Anne C. Conibear

The chemistry of cyclotides.

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.

Structural characterization of the cyclic cystine ladder motif of θ-defensins.

The θ-defensins are, to date, the only known ribosomally synthesized cyclic peptides in mammals, and they have promising antimicrobial bioactivities. The characteristic structural motif of the θ-defensins is the cyclic cystine ladder, comprising a cyclic peptide backbone and three parallel disulfide bonds. In contrast to the cyclic cystine knot, which characterizes the plant cyclotides, the cyclic cystine ladder has not been as well described as a structural motif. Here we report the solution structures and nuclear magnetic resonance relaxation properties in aqueous solution of three representative θ-defensins from different species. Our data suggest that the θ-defensins are more rigid and structurally defined than previously thought. In addition, all three θ-defensins were found to self-associate in aqueous solution in a concentration-dependent and reversible manner, a property that might have a role in their mechanism of action. The structural definition of the θ-defensins and the cyclic cystine ladder will help to guide exploitation of these molecules as structural frameworks for the design of peptide drugs.

The Cyclic Cystine Ladder in θ-Defensins Is Important for Structure and Stability, but Not Antibacterial Activity

Background: θ-Defensins are antimicrobial peptides comprising a cyclic backbone and three disulfide bonds. Results: θ-Defensins retain antibacterial and membrane-binding activity, but lose structure and stability when the disulfide bonds are removed. Conclusion: Disulfide bonds are important for the stability and structure of θ-defensins, but not antibacterial activity. Significance: Understanding the role of disulfide bonds and cyclization in θ-defensins facilitates applications as peptide drug scaffolds. θ-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 θ-defensins. In this study, we explore the role of the cyclic peptide backbone and cystine ladder in the structure, stability, and activity of θ-defensins. θ-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 θ-defensins and illustrate the potential of θ-defensin analogues as scaffolds for peptide drug design.

The Chemistry and Biology of Theta Defensins

Abstract 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.

The cyclic cystine ladder of theta-defensins as a stable, bifunctional scaffold: A proof-of-concept study using the integrin-binding RGD motif.

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. θ‐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 θ‐defensin RTD‐1. The most active analogue had an IC50 of 18 nM for the αvβ3 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 θ‐defensins can provide a stable and conformationally restrained scaffold for bioactive epitopes in a β‐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.

Synthesis and evaluation of phosphonated N-heteroarylcarboxamides as DOXP-reductoisomerase (DXR) inhibitors

The diethyl esters and disodium salts of a range of heteroarylcarbamoylphosphonic acids have been prepared and evaluated as analogues of the highly active DOXP-reductoisomerase (DXR) inhibitor, fosmidomycin. Computer-simulated docking studies, Saturation Transfer Difference (STD) NMR analysis and enzyme inhibition assays have been used to explore enzyme-binding and -inhibition potential, while in silico analysis of the DXR active site has highlighted the importance of including a well-parameterised metal co-factor in docking studies and has revealed the availability of an additional binding pocket to guide future drug design.

Mirror images of antimicrobial peptides provide reflections on their functions and amyloidogenic properties

Enantiomeric forms of BTD-2, PG-1, and PM-1 were synthesized to delineate the structure and function of these β-sheet antimicrobial peptides. Activity and lipid-binding assays confirm that these peptides act via a receptor-independent mechanism involving membrane interaction. The racemic crystal structure of BTD-2 solved at 1.45 Å revealed a novel oligomeric form of β-sheet antimicrobial peptides within the unit cell: an antiparallel trimer, which we suggest might be related to its membrane-active form. The BTD-2 oligomer extends into a larger supramolecular state that spans the crystal lattice, featuring a steric-zipper motif that is common in structures of amyloid-forming peptides. The supramolecular structure of BTD-2 thus represents a new mode of fibril-like assembly not previously observed for antimicrobial peptides, providing structural evidence linking antimicrobial and amyloid peptides.

Approaches to the stabilization of bioactive epitopes by grafting and peptide cyclization

Peptides are attracting increasing interest from the pharmaceutical industry because of their specificity and ability to address novel targets, including protein–protein interactions. However, typically they require stabilization for therapeutic applications owing to their susceptibility to degradation by proteases. Advances in the ability to chemically synthesize peptides and the development of new side‐chain and backbone ligation strategies provide new tools to stabilize bioactive peptide epitopes. Two such epitopes are LyP1, a nine residue peptide that localizes to tumor cells and has potential as an anticancer therapeutic, and RGDS, a tetrapeptide shown to bind to survivin and induce apoptosis. Here we applied a variety of strategies for the stabilization of LyP1 and RGDS, including side‐chain cyclization using “click” chemistry and “grafting” the epitopes into two naturally occurring cyclic peptide scaffolds, i.e., θ‐defensins and cyclotides. NMR data showed that the three‐disulfide θ‐defensin and cyclotide scaffolds accommodated the LyP1 and RGDS epitopes but that scaffolds with fewer disulfide bonds were structurally compromised by inclusion of the LyP1 epitope. LyP1, LyP1‐, and RGDS‐grafted peptides that were largely unstructured also had reduced resistance to degradation in human serum, showing that grafting into a stable cyclic scaffold is an effective strategy for increasing the stability of a bioactive peptide epitope. Overall, the study demonstrates several methods for stabilizing peptide epitopes using side‐chain or backbone cyclization and illustrates their potential in peptide drug design.

Quantification of small cyclic disulfide-rich peptides.

Cyclic disulfide‐rich peptides ranging in size from ∼14 to 29 amino acids have been found in a wide variety of organisms and have exciting biological and medicinal applications due to their stability and structure. Many of these peptides can be chemically synthesized, but their small size and limited number of chromophore‐containing amino acids make them difficult to quantify by methods routinely used for large proteins. A comparison of the precision and accuracy of gravimetric, UV‐ and NMR‐based methods in current use for the quantification of small peptides is presented for a representative set of cyclic disulfide‐rich peptides. The study shows that gravimetric and UV absorbance methods should be used with caution for small peptides and all methods should be carefully validated. For the routine quantification of small disulfide‐rich peptides, we recommend comparison of the analytical reverse‐phase high‐performance liquid chromatography trace or UV absorbance at 214 nm with that of a standard peptide solution quantified by amino acid analysis. An accurate quantification method that is simple and cost effective will assist in comparison of inhibition and activity data between different laboratories and peptides and correct calculation of synthesis yields.

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.