Biosynthesis and Biological Screening of a Genetically Encoded Library Based on the Cyclotide MCoTI‐I

Abstract

Cyclotides are fascinating micro-proteins present in plants from the Violaceae, Rubiaceae and also Cucurbitaceae and feature various biological actions such as protease inhibitory, anti-microbial, insecticidal, cytotoxic, anti-HIV or hormone-like activity.[1, 2] They 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 (Scheme 1A). Cyclotides have several characteristics that make them ideal drug development tools.[3, 4] The cystine knot and cyclic backbone topology makes them exceptionally resistant to thermal, chemical, and enzymatic degradation compared with other peptides of similar size.[5] 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,[6] and other cyclotides have been shown to cross the cell membrane through macropinocytosis.[7] Immunogenicity is generally considered not to be a major issue for small-sized and stable microproteins.[8, 9] Cyclotides are also amenable to substantial sequence variation and they can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization [2, 10]. Cyclotides can also be chemically synthesized thus allowing the introduction of specific chemical modifications or biophysical probes.[11–14] More importantly, cyclotides can be now biosynthesized in E. coli cells by using a biomimetic approach that involves the use of modified protein splicing units[15, 16] (Fig. 1), and therefore making them ideal scaffolds for molecular evolution strategies to enable generation and selection of compounds with optimal binding and inhibitory characteristics against particular molecular targets. Cyclotides thus appear as promising leads or frameworks for peptide drug design.[3, 4] Figure 1 Biosynthetic approach for the production of cyclotide MCoTI-I libraries inside living E. coli cells. Backbone cyclization of the linear precursor is mediated by a modified protein splicing unit or intein. The cyclized peptide then folds spontaneously ... Scheme 1 A. Primary and tertiary structure of MCoTI and Kalata cyclotides isolated from Momordica cochinchinensis and Oldenlandia affinis, respectively [6, 20, 21]. B. Multiple sequence alignment of cyclotide MCoTI-I with other squash trypsin inhibitors. Multiple ... Investigation of the contribution of individual residues to the structural integrity and biological activities of particular cyclotides is therefore crucial for their use in any potential pharmaceutical application.[17] A better understanding of the structural limitations of the cyclotide scaffold can greatly assist in the correct design of cyclotide-based libraries for molecular screening and selection of de-novo sequences with new biological activities or developing grafted analogues for use as peptide-based drugs,[14, 18] so that sequence modifications in structurally important regions are avoided. Understanding the molecular basis for bioactivity also may allow the minimization or avoidance of undesirable properties such as cytotoxic or hemolytic activity found in some cyclotides.[17] The cyclotides MCoTI-I/II are powerful trypsin inhibitors which have been recently isolated from the dormant seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family.[19] Although MCoTI cyclotides do not share significant sequence homology with other cyclotides beyond the presence of the three cystine bridges, solution NMR has shown that they adopt a similar backbone-cyclic cystine-knot topology [20, 21] (Scheme 1A). MCoTI cyclotides, however, share a high sequence homology with related cystine-knot trypsin inhibitors found in squash such as EETI, and it is likely they have a similar binding to that of the EETI-family (Scheme 1B).[19] Hence, cyclic MCoTIs represent interesting candidates for drug design, either by changing their specificity of inhibition or by using their structure as natural scaffolds possessing new binding activities.