Characterization of a novel alpha-conotoxin TxID from Conus textile that potently blocks rat alpha3/beta4 nicotinic acetylcholine receptors

The alpha 3 beta 4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an alpha-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 alpha 3 beta 4 nAChRs with a 12.5 nM IC50, which places it among the most potent alpha 3 beta 4 nAChR antagonists. TxID also blocked the closely related alpha 6/alpha 3 beta 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 alpha-CTx fold but with different surface charge distribution to other 4/6 family members. alpha-CTx TxID is a novel tool with which to probe the structure and function of alpha 3 beta 4 nAChRs

Design and discovery of cyclic peptides with applications in drug development

In recent years, the use of peptides in drug design and development applications has gained increasing prominence. Cyclotides are plant-derived disulfide-rich peptides with high stability, and their cyclic cystine knot motif makes them very useful scaffolds for protein engineering purposes. Conotoxins are another class of disulfide rich peptides. They are obtained from the venoms of marine cone snails and some of them have similar topologies to cyclotides. Because of their highly selective and potent activities, several conotoxins are being used to develop novel drugs. However, being peptides their susceptibility to proteolysis potentially limits their use as drugs. It is proposed in this thesis that chimeric cyclotide-conotoxin peptides might have enhanced resistance to enzymatic digestions. Thus, the overall aim of this thesis is to develop new cyclic peptides with potential therapeutic applications. The thesis comprises seven chapters that describe a range of studies on different aspects of the challenges of using natural peptides to develop drugs. Chapter 1 introduces the peptides studied in the thesis and Chapter 2 describes the materials and methods used throughout the thesis. The remaining chapters describe the experimental findings. Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides, but until recently had not been applied to peptides with multiple disulfide bonds. In Chapter 3, the backbone cyclization of ω-conotoxins MVIIA and CVID and the P-superfamily conotoxins Gm9a and Bru9a was studied by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclised derivatives of MVIIA have potent activity at N-type calcium channels, similar to the native peptide, highlighting the potential of this approach in developing active ω-conotoxin analogues. Backbone cyclization of Gm9a and Bru9a resulted in correctly folded cyclic peptides with high serum stability. x In Chapter 4, the aim was to re-engineering of a scorpion venom peptide, chlorotoxin (CTX), by substituting Lys15 and Lys23 with Ala or Arg to produce a mono-labeled peptide for regulatory purposes and to synthesize a cyclic version with high stability. The most remarkable feature of CTX is that it selectively binds to glioma, a type of tumor cells in the brain. An optical imaging contrast agent called “Tumor Paint” is being developed to enable surgeons to distinguish cancer cells from adjacent normal tissue. Tumour paint is a bioconjugated form of CTX with a near infrared fluorescent (NIRF) molecule Cy5.5. The studies described in this chapter were aimed to improve the stability and labeling efficiency of conjugated CTX. Encouragingly, the results showed that the bioconjugates of the engineered peptide are functionally equivalent to the native CTX:Cy.5.5 and backbone cyclization resulted in a more stable peptide. In Chapter 5, an alanine scanning mutagenesis of CTX was carried out to determine which residues are important for binding to tumor cells. All alanine mutants were synthesized by Fmoc solid phase peptide chemistry and tested for binding to tumors in a ND2:SmoA1 medulloplastoma mouse model after tail-vein injection. Biophotonic images of mice brains were obtained with a Xenogen near infrared imaging system. Twenty six alanine mutants were tested, with six showing an increase in tumor targeting. The results of this study will be used in the design and synthesis of more active mutants and cyclization will be used to increase the stability of the active analogues. In a complementary approach to the backbone cyclization of peptides described in Chapters 3-5, Chapter 6 explores a grafting approach to transfer the bioactive residues of a conotoxin onto a more stable cyclotide framework. The second loop of MVIA, containing the most active residue Tyr13, was grafted onto the second loop of kalata B1. The grafted peptide has a native fold and higher serum stability than MVIIA. However, no inhibition of N-type VGCCs was observed. xi In summary, this thesis has provided fundamental new insights into the backbone cyclization of disulfide-rich peptides and grafting of the bioactive epitopes to produce drug leads with enhanced stability. The findings from the structure activity studies have the potential to facilitate the development of stable and more effective molecular imaging agents. Overall, the study has provided new knowledge on the tolerance of disulfide-rich peptide scaffolds to chemical reengineering

Synthesis of cyclic disulfide-rich peptides

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

The three-dimensional solution structure of mini-M conotoxin BtIIIA reveals a disconnection between disulfide connectivity and peptide fold

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 angstrom. 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. (C) 2013 Elsevier Ltd. All rights reserved

Less is more: design of a highly stable disulfide-deleted mutant of analgesic cyclic α-conotoxin Vc1.1

Cyclic α-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H,C8F]cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human α9α10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 μM and rat N-type (Cav2.2) and recombinant human Cav2.3 calcium channels via GABAB receptor activation, with an IC50 of ~900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis

Cyclization of conotoxins to improve their biopharmaceutical properties

Conotoxins are disulfide-rich peptides from the venoms of marine cone snails that are used in prey capture. Due to their exquisite potency and selectivity for different ion channels, receptors and transporters they have attracted much interest as leads in drug design. This article gives a brief background on conotoxins, describes their structures and highlights methods for synthetic cyclization to improve their biopharmaceutical properties. The proximity of the N and C termini of many conotoxins makes them particularly suitable for cyclization with linkers of on average five to seven amino acids. By linking the ends of conotoxins it is possible to significantly decrease their susceptibility to proteolysis without loss of their intrinsic biological activity. Here, the principles of conotoxin cyclization are illustrated with applications to the α- and χ- conotoxin classes, which have been implicated as leads for the treatment of pain and a range of other disorders including neuroprotection, schizophrenia, depression and cancer

Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition

Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases

Less is More: Design of a Highly Stable Disulfide-Deleted Mutant of Analgesic Cyclic α-Conotoxin Vc1.1

Cyclic alpha-conotoxin Vc1.1 (cVc1.1) is an orally active peptide with analgesic activity in rat models of neuropathic pain. It has two disulfide bonds, which can have three different connectivities, one of which is the native and active form. In this study we used computational modeling and nuclear magnetic resonance to design a disulfide-deleted mutant of cVc1.1, [C2H, C8F] cVc1.1, which has a larger hydrophobic core than cVc1.1 and, potentially, additional surface salt bridge interactions. The new variant, hcVc1.1, has similar structure and serum stability to cVc1.1 and is highly stable at a wide range of pH and temperatures. Remarkably, hcVc1.1 also has similar selectivity to cVc1.1, as it inhibited recombinant human alpha 9 alpha 10 nicotinic acetylcholine receptor-mediated currents with an IC50 of 13 mu M and rat N-type (Ca(v)2.2) and recombinant human Ca(v)2.3 calcium channels via GABA(B) receptor activation, with an IC50 of similar to 900 pM. Compared to cVc1.1, the potency of hcVc1.1 is reduced three-fold at both analgesic targets, whereas previous attempts to replace Vc1.1 disulfide bonds by non-reducible dicarba linkages resulted in at least 30-fold decreased activity. Because it has only one disulfide bond, hcVc1.1 is not subject to disulfide bond shuffling and does not form multiple isomers during peptide synthesis