 01-14-2014
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Disulfide-rich cyclic peptides have generated great interest in the development of peptide-based therapeutics due to their exceptional stability toward chemical, enzymatic, or thermal attack. In particular, they have been used as scaffolds onto which bioactive epitopes can be grafted to take advantage of the favorable biophysical properties of disulfide-rich cyclic peptides. To date, the most commonly used method for the head-to-tail cyclization of peptides has been native chemical ligation. In recent years, however, enzyme-mediated cyclization has become a promising new technology due to its efficiency, safety, and cost-effectiveness. Sortase A (SrtA) is a bacterial enzyme with transpeptidase activity. It recognizes a C-terminal penta-amino acid motif, LPXTG, and cleaves the amide bond between Thr and Gly to form a thioacyl-linked intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence to form an amide bond between the Thr and N-terminal Gly. Here, we demonstrate that sortase A can successfully be used to cyclize a variety of small disulfide-rich peptides, including the cyclotide kalata B1, ?-conotoxin Vc1.1, and sunflower trypsin inhibitor 1. These peptides range in size from 14 to 29 amino acids and contain three, two, or one disulfide bond, respectively, within their head-to-tail cyclic backbones. Our findings provide proof of concept for the potential broad applicability of enzymatic cyclization of disulfide-rich peptides with therapeutic potential.
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Disulfide-rich macrocyclic peptides as templates in drug design.
Eur J Med Chem
PUBLISHED: 01-10-2014
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Recently disulfide-rich head-to-tail cyclic peptides have attracted the interest of medicinal chemists owing to their exceptional thermal, chemical and enzymatic stability brought about by their constrained structures. Here we review current trends in the field of peptide-based pharmaceuticals and describe naturally occurring cyclic disulfide-rich peptide scaffolds, discussing their pharmaceutically attractive properties and benefits. We describe how we can utilise these stable frameworks to graft and/or engineer pharmaceutically interesting epitopes to increase their selectivity and bioactivity, opening up new possibilities for addressing 'difficult' pharmaceutical targets.
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A novel ?4/7-conotoxin LvIA from Conus lividus that selectively blocks ?3?2 vs. ?6/?3?2?3 nicotinic acetylcholine receptors.
FASEB J.
PUBLISHED: 01-07-2014
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This study was performed to discover and characterize the first potent ?3?2-subtype-selective nicotinic acetylcholine receptor (nAChR) ligand. A novel ?4/7-conotoxin, ?-CTxLvIA, was cloned from Conus lividus. Its pharmacological profile at Xenopus laevis oocyte-expressed rat nAChR subtypes was determined by 2-electrode voltage-clamp electrophysiology, and its 3-dimensional (3D) structure was determined by NMR spectroscopy. ?-CTx LvIA is a 16-aa C-terminally-amidated peptide with 2-disulfide bridges. Using rat subunits expressed in Xenopus oocytes, we found the highest affinity of ?-CTxLvIA was for ?3?2 nAChRs (IC50 8.7 nM), where blockade was reversible within 2 min. IC50 values were >100 nM at ?6/?3?2?3, ?6/?3?4, and ?3?4 nAChRs, and ?3 ?M at all other subtypes tested. ?3?2 vs. ?6?2 subtype selectivity was confirmed for human-subunit nAChRs with much greater preference (300-fold) for ?3?2 over ?6?2 nAChRs. This is the first ?-CTx reported to show high selectivity for human ?3?2 vs. ?6?2 nAChRs. ?-CTxLvIA adopts two similarly populated conformations water: one (assumed to be bioactive) is highly structured, whereas the other is mostly random coil in nature. Selectivity differences with the similarly potent, but less selective, ?3?2 nAChR antagonist ?-CTx PeIA probably reside within the three residues, which differ in loop 2, given their otherwise similar 3D structures. ?4/7-CTx LvIA is a new, potent, selective ?3?2 nAChR antagonist, which will enable detailed studies of ?3?2 nAChR structure, function, and physiological roles.
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The cyclic cystine ladder of theta-defensins as a stable, bifunctional scaffold: A proof-of-concept study using the integrin-binding RGD motif.
Chembiochem
PUBLISHED: 01-02-2014
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Peptides have the specificity and size required to target the protein-protein interactions involved in many diseases. Some cyclic peptides have been utilised as scaffolds for peptide drugs because of their stability; however, other cyclic peptide scaffolds remain to be explored. ?-Defensins are cyclic peptides from mammals; they are characterised by a cyclic cystine ladder motif and have low haemolytic and cytotoxic activity. Here we demonstrate the potential of the cyclic cystine ladder as a scaffold for peptide drug design by introducing the integrin-binding Arg-Gly-Asp (RGD) motif into the ?-defensin RTD-1. The most active analogue had an IC50 of 18 nM for the ?v ?3 integrin as well as high serum stability, thus demonstrating that a desired bioactivity can be imparted to the cyclic cystine ladder. This study highlights how ?-defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a ?-strand or turn conformation. Furthermore, the symmetry of the cyclic cystine ladder presents the opportunity to design peptides with dual bioactive epitopes to increase activity and specificity.
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Characterization of a Novel ?-Conotoxin TxID from Conus textile That Potently Blocks Rat ?3?4 Nicotinic Acetylcholine Receptors.
J. Med. Chem.
PUBLISHED: 11-22-2013
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The ?3?4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an ?-4/6-conotoxin (CTx) TxID from Conus textile. The new toxin consists of 15 amino acid residues with two disulfide bonds. TxID was synthesized using solid phase methods, and the synthetic peptide was functionally tested on nAChRs heterologously expressed in Xenopus laevis oocytes. TxID blocked rat ?3?4 nAChRs with a 12.5 nM IC50, which places it among the most potent ?3?4 nAChR antagonists. TxID also blocked the closely related ?6/?3?4 with a 94 nM IC50 but showed little activity on other nAChR subtypes. NMR analysis showed that two major structural isomers exist in solution, one of which adopts a regular ?-CTx fold but with different surface charge distribution to other 4/6 family members. ?-CTx TxID is a novel tool with which to probe the structure and function of ?3?4 nAChRs.
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Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-18-2013
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Cyclotides are plant peptides comprising a circular backbone and three conserved disulfide bonds that confer them with exceptional stability. They were originally discovered in Oldenlandia affinis based on their use in traditional African medicine to accelerate labor. Recently, cyclotides have been identified in numerous plant species of the coffee, violet, cucurbit, pea, potato, and grass families. Their unique structural topology, high stability, and tolerance to sequence variation make them promising templates for the development of peptide-based pharmaceuticals. However, the mechanisms underlying their biological activities remain largely unknown; specifically, a receptor for a native cyclotide has not been reported hitherto. Using bioactivity-guided fractionation of an herbal peptide extract known to indigenous healers as "kalata-kalata," the cyclotide kalata B7 was found to induce strong contractility on human uterine smooth muscle cells. Radioligand displacement and second messenger-based reporter assays confirmed the oxytocin and vasopressin V1a receptors, members of the G protein-coupled receptor family, as molecular targets for this cyclotide. Furthermore, we show that cyclotides can serve as templates for the design of selective G protein-coupled receptor ligands by generating an oxytocin-like peptide with nanomolar affinity. This nonapeptide elicited dose-dependent contractions on human myometrium. These observations provide a proof of concept for the development of cyclotide-based peptide ligands.
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Molecular Grafting onto a Stable Framework Yields Novel Cyclic Peptides for the Treatment of Multiple Sclerosis.
ACS Chem. Biol.
PUBLISHED: 11-07-2013
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Multiple sclerosis (MS) is an inflammatory disease of the central nervous system (CNS) and is characterized by the destruction of myelin and axons leading to progressive disability. Peptide epitopes from CNS proteins, such as myelin oligodendrocyte glycoprotein (MOG), possess promising immunoregulatory potential for treating MS; however, their instability and poor bioavailability is a major impediment for their use clinically. To overcome this problem, we used molecular grafting to incorporate peptide sequences from the MOG35-55 epitope onto a cyclotide, which is a macrocyclic peptide scaffold that has been shown to be intrinsically stable. Using this approach, we designed novel cyclic peptides that retained the structure and stability of the parent scaffold. One of the grafted peptides, MOG3, displayed potent ability to prevent disease development in a mouse model of MS. These results demonstrate the potential of bioengineered cyclic peptides for the treatment of MS.
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Structural insights into the role of the cyclic backbone in a squash trypsin inhibitor.
J. Biol. Chem.
PUBLISHED: 10-29-2013
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MCoTI-II is a head-to-tail cyclic peptide with potent trypsin inhibitory activity and, on the basis of its exceptional proteolytic stability, is a valuable template for the design of novel drug leads. Insights into inhibitor dynamics and interactions with biological targets are critical for drug design studies, particularly for protease targets. Here, we show that the cyclization and active site loops of MCoTI-II are flexible in solution, but when bound to trypsin, the active site loop converges to a single well defined conformation. This finding of reduced flexibility on binding is in contrast to a recent study on the homologous peptide MCoTI-I, which suggested that regions of the peptide are more flexible upon binding to trypsin. We provide a possible explanation for this discrepancy based on degradation of the complex over time. Our study also unexpectedly shows that the cyclization loop, not present in acyclic homologues, facilitates potent trypsin inhibitory activity by engaging in direct binding interactions with trypsin.
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Chemical Synthesis, 3D Structure, and ASIC Binding Site of the Toxin Mambalgin-2.
Angew. Chem. Int. Ed. Engl.
PUBLISHED: 10-12-2013
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Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid-sensing ion channels (ASICs). The 57-residue polypeptide mambalgin-2 (Ma-2) was synthesized by using a combination of solid-phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three-finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma-2 on wild-type and mutant ASIC1a receptors allowed us to identify ?-helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma-2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure-activity relationship (SAR) studies and further development of this promising analgesic peptide.
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Identifying Key Amino Acid Residues That Affect ?-Conotoxin AuIB Inhibition of ?3?4 Nicotinic Acetylcholine Receptors.
J. Biol. Chem.
PUBLISHED: 10-07-2013
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?-Conotoxin AuIB is a selective ?3?4 nicotinic acetylcholine receptor (nAChR) subtype inhibitor. Its analgesic properties are believed to result from it activating GABAB receptors and subsequently inhibiting CaV2.2 voltage-gated calcium channels. The structural determinants that mediate diverging AuIB activity at these targets are unknown. We performed alanine scanning mutagenesis of AuIB and ?3?4 nAChR, homology modeling, and molecular dynamics simulations to identify the structural determinants of the AuIB·?3?4 nAChR interaction. Two alanine-substituted AuIB analogues, [P6A]AuIB and [F9A]AuIB, did not inhibit the ?3?4 nAChR. NMR and CD spectroscopy studies demonstrated that [F9A]AuIB retains its native globular structure, so its activity loss is probably due to loss of specific toxin-receptor residue pairwise contacts. Compared with AuIB, the concentration-response curve for inhibition of ?3?4 by [F9A]AuIB shifted rightward more than 10-fold, and its subtype selectivity profile changed. Homology modeling and molecular dynamics simulations suggest that Phe-9 of AuIB interacts with a two-residue binding pocket on the ?4 nAChR subunit. This hypothesis was confirmed by site-directed mutagenesis of the ?4-Trp-59 and ?4-Lys-61 residues of loop D, which form a putative binding pocket. AuIB analogues with Phe-9 substitutions corroborated the finding of a binding pocket on the ?4 subunit and gave further insight into how AuIB Phe-9 interacts with the ?4 subunit. In summary, we identified critical residues that mediate interactions between AuIB and its cognate nAChR subtype. These findings might help improve the design of analgesic conopeptides that selectively "avoid" nAChR receptors while targeting receptors involved with nociception.
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Mining the genome of Arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance.
J. Exp. Bot.
PUBLISHED: 09-16-2013
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Although evidence has accumulated on the role of plant peptides in the response to external conditions, the number of peptide-encoding genes in the genome is still underestimated. Using tiling arrays, we identified 176 unannotated transcriptionally active regions (TARs) in Arabidopsis thaliana that were induced upon oxidative stress generated by the herbicide paraquat (PQ). These 176 TARs could be translated into 575 putative oxidative stress-induced peptides (OSIPs). A high-throughput functional assay was used in the eukaryotic model organism Saccharomyces cerevisiae allowing us to test for bioactive peptides that increase oxidative stress tolerance. In this way, we identified three OSIPs that, upon overexpression in yeast, resulted in a significant rise in tolerance to hydrogen peroxide (H2O2). For one of these peptides, the decapeptide OSIP108, exogenous application to H2O2-treated yeast also resulted in significantly increased survival. OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the reactive oxygen species-inducer PQ and the necrotrophic fungal pathogen Botrytis cinerea. Moreover, infiltration and overexpression of OSIP108 in A. thaliana leaves resulted in increased tolerance to treatment with PQ. In conclusion, the identification and characterization of OSIP108 confirms the validity of our high-throughput approach, based on tiling array analysis in A. thaliana and functional screening in yeast, to identify bioactive peptides.
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Synthesis of cyclic disulfide-rich peptides.
Methods Mol. Biol.
PUBLISHED: 08-15-2013
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In this chapter we describe two SPPS approaches for producing cyclic disulfide-rich peptides in our laboratory, including cyclotides from plants, cyclic conotoxins from cone snail venoms, chlorotoxin from scorpion venom, and the sunflower trypsin inhibitor peptide, SFTI-1.
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Functional evolution of scorpion venom peptides with an inhibitor cystine knot fold.
Biosci. Rep.
PUBLISHED: 06-01-2013
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The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named ?-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. ?-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel ?-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of ?-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of ?-MeuKTx-1. The presence of a functional dyad in ?-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon-intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.
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The future of peptide-based drugs.
Chem Biol Drug Des
PUBLISHED: 05-23-2013
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The suite of currently used drugs can be divided into two categories - traditional small molecule drugs with typical molecular weights of <500 Da but with oral bioavailability, and much larger biologics typically >5000 Da that are not orally bioavailable and need to be delivered via injection. Due to their small size, conventional small molecule drugs may suffer from reduced target selectivity that often ultimately manifests in human side-effects, whereas protein therapeutics tend to be exquisitely specific for their targets due to many more interactions with them, but this comes at a cost of low bioavailability, poor membrane permeability, and metabolic instability. The time has now come to reinvestigate new drug leads that fit between these two molecular weight extremes, with the goal of combining advantages of small molecules (cost, conformational restriction, membrane permeability, metabolic stability, oral bioavailability) with those of proteins (natural components, target specificity, high potency). This article uses selected examples of peptides to highlight the importance of peptide drugs, some potential new opportunities for their exploitation, and some difficult challenges ahead in this field.
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The antimicrobial activity of Sub3 is dependent on membrane binding and cell-penetrating ability.
Chembiochem
PUBLISHED: 05-02-2013
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Because of their high activity against microorganisms and low cytotoxicity, cationic antimicrobial peptides (AMPs) have been explored as the next generation of antibiotics. Although they have common structural features, the modes of action of AMPs are extensively debated, and a single mechanism does not explain the activity of all AMPs reported so far. Here we investigated the mechanism of action of Sub3, an AMP previously designed and optimised from high-throughput screening with bactenecin as the template. Sub3 has potent activity against Gram-negative and Gram-positive bacteria as well as against fungi, but its mechanism of action has remained elusive. By using AFM imaging, ? potential, flow cytometry and fluorescence methodologies with model membranes and bacterial cells, we found that, although the mechanism of action involves membrane targeting, Sub3 internalises inside bacteria at lethal concentrations without permeabilising the membrane, thus suggesting that its antimicrobial activity might involve both the membrane and intracellular targets. In addition, we found that Sub3 can be internalised into human cells without being toxic. As some bacteria are able to survive intracellularly and consequently evade host defences and antibiotic treatment, our findings suggest that Sub3 could be useful as an intracellular antimicrobial agent for infections that are notoriously difficult to treat.
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Joseph Rudinger memorial lecture: discovery and applications of cyclotides.
J. Pept. Sci.
PUBLISHED: 05-01-2013
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Cyclotides are plant-derived peptides of approximately 30 amino acids that have the characteristic structural features of a head-to-tail cyclized backbone and a cystine knot arrangement of their three conserved disulfide bonds. This article gives a personal account of the discovery of cyclotides, their characterization and their applications, based on work carried out in my laboratory over the last 20?years. It describes some of the background to their discovery and focuses on how their unique structural features lead to exceptional stability. This stability and their amenability to chemical synthesis have made it possible to use cyclotides as templates in protein engineering and drug design applications. These applications complement the interest in cyclotides deriving from their unique structures and natural function as host defense molecules.
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Determination of the ?-conotoxin Vc1.1 binding site on the ?9?10 nicotinic acetylcholine receptor.
J. Med. Chem.
PUBLISHED: 04-29-2013
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?-Conotoxin Vc1.1 specifically and potently inhibits the nicotinic acetylcholine receptor subtype ?9?10 (?9?10 nAChR) and is a potential novel treatment for neuropathic pain. Here, we used a combination of computational modeling and electrophysiology experiments to determine the Vc1.1 binding site on the ?9?10 nAChR. Interactions of Vc1.1 with two probable binding sites, ?9?10 and ?10?9, were modeled. Mutational energies calculated by assuming specific interactions in the ?10?9 binding site correlated better with electrophysiological recordings than those assuming interactions with the ?9?10 binding site. Two novel Vc1.1 analogues, [N9F]Vc1.1 and [N9W]Vc1.1, were predicted to have large differences in affinity between the two binding sites. Data from functional studies were consistent with computational predictions that assumed preferred binding of Vc1.1 to the ?10?9 pocket. Moreover, our modeling study suggested that a single hydrogen bond formed between Vc1.1 and position 59 of the ?10?9 pocket confers specificity to rat versus human ?9?10 nAChRs.
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A radish seed antifungal peptide with a high amyloid fibril-forming propensity.
Biochim. Biophys. Acta
PUBLISHED: 04-12-2013
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The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.
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Design of a Cyclotide Antagonist of Neuropilin-1 and -2 That Potently Inhibits Endothelial Cell Migration.
ACS Chem. Biol.
PUBLISHED: 04-12-2013
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Neuropilin-1 and -2 are critical regulators of angiogenesis, lymphangiogenesis, and cell survival as receptors for multiple growth factors. Disulfide-rich peptides that antagonize the growth factor receptors neuropilin-1 and neuropilin-2 were developed using bacterial display libraries. Peptide ligands specific for the VEGFA binding site on neuropilin-1 were identified by screening a library of disulfide-rich peptides derived from the thermostable, protease-resistant cyclotide kalata B1. First generation ligands were subjected to one cycle of affinity maturation to yield acyclic peptides with affinities of 40-60 nM and slow dissociation rate constants (?1 × 10(-3) s(-1)). Peptides exhibited equivalent affinities for human and mouse neuropilin-1 and cross-reacted with human neuropilin-2 with lower affinity. A C-to-N cyclized variant (cyclotide) of one neuropilin ligand retained high affinity, exhibited increased protease resistance, and conferred improved potency for inhibiting endothelial cell migration in vitro (EC50 ? 100 nM). These results demonstrate that potent, target-specific cyclotides can be created by evolutionary design and that backbone cyclization can confer improved pharmacological properties.
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High-affinity cyclic peptide matriptase inhibitors.
J. Biol. Chem.
PUBLISHED: 04-02-2013
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Sunflower trypsin inhibitor-1 (SFTI-1) and Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) are potent protease inhibitors comprising a cyclic backbone.
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A systematic approach to document cyclotide distribution in plant species from genomic, transcriptomic, and peptidomic analysis.
Biopolymers
PUBLISHED: 03-26-2013
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Cyclotides are a large family of plant peptides characterized by their cyclic cystine knot composed of a circular backbone and three disulfide bonds that impart exceptional stability. They, and several acyclic variants, have been isolated from plants within the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, Solanaceae, and Poaceae families. A variety of chemical and genetic approaches have been applied for the discovery and characterization of cyclotides. As investigations of cyclotide expression, distribution, and phylogeny rapidly increase, the authors have proposed the inclusion of information pertaining to plant species that have been analyzed but do not appear to express cyclotides into the CyBase database. CyBase is dedicated to providing web tools and information about cyclic peptides and proteins to the scientific community. Including detailed information about sampling and analysis parameters of plant species that have been investigated but not published elsewhere should assist in the process of selecting species for establishing new cyclotide discovery projects, as well as for detailed reanalysis using alternative technical approaches. In summary, the collection and deposition of all plant species that have been examined (whether cyclotides have been found or not) would help to impart a deeper understanding of cyclotide discovery, evolution, and physiological function. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 433-437, 2013.
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Isolation and characterization of ?-conotoxin LsIA with potent activity at nicotinic acetylcholine receptors.
Biochem. Pharmacol.
PUBLISHED: 03-21-2013
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A new ?-conotoxin LsIA was isolated from the crude venom of Conus limpusi using assay-guided RP-HPLC fractionation. Synthetic LsIA was a potent antagonist of ?3?2, ?3?5?2 and ?7 nAChRs, with half-maximal inhibitory concentrations of 10, 31 and 10 nM, respectively. The structure of LsIA determined by NMR spectroscopy comprised a characteristic disulfide bond-stabilized ?-helical structure and disordered N-terminal region. Potency reductions of up to 9-fold were observed for N-terminally truncated analogues of LsIA at ?7 and ?3?2 nAChRs, whereas C-terminal carboxylation enhanced potency 3-fold at ?3?2 nAChRs but reduced potency 3-fold at ?7 nAChRs. This study gives further insight into ?-conotoxin pharmacology and the molecular basis of nAChR selectivity, highlighting the influence of N-terminal residues and C-terminal amidation on conotoxin pharmacology.
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Making ends meet: chemically mediated circularization of recombinant proteins.
Chembiochem
PUBLISHED: 02-26-2013
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A selective N?S acyl transfer reaction facilitates semi-synthesis of the plant cyclotide kalata B1 from a linear precursor peptide of bacterial origin, through simple appendage of N-terminal cysteine and a thiol-labile C-terminal Gly-Cys motif. This constitutes the first synthesis of a ribosomally derived circular miniprotein, without recourse to protein splicing elements.
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Cyclotide biosynthesis.
Curr Opin Chem Biol
PUBLISHED: 02-25-2013
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Cyclotides are bioactive macrocyclic peptides from plants that are characterized by their exceptional stability and potential applications as protein engineering or drug design frameworks. Their stability arises from their unique cyclic cystine knot structure, which combines a head-to-tail cyclic peptide backbone with three conserved disulfide bonds having a knotted topology. Cyclotides are ribosomally synthesized by plants and expressed in a wide range of tissues, including leaves, flowers, stems and roots. Here we describe recent studies that have examined the biosynthesis of cyclotides and in particular the mechanism associated with post-translational backbone cyclization.
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The cyclic cystine ladder in ?-defensins is important for structure and stability, but not antibacterial activity.
J. Biol. Chem.
PUBLISHED: 02-21-2013
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?-Defensins are ribosomally synthesized cyclic peptides found in the leukocytes of some primate species and have promising applications as antimicrobial agents and scaffolds for peptide drugs. The cyclic cystine ladder motif, comprising a cyclic peptide backbone and three parallel disulfide bonds, is characteristic of ?-defensins. In this study, we explore the role of the cyclic peptide backbone and cystine ladder in the structure, stability, and activity of ?-defensins. ?-Defensin analogues with different numbers and combinations of disulfide bonds were synthesized and characterized in terms of their NMR solution structures, serum and thermal stabilities, and their antibacterial and membrane-binding activities. Whereas the structures and stabilities of the peptides were primarily dependent on the number and position of the disulfide bonds, their antibacterial and membrane-binding properties were dependent on the cyclic backbone. The results provide insights into the mechanism of action of ?-defensins and illustrate the potential of ?-defensin analogues as scaffolds for peptide drug design.
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Anthelminthic activity of the cyclotides (kalata B1 and B2) against schistosome parasites.
Biopolymers
PUBLISHED: 02-15-2013
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The risk of reduced sensitivity of the human schistosomes to praziquantel has led to efforts to find new therapies. Here, the cyclotides kalata B1 (kB1), kalata B2 (kB2), MCoCC-1, and MCoTI-II, cyclic peptides extracted from plants and shown to be potent against nematodes and insects, were tested for antischistosome activity. In vitro assays showed that high concentrations (500-1000 ?g/mL) of either kB1 or kB2 killed Schistosoma japonicum and Schistosoma mansoni adults within 5 min, whereas MCoTI-II and MCoCC-1 had no effect. Lethal concentrations to kill 50% of the population for kB2 was 15.5?±?7.4 ?g/mL at 1 h for male S. japonicum (Philippine strain). Males were more susceptible than females. kB2 showed higher antischistosome activity than kB1 and killing time was concentration-dependent. Mode of action studies revealed that kB1 and 2 lysed the tegument of adult worms. Lysis of myofibrils was not demonstrated, but longitudinal and radial muscle fibers were distorted, an observation consistent with strong coiling of the parasites after drug exposure. A single dose of kB2 administered either orally or intravenously, reduced worm burdens in S. japonicum-infected mice from 15% to 60%. However, treatment of S. mansoni-infected mice did not result in reduction in worm burdens. Our studies show that kB2 acts as a promising antischistosomal against Philippine S. japonicum, and it or other cyclotides may be developed further as general anthelminthics. With thousands of cyclotides predicted to occur in plants, and the amenability of these peptides to combinatorial variation, there is potential for their exploitation as wide-spectrum anthelminthics. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 461-470, 2013.
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Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature.
Nat Prod Rep
PUBLISHED: 02-09-2013
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This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
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Prediction and characterization of cyclic proteins from sequences in three domains of life.
Biochim. Biophys. Acta
PUBLISHED: 02-02-2013
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Cyclic proteins (CPs) have circular chains with a continuous cycle of peptide bonds. Their unique structural traits result in greater stability and resistance to degradation when compared to their acyclic counterparts. They are also promising targets for pharmaceutical/therapeutic applications. To date, only a few hundred CPs are known, although recent studies suggest that their numbers might be substantially higher. Here we developed a first-of-its-kind, accurate and high-throughput method called CyPred that predicts whether a given protein chain is cyclic. CyPred considers currently well-represented CP families: cyclotides, cyclic defensins, bacteriocins, and trypsin inhibitors. Empirical tests demonstrate that CyPred outperforms commonly used alignment methods. We used CyPred to estimate the incidence of CPs and found ~3500 putative CPs among 5.7+ million chains from 642 fully sequenced proteomes from archaea, bacteria, and eukaryotes. The median number of putative CPs per species ranges from three for archaea proteomes to two for eukaryotes/bacteria, with 7% of archaea, 11% of bacterial, and 16% of eukaryotic proteomes having 10+ CPs. The differences in the estimated fractions of CPs per proteome are as large as three orders of magnitude. Among eukaryotes, animals have higher ratios of CPs compared to fungi, while plants have the largest spread of the ratios. We also show that proteomes enriched in cyclic proteins evolve more slowly than proteomes with fewer cyclic chains. Our results suggest that further research is needed to fully uncover the scope and potential of cyclic proteins. A list of putative CPs and the CyPred method are available at http://biomine.ece.ualberta.ca/CyPred/. This article is part of a Special Issue entitled: Computational Proteomics, Systems Biology & Clinical Implications. Guest Editor: Yudong Cai.
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Cyclotides as grafting frameworks for protein engineering and drug design applications.
Biopolymers
PUBLISHED: 01-26-2013
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Cyclotides are a family of naturally occurring backbone-cyclized macrocyclic mini-proteins from plants that have a knotted trio of intramolecular disulfide bonds. Their structural features imbue cyclotides with extraordinary stability against degradation at elevated temperatures or in the presence of proteolytic enzymes. The plasticity of their intracysteine loop sequences is exemplified by the more than 250 natural cyclotides sequenced to date, and this tolerance to sequence variation, along with their diverse bioactivities, underpins the suitability of the cyclic cystine knot motif as a valuable drug design scaffold and research tool for protein engineering studies. Here, we review the recent literature on applications of cyclotides for the stabilization of peptide epitopes and related protein engineering studies. Possible future directions in this field are also described. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 480-491, 2013.
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The self-association of the cyclotide kalata B2 in solution is guided by hydrophobic interactions.
Biopolymers
PUBLISHED: 01-26-2013
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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.
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Recent progress towards pharmaceutical applications of disulfide-rich cyclic peptides.
Curr. Protein Pept. Sci.
PUBLISHED: 01-24-2013
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Cyclotides and conotoxins are two classes of disulfide-rich peptides that occur in plants and animals respectively and are the major focus of study in our laboratory. In the last three years there has been significant progress in studies of these two classes of compounds and in this article we provide an overview of the findings from our laboratory in this period. Highlights include the discovery of cyclotides in the Fabaceae and Solanaceae plant families, members of which are widely used in human nutrition, and the discovery of new classes of cyclotide precursors. These discoveries confirm the widespread distribution of cyclotides in the plant kingdom and the diversity of precursor proteins involved in their biosynthesis. Other studies have delineated the mode of action of naturally occurring cyclotides and have demonstrated the versatility of synthetic cyclotides as stable protein engineering frameworks, with applications in drug design. Conotoxins continue to be a rich source of inspiration for drug design programs, and we summarize here a range of recent studies from our laboratory focusing on the development of novel synthetic strategies and the delineation of structure-activity relationships. A major highlight was the development of an orally active cyclized conotoxin derivative that is highly efficacious in a rat model of neuropathic pain. Overall the studies described herein provide much encouragement for continuing efforts to develop peptides as drugs.
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Cyclization of the antimicrobial peptide gomesin with native chemical ligation: influences on stability and bioactivity.
Chembiochem
PUBLISHED: 01-22-2013
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Gomesin is an 18-residue peptide originally isolated from the hemocytes of the Brazilian spider Acanthoscurria gomesiana. A broad spectrum of bioactivities have been attributed to gomesin, including in vivo and in vitro cytotoxicity against tumour cells, antimicrobial, antifungal, anti-Leishmania and antimalarial effects. Given the potential therapeutic applications of gomesin, it was of interest to determine if an engineered version with a cyclic backbone has improved stability and bioactivity. Cyclization has been shown to confer enhanced stability and activity to a range of bioactive peptides and, in the case of a cone snail venom peptide, confer oral activity in a pain model. The current study demonstrates that cyclization improves the in vitro stability of gomesin over a 24 hour time period and enhances cytotoxicity against a cancer cell line without being toxic to a noncancerous cell line. In addition, antimalarial activity is enhanced upon cyclization. These findings provide additional insight into the influences of backbone cyclization on the therapeutic potential of peptides.
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The three-dimensional solution structure of mini-M conotoxin BtIIIA reveals a disconnection between disulfide connectivity and peptide fold.
Bioorg. Med. Chem.
PUBLISHED: 01-15-2013
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Conotoxins are bioactive peptides from the venoms of marine snails and have been divided into several superfamilies based on homologies in their precursor sequences. The M-superfamily conotoxins can be further divided into five branches based on the number of residues in the third loop of the peptide sequence. Recently two M-1 branch conotoxins (tx3a and mr3e) with a C1-C5, C2-C4, C3-C6 disulfide connectivity and one M-2 branch conotoxin (mr3a) with a C1-C6, C2-C4, C3-C5 disulfide connectivity were described. Here we report the disulfide connectivity, chemical synthesis and the three-dimensional NMR structure of the novel 14-residue conotoxin BtIIIA, extracted from the venom of Conus betulinus. It has the same disulfide connectivity as mr3a, which puts it in the M-2 branch conotoxins but has a distinctly different structure from other M-2 branch conotoxins. 105 NOE distance restraints and seven dihedral angle restraints were used for the structure calculations. The three-dimensional structure was determined with CYANA based on torsion angle dynamics and refinement in a water solvent box was carried out with CNS. Fifty structures were calculated and the 20 lowest energy structures superimposed with a RMSD of 0.49±0.16 Å. Even though it has the M-2 branch disulfide connectivity, BtIIIA was found to have a flying bird backbone motif depiction that is found in the M-1 branch conotoxin mr3e. This study shows that conotoxins with the same cysteine framework can have different disulfide connectivities and different peptide folds.
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Design, synthesis, and characterization of cyclic analogues of the iron regulatory peptide hormone hepcidin.
Biopolymers
PUBLISHED: 01-06-2013
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The peptide hormone hepcidin is a key regulator of iron homeostasis in vertebrates. Hepcidin acts by binding to ferroportin, the sole known iron exporter, causing it to be internalized and thus trapping iron within the cell. Dysregulation of hepcidin concentrations is associated with a range of iron-related diseases and hepcidin-based therapeutics could be developed as candidate treatments for these diseases. However peptide-based drugs, despite their many advantages, are often limited by their susceptibility to degradation within the body. Here we describe the design, synthesis and characterization of a series of backbone cyclized hepcidin analogues as an approach to produce stable hepcidin-based leads. The cyclic peptides were shown by NMR to be structurally analogous to native hepcidin. Comparison of the stability of hepcidin with one of the cyclic analogues in human serum revealed that 77% of the cyclic peptide but only 18% of linear hepcidin remained after 24 h. The cyclic peptides were tested for their ability to induce internalization of GFP-ferroportin in vitro but were all found to be inactive. This study demonstrates that backbone cyclization of disulfide-rich peptides is a suitable approach for increasing stability. However, careful consideration of a number of factors, including location of important residues and their bioactive conformation, is required to generate biologically active lead molecules. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 519-526, 2013.
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A novel quantitative kinase assay using bacterial surface display and flow cytometry.
PLoS ONE
PUBLISHED: 01-01-2013
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The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli) to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.
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Novel inhibitor cystine knot peptides from Momordica charantia.
PLoS ONE
PUBLISHED: 01-01-2013
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Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.
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Cyclotides Suppress Human T-Lymphocyte Proliferation by an Interleukin 2-Dependent Mechanism.
PLoS ONE
PUBLISHED: 01-01-2013
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Cyclotides are a diverse and abundant group of ribosomally synthesized plant peptides containing a unique cyclic cystine-knotted topology that confers them with remarkable stability. Kalata B1, a representative member of this family of mini-proteins, has been found to inhibit the proliferation of human peripheral blood mononuclear cells. Analysis of T-cell proliferation upon treatment with chemically synthesized kalata B1 mutants revealed a region comprising inter-cysteine loops 1 and 2 of the cyclotide framework to be important for biological activity. Cytokine signaling analysis using an active kalata B1 mutant [T20K], and the reference drug cyclosporin A (CsA) demonstrated that treatment of activated T-lymphocytes with these compounds decreased the expression of the interleukin-2 (IL-2) surface receptor as well as IL-2 cytokine secretion and IL-2 gene expression, whereas the inactive kalata B1 mutant [V10K] did not cause any effects. The anti-proliferative activity of [T20K] kalata B1 was antagonized by addition of exogenous IL-2. Furthermore, treatment with [T20K] kalata B1 led to an initial reduction of the effector function, as indicated by the reduced IFN-? and TNF-? production, but the levels of both cytokines stabilized over time and returned to their normal levels. On the other hand, the degranulation activity remained reduced. This indicated that cyclotides interfere with T-cell polyfunctionality and arrest the proliferation of immune-competent cells through inhibiting IL-2 biology at more than one site. The results open new avenues to utilize native and synthetically-optimized cyclotides for applications in immune-related disorders and as immunosuppressant peptides.
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Identification and structural characterization of novel cyclotide with activity against an insect pest of sugar cane.
J. Biol. Chem.
PUBLISHED: 11-10-2011
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Cyclotides are a family of plant-derived cyclic peptides comprising six conserved cysteine residues connected by three intermolecular disulfide bonds that form a knotted structure known as a cyclic cystine knot (CCK). This structural motif is responsible for the pronounced stability of cyclotides against chemical, thermal, or proteolytic degradation and has sparked growing interest in this family of peptides. Here, we isolated and characterized a novel cyclotide from Palicourea rigida (Rubiaceae), which was named parigidin-br1. The sequence indicated that this peptide is a member of the bracelet subfamily of cyclotides. Parigidin-br1 showed potent insecticidal activity against neonate larvae of Lepidoptera (Diatraea saccharalis), causing 60% mortality at a concentration of 1 ?m but had no detectable antibacterial effects. A decrease in the in vitro viability of the insect cell line from Spodoptera frugiperda (SF-9) was observed in the presence of parigidin-br1, consistent with in vivo insecticidal activity. Transmission electron microscopy and fluorescence microscopy of SF-9 cells after incubation with parigidin-br1 or parigidin-br1-fluorescein isothiocyanate, respectively, revealed extensive cell lysis and swelling of cells, consistent with an insecticidal mechanism involving membrane disruption. This hypothesis was supported by in silico analyses, which suggested that parigidin-br1 is able to complex with cell lipids. Overall, the results suggest promise for the development of parigidin-br1 as a novel biopesticide.
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ConoServer: updated content, knowledge, and discovery tools in the conopeptide database.
Nucleic Acids Res.
PUBLISHED: 11-03-2011
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ConoServer (http://www.conoserver.org) is a database specializing in the sequences and structures of conopeptides, which are toxins expressed by marine cone snails. Cone snails are carnivorous gastropods, which hunt their prey using a cocktail of toxins that potently subvert nervous system function. The ability of these toxins to specifically target receptors, channels and transporters of the nervous system has attracted considerable interest for their use in physiological research and as drug leads. Since the founding publication on ConoServer in 2008, the number of entries in the database has nearly doubled, the interface has been redesigned and new annotations have been added, including a more detailed description of cone snail species, biological activity measurements and information regarding the identification of each sequence. Automatically updated statistics on classification schemes, three-dimensional structures, conopeptide-bearing species and endoplasmic reticulum signal sequence conservation trends, provide a convenient overview of current knowledge on conopeptides. Transcriptomics and proteomics have began generating massive numbers of new conopeptide sequences, and two dedicated tools have been recently implemented in ConoServer to standardize the analysis of conopeptide precursor sequences and to help in the identification by mass spectrometry of toxins whose sequences were predicted at the nucleic acid level.
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Engineering pro-angiogenic peptides using stable, disulfide-rich cyclic scaffolds.
Blood
PUBLISHED: 10-28-2011
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Fragments from the extracellular matrix proteins laminin and osteopontin and a sequence from VEGF have potent proangiogenic activity despite their small size (< 10 residues). However, these linear peptides have limited potential as drug candidates for therapeutic angiogenesis because of their poor stability. In the present study, we show that the therapeutic potential of these peptides can be significantly improved by "grafting" them into cyclic peptide scaffolds. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1), naturally occurring, plant-derived cyclic peptides of 34 and 14 residues, respectively, were used as scaffolds in this study. Using this approach, we have designed a peptide that, in contrast to the small peptide fragments, is stable in human serum and at nanomolar concentration induces angiogenesis in vivo. This is the first report of using these scaffolds to improve the activity and stability of angiogenic peptide sequences and is a promising approach for promoting angiogenesis for therapeutic uses.
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Cyclotides: a patent review.
Expert Opin Ther Pat
PUBLISHED: 10-25-2011
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Cyclotides are bioactive mini-proteins from plants that have the unique topological feature of a head-to-tail cyclic backbone combined with a cystine knot. Because of this structure they are ultra-stable and have attracted interest as peptide-based templates for drug design applications. Cyclotide biosynthesis involves processing from a genetically encoded precursor protein but methods have been developed for their man-made synthesis using solid phase peptide synthesis as well as recombinant methods. Their natural function in plants is as insecticidal agents and thus they have potential applications in agriculture. However, they have a range of pharmaceutically relevant activities, including anti-HIV, antimicrobial and uterotonic activity. Their exceptional stability and facile synthesis lend them to uses as pharmaceutical templates into which bioactive peptide sequences can be grafted.
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?-Conotoxin ImI incorporating stable cystathionine bridges maintains full potency and identical three-dimensional structure.
J. Am. Chem. Soc.
PUBLISHED: 09-15-2011
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The two disulfide bonds of ?-conotoxin ImI, a peptide antagonist of the ?7 nicotinic acetylcholine receptor (nAChR), were systematically replaced with isosteric redox-stable cystathionine thioethers. Regioselective thioether formation was accomplished on solid support through substitution of a ?-chlorohomoalanine by an intramolecular cysteine thiol to produce hybrid thioether/disulfide analogues (2 and 3) as well as a dual cystathionine analogue (4) that were found to be structurally homologous to ?-conotoxin ImI by (1)H NMR. The antagonistic activity at the ?7 nAChR of cystathionine analogue 3 (pIC(50) = 6.41 ± 0.09) was identical to that of ?-conotoxin ImI (1, pIC(50) = 6.41 ± 0.09), whereas those of 2 (pIC(50) = 5.96 ± 0.09) and 4 (pIC(50) = 5.89 ± 0.09) showed a modest decrease. The effect of oxidation of the thioethers to sulfoxides was also investigated, with significant changes in the biological activities observed ranging from a >30-fold reduction (2S?O) to a 3-fold increase (3S?O(B)) in potencies.
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Effects of cyclization on stability, structure, and activity of ?-conotoxin RgIA at the ?9?10 nicotinic acetylcholine receptor and GABA(B) receptor.
J. Med. Chem.
PUBLISHED: 09-15-2011
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?-Conotoxin RgIA is of interest as a lead in the development of drugs for neuropathic pain. It modulates the ?9?10 nicotinic acetylcholine receptor (nAChR) and the GABA(B) receptor, both of which are implicated in antinociception. However, because of its peptidic nature, RgIA is potentially susceptible to generic problems encountered by peptide-based drugs of poor oral bioavailability, short biological half-life, and low stability. Here, we improved the biopharmaceutical properties of RgIA by backbone cyclization using 3-7 residue peptidic linkers. Cyclization with a six-residue linker does not perturb the overall structure of RgIA, improves selectivity for the GABA(B) receptor over the ?9?10 nAChR, and improves stability in human serum. The results provide insights to further improve the therapeutic properties of RgIA and other conotoxins being considered as drug leads and confirm that cyclization is a readily applicable strategy to improve the stability of peptides with proximate N- and C-termini.
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RegIIA: an ?4/7-conotoxin from the venom of Conus regius that potently blocks ?3?4 nAChRs.
Biochem. Pharmacol.
PUBLISHED: 08-27-2011
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Neuronal nicotinic acetylcholine receptors (nAChRs) play pivotal roles in the central and peripheral nervous systems. They are implicated in disease states such as Parkinsons disease and schizophrenia, as well as addictive processes for nicotine and other drugs of abuse. Modulation of specific nAChRs is essential to understand their role in the CNS. ?-Conotoxins, disulfide-constrained peptides isolated from the venom of cone snails, potently inhibit nAChRs. Their selectivity varies markedly depending upon the specific nAChR subtype/?-conotoxin pair under consideration. Thus, ?-conotoxins are excellent probes to evaluate the functional roles of nAChRs subtypes. We isolated an ?4/7-conotoxin (RegIIA) from the venom of Conus regius. Its sequence was determined by Edman degradation and confirmed by sequencing the cDNA of the protein precursor. RegIIA was synthesized using solid phase methods and native and synthetic RegIIA were functionally tested using two-electrode voltage clamp recording on nAChRs expressed in Xenopus laevis oocytes. RegIIA is among the most potent antagonist of the ?3?4 nAChRs found to date and is also active at ?3?2 and ?7 nAChRs. The 3D structure of RegIIA reveals the typical folding of most ?4/7-conotoxins. Thus, while structurally related to other ?4/7 conotoxins, RegIIA has an exquisite balance of shape, charge, and polarity exposed in its structure to potently block the ?3?4 nAChRs.
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Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides.
J. Biol. Chem.
PUBLISHED: 08-26-2011
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Cell-penetrating peptides can translocate across the plasma membrane of living cells and thus are potentially useful agents in drug delivery applications. Disulfide-rich cyclic peptides also have promise in drug design because of their exceptional stability, but to date only one cyclic peptide has been reported to penetrate cells, the Momordica cochinchinensis trypsin inhibitor II (MCoTI-II). MCoTI-II belongs to the cyclotide family of plant-derived cyclic peptides that are characterized by a cyclic cystine knot motif. Previous studies in fixed cells showed that MCoTI-II could penetrate cells but kalata B1, a prototypic cyclotide from a separate subfamily of cyclotides, was bound to the plasma membrane and did not translocate into cells. Here, we show by live cell imaging that both MCoTI-II and kalata B1 can enter cells. Kalata B1 has the same cyclic cystine knot structural motif as MCoTI-II but differs significantly in sequence, and the mechanism by which these two peptides enter cells also differs. MCoTI-II appears to enter via macropinocytosis, presumably mediated by interaction of positively charged residues with phosphoinositides in the cell membrane, whereas kalata B1 interacts directly with the membrane by targeting phosphatidylethanolamine phospholipids, probably leading to membrane bending and vesicle formation. We also show that another plant-derived cyclic peptide, SFTI-1, can penetrate cells. SFTI-1 includes just 14 amino acids and, with the exception of its cyclic backbone, is structurally very different from the cyclotides, which are twice the size. Intriguingly, SFTI-1 does not interact with any of the phospholipids tested, and its mechanism of penetration appears to be distinct from MCoTI-II and kalata B1. The ability of diverse disulfide-rich cyclic peptides to penetrate cells enhances their potential in drug design, and we propose a new classification for them, i.e. cyclic cell-penetrating peptides.
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Engineering of conotoxins for the treatment of pain.
Curr. Pharm. Des.
PUBLISHED: 08-17-2011
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The peptides present in the venoms of marine snails are used by the snails to capture prey, but they have also attracted the interest of drug designers because of their potent activity against therapeutically important targets. These peptides are typically disulfiderich and target a wide range of ion channels, transporters and receptors with exquisite selectivity. In this article, we discuss structural and biological studies on several classes of conotoxins that have potential as drug leads for the treatment of pain. The chemical re-engineering of conotoxins via cyclization has been particularly valuable in improving their biopharmaceutical properties. An excellent example is the ?-conotoxin Vc1.1, for which several cyclized analogs have been made. One of them was shown to be orally active in a rat pain model and this analog is currently undergoing pre-clinical development for the treatment of neuropathic pain. Several other ?-conotoxins, including ImI, AuIB and MII, have proved amenable to cyclization and in all cases improvements in stability are obtained upon cyclization, suggesting that cyclization is a generally applicable approach to conotoxin stabilization. A variety of other chemical re-engineering approaches have also been used. Minor re-engineering of ?-conotoxin MrIa to convert its N-terminal residue to pyroglutamic acid proved particularly successful and the modified derivative, Xen2174, is currently in clinical trials for neuropathic pain.
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A Synthetic mirror image of kalata B1 reveals that cyclotide activity is independent of a protein receptor.
Chembiochem
PUBLISHED: 07-14-2011
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Featuring a circular, knotted structure and diverse bioactivities, cyclotides are a fascinating family of peptides that have inspired applications in drug design. Most likely evolved to protect plants against pests and herbivores, cyclotides also exhibit anti-cancer, anti-HIV, and hemolytic activities. In all of these activities, cell membranes appear to play an important role. However, the question of whether the activity of cyclotides depends on the recognition of chiral receptors or is primarily modulated by the lipid-bilayer environment has remained unknown. To determine the importance of lipid membranes on the activity of the prototypic cyclotide, kalata B1, we synthesized its all-D enantiomer and assessed its bioactivities. After the all-D enantiomer had been confirmed by (1)H NMR to be the structural mirror image of the native kalata B1, it was tested for anti-HIV activity, cytotoxicity, and hemolytic properties. The all-D peptide is active in these assays, albeit with less efficiency; this reveals that kalata B1 does not require chiral recognition to be active. The lower activity than the native peptide correlates with a lower affinity for phospholipid bilayers in model membranes. These results exclude a chiral receptor mechanism and support the idea that interaction with phospholipid membranes plays a role in the activity of kalata B1. In addition, studies with mixtures of L and D enantiomers of kalata B1 suggested that biological activity depends on peptide oligomerization at the membrane surface, which determines affinity for membranes by modulating the association-dissociation equilibrium.
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The chemistry of cyclotides.
J. Org. Chem.
PUBLISHED: 05-19-2011
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Cyclotides are head-to-tail cyclic peptides that contain a cystine knot motif built from six conserved cysteine residues. They occur in plants of the Rubiaceae, Violaceae, Cucurbitaceae, and Fabaceae families and, aside from their natural role in host defense, have a range of interesting pharmaceutical activities, including anti-HIV activity. The variation seen in sequences of their six backbone loops has resulted in cyclotides being described as a natural combinatorial template. Their exceptional stability and resistance to enzymatic degradation has led to their use as scaffolds for peptide-based drug design. To underpin such applications, methods for the chemical synthesis of cyclotides have been developed and are described here. Cyclization using thioester chemistry has been instrumental in the synthesis of cyclotides for structure-activity studies. This approach involves a native chemical ligation reaction between an N-terminal Cys and a C-terminal thioester in the linear cyclotide precursor. Since cyclotides contain six Cys residues their syntheses can be designed around any of six linear precursors, thus providing flexibility in synthesis. The ease with which cyclotides fold, despite their topologically complex knot motif, as well as the ability to introduce combinatorial variation in the loops, makes cyclotides a promising drug-design scaffold.
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Discovery of an unusual biosynthetic origin for circular proteins in legumes.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-18-2011
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Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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