Disulfide-rich Peptides Research Articles

Evaluation of Efficient Non-reducing Enzymatic and Chemical Ligation Strategies for Complex Disulfide-Rich Peptides.

Double-knotted peptides identified in venoms and synthetic bivalent peptide constructs targeting ion channels are emerging tools for the study of ion channel pharmacology and physiology. These highly complex and disulfide-rich peptides contain two individual cystine knots, each comprising six cysteines and three disulfide bonds. Until now, native double-knotted peptides, such as Hi1a and DkTx, have only been isolated from venom or produced recombinantly, whereas engineered double-knotted peptides have successfully been produced through enzymatic ligation using sortase A to form a seamless amide bond at the ligation site between two knotted toxins, and by alkyne/azide click chemistry, joining two peptide knots via a triazole linkage. To further pursue these double-knotted peptides as pharmacological tools or probes for therapeutically relevant ion channels, we sought to identify a robust methodology resulting in a high yield product that lends itself to rapid production and facile mutational studies. In this study, we evaluated the ligation efficiency of enzymatic (sortase A5°, butelase 1, wild-type OaAEP 1, C247A-OaAEP 1, and peptiligase) and mild chemical approaches (α-ketoacid-hydroxylamine, KAHA) for forming a native amide bond linking the toxins while maintaining the native disulfide connectivity of each pre-folded peptide. We used two NaV1.7 inhibitors: PaurTx3, a spider-derived gating modifier peptide, and KIIIA, a small cone snail-derived pore blocker peptide, which have previously been shown to increase affinity and inhibitory potency on hNaV1.7 when ligated together. Correctly folded peptides were successfully ligated in varying yields, without disulfide bond shuffling or reduction, with sortase A5° being the most efficient, resulting in 60% ligation conversion within 15 min. In addition, electrophysiology studies demonstrated that for these two peptides, the amino acid composition of the linker did not affect the activity of the double-knotted peptides. This study demonstrates the powerful application of enzymes in efficiently ligating complex disulfide-rich peptides, paving the way for facile production of double-knotted peptides.

Rigidity of loop 1 contributes to equipotency of globular and ribbon isomers of \u03b1-conotoxin AusIA

α-Conotoxins are small disulfide-rich peptides targeting nicotinic acetylcholine receptors (nAChRs) characterised by a CICII-Xm-CIII-Xn-CIV framework that invariably adopt the native globular conformations which is typically most potent. α-Conotoxins are divided into several structural subgroups based on the number of residues within the two loops braced by the disulfide bonds (m/n), with the 4/7 and 4/3 subgroups dominating. AusIA is a relatively rare α5/5-conotoxin isolated from the venom of Conus australis. Surprisingly, the ribbon isomer displayed equipotency to the wild-type globular AusIA at human α7-containing nAChR. To understand the molecular basis for equipotency, we determined the co-crystal structures of both isomers at Lymnea stagnalis acetylcholine binding protein. The additional residue in the first loop of AusIA was found to be a critical determinant of equipotency, with 11-fold and 86-fold shifts in potency in favour of globular AusIA over ribbon AusIA observed following deletion of Ala4 or Arg5, respectively. This divergence in the potency between globular AusIA and ribbon AusIA was further enhanced upon truncation of the non-conserved Val at the C-termini. Conversely, equipotency could be replicated in LsIA and TxIA [A10L] following insertion of an Ala in the first loop. These findings provide a new understanding of the role the first loop in ribbon and globular α-conotoxins can play in directing α-conotoxin nAChR pharmacology.

Structural Characterization of the PawL-Derived Peptide Family, an Ancient Subfamily of Orbitides.

Plants are an excellent source of bioactive peptides, often with disulfide bonds and/or a cyclic backbone. While focus has predominantly been directed at disulfide-rich peptides, a large family of small, cyclic plant peptides lacking disulfide bonds, known as orbitides, has been relatively ignored. A recently discovered subfamily of orbitides is the PawL-derived peptides (PLPs), produced during the maturation of precursors for seed storage albumins. Although their evolutionary origins have been dated, in-depth exploration of the family's structural characteristics and potential bioactivities remains to be conducted. Here we present an extensive and systematic characterization of the PLP family. Nine PLPs were chosen and prepared by solid phase peptide synthesis. Their structural features were studied using solution NMR spectroscopy, and seven were found to possess regions of backbone order. Ordered regions consist of β-turns, with some PLPs adopting two well-defined β-turns within sequences as short as seven residues, which are largely the result of side chain interactions. Our data highlight that the sequence diversity within this family results in equally diverse structures. None of these nine PLPs showed antibacterial or antifungal activity.

Insight on the Order of Regioselective Ultrafast Formation of Disulfide Bonds in (Antimicrobial) Peptides and Miniproteins.

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets have prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one-pot disulfide bond formation, tackling most of the previous limitations. However, the effect of the order of oxidation remained an underexplored issue. Herein we report on the complete synthetic flexibility of the approach with respect to the order of oxidation of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistant Staphylococcus aureus (MRSA), a notorious human pathogen.

Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin.

Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (K d for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Venom composition of the endoparasitoid wasp Cotesia flavipes (Hymenoptera: Braconidae) and functional characterization of a major venom peptide

Endoparasitoid wasps use complex biochemical arsenals to suppress the normal humoral and cellular immune responses of their hosts in order to transform them into a suitable environment for development of their eggs and larvae. Venom injected during oviposition is a key component of this arsenal, but the functions of individual venom toxins are still poorly understood. Furthermore, there has been little investigation of the potential biotechnological use of these venom toxins, for example for control of agricultural pests. The endoparasitoid Cotesia flavipes (Hymenoptera: Braconidae) is a biocontrol agent reared in biofactories and released extensively in Brazil to control the sugarcane borer Diatraea saccharalis (Lepidoptera: Crambidae). The objectives of this work were to reveal venom components produced by C. flavipes and explore the function of a major venom peptide, Cf4. Using a combined proteomic/transcriptomic approach, we identified 38 putative venom toxins including both linear and disulfide-rich peptides, hydrolases, protease inhibitors, apolipophorins, lipid-binding proteins, and proteins of the odorant binding families. Because of its high abundance in the venom, we selected Cf4, a 33-residue peptide with three disulfide bonds, for synthesis and further characterization. We found that synthetic Cf4 reduced the capacity of D. saccharalis hemocytes to encapsulate foreign bodies without any effect on phenoloxidase activity, consistent with a role in disruption of the cellular host immune response. Feeding leaves coated with Cf4 to neonate D. saccharalis resulted in increased mortality and significantly reduced feeding compared to caterpillars fed untreated leaves, indicating that Cf4 is a potential candidate for insect pest control through ingestion. This study adds to our knowledge of endoparasitoid wasp venoms composition, host regulation mechanisms and their biotechnological potential for pest management.

Design and In Silico Evaluation of a Novel Cyclic Disulfide-Rich anti-VEGF Peptide as a Potential Antiangiogenic Drug

Design and In Silico Evaluation of a Novel Cyclic Disulfide-Rich anti-VEGF Peptide as a Potential Antiangiogenic Drug

Plant-Derived Cyclotides Modulate κ-Opioid Receptor Signaling.

Cyclotides are plant-derived disulfide-rich peptides comprising a cyclic cystine knot, which confers remarkable stability against thermal, proteolytic, and chemical degradation. They represent an emerging class of G protein-coupled receptor (GPCR) ligands. In this study, utilizing a screening approach of plant extracts and pharmacological analysis we identified cyclotides from Carapichea ipecacuanha to be ligands of the κ-opioid receptor (KOR), an attractive target for developing analgesics with reduced side effects and therapeutics for multiple sclerosis (MS). This prompted us to verify whether [T20K]kalata B1, a cyclotide in clinical development for the treatment of MS, is able to modulate KOR signaling. T20K bound to and fully activated KOR in the low μM range. We then explored the ability of T20K to allosterically modulate KOR. Co-incubation of T20K with KOR ligands resulted in positive allosteric modulation in functional cAMP assays by altering either the efficacy of dynorphin A1–13 or the potency and efficacy of U50,488 (a selective KOR agonist), respectively. In addition, T20K increased the basal response upon cotreatment with U50,488. In the bioluminescence resonance energy transfer assay T20K negatively modulated the efficacy of U50,488. This study identifies cyclotides capable of modulating KOR and highlights the potential of plant-derived peptides as an opportunity to develop cyclotide-based KOR modulators.

Discovery of Methylene Thioacetal-Incorporated α-RgIA Analogues as Potent and Stable Antagonists of the Human α9α10 Nicotinic Acetylcholine Receptor for the Treatment of Neuropathic Pain.

α9-Containing nicotinic acetylcholine receptors (nAChRs) are key targets for the treatment of neuropathic pain. α-Conotoxin RgIA4 is a peptide antagonist of human α9α10 nAChRs with high selectivity. However, structural rearrangement reveals a potential liability for clinical applications. We herein report our designer RgIA analogues stabilized by methylene thioacetal as nonopioid analgesic agents. We demonstrate that replacing disulfide loop I [CysI-CysIII] with methylene thioacetal in the RgIA skeleton results in activity loss, whereas substitution of loop II [CysII-CysIV] can be accommodated. The lead molecule, RgIA-5524, exhibits highly selective inhibition of α9α10 nAChRs with an IC50 of 0.9 nM and much reduced degradation in human serum. In vivo studies showed that RgIA-5524 relieves chemotherapy-induced neuropathic pain in wild type but not α9 knockout mouse models, demonstrating that α9-containing nAChRs are necessary for the therapeutic effects. This work highlights the application of methylene thioacetal as a disulfide surrogate in conotoxin-based, disulfide-rich peptide drugs.

α-Conotoxin Bt1.8 from Conus betulinus selectively inhibits α6/α3β2β3 and α3β2 nicotinic acetylcholine receptor subtypes.

α-Conotoxins are small disulfide-rich peptides found in the venom of marine cone snails and are potent antagonists of nicotinic acetylcholine receptors (nAChRs). They are valuable pharmacological tools and have potential therapeutic applications for the treatment of chronic pain or neurological diseases and disorders. In the present study, we synthesized and functionally characterized a novel α-conotoxin Bt1.8, which was cloned from Conus betulinus. Bt1.8 selectively inhibited ACh-evoked currents in Xenopus oocytes expressing rat(r) α6/α3β2β3 and rα3β2 nAChRs with an IC50 of 2.1nM and 9.4nM, respectively, and similar potency for human (h) α6/α3β2β3 and hα3β2 nAChRs. Additionally, Bt1.8 had higher binding affinity with a slower dissociation rate for the rα6/α3β2β3 subtype compared to rα3β2. The amino acid sequence of Bt1.8 is significantly different from other reported α-conotoxins targeting the two nAChR subtypes. Further Alanine scanning analyses demonstrated that residues Ile9, Leu10, Asn11, Asn12 and Asn14 are critical for its inhibitory activity at the α6/α3β2β3 and α3β2 subtypes. Moreover, the NMR structure of Bt1.8 indicated the presence of a relatively larger hydrophobic zone than other α4/7-conotoxins which may explain its potent inhibition at α6/α3β2β3 nAChRs.

Molecular Dynamics Simulation Study on Interactions of Cycloviolacin with Different Phospholipids.

Cyclotides are disulfide-rich cyclic peptides isolated from plants, which are extremely stable against thermal and proteolytic degradation, with a variety of biological activities including antibacterial, hemolytic, anti-HIV, and anti-tumor. Most of these bioactivities are related to their preference for binding to certain types of phospholipids and subsequently disrupt lipid membranes. In the present study, we use a cyclotide, cycloviolacin O2 (cyO2), as a model system to investigate its interactions with three lipid bilayers 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)-doped POPE, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), to help understand its potential mechanism of action toward the membranes at the molecular level using molecular dynamics simulations. In our simulations, cyO2 repeatedly forms stable binding complexes with the POPE-containing bilayers, while within the same simulation time scale, it "jumps" back and forth on the surface of the POPC bilayer without a strong binding. Detailed analyses reveal that the electrostatic attraction is the main driving force for the initial bindings between cyO2 and the lipids, but with strikingly different strengths in different bilayers. For the POPE-containing bilayers, the charged residues of cyO2 attract both POPE amino and phosphate head groups favorably; meanwhile, its hydrophobic residues are deeply inserted into the lipid hydrophobic tails (core) of the membrane, thus forming stable binding complexes. In contrast, POPC lipids with three methyl groups on the amino head group create a steric hindrance when interacting with cyO2, thus resulting in a relatively difficult binding of cyO2 on POPC compared to POPE. Our current findings provide additional insights for a better understanding of how cyO2 binds to the POPE-containing membrane, which should shed light on the future cyclotide-based antibacterial agent design.

Characterization of 2 linear peptides without disulfide bridges from the venom of the spider Lycosa poonaensis (Lycosidae).

Spider venom is a complex mixture of bioactive components, in which peptides play an important role by showing neurotoxicity or cytotoxicity. Disulfide-rich peptides are major components in the venom, but linear peptides without disulfide bridges are also present and often show antimicrobial activity. In this study, we analyzed the venom of the spider Lycosa poonaensis (Lycosidae) to find novel antimicrobial peptides using mass spectrometry. The result revealed that 120 out of 401 detected components were nondisulfide-bridged peptides. From them, the sequence of 2 peptides (lyp2370 and lyp1987) were determined by MS/MS analysis. The biological activity test revealed that lyp2370 has only weak antibacterial activity. On the other hand, lyp1987, which is identical to M-lycotoxin-Ls3b from the Lycosa singoriensi venom, showed significant antibacterial activity. The weak activity of lyp2370 was found to be due to the presence of a Glu residue on the hydrophilic face of its amphipathic α-helical structure.

Abstract PO030: A novel integrin-targeted therapeutic agent for prostate cancer

Abstract Disintegrins are disulfide-rich peptides, many containing an Arg-Gly-Asp (RGD) motif. They hold significant translational potential as anti-cancer agents based on their high-affinity/high-specificity interaction with tumor integrins and desirable pharmacological attributes. The integrin-binding activity of disintegrins depends on a mobile 11-amino acid loop displaying the RGD tripeptide motif or some other tripeptide motif. They bind only to the activated conformation of integrins on motile cancer cells and angiogenic endothelial cells. We designed a sequence-engineered RGD-disintegrin, vicrostatin (VCN), that can be produced at ~200mg/L from cell lysates of a bacterial recombinant system. VCN, a monomer (MW=7146), inhibits tumor cell adhesion and dissemination, and angiogenic endothelial cell invasion and tube formation. Integrins are cell surface adhesion receptors (containing a and b chains) operating at the interface between the extracellular matrix and cytoskeletal apparatus. A sub-group of integrins recognize the RGD sequence present in key extracellular matrix proteins involved in prostate cancer (PC) growth and dissemination. Integrins are involved in bi-directional signaling interactions that alter cellular functions. Among these interactions are those involved in PC growth, metastasis and angiogenesis. Integrin avb3 plays an important role in the development of bone metastases, supporting our belief that it, as well as other RGD-integrins, are important therapeutic targets in advanced PC. We examined the effect of treatment of a xenograft model of human PC using a liposomal formulation of VCN (LVCN). PC3 cells were implanted subcutaneously in 5-week old male nude mice. LVCN and VCN (100µg VCN equivalent per dose) administration was initiated IV when tumors became palpable (14 days). Tumor size was measured weekly by caliper in a blinded study. There was a 75% reduction in tumor growth in LVCN-treated animals compared to controls. VCN displayed little anti-tumor activity most likely due to its lack of availability to the PC cells. We also examined the anti-angiogenic activity of LVCN in this model using CD31/PECAM immunohistochemistry and observed a significant decrease in tumor-associated microvessel density. We then evaluated therapeutic efficacy of LVCN in treating bone metastases. CWR22rV1 PC cells were suspended in a solution of matrigel and injected into a drilled hole in the proximal left tibia of immunodeficient mice (100,000 cells/10µL). Following injection, the diameters of both tibias were measured by caliper twice weekly. Since CWR22rV1 cells are androgen dependent, animals received daily injections of testosterone. Tumors grew slowly initially, but at 20 days tumors were evident and treatment began. LVCN and VCN were administered twice weekly via IV injection (100µg per dose) for 5 weeks. LVCN displayed ~81% inhibition of tumor growth compared to VCN, PBS or empty liposomes. Our findings provide strong support for the effectiveness of LVCN as an inhibitor of PC growth and metastases. Citation Format: Francis S. Markland, Stephen Swenson, Radu Minea, Jacek Pinski. A novel integrin-targeted therapeutic agent for prostate cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO030.

Yeast-based bioproduction of disulfide-rich peptides and their cyclization via asparaginyl endopeptidases.

Cyclic disulfide-rich peptides have attracted significant interest in drug development and biotechnology. Here, we describe a protocol for producing cyclic peptide precursors in Pichia pastoris that undergo in vitro enzymatic maturation into cyclic peptides using recombinant asparaginyl endopeptidases (AEPs). Peptide precursors are expressed with a C-terminal His tag and secreted into the media, enabling facile purification by immobilized metal affinity chromatography. After AEP-mediated cyclization, cyclic peptides are purified by reverse-phase high-performance liquid chromatography and characterized by mass spectrometry, peptide mass fingerprinting, NMR spectroscopy, and activity assays. We demonstrate the broad applicability of this protocol by generating cyclic peptides from three distinct classes that are either naturally occurring or synthetically backbone cyclized, and range in size from 14 amino acids with one disulfide bond, to 34 amino acids with a cystine knot comprising three disulfide bonds. The protocol requires 14 d to identify and optimize a high-expressing Pichia clone in small-scale cultures (24 well plates or 50 mL tubes), after which large-scale production in a bioreactor and peptide purification can be completed in 10 d. We use the cyclotide Momordica cochinchinensis trypsin inhibitor II as an example. We also include a protocol for recombinant AEP production in Escherichia coli as AEPs are emerging tools for orthogonal peptide and protein ligation. We focus on two AEPs that preferentially cyclize different peptide precursors, namely an engineered AEP with improved catalytic efficiency [C247A]OaAEP1b and the plant-derived MCoAEP2. Rudimentary proficiency and equipment in molecular biology, protein biochemistry and analytical chemistry are needed.

Chemical Synthesis and NMR Solution Structure of Conotoxin GXIA from Conus geographus.

Conotoxins are disulfide-rich peptides found in the venom of cone snails. Due to their exquisite potency and high selectivity for a wide range of voltage and ligand gated ion channels they are attractive drug leads in neuropharmacology. Recently, cone snails were found to have the capability to rapidly switch between venom types with different proteome profiles in response to predatory or defensive stimuli. A novel conotoxin, GXIA (original name G117), belonging to the I3-subfamily was identified as the major component of the predatory venom of piscivorous Conus geographus. Using 2D solution NMR spectroscopy techniques, we resolved the 3D structure for GXIA, the first structure reported for the I3-subfamily and framework XI family. The 32 amino acid peptide is comprised of eight cysteine residues with the resultant disulfide connectivity forming an ICK+1 motif. With a triple stranded β-sheet, the GXIA backbone shows striking similarity to several tarantula toxins targeting the voltage sensor of voltage gated potassium and sodium channels. Supported by an amphipathic surface, the structural evidence suggests that GXIA is able to embed in the membrane and bind to the voltage sensor domain of a putative ion channel target.

Tracking internal and external ions for constrained peptides leads to enhanced sequence coverage and disulfide bond deciphering

Top-down characterization of disulfide-rich peptides and proteins presents many challenges due to the constrained and protected amino acid backbone. Typically, chemical reduction is required to reduce the disulfide bonds and/or enzymatic digestion (bottom-up analysis) is utilized to selectively cleave the amino acid sequence prior to mass spectrometry analysis owing to the challenges associated with intact, top-down analysis of these biomolecules. While extravagant top-down characterization techniques such as ultraviolet photodissociation (UVPD) or electron capture dissociation (ECD), have demonstrated the ability to break disulfide bonds in top-down workflows, implementation of these technologies and analysis of the resulting fragmentation spectra is not trivial and often inaccessible to many laboratories and users. In the study presented herein, traditional collision induced dissociation (CID) of disulfide-rich peptides is performed to confirm the disulfide bond connectivity and localize chemical modifications for these synthetic therapeutic peptides. While collisional activation does not fragment the peptide backbone linearly (typical N- and C-terminal fragment ions) within the disulfide-bonded regions, internal and external ions are consistently produced throughout the sequence via secondary fragmentation pathways. In this study, seven disulfide-rich peptides (Peptides A – G) with similar disulfide connectivity but varying amino acid composition were subjected to collisional activation for sequencing and disulfide bond confirmation. While only four linear b- and y-type fragment ions are produced for these peptides, fragmentation throughout the amino acid sequence is observed when searching for internal and external fragment ions. These ions are typically not considered during traditional top-down sequencing experiments due to the computational challenge of having an increased search space for fragment ion identification. Through the identification of reproducible internal and external fragment ions, site-specific modifications can also be localized, such as oxidation on the 18th residue in Peptide A. Ultimately, this observation and identification of internal and external ions simplifies the experimental process and wet-chemistry required to accurately depict the disulfide connectivity and the sequencing of these traditionally challenging biomolecules. Further consideration to these non-traditional fragment ions should be given during top-down intact peptide and protein analysis, especially when non-linear sequences are involved.

Enzymatic Ligation of Disulfide-Rich Animal Venom Peptides: Using Sortase A to Form Double-Knotted Peptides.

Sortase A is a thiol transpeptidase expressed by Gram-positive bacteria. This enzyme is capable of site-specifically ligating peptides containing the C-terminal recognition motif LPXTG to peptides containing an N-terminal polyglycine sequence, forming a native peptide bond. Here, we describe the preparation and application of sortase A to the ligation of two individually folded disulfide-rich animal venom peptides in order to form a heterodimeric double-knotted peptide with a native peptide linker. This method is mild enough to preserve the structures and disulfide connectivities of the peptides during ligation. We employed a highly efficient sortase A pentamutant (SrtA5°), which brings the reaction to completion within 15min with a ~50-80% yield of ligated peptide.

An evolution-inspired strategy to design disulfide-rich peptides tolerant to extensive sequence manipulation

Natural disulfide-rich peptides (DRPs) are valuable scaffolds for the development of new bioactive molecules and therapeutics. However, there are only a limited number of topologically distinct DRP folds in nature, and most of them suffer from the problem of in vitro oxidative folding. Thus, strategies to design DRPs with new constrained topologies beyond the scope of natural folds are desired. Herein we report a general evolution-inspired strategy to design new DRPs with diverse disulfide frameworks, which relies on the incorporation of two cysteine residues and a random peptide sequence into a precursor disulfide-stabilized fold. These peptides can spontaneously fold in redox buffers to the expected tricyclic topologies with high yields. Moreover, we demonstrated that these DRPs can be used as templates for the construction of phage-displayed peptide libraries, enabling the discovery of new DRP ligands from fully randomized sequences. This study thus paves the way for the development of new DRP ligands and therapeutics with structures not derived from natural DRPs.

Solution NMR and racemic crystallography provide insights into a novel structural class of cyclic plant peptides.

Head-to-tail cyclic and disulfide-rich peptides are natural products with applications in drug design. Among these are the PawS-Derived Peptides (PDPs) produced in seeds of the daisy plant family. PDP-23 is a unique member of this class in that it is twice the typical size and adopts two β-hairpins separated by a hinge region. The β-hairpins, both stabilised by a single disulfide bond, fold together into a V-shaped tertiary structure creating a hydrophobic core. In water two PDP-23 molecules merge their hydrophobic cores to form a square prism quaternary structure. Here, we synthesised PDP-23 and its enantiomer comprising d-amino acids and achiral glycine, which allowed us to confirm these solution NMR structural data by racemic crystallography. Furthermore, we discovered the related PDP-24. NMR analysis showed that PDP-24 does not form a dimeric structure and it has poor water solubility, but in less polar solvents adopts near identical secondary and tertiary structure to PDP-23. The natural role of these peptides in plants remains enigmatic, as we did not observe any antimicrobial or insecticidal activity. However, the plasticity of these larger PDPs and their ability to change structure under different conditions make them appealing peptide drug scaffolds.

Cysteine protecting groups: applications in peptide and protein science.

Protecting group chemistry for the cysteine thiol group has enabled a vast array of peptide and protein chemistry over the last several decades. Increasingly sophisticated strategies for the protection, and subsequent deprotection, of cysteine have been developed, facilitating synthesis of complex disulfide-rich peptides, semisynthesis of proteins, and peptide/protein labelling in vitro and in vivo. In this review, we analyse and discuss the 60+ individual protecting groups reported for cysteine, highlighting their applications in peptide synthesis and protein science.