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Towards a novel class of non-antibody based protein-capture reagents

By Julio Camarero, Associate Professor, Department of Pharmaceutical Sciences

High-throughput assays are indispensable for comprehensive functional proteome research. The development of these techniques has been driven by the complete mapping of many genomes, including the human. Of great importance for achieving this goal is the development of new protein capture tools for the detection and identification of specific proteins. New capture reagents should be stable to thermal and proteolytic degradation, have high affinity, easy to produce and present low cross-reactivity.

To achieve this objective we are using a cyclotide-based molecular scaffold for generating molecular libraries that will be screened and selected in vivo for potential antagonists against specific protein targets or protein interactions. In this innovative approach, we are using a cell-based library (E. coli cell libraries) where every single cell will express a different cyclotide, in what we could call a single cell-single compound approach. These compounds are then screened and selected for their ability to inhibit a particular interaction inside the bacterial cell using a genetically-encoded reporter in combination with high throughput flow cytometry to identify bacteria encoding specific cyclotide-based antagonists (Scheme 1). Cyclotides, a novel type of peptide-based protein-capture reagent. Cyclotides are fascinating micro-proteins present in plants from the Violaceae, Rubiaceae and also Cucurbitacea and featuring various biological actions such as toxic, 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 (Fig. 1 Cyclotides belong to the family of knottins, a group of microproteins that also includes conotoxins (389 sequences) and spider toxins (257 sequences). Basically, cyclotides are knottins with a head-to-tail circular topology. These micro-proteins can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold [2] and head-to-tail cyclization but in which hypermutation of essentially all residues are permitted with the exception of the strictly conserved cysteines of the knot. The main features of cyclotides and knottins in general are therefore a remarkable stability due to the cystine knot, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations. Cyclotides and knottins thus appear as promising leads or frameworks for peptide drug design [3, 4] and as extremely versatile and stable protein capture reagents.

Cyclotides are ribosomally produced in plants from precursors that comprise between one and three cyclotide domains, however the mechanism of excision of the cyclotide domains and ligation of the free N- and C-termini to produce the circular peptides has not been elucidated. Our group has recently developed and successfully used a bio-mimetic approach for the biosynthesis of folded cyclotides inside cells by making use of modified protein splicing units (Fig. 2). Our important finding makes possible the generation of large libraries of cyclotides (≈109) for high throughput cell-based screening and selection of specific sequences able to recognize particular biomolecular targets [5-7]. We are using this unique set of technologies for cell-based screening and selection of genetically-encoded libraries of cyclotides against particular protein targets.

Cell-based screening: Available methods for producing and screening high-affinity ligands against particular molecular targets are either based in rational or combinatorial approaches. The rational approach usually requires the molecular structure of the biomolecular target, and then potential binders are selected from a virtual library of compounds using docking software [8]. Despite recent advances in computing technology and the development of adaptive docking software [8], this is still a slow, although promising, process. Combinatorial approaches, on the other hand, use random generation of a large number of compounds that are then screened against a biomolecular target. Most of the methods for library screening, however, are performed in vitro, which is a long and laborious process. Cell-based screening, on the other hand, opens the possibility of using single cells as microfactories where the biosynthesis and screening of particular ligands can take place in a single process within the same cellular cytoplasm [9]. The use of a complex molecular environment, such as the cellular cytoplasm, provides the ideal background to identify highly specific inhibitors. Furthermore, the recent introduction of genetically encoded fluorescence-based assays [10] allows the use of high-throughput screening methods such as fluorescence-activated cell sorting (FACS) to study molecular interactions inside living cells [11]. Our group has recently reported a cell-based screening approach for Anthrax Lethal Factor antagonists [12]. This approach used the CyPet and YPet fluorescent proteins as a FRET-couple to screen genetically-encoded libraries of cyclotides inside living bacterial cells [11-13] (Fig. 3). This screening approach is optimized for use in E. coli in combination with FACS, and it is designed to screen large libraries meanwhile minimizing the number of false positives.

We are combining this set of unique technologies for finding specific de novo sequences of cyclotides able to bind to particular serum proteins markers for early detection of ovarian cancer as well as inactivate some key interactions involved in tumor cell proliferation and suppression.

Conclusions and outlook

Cyclotides are small globular micro proteins with a unique head-to-tail cyclized backbone, which is stabilized by three disulfide bonds [14]. The number and positions of cysteine residues are conserved throughout the family, forming the cyclic cystine-knot motif (CCK) [14] that acts as a highly stable and versatile scaffold on which hyper-variable loops are arranged. This CCK framework gives the cyclotides exceptional resistance to thermal and chemical denaturation and enzymatic degradation. This is particularly important for the selection of protein-capture reagents able to work in biologically complex samples such as tears, blood, plasma and other biological fluids, which high content in proteases. Together, these characteristics make cyclotides ideal candidates to be used as molecular scaffolds for the discovery of stable high affinity ligands against particular biomolecular targets, thus replacing the less stable antibody-based scaffolds which have been traditionally used as the protein capture reagents of choice.

Dr. Julio A. Camarero is Associate Professor at the Department of Pharmaceutical Sciences in the University of Southern California since 2008. He studied chemistry at the University of Barcelona, received Masters Degree in 1992 and completed his PhD Thesis in 1996. Then he joined the group of Professor Tom W. Muir at The Rockefeller University as a Burroughs Wellcome Fellow where he contributed to the development of new chemo selective ligation techniques for the chemical engineering of proteins to study bacterial transcription. In 2000, he moved to the Lawrence Livermore National Laboratory as a Distinguished Lawrence Fellow where he became staff scientist and head of laboratory in 2003. He finally joined the School of Pharmacy at the University of Southern California in 2008 as Associate Professor. His current research interests are focused in the development of new bioorganic approaches using protein splicing and synthetic protein chemistry for studying biological processes involved in bacterial pathogenicity and cancer and how can be modulated or inhibited by highly constrained cyclic peptides. Dr. Camarero has authored over 40 peer-reviewed publications and four invited book chapters. For article feedback, contact Dr. Camarero at   jcamarer@pharmacy.usc.edu


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