Design and modification of frog skin peptide brevinin-1GHa with enhanced antimicrobial activity on Gram-positive bacterial strains.

Naturally occurring frog skin peptides are one of largest sources of antimicrobial peptides that have many advantages including high potency, broad spectrum of targets and low susceptibility to multiple drug-resistance bacteria. However, they also have disadvantages such as hemolytic activity, low stability and high production costs. For these reasons, various strategies have been applied to overcome these drawbacks restricting their use in clinical trials. Previously reported brevinin-1GHa (BR-1GHa) is a 24 amino acid long antimicrobial peptide isolated from Hylarana guentheri with hemolytic activity. To enhance the antimicrobial activity of this peptide and to reduce its hemolytic activity, we designed five new temporin like analogues and examined their bioactivities. Temporins are another class of frog skin peptides without hemolytic activity and shorter than brevinins. When the antimicrobial activities of new analogues were examined against a panel of microorganisms, BR-1GHa-3, in which two alanine residues in the truncated version of BR-1GHa were replaced with leucine, exhibited significantly improved antimicrobial activity against Gram-positive bacterial strains (e.g., S. aureus ATCC 29213 and E. casseliflavus ATCC 700327) with lower hemolytic activity compared to the BR-1GHa peptide. Furthermore, BR-1GHa-4 analogue, in which Gly3 was replaced with Pro, did not show any hemolytic activity except for highest (128 µM) concentration tested and have a strong antimicrobial effect on Gram-positive bacteria (e.g., E. faecalis ATCC 51299 and B. cereus ATCC 13061).

Cyclotide-rich fractions containing nanofibers by electrospinning: preparation, characterization and examination of antimicrobial activity.

In this study, antimicrobial nanofibers were produced with the mixtures of polyvinyl alcohol (PVA) and cyclotide-rich fractions by electrospinning method. After extraction, the first separation was carried out with C18 flash chromatography and then fractioned into five separate parts by reversed-phase high-pressure liquid chromatography (RP-HPLC). The molecular weights of cyclotides in each fraction were determined by quadrupole time-of-flight liquid chromatography-mass spectrometry (Q-TOF LC-MS). Cyclotide-rich fractions were mixed with 10% of PVA solution and nanofibers were produced from this biocomposite mixture by electrospinning method. The nanofibers were characterized by field emission scanning electron microscopy (FE-SEM), and it was observed that 100% peptide-containing nanofibers (cyclotide-rich fraction/10% PVA, w/v) had more regular fiber textures. The presence of the peptides in the nanofiber was also confirmed by analytical RP-HPLC, as the peptides in both peptide fractions and nanofiber solutions have the same retention times. The nanofibers produced with the fourth cyclotide-rich fraction showed activity against gram-positive bacteria (Bacillus cereus) in antimicrobial susceptibility test. As a result of these findings, cyclotide-containing nanofibers with antimicrobial activity can be produced for pharmaceutical research and development studies.

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.

Transforming conotoxins into cyclotides: Backbone cyclization of P-superfamily conotoxins.

Peptide backbone cyclization is a widely used approach to improve the activity and stability of small peptides but until recently it had not been applied to peptides with multiple disulfide bonds. Conotoxins are disulfide-rich conopeptides derived from the venoms of cone snails that have applications in drug design and development. However, because of their peptidic nature, they can suffer from poor bioavailability and poor stability in vivo. In this study two P-superfamily conotoxins, gm9a and bru9a, were backbone cyclized by joining the N- and C-termini with short peptide linkers using intramolecular native chemical ligation chemistry. The cyclized derivatives had conformations similar to the native peptides showing that backbone cyclization can be applied to three disulfide-bonded peptides with cystine knot motifs. Cyclic gm9a was more potent at high voltage-activated (HVA) calcium channels than its acyclic counterpart, highlighting the value of this approach in developing active and stable conotoxins containing cyclic cystine knot motifs.

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.

Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro.

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 µM) compared with wild-type EGF-A (KD = 1.2 µM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.

Characterization of a novel α-conotoxin TxID from Conus textile that potently blocks rat α3ß4 nicotinic acetylcholine receptors.

The α3ß4 nAChRs are implicated in pain sensation in the PNS and addiction to nicotine in the CNS. We identified an α-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 α3ß4 nAChRs with a 12.5 nM IC50, which places it among the most potent α3ß4 nAChR antagonists. TxID also blocked the closely related α6/α3ß4 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 α-CTx fold but with different surface charge distribution to other 4/6 family members. α-CTx TxID is a novel tool with which to probe the structure and function of α3ß4 nAChRs.

Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design.

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.

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 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.

Characterization of a novel α-conotoxin from conus textile that selectively targets α6/α3ß2ß3 nicotinic acetylcholine receptors.

α6ß2 Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic neurons in the CNS are potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine addiction and Parkinson disease. However, recent studies indicate that the α6 subunit can also associate with the ß4 subunit to form α6ß4 nAChRs that are difficult to pharmacologically distinguish from α6ß2, α3ß4, and α3ß2 subtypes. The current study characterized a novel 16-amino acid α-conotoxin (α-CTx) TxIB from Conus textile whose sequence is GCCSDPPCRNKHPDLC-amide as deduced from gene cloning. The peptide and an analog with an additional C-terminal glycine were chemically synthesized and tested on rat nAChRs heterologously expressed in Xenopus laevis oocytes. α-CTx TxIB blocked α6/α3ß2ß3 nAChR with an IC(50) of 28 nm. In contrast, the peptide showed little or no block of other tested subtypes at concentrations up to 10 µm. The three-dimensional solution structure of α-CTx TxIB was determined using NMR spectroscopy. α-CTx TxIB represents a uniquely selective ligand for probing the structure and function of α6ß2 nAChRs.

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.

Chemical re-engineering of chlorotoxin improves bioconjugation properties for tumor imaging and targeted therapy.

Bioconjugates composed of chlorotoxin and near-infrared fluorescent (NIRF) moieties are being advanced toward human clinical trials as intraoperative imaging agents that will enable surgeons to visualize small foci of cancer. In previous studies, the NIRF molecules were conjugated to chlorotoxin, which results in a mixture of mono-, di-, and trilabeled peptide. Here we report a new chemical entity that bound only a single NIRF molecule. The lysines at positions 15 and 23 were substituted with either alanine or arginine, which resulted in only monolabeled peptide that was functionally equivalent to native chlorotoxin/Cy5.5. We also analyzed the serum stability and serum half-life of cyclized chlorotoxin, which showed an 11 h serum half-life and resulted in a monolabeled product. Based on these data, we propose to advance a monolabeled chlorotoxin to human clinical trials.