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Polypeptide conjugates for intracellular delivery of stapled peptides

A conjugate and conjugation technology, applied in the field of polypeptide conjugates, can solve problems such as poor cell membrane permeability

Pending Publication Date: 2020-07-24
OHIO STATE INNOVATION FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is clear from these studies that many stapled peptides are either impermeable or poorly permeable to cell membranes, which limits the usefulness of stapled peptides as therapeutic agents

Method used

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  • Polypeptide conjugates for intracellular delivery of stapled peptides
  • Polypeptide conjugates for intracellular delivery of stapled peptides
  • Polypeptide conjugates for intracellular delivery of stapled peptides

Examples

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example 1

[0246] Example 1: Design strategy and synthesis of cyclic CPP binding peptide conjugate

[0247] We chose to prepare cCPP binding peptide conjugates by using a pooled synthesis method (Figure 1). First, the cargo peptide was synthesized by standard solid phase peptide synthesis (SPPS), in which two homocysteine ​​residues were incorporated at positions i and i+4. After cleavage from the resin and side chain deprotection, the peptide was treated with 1.5 equivalents of 1,3-dichloroacetone (DCA) to bind the peptide into an α-helical conformation. The binding process also incorporates ketone groups into the binding peptide for subsequent bioorthogonal conjugation with cCPP. Next, cCPP [e.g., CPP9] is synthesized by SPPS using a miniature PEG-Lys (Mtt) linker attached to the side chain of Gln. While still on the resin, the Mtt group on the side chain of Lys was selectively removed by treatment with 5% trifluoroacetic acid (TFA), and the exposed amine was partially acylated with Boc...

example 2

[0248] Example 2: Cell-permeable binding peptides against MDM2-p53 interaction

[0249] As a proof of concept, we synthesized a cell-permeable binding peptide that is resistant to MDM2-p53 interaction. The activation of the p53 protein can protect the organism from the proliferation of cells carrying damaged DNA with potentially carcinogenic mutations. MDM2 is a p53-specific E3 ubiquitin ligase and the main cell antagonist of p53, which acts to limit the growth inhibitory function of p53 in cancer cells. MDM2 mediates p53 monoubiquitination and proteasomal degradation. The use of small molecules and binding peptides to disrupt the p53-MDM2 complex has become a popular method of treating cancer with WT p53 protein. See Wade, M. et al., Nature Reviews Cancer 13, 83-96 (2013).

[0250] We selected the previously reported MDM2 ligand Ac-LTFEHYWAQLTS (SEQ ID NO:1) ("PDI"; see Phan, J. et al., J. Biol. Chem. 285, 2174-2183 (2010)), and passed The mini PEG-Lys linker is labeled with f...

example 3

[0256] Example 3: Peptide binding and conjugation with 3,5-bis(bromomethyl)benzoic acid

[0257] The main limitation of oxime-based conjugation methods is the formation of two different stereoisomers, which can complicate product separation and further clinical development. To overcome this limitation, we next adopted 3,5-bis(bromomethyl)benzoic acid ("BBA") as the binding agent. The structurally similar compound meta-xylene dibromide has previously been used to bind α-helical peptides. See Jo, H. et al., J Am Chem Soc. 134, 17704-17713 (2012). The m-xylene dibromide reacts rapidly with two cysteines within spatial proximity, thereby forming a single bound peptide product with high yield and low reagent / peptide stoichiometry. We have developed two methods for binding / conjugating α-helical peptides to BBA. In the first method (Figure 3A), first, a cargo peptide containing two acetamidomethyl (Acm) protected cysteine ​​is synthesized on a solid support by standard solid phase pe...

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Abstract

The present disclosure provides novel polypeptide conjugates. The polypeptide conjugates disclosed herein comprise a stapled peptide comprising a peptide and at least one staple which holds the peptide in an alpha-helical confirmation, and a cyclic cell-penetrating peptide (cCPP) conjugated, directly or indirectly, to the stapled peptide. The present disclosure demonstrates that cCPPs can be usedto confer consistent cell-permeability to stapled peptides.

Description

[0001] Cross reference to related patent applications [0002] This application claims the priority rights of U.S. Provisional Application No. 62 / 578213 filed on October 27, 2017, the entire content of which is incorporated by introduction in its entirety for all purposes. [0003] Statement on government funding [0004] The present invention was completed under the support of government grant numbers R01-GM110208 and R35-GM122459 awarded by the National Institute of General Medical Sciences (NIHMS) of the National Institutes of Health. The government has certain rights in this invention. Background technique [0005] As an exciting class of therapeutic agents targeting intracellular protein interaction (PPI), binding peptides have become a challenging target for traditional small molecules and biological agents. Verdine G.L. et al., Methods Enzymol. 503, 3-33 (2012); Walensky, L.D. et al., J. Med. Chem. 57, 6275-6288 (2014). They outline the structure and specificity of biological...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K47/42C07K19/00
CPCC07K7/08C07K7/64A61K47/64A61K47/60A61P35/00A61P29/00A61P37/02C07K2319/10A61K38/00C07K7/52C07K14/001A61K47/58C07K14/4703
Inventor 裴德华
Owner OHIO STATE INNOVATION FOUND
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