Teshome Leta Aboye

Ultra-stable peptide scaffolds for protein engineering-synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2.

The cyclic cystine knot motif, as defined by the cyclotide peptide family, is an attractive scaffold for protein engineering. To date, however, the utilisation of this scaffold has been limited by the inability to synthesise members of the most diverse and biologically active subfamily, the bracelet cyclotides. This study describes the synthesis and first direct oxidative folding of a bracelet cyclotide—cycloviolacin O2—and thus provides an efficient method for exploring the most potent cyclic cystine knot peptides. The linear chain of cycloviolacin O2 was assembled by solid‐phase Fmoc peptide synthesis and cyclised by thioester‐mediated native chemical ligation, and the inherent difficulties of folding bracelet cyclotides were successfully overcome in a single‐step reaction. The folding pathway was characterised and was found to include predominating fully oxidised intermediates that slowly converted to the native peptide structure.

Interlocking Disulfides in Circular Proteins: Toward Efficient Oxidative Folding of Cyclotides

Cyclotides are ultrastable plant proteins characterized by the presence of a cyclic amide backbone and three disulfide bonds that form a cystine knot. Because of their extreme stability, there has been significant interest in developing these molecules as a drug design scaffold. For this potential to be realized, efficient methods for the synthesis and oxidative folding of cyclotides need to be developed, yet we currently have only a basic understanding of the folding mechanism and the factors influencing this process. In this study, we determine the major factors influencing oxidative folding of the different subfamilies of cyclotides. The folding of all the cyclotides examined was heavily influenced by the concentration of redox reagents, with the folding rate and final yield of the native isomer greatly enhanced by high concentrations of oxidized glutathione. Addition of hydrophobic solvents to the buffer also enhanced the folding rates and appeared to alter the folding pathway. Significant deamidation and isoaspartate formation were seen when oxidation conditions were conducive to slow folding. The identification of factors that influence the folding and degradation pathways of cyclotides will facilitate the development of folding screens and optimized conditions for producing cyclotides and grafted analogs as stable peptide-based therapeutics.

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.

An Efficient Approach for the Total Synthesis of Cyclotides by Microwave Assisted Fmoc-SPPS

Cyclotides are mini-proteins of approximately 30 amino acid residues that have a unique structure consisting of a head-to-tail cyclized backbone and a knotted arrangement of three disulfide bonds. This unique cyclotide structure provides exceptional stability to chemical, enzymatic and thermal treatments and has been implicated as an ideal drug scaffold for the development into agricultural and biotechnological agents. In the current work, we present the first method for microwave assisted Fmoc-SPPS of cyclotides. This protocol adopts a strategy that combines optimized microwave assisted chemical reactions for Fmoc-SPPS of the peptide backbone, the cleavage of the protected peptide and the introduction of a thioester at the C-terminal carboxylic acid to obtain the head-to-tail cyclized cyclotide backbone by native chemical ligation. To exemplify the utility of this protocol in the synthesis of a wide array of different cyclotide sequences we synthesized representative members from the three cyclotide subfamilies—the Möbius kalata B1, the bracelet cycloviolacin O2 and the trypsin inhibitory MCoTI-II. In addition, a “one pot” reaction promoting both cyclization and oxidative folding of crude peptide thioester was adapted for kalata B1 and MCoTI-II.

A Cactus-Derived Toxin-Like Cystine Knot Peptide with Selective Antimicrobial Activity

Naturally occurring cystine knot peptides show a wide range of biological activity, and as they have inherent stability they represent potential scaffolds for peptide‐based drug design and biomolecular engineering. Here we report the discovery, sequencing, chemical synthesis, three‐dimensional solution structure determination and bioactivity of the first cystine knot peptide from Cactaceae (cactus) family: Ep‐AMP1 from Echinopsis pachanoi. The structure of Ep‐AMP1 (35 amino acids) conforms to that of the inhibitor cystine knot (or knottin) family but represents a novel diverse sequence; its activity was more than 500 times higher against bacterial than against eukaryotic cells. Rapid bactericidal action and liposome leakage implicate membrane permeabilisation as the mechanism of action. Sequence homology places Ec‐AMP1 in the plant C6‐type of antimicrobial peptides, but the three dimensional structure is highly similar to that of a spider neurotoxin.

An in vitro Study on the DNA Damaging Effects of Phytochemicals Partially Isolated from an Extract of Glinus lotoides

An extract of Glinus lotoides, a medicinal plant used in Africa and Asia for various therapeutic purposes, was recently shown to cause DNA damage in vitro. To further explore the potential genotoxicity of this plant, fractionation of the crude extract was performed using reverse phase solid‐phase extraction and a stepwise gradient elution of methanol in water. Four fractions were collected and subsequently analysed for their DNA damaging effects in mouse lymphoma cells using an alkaline version of the comet assay. To identify potential genotoxic and non‐genotoxic principles, each fraction was then subjected to liquid chromatography coupled to mass spectrometry, LC‐MS/MS. 1D and 2D nuclear magnetic resonance analyses were used to confirm the identity of some saponins. Although fractions containing a mixture of flavonoids and oleanane‐type saponins or oleanane‐type saponins alone produced no DNA damage, those containing hopane‐type saponins exhibited a pronounced DNA damaging effect without affecting the viability of the cells. To conclude, even if this study presents evidence that hopane‐type of saponins are endowed with a DNA damaging ability, further studies are needed before individual saponins can be cited as a culprit for the previously reported genotoxicity of the crude extract of G. lotoides. Copyright