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Powell, Michael F.; Stewart, Tracy L.; Ötvös, László; Ürge, László; Gaeta, F. C. A.; Sette, Alessandro; Arrhenius, Thomas; Thomson, David; Soda, Ken; Colón, S M

doi:10.1023/a:1018953309913

Cited by 172 publications

(57 citation statements)

References 25 publications

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“…Serum generally degrades peptides very quickly24 due to the presence of multiple proteases with differing specificity (e.g., thrombin, plasmin, and kallikrein). In line with that view, linear GiBP is undetectable at the first time point after incubation (t 1/2 < 1 min) and cycGiBP shows a half-life of ~20 minutes.…”

Section: Resultsmentioning

confidence: 99%

Serum Stable Natural Peptides Designed by mRNA Display

Howell

Fiacco

Takahashi

et al. 2014

Sci Rep

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Peptides constructed with the 20 natural amino acids are generally considered to have little therapeutic potential because they are unstable in the presence of proteases and peptidases. However, proteolysis cleavage can be idiosyncratic, and it is possible that natural analogues of functional sequences exist that are highly resistant to cleavage. Here, we explored this idea in the context of peptides that bind to the signaling protein Gαi1. To do this, we used a two-step in vitro selection process to simultaneously select for protease resistance while retaining function–first by degrading the starting library with protease (chymotrypsin), followed by positive selection for binding via mRNA display. Starting from a pool of functional sequences, these experiments revealed peptides with 100–400 fold increases in protease resistance compared to the parental library. Surprisingly, selection for chymotrypsin resistance also resulted in similarly improved stability in human serum (~100 fold). Mechanistically, the decreases in cleavage results from both a lower rate of cleavage (kcat) and a weaker interaction with the protease (Km). Overall, our results demonstrate that the hydrolytic stability of functional, natural peptide sequences can be improved by two orders of magnitude simply by optimizing the primary sequence.

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Section: Resultsmentioning

confidence: 99%

Serum Stable Natural Peptides Designed by mRNA Display

Howell

Fiacco

Takahashi

et al. 2014

Sci Rep

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“…[23][24][25]. Triplicate samples of native and cyclic peptides were assayed simultaneously at a concentration of 20 M. At 0, 4, 8, 12, 16, and 24 h, 200 l of the mixture was removed and added to 100 l of 15% aqueous trichloroacetic acid to precipitate serum proteins.…”

Section: Methodsmentioning

confidence: 99%

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

Clark

Fischer

Dempster

et al. 2005

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

215

222

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Conotoxins (CTXs), with their exquisite specificity and potency, have recently created much excitement as drug leads. However, like most peptides, their beneficial activities may potentially be undermined by susceptibility to proteolysis in vivo. By cyclizing the ␣-CTX MII by using a range of linkers, we have engineered peptides that preserve their full activity but have greatly improved resistance to proteolytic degradation. The cyclic MII analogue containing a seven-residue linker joining the N and C termini was as active and selective as the native peptide for native and recombinant neuronal nicotinic acetylcholine receptor subtypes present in bovine chromaffin cells and expressed in Xenopus oocytes, respectively. Furthermore, its resistance to proteolysis against a specific protease and in human plasma was significantly improved. More generally, to our knowledge, this report is the first on the cyclization of disulfide-rich toxins. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.conotoxins ͉ drug delivery ͉ molecular engineering V enoms from marine snails of the Conus genus comprise a myriad of peptides called conotoxins (CTXs) for the rapid immobilization of prey (1, 2). These 12-to 30-aa peptides target membrane receptors with exquisite selectivity and potency and have become invaluable neurophysiological probes and drug leads. Recently, the CTX ziconotide (MVIIA) was approved for use in the treatment of severe chronic pain by the FDA, and other CTXs have entered clinical trials as treatments for pain (3,4). In addition, CTXs have played a critical role in dissecting the molecular mechanisms of ion channel and transporter functions in the nervous system (2). One family of CTXs, the ␣-CTXs, consists of members that antagonize the nicotinic acetylcholine receptors (nAChRs). Ranging in size from 12 to 19 residues, ␣-CTXs are the smallest of all of the CTXs, yet this family is the most widely distributed among Conus venoms (5).Despite their exciting applications, many peptide toxins are susceptible to enzymatic degradation by proteases. This characteristic may limit the therapeutic applications of CTXs, and, hence, methods that provide improvements in biological half-life are valuable. Cyclization has been used in the past as a strategy in the pharmaceutical industry for stabilizing and locking the conformation of small peptides (6). Similarly, microorganisms are known to produce cyclized peptides, such as cyclosporin A, which is now in widespread use as an immunosuppressant. Such a strategy has not been applied in the past to disulfide-rich proteins, but with the recent discovery of the cyclotide family of macrocyclic miniproteins (7), it is clear that the approach can be applied to disulfide-rich toxins to produce additional stabilization with the potential to dramatically increase the therapeutic potential of these molecules when limited by poor in vivo stability.This stu...

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“…24 and see below). The non-natural residues we used (D-amino acids, N-methyl-amino acids, and Aib) are efficient inhibitors of exopeptidase (aminopeptidase and ACE) activities (35). We may reasonably postulate that the disubstituted analogues should also be peptidase-resistant in vivo (18).…”

Section: A1 and Its Substituted Analoguesmentioning

confidence: 99%

A Structure-based Approach to Designing Non-natural Peptides That Can Activate Anti-melanoma Cytotoxic T Cells

Ayyoub

Mazarguil

Monsarrat

et al. 1999

Journal of Biological Chemistry

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Tumor antigens presented by major histocompatibility complex (MHC) class I molecules and recognized by CD8؉ cytotoxic T lymphocytes (CTLs) may generate an efficient antitumor immune response after appropriate immunization. Antigenic peptides can be used in vivo to induce antitumor or antiviral immunity. The efficiency of naked peptides may be greatly limited by their degradation in the biological fluids. We present a rational, structure-based approach to design structurally modified, peptidase-resistant and biologically active analogues of human tumor antigen MAGE-1.A1. This approach is based on our understanding of the peptide interaction with the MHC and the T cell receptor and its precise degradation pathway. Knowledge of these mechanisms led to the design of a non-natural, minimally modified analogue of MAGE-1.A1, [Aib 2 ,NMeSer 8 ]MAGE-1.A1, which was highly peptidase-resistant and bound to MHC and activated MAGE-1.A1-specific anti-melanoma CTLs. Thus, we showed that it is possible to structurally modify peptide epitopes to obtain analogues that are still specifically recognized by CTLs. Such analogues may represent interesting leads for antitumor synthetic vaccines.

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Untitled

Cited by 172 publications

References 25 publications

Serum Stable Natural Peptides Designed by mRNA Display

Serum Stable Natural Peptides Designed by mRNA Display

Engineering stable peptide toxins by means of backbone cyclization: Stabilization of the α-conotoxin MII

A Structure-based Approach to Designing Non-natural Peptides That Can Activate Anti-melanoma Cytotoxic T Cells

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