Krishnappa Jagadish

Cyclotides, a promising molecular scaffold for peptide‐based therapeutics

Cyclotides are a new emerging family of large plant‐derived backbone‐cyclized polypeptides (≈30 amino acids long) that share a disulfide‐stabilized core (three disulfide bonds) characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds make them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Currently, more than 100 sequences of different cyclotides have been characterized, and the number is expected to increase dramatically in the coming years. Considering their stability and biological activities like anti‐HIV, uterotonic, and insecticidal, and also their abilities to cross the cell membrane, cyclotides can be exploited to develop new stable peptide‐based drugs. We have recently demonstrated the intriguing possibility of producing libraries of cyclotides inside living bacterial cells. This opens the possibility to generate large genetically encoded libraries of cyclotides that can then be screened inside the cell for selecting particular biological activities in a high‐throughput fashion. The present minireview reports the efforts carried out toward the selection of cyclotide‐based compounds with specific biological activities for drug design.

Backbone dynamics of cyclotide MCoTI-I free and complexed with trypsin

Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides ( 28–37 amino acids long) that share a disulfide-stabilized core (3 disulfide bonds) characterized by an unusual knotted.[1] Cyclotides contrast with other circular polypeptides in that they have a well-defined three-dimensional structure, and despite their small size, can be considered as micro-proteins. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal and enzymatic degradation (Scheme 1). Furthermore, their well-defined structures have been associated with wide range of biological functions.[2,3] Cyclotides MCoTI-I/II are powerful trypsin inhibitors (Ki 20 – 30 pM) which have been recently isolated from the dormant seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family.[4] Although MCoTI cyclotides do not share significant sequence homology with other cyclotides beyond the presence of the three cystine bridges, structural analysis by NMR has shown that they adopt a similar backbonecyclic cystine-knot topology.[5,6] MCoTI cyclotides, however, show high sequence homology with related cystine-knot squash trypsin inhibitors,[4] and therefore represent interesting molecular scaffolds for drug design.[7–10] Determination of the backbone dynamics of these fascinating micro-proteins is key for understanding their physical and biological properties. Internal motions of a protein on different time scales, extending from picoseconds to second, have been suggested to play an important role in its biological function.[11] A better understanding of the backbone dynamics of the cyclotide scaffold will be extremely helpful for evaluating its utility as a scaffold for peptide-based drug discovery. Such insight will help in the design of optimal focused libraries than can be used for the discovery of new cyclotides sequences with novel biological activities. [12,13] In the current study we report for the first time the determination of internal dynamics of the cyclotide MCoTI-I in free state and complexed with trypsin. Uniformly 15 N-labeled natively folded cyclotide MCoTI-I was recombinantly produced in E. coli growing in minimal M9 medium containing 15 NH4Cl as only source of nitrogen. Concomitant backbone cyclization

Expression of Fluorescent Cyclotides using Protein Trans-Splicing for Easy Monitoring of Cyclotide–Protein Interactions†

Cyclotides are fascinating natural plant micro-proteins ranging from 28 to 37 amino acid residues long and exhibit various biological actions including anti-microbial, insecticidal, cytotoxic, antiviral (against HIV), protease inhibitory, and hormone-like activities.[1–4] They share a unique head-to-tail circular knotted topology of three disulfide bridges; one disulfide penetrates through a macrocycle formed by the other two disulfides, thereby inter-connecting the peptide backbone to form what is called a cystine knot topology (Fig. 1). This cyclic cystine knot (CCK) framework gives the cyclotides exceptional rigidity[5], resistance to thermal and chemical denaturation, and enzymatic stability against degradation.[4, 6] In fact, some cyclotides have been shown to be orally bioavailable. For example, the first cyclotide to be discovered, kalata B1, was found to be an orally effective uterotonic,[7] and other cyclotides have been shown to cross the cell membrane through macropinocytosis.[8–10] All of these features make cyclotides ideal tools for drug development.[11–14]

Recombinant Expression and Phenotypic Screening of a Bioactive Cyclotide Against α-Synuclein-Induced Cytotoxicity in Baker′s Yeast†

We report for the first time the recombinant expression of fully folded bioactive cyclotides inside live yeast cells by using intracellular protein trans-splicing in combination with a highly efficient split-intein. This approach was successfully used to produce the naturally occurring cyclotide MCoTI-I and the engineered bioactive cyclotide MCoCP4. Cyclotide MCoCP4 was shown to reduce the toxicity of human α-synuclein in live yeast cells. Cyclotide MCoCP4 was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1:5×10(4). This demonstrates the potential for using yeast to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells.

Recombinant Expression of Cyclotides Using Split Inteins

Cyclotides are fascinating microproteins (≈30 residues long) present in several families of plants that share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology. Naturally occurring cyclotides have shown to posses various pharmacologically relevant activities and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes. In this chapter we describe how to express a native folded cyclotide using intein-mediated protein trans-splicing in live Escherichia coli cells.