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

Lawrence Livermore National Laboratory, Livermore, CA 94550, USA.
ChemBioChem (Impact Factor: 3.09). 11/2009; 10(16):2663-70. DOI: 10.1002/cbic.200900534
Source: PubMed


(Figure Presented) Cell-ing point: This study shows that MCoTI-cyclotides can provide an ideal scaffold for the biosynthesis of large combinatorial libraries inside living E. coli cells. Coupled to an appropriate in vivo reporter system, this library may rapidly be screened, for example, by fluorescence-activated cell sorting.

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Available from: Julio A Camarero
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    • "To engineer the cyclotide MCoTI-I to have AT1-7 activity, we grafted a derivative of the peptide AT1-7 peptide onto the cyclotide scaffold using loop 6 (Figure 1). This loop has been shown previously to be more disordered in solution[9]and amenable to sequence variation[30]. The peptide was grafted using the side-chains of residues 1 and 7. "
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    ABSTRACT: We report for the first time the design and synthesis of a novel cyclotide able to activate the unique receptor of angiotensin (1-7) (AT1-7), the MAS1 receptor. This was accomplished by grafting an AT1-7 peptide analog onto loop 6 of cyclotide MCoTI-I using isopeptide bonds to preserve the α-amino and C-terminal carboxylate groups of AT1-7, which are required for activity. The resulting cyclotide construct was able to adopt a cyclotide-like conformation and showed similar activity to that of AT1-7. This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based in the treatment of cancer and myocardial infarction.
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    • "Numerous studies have detailed the successful incorporation of linear bioactive peptides grafted onto the cyclotide framework, resulting in improved stability while maintaining potency and selectivity for their targets (Poth et al., 2013). In addition to rational design, high-throughput screening approaches that build combinatorial libraries of cyclotide analogs are being used for drug lead selection (Austin et al., 2009; Getz et al., 2011, 2013). "
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    ABSTRACT: Cyclotides combine the stability of disulfide-rich peptides with the intracellular accessibility of cell-penetrating peptides, giving them outstanding potential as drug scaffolds with an ability to inhibit intracellular protein-protein interactions. To realize and optimize the application of cyclotides as a drug framework and delivery system, we studied the ability of the prototypic cyclotide, kalata B1, to enter mammalian cells. We show that kalata B1 can enter cells via both endocytosis and direct membrane translocation. Both pathways are initiated by targeting phosphatidylethanolamine phospholipids at the cell surface and inducing membrane curvature. This unusual approach to initiate internalization might be harnessed to deliver drugs into cells and, in particular, cancer cells, which present a higher proportion of surface-exposed phosphatidylethanolamine phospholipids. Our findings highlight the potential of these peptides as drug leads for the modulation of traditionally "undruggable" targets, such as intracellular protein-protein interactions. Copyright © 2015 Elsevier Ltd. All rights reserved.
    No preview · Article · Aug 2015 · Chemistry & biology
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    • "These alterations may serve as a basis for the identification of sites that have target binding potential, i.e. sites 9 in Loop 1; 24, 26, 28 in Loop 5; and 30, 31, 33 in Loop 6. This prediction agrees with a study that showed that the aforementioned sites in Loops 1 and 5 play a role in binding with trypsin (Austin et al. 2009). "
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    ABSTRACT: Cyclic proteins have evolved for millions of years across all kingdoms of life to confer structural stability over their acyclic counterparts while maintaining intrinsic functional properties. Here, we show that cyclic miniproteins (or peptides) from Momordica (Cucurbitaceae) seeds evolved in species that diverged from an African ancestor around 19 Ma. The ability to achieve head-to-tail cyclization of Momordica cyclic peptides appears to have been acquired through a series of mutations in their acyclic precursor coding sequences following recent and independent gene expansion event(s). Evolutionary analysis of Momordica cyclic peptides reveals sites that are under selection, highlighting residues that are presumably constrained for maintaining their function as potent trypsin inhibitors. Molecular dynamics of Momordica cyclic peptides in complex with trypsin reveals site-specific residues involved in target binding. In a broader context, this study provides a basis for selecting Momordica species to further investigate the biosynthesis of the cyclic peptides and for constructing libraries that may be screened against evolutionarily related serine proteases implicated in human diseases.
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