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Broad-spectrum proteome editing with an engineered bacterial ubiquitin ligase mimic

A protein and bacterial technology, applied in the field of broad-spectrum proteome editing, can solve the problem of low efficacy of PROTAC

Pending Publication Date: 2021-01-05
CORNELL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

An attractive feature of these compounds is their drug-like properties, including cell permeability; however, many peptide and small molecule-based PROTACs have low potency—concentrations as high as 25 μM are often required to induce sufficient degradation (Buckley et al. "Small-Molecule Control of Intracellular Protein Levels Through Modulation of the UbiquitinProteasome System," Angew Chem.Int.Ed.Engl.53(9):2312-30(2014))—and the generation of custom PROTACs is limited by the relative absence of E3 ubiquitin linkages Limitations on available ligands for enzymes and desired protein targets and technical challenges associated with de novo generation of such ligands (Osherovich, L., "Degradation From Within," Science-Business Exchange 7:10-11 (2014))

Method used

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  • Broad-spectrum proteome editing with an engineered bacterial ubiquitin ligase mimic
  • Broad-spectrum proteome editing with an engineered bacterial ubiquitin ligase mimic
  • Broad-spectrum proteome editing with an engineered bacterial ubiquitin ligase mimic

Examples

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example

[0236] The following examples are provided to illustrate embodiments of the application, but they are in no way intended to limit the scope thereof.

[0237] The following examples are included to illustrate illustrative embodiments of the present application. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of this application, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present application, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or a similar result without departing from the spirit and scope of the application.

[0238] experimental method

[0239] plasmid. Table 1 provides all plasmids used in this study.

[0240] Table 1. Strains, cell lines and plasmids used in thi...

example 1

[0275] Example 1 - Engineered IpaH9.8 efficiently silences GFP in mammalian cells

[0276] To determine whether E3 ubiquitin ligase mimics from pathogenic bacteria could be redesigned to silence non-native targets, we focused on a panel of 14 candidate enzymes that represent the major E3 classes found in bacteria to date (Table 1, below). 2). Maculins et al., “Bacteria-Host Relationship: Ubiquitin Ligases as Weapons of Invasion. Cell Res. 26(4):499-5l0 (2016) and Lin et al., “Exploitation of the Host Cell Ubiquitin Machinery by Microbial Effector Proteins,” J. Cell Sci. 130(12):1985-96(2017), the entire contents of which are hereby incorporated by reference.

[0277] Table 2. Bacterial E3 ubiquitin ligases evaluated in this study.

[0278]

[0279]

[0280] 1 References listed in Table 2 provide clear evidence or annotations of the catalytic domain of each E3 ubiquitin ligase.

[0281] Abbreviations in Table 2: NEL, novel E3 ligase; HECT, homology to E6AP C-terminus;...

example 3

[0305] Example 3 - GS2-IpaH9.8-mediated proteome editing is flexible and modular.

[0306] An attractive feature of uAbs is their highly modular structure—the E3 catalytic domain and synthetic binding protein domain can be interchanged to reprogram activity and specificity. Indeed, the above results reveal the ease with which different bacterial and eukaryotic E3 domains can be chimerized to form functional uAbs. To investigate the interchangeability of synthetic binding protein domains in IpaH9.8-based uAbs, other high-affinity GFP-binding proteins such as the FN3 antibody analog GS5 (K d = 62nM) (Koide et al., "Teaching an Old Scaffold New Tricks: Monobodies Constructed Using Alternative Surfaces of the FN3 Scaffold," J Mol. Biol. 415(2):393-405 (2012), the entire contents of which are hereby incorporated by reference entered this article) or cAbGFP4 (K d = 0.32nM) to replace GS2 (Saerens et al., "Identification of a Universal VHH Framework to Graft Non-Canonical Antigen-B...

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Abstract

The present application relates to an isolated chimeric molecule comprising a degradation domain comprising an E3 ubiquitin ligase (E3) motif and a targeting domain capable of specifically directing the degradation domain to a substrate, where the targeting domain is heterologous to the degradation domain. A linker couples the degradation domain to the targeting domain. Also disclosed are compositions as well as methods of treating a disease, substrate silencing, screening agents for therapeutic efficacy against a disease, and methods of screening for disease biomarkers.

Description

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 62 / 644,055, filed March 16, 2018, the entire contents of which are hereby incorporated by reference. technical field [0002] The present application relates generally to broad-spectrum proteome editing with engineered bacterial ubiquitin ligase mimics. Background technique [0003] Protein function has traditionally been studied by disrupting the expression of the target gene encoding the protein and analyzing the resulting phenotypic results. Gene silencing and genome editing technologies such as antisense oligonucleotides ("ASO"), RNA interference ("RNAi"), zinc finger nucleases ("ZFN"), transcription activator-like effector nucleases (" TALEN") and clustered regularly interspaced short palindromic repeats ("CRISPR")-Cas systems for such loss-of-function experiments. McManus et al., "GeneSilencing in Mammals by Small Interfering RNAs," Nat. Rev. Genet. 3(10):737-47 (2...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/11C07K19/00C07K14/78
CPCC12N2310/20C12Q1/37C12N15/62C12Y203/02C12N9/104C07K2319/95C07K2319/01C12N9/93A61K38/00C07K16/46C07K2317/569C07K2318/20A61K45/06C07K16/2863C12Q1/48G01N33/5005
Inventor M·P·德利萨M·B·鲁德维奇P·T·哈蒙德
Owner CORNELL UNIVERSITY
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