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Compositions and methods for enhancing proteasome activity

A compound and hydrate technology, applied in the field of compositions and methods for improving proteasome activity, can solve problems such as affecting the degradation rate of substrates

Inactive Publication Date: 2012-12-26
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since longer chains of ubiquitin interact more strongly with the proteasome than shorter chains of ubiquitin (Thrower et al. (2000), EMBO J.19, 94-102), processes that frequently change the length of will affect the degradation rate of the substrate

Method used

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  • Compositions and methods for enhancing proteasome activity
  • Compositions and methods for enhancing proteasome activity
  • Compositions and methods for enhancing proteasome activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0505] Embodiment 1--the synthesis of inhibitor

[0506] Figure 29 One method of preparing the pyrroles of the invention is described. Similar oxadiazole compounds can be prepared by forming a 1,3-oxadiazole instead of an pyrrole. By varying the ring substitution on the arylamine ring 1a, or substituting alkylamines, heteroarylamines, aralkylamines, etc., a wide variety of compounds can be synthesized. Likewise, compound 1e can be reacted with a variety of nucleophiles to provide a variety of compounds. provided below Figure 29 The experimental procedures for the corresponding compounds are shown.

[0507] Synthesis of 1-(4-chlorophenyl)-2,5-dimethylpyrrole (1c). The mixture of 1a (7.65g, 60.0mmol) and 1b (34.2g, 300.0mmol) was heated to 100°C in glacial acetic acid (40mL) for 1 hour, then the solvent was evaporated to dryness, and the residue was purified by silica gel column chromatography to obtain 1c (11.07g , yield: 89.8%).

[0508] Synthesis of 2-chloro-1-[1-(4-...

Embodiment 2

[0510] Example 2 - Usp14 mediates deubiquitination of substrates

[0511] To test whether Usp14 is a potent inhibitor of the human proteasome, a purification procedure was developed to generate proteasomes lacking detectable levels of the deubiquitinating enzyme Usp14 (modified from Wang et al., (2007), Biochemistry, 46, 3553-3565). Briefly, human proteasomes were large-scale affinity purified from a stable HEK293 cell line harboring HTBH-tagged hRpn11. Cells were lysed in a Dunes homogenizer using lysis buffer containing protease inhibitors (50 mM NaH 2 PO 4 [pH 7.5], 100mM NaCl, 10% glycerol, 5mM MgCl 2 , 0.5% NP-40, 5mM ATP, and 1mM DTT). Washed lysates were incubated overnight at 4°C on NeutrAvidin agarose resin (Thermo Scientific). Subsequently, the beads were washed with excess lysis buffer, followed by wash buffer (50 mM Tris-HCl [pH 7.5], 1 mM MgCl 2 and 1mM ATP). To generate VS-proteasomes, 1 to 1.5 μM Ub-VS (Boston Biochem) was added to the resin and incubated...

Embodiment 3

[0516] Example 3 - Usp14 inhibits proteasomal degradation

[0517] The effect of Usp14 on the degradation of ubiquitinated substrates was examined in an in vitro degradation assay using the ubiquitin-dependent proteasome substrate polyubiquitinated cyclin B (Ub n -ClnB). In these experiments, Ub n -ClnB was co-incubated with human proteasome (4nM) containing wild-type or catalytically inactive Usp14 (60nM). The catalytically inactive Usp used in these experiments was Usp14-C114A, which contains a mutation in the active deubiquitinating site of Usp14. Notably, both wild-type Usp14 and Usp14-C114A were able to bind to mammalian proteasome 26S ( figure 2 ). Such as Figure 5As shown, Usp14 strongly inhibits the degradation of cyclin B, while the active site mutant of Usp14 has no inhibitory effect. Active site mutants deficient for Ub n -Inhibition of ClnB degradation suggests that the activity of wild-type Usp14 to trim ubiquitin chains is required for Usp14 to inhibit p...

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Abstract

Proteinopathies result from the proteasome not acting efficiently enough to eliminate harmful proteins and prevent the formation of the pathogenic aggregates. As described herein, inhibition of proteasome-associated deubiquitinase Usp 14 results in increased proteasome efficiency. The present invention therefore provides novel compositions and methods for inhibition of Uspl4, enhancement of proteasome activity and treatment of proteinopathies.

Description

[0001] related application [0002] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 373,404, filed August 13, 2010, and U.S. Provisional Patent Application No. 61 / 336,959, filed January 28, 2010, both of which are hereby incorporated by reference in their entirety enter. [0003] governmental support [0004] This invention was made with US Government support under Grant Nos. GM065592, GM66492 and DK082906 from the National Institutes of Health. The US Government has certain rights in this invention. Background technique [0005] The proteasome is a large protein complex containing 33 different subunits. The proteasome complex acts as a protease that partially degrades unwanted or misfolded proteins. Proteasome can regulate many aspects of cell physiology, and its dysfunction can cause various diseases, including cancer and neurodegenerative diseases (Finley D., (2009), Annu.Rev.Biochem., 78,477-513; Hoeller and Dikic, (2009), Nature, 458...

Claims

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Application Information

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IPC IPC(8): C07D401/06C07D403/06C07D207/323A61K31/4439A61K31/4025A61P25/28
CPCC07D401/12C07D471/04C07D403/04C07D207/333C07D401/06C07D233/64C07D207/335C07D487/04C07D401/10C07D209/44C07D403/06A61P3/10A61P7/00A61P17/00A61P19/08A61P21/00A61P25/02A61P25/14A61P25/28A61P27/02A61P27/12A61P35/00A61P43/00C07D209/08C07D401/04A61K31/4439C07B2200/07C07D401/14G01N33/573G01N2333/948G01N2500/04
Inventor D·芬利R·W·金李秉宪李民杰T·C·甘曼
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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