Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic β-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.
Pathogenic microbes are developing resistance to established antibiotics, making the development of novel antimicrobial molecules paramount. One major resource for discovery of antimicrobials is the arsenal of innate immunity molecules that are part of the first line of pathogen defense in many organisms. Gene encoded cationic antimicrobial peptides are a major constituent of innate immune arsenals. Many of these peptides exhibit potent antimicrobial activity in vitro . However, a major hurdle that has impeded their development for use in the clinic is the loss of activity at physiological salt concentrations, attributed to weakening of the electrostatic interactions between the cationic peptide and anionic surfaces of the microbial cells in the presence of salt. Using plant defensins we have investigated the relationship between the charge of an antimicrobial peptide and its activity in media with elevated salt concentrations. Plant defensins are a large class of antifungal peptides that have remarkable stability at extremes of pH and temperature as well as resistance to protease digestion. A search of a database of over 1200 plant defensins identified ZmD32, a defensin from Zea mays , with a predicted charge of +10.1 at pH 7, the highest of any defensin in the database. Recombinant ZmD32 retained activity against a range of fungal species in media containing elevated concentrations of salt. In addition, ZmD32 was active against Candida albicans biofilms as well as both Gram negative and Gram-positive bacteria. This broad spectrum antimicrobial activity, combined with a low toxicity on human cells make ZmD32 an attractive lead for development of future antimicrobial molecules.
A series of monomeric [AgX(P(C,H, complexes have been prepared for X = CN, I, Br, C1, SCN, NCO, NO, or CIO, and characterized by single-crystal X-ray determinations, solid-state cross polarization magic angle spinning (CP MAS) ,'P NMR, solution 31P NMR and far-infrared spectroscopy. For X = CN, I, Br, C1 or SCN the crystal structures are isomorphous, crystallizing in a monoclinic C2jc cell with a z 17, b = 9.3, c' z 25 A. p z 1 lo", 2 = 4; the SCN complex exhibits anion disorder/space group ambiguity. For X = NCO or NO, (redetermined) the structures are isomorphous with the previously studied perchlorate, crystallizing in a triclinic P I cell derivative of the C2jc array with a z 9.3, b e 9.8, c = 23 A, CX z 95, p = 96, y z 116". The Ag-P bond lengths correlate inversely with the P-Ag-P angles and depend on the donor properties of the anion X, with, however, a reverse trend noted for the halide complexes. The solid-state CP MAS ,'P NMR spectra show splitting due to 'J(P-Ag) coupling which progressively increases from 322 Hz for X = CN to 505 Hz for X = C10,. For the triclinic species further splitting is observed and assigned to 'J(P-P) coupling between the crystallographically inequivalent phosphorus atoms. These NMR results are consistent with the structural results except for the chloride and perchlorate systems where experiments suggest that both triclinic and monoclinic phases co-crystallize in proportions which depend on the recrystallization procedures. Solution 'lP NMR spectra show doublets assignable to 1 : 2 species. For X = Br, C1, NCO or SCN signals assignable to 1 : 1 species are also observed. Comparison with solid-state data show differences in 'J(P-Ag) that are ascribed to differences in B(P-Ag-P) in the solution and solid states. Far-IR spectra of the halide and pseudo-halide complexes in the series exhibit single bands due to v(AgX) vibrational modes at 207, 149, 121 for X = C1, Br or I and at 288, 241 and 174 cm-' for X = CN, NCO or SCN, these latter results providing rare data for the vibrational frequencies of terminal Ag-C, Ag-N and Ag--S bonds.
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 α-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 α-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 χ-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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