The Engineering of an Orally Active Conotoxin for the Treatment of Neuropathic Pain

Clark, Richard J.; Jensen, Jonas E.; Nevin, Simon T.; Callaghan, Brid; Adams, David J.; Craik, David J.

doi:10.1002/anie.201000620

Cited by 284 publications

(259 citation statements)

References 25 publications

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“…Cyclic proteins are also resistant to exoproteolytic attack (25,26), a feature that may enhance utility of any therapeutic proteins exposed to exoproteases, for example upon receptor mediated internalization. In addition, cyclization of proteins and peptides has been shown to improve potency, stability, and oral bioavailability (27)(28)(29)(30).…”

Section: Discussionmentioning

confidence: 99%

Sortase-catalyzed transformations that improve the properties of cytokines

Popp

Dougan

Chuang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

172

149

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Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortasemediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.M any clinically relevant cytokines share a four-helix bundle structure, typified by IFNα2, Granulocyte colony-stimulating factor 3 (GCSF-3), Erythropoietin (EPO), IL-2, IL-4, IL-7, IL-9, and IL-15. Cocrystal structures of cytokines with receptor fragments and biochemical studies that map residues critical for interaction of a cytokine with its receptor show that the receptor contacts the sides of the helical bundles (1). This mode of interaction positions the N and C termini of the cytokine away from the receptor.Polyethylene glycol chains attached to therapeutically important proteins increase circulatory half-life, reduce clearance by kidney filtration, reduce proteolysis, and reduce the generation of neutralizing antibodies (2-4). The attachment of PEG commonly employs standard chemistries that target reactive amino acid side chains (e.g., cysteine and lysine). This strategy often generates a heterogeneous mixture in which multiple amino acids in the target are modified with a PEG chain, necessitating cumbersome separations and characterization (5). Such PEGylated molecules often show decreased biological activity, likely due to attachment of a PEG chain to a residue important for interaction with the receptor (6)-this problem may be overcome by sitespecific PEGylation. Although engineering of a carefully placed unpaired cysteine residue allows site-specific PEGylation (7, 8), this method must be tailored to the specific protein target.Sortase A from Staphylcoccus aureus (SrtA Staph ) is a thiolcontaining transpeptidase that recognizes an LPXTG motif in multiple structurally unrelated substrates (9). SrtA Staph cleaves the peptide bond between the threonine and glycine residues with concomitant formation of a thioacyl enzyme intermediate that involves the catalytic cysteine and the substrate threonine. This acyl-enzyme is resolved by nucleophilic attack by the N terminus of an oligo...

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Section: Discussionmentioning

confidence: 99%

Sortase-catalyzed transformations that improve the properties of cytokines

Popp

Dougan

Chuang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

172

149

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“…Additionally, anecdotal evidence of some of them being orally bioavailable in indigenous medicine applications (2) has recently received support from studies demonstrating oral activity of engineered cyclic peptides in animal pain models (3,4). Several classes of cyclic peptides, including the sunflower trypsin inhibitors and cyclotides, have now been discovered in a variety of plant species (5).…”

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confidence: 99%

“…Several classes of cyclic peptides, including the sunflower trypsin inhibitors and cyclotides, have now been discovered in a variety of plant species (5). In addition to the plant-derived cyclic peptides, disulfide-rich peptides isolated from cone snails, including ␣-conotoxins, have successfully been cyclized (3,6) and are showing promise as potential treatments for neuropathic pain (3).…”

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confidence: 99%

“…As well as being valuable scaffolds, naturally occurring cyclic peptides, such as cyclotides and SFTI-1, have inspired the concept of using artificial head-to-tail backbone cyclization as a stabilizing strategy for peptides and proteins of therapeutic interest (3,6). For example, Clark et al (3) showed that cyclization of ␣-conotoxin Vc1.1, a potential neuropathic pain treatment, endowed it with oral activity.…”

mentioning

confidence: 99%

“…Cleavage of the peptide from the resin was achieved using trifluoroacetic acid (TFA)/triisopropylsilane/water (95:2.5:2.5) at room temperature for 2 h. The TFA was evaporated, and the peptide was precipitated with ice-cold diethyl ether and dissolved in 50% acetonitrile (ACN) containing 0.05% TFA and lyophilized. Wild type Vc1.1 and SFTI-1 were prepared as described previously (3,49).…”

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confidence: 99%

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Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A

Jia

Kwon

Wang

et al. 2014

Journal of Biological Chemistry

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Background: Sortase A (SrtA) is a transpeptidase capable of catalyzing the formation of amide bonds. Results: SrtA was used to backbone-cyclize disulfide-rich peptides, including kalata B1, ␣-conotoxin Vc1.1, and SFTI-1. Conclusion: SrtA-mediated cyclization is applicable to small disulfide-rich peptides. Significance: SrtA-mediated cyclization is an alternative to native chemical ligation for the cyclization of small peptides of therapeutic interest.

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Discovery of Peptide Drugs from Natural Sources

Henriques

Craik

2015

Peptide Chemistry and Drug Design

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The Engineering of an Orally Active Conotoxin for the Treatment of Neuropathic Pain

Cited by 284 publications

References 25 publications

Sortase-catalyzed transformations that improve the properties of cytokines

Sortase-catalyzed transformations that improve the properties of cytokines

Semienzymatic Cyclization of Disulfide-rich Peptides Using Sortase A

Discovery of Peptide Drugs from Natural Sources

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