Ligation of stapled polypeptides

Abstract

The present invention provides technology for making large (e.g., greater than 50 amino acids), semi-synthetic, stapled or stitched proteins. The method essentially involves ligating a synthetically produced stapled or stitched peptide to a larger protein. Modified version of IL-13 and MYC are provided as illustrative examples.

Description

RELATED APPLICATIONS[0001]The present application claims priority under 35 U.S.C. §119(e) to U.S. provisional patent applications, U.S. Ser. No. 61 / 082,935, filed Jul. 23, 2008, and U.S. Ser. No. 61 / 225,191, filed Jul. 13, 2009, each of which is incorporated herein by reference.BACKGROUND OF THE INVENTION[0002]Protein therapeutics represent the most rapidly expanding class of drugs, allowing for the treatment of patients with diabetes, cancer, neurological diseases, anemia, infectious diseases, and immunological diseases, among others. Proteins in their natural state are folded into regions of secondary structure, such as helices, sheets, and turns. The α-helix is one of the most common structural motifs found in proteins, and many biologically important protein interactions are mediated by the interaction of an α-helical region of one protein with another protein. However, α-helices have a propensity for unraveling and forming random coils, which are, in most cases, biologically le...

AI Technical Summary

Benefits of technology

[0006]The present invention stems from the recognition that it would be desirable to produce large semi-synthetic stitched or stapled proteins. Such proteins may find use as therapeutics, as diagnostics, or as research tools. Typically, synthetic peptide technology only allows for the preparation of peptides of approximately 50 amino acids or less. Although larger proteins could theoretically be produced by current peptide synthesis methodology, it would be a monumental undertaking and would certainly not be feasible for producing large amounts of a protein (e.g., for use in the clinic). The present invention provides technology for producing large proteins with a stitched or stapled portion. In certain embodiments, the stitched or stapled portion is an α-helical portion. In some embodiments, the stitched or stapled portion is a bifunctional peptide as described in U.S. provisional patent application, U.S. Ser. No. 61 / 225,191, filed Jul. 13, 2009, which is incorporated herein by reference. The inventive method essentially involves ligating a synthetically produced stapled or stitched peptide to a larger protein that may have been produced recombinantly, purified from natural sources, or obtained by other means. The inventive method allows for the production of modified versions of cytokines (e.g., IL-13), transcription factors (e.g., myc), enzymes (e.g., streptokinase, urokinase), receptors, and hormones (e.g., insulin, erythropoietin). The modified protein may have altered biological activity (e.g., gain of function, increased activity, decreased activity, agonist to antagonist) or may simply have increased stability.

[0014]“Stapling” and “hydrocarbon-stapling” as used herein introduces into a peptide at least two moieties capable of undergoing reaction to promote carbon-carbon bond formation when contacted with a reagent to generate at least one cross-linker between the at least two moieties. Stapling provides a constraint on a secondary structure, such as an alpha-helical structure. The length and geometry of the cross-linker can be optimized to improve the yield of the desired secondary structure content. The constraint provided can, for example, prevent the secondary structure to unfold and / or can reinforce the shape of the secondary structure. A secondary structure that is prevented from unfolding is, for example, more stable.

[0015]A “stapled” peptide is a peptide comprising a selected number of standard or non-standard amino acids, further comprising at least two moieties capable of undergoing reaction to promote carbon-carbon bond formation, that has been contacted with a reagent to generate at least one cross-linker between the at least two moieties, which modulates, for example, peptide stability.

Problems solved by technology

Although larger proteins could theoretically be produced by current peptide synthesis methodology, it would be a monumental undertaking and would certainly not be feasible for producing large amounts of a protein (e.g., for use in the clinic).

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