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Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII.

ABSTRACT: 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 alpha-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.

SUBMITTER: Clark RJ

PROVIDER: S-EPMC1236553 | biostudies-literature | 2005 Sep

REPOSITORIES: biostudies-literature

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Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII.

Clark Richard J RJ Fischer Harald H Dempster Louise L Daly Norelle L NL Rosengren K Johan KJ Nevin Simon T ST Meunier Frederic A FA Adams David J DJ Craik David J DJ

Proceedings of the National Academy of Sciences of the United States of America 20050914 39

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 alpha-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 ...[more]

PMID: 16162671

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Project description:Peptide cyclization is a strategy used to improve stability and activity of peptides. The most commonly used cyclization method is disulfide bridge formation of cysteine-containing peptides, as is typically found in nature. Over the years, an increasing number of alternative chemistries for peptide cyclization with improved efficiency, kinetics, orthogonality, and stability have been reported. However, there has been less appreciation for the opportunity to fine-tune peptide activity via the diverse chemical entities introduced at the site of linkage by different cyclization strategies. Here, we demonstrate how cyclization optimization of an M2 "anti-inflammatory" macrophage-binding peptide (M2pep) resulted in a significant increase in binding affinity of the optimized analog to M2 macrophages while maintaining binding selectivity compared to M1 "pro-inflammatory" macrophages. In this study, we report synthesis and evaluation of four cyclic M2pep(RY) analogs with diverse cyclization strategies: (1) Asp-[amide]-Lys, (2) azido-Lys-[triazole(copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC))]-propargyl-Gly, (3) Cys-[decafluorobiphenyl (DFBP)]-Cys, and (4) Cys-[decafluorobiphenyl sulfone (DFS)]-Cys, whereby the chemical entity or linker at the linkage site is shown in the square bracket and is between the residues involved in cyclization. These peptides are compared to a disulfide-cyclized M2pep(RY) that we previously reported as a serum-stable, affinity-enhanced analog to the original linear M2pep. DFBP-cyclized M2pep(RY) exhibits the highest binding activity to M2 macrophages with apparent dissociation constant (KD) about 2.03 μM compared to 36.3 μM for the original disulfide-cyclized M2pep(RY) and 220 μM for the original linear peptide. DFS-cyclized M2pep(RY) also binds more strongly than the original cyclized analog, whereas amide- and triazole-cyclized M2pep(RY) analogs bind less strongly. We verified that DFBP alone has negligible binding to M2 macrophages and the incorporation of diphenylalanine to the original sequence improves binding activity at the expense of solubility and increased toxicity. In conclusion, we report development of cyclic M2pep(RY) analogs with diverse cyclization strategies leading to the discovery of DFBP-cyclized M2pep(RY) with enhanced M2 macrophage-binding activity.

Project description:Proteins with high catalytic efficiency and selectivity under mild conditions have long been appreciated by industrial and medicinal fields. These proteins, which are commonly multimeric, often possess low stability, impeding wider application. Currently, strategies to improve the stability of multimeric proteins concentrate on enhancing the interaction at internal interface of the subunits. In this report, we confirmed that the largely underestimated subunit terminal ends are as significant as the internal interface for protein stability. By connecting both the terminal ends and internal interface of subunits, the tetrameric Leifsonia alcohol dehydrogenase (LnADH) protein can been cyclized into a rigid form with significantly improved thermostability and resilience. The improvement in the temperature at which enzyme activity is reduced to 50% after a 15-min heat treatment (T5015) and melting temperature (Tm ) of the modified protein was 18°C and 23.3°C, respectively, which is superior to the results achieved by normal protein engineering. Our study provided a novel strategy to effectively improve the stability of multimeric proteins, which is suitable not only for the short-chain dehydrogenase/reductase (SDR) family but also other classes of proteins with close terminal ends.IMPORTANCE Industrially interesting proteins are generally multimeric proteins; however, their applications are often restricted due to low stability caused by the natural tendency of subunit disassociation. Current approaches targeting this problem mainly focus on enhancing the internal interfaces of the subunits to avoid their disassociation. In this study, we identified and confirmed the external interface to be significant for improving the stability of multimeric proteins. By connecting the terminal ends and internal interface with disulfide bonds, we found that the multimeric protein LnADH cyclized into a robust monomeric-like form, resulting in superior thermostability compared to traditional protein engineering. This intersubunit cyclization approach is efficient and easy to perform, providing a novel method for engineering many important classes of multimeric proteins.

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Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII.

Publications

Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII.

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