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US-12618847-B2 - Activity-based probes with unnatural amino acids to monitor proteasome activity

US12618847B2US 12618847 B2US12618847 B2US 12618847B2US-12618847-B2

Abstract

The disclosure relates to a compound of the formula (I) and the use of such compounds.

Inventors

  • Darci J. Trader
  • Andres Salazar-Chaparro
  • Breanna Zerfas

Assignees

  • PURDUE RESEARCH FOUNDATION

Dates

Publication Date
20260505
Application Date
20210311

Claims (13)

  1. 1 . A compound of formula (I): wherein: R is A 1 -A 2 -A 3 -A 4 * (SEQ ID NO: 2) or A 1 -A 2 -A 3 -A 4 -A 5 * (SEQ ID NO: 2); R 2 is: each R 3 is independently —N(CH 3 ) 2 or —OCH 3 ; A 1 is Leu or Ac; A 2 is Leu, Gly, or Arg; A 3 is Val, Pro, or Leu; A 4 is Tyr, Leu, or Arg; A 5 is Asp; each instance of n is independently 1, 2, or 3; and m is 1, 2, or 3.
  2. 2 . The compound of claim 1 , wherein at least one instance of R 3 is N(CH 3 ) 2 .
  3. 3 . The compound of claim 1 , wherein all instances of R 3 are —N(CH 3 ) 2 .
  4. 4 . The compound of claim 1 , wherein at least one instance of R 3 is —OCH 3 .
  5. 5 . The compound of claim 1 , wherein all instances of R 3 are —OCH 3 .
  6. 6 . The compound of claim 1 , wherein R 2 is
  7. 7 . The compound of claim 1 , wherein the compound is selected from the group consisting of: or selected from the group consisting of:
  8. 8 . The compound of claim 1 , wherein the compound is:
  9. 9 . A method of monitoring a real time increase in proteasome activity in cells, the method comprising contacting the cells with a compound of formula (I) wherein: R 1 is A 1 -A 2 -A 3 -A 4 * (SEQ ID NO: 2) or A 1 -A 2 -A 3 -A 4 -A 5 -* (SEQ ID NO: 2); R 2 is: each R 3 is independently —N(CH 3 ) 2 or —OCH 3 ; A 1 is Leu or Ac; A 2 is Leu, Gly, or Arg; A 3 is Val, Pro, or Leu; A 4 is Tyr, Leu, or Arg; A 5 is Asp; each instance of n is independently 1 2, 3; and m is 1, 2, 3, and measuring proteasome activity by the accumulation of a fluorescent signal.
  10. 10 . The method of claim 9 , wherein the cells are cells from different cancer cell lines and the relative of levels of proteasome activities between the different cancer cell lines are differentiated.
  11. 11 . The method of claim 10 , wherein the method further comprises contacting the cells with small molecule stimulators and monitoring the effects of small molecule stimulators on the relative levels of proteasome activities between the different cancer cell lines.
  12. 12 . The method of claim 10 , wherein the method further comprises contacting the cells with small molecule inhibitors and small molecules stimulators of the proteasome and evaluating the small molecule inhibitors and the small molecule stimulators on the relative levels of proteasome activities between the different cancer cell lines.
  13. 13 . A method of determining the threshold of proteasome activity required for a cancer cell type to be highly susceptible to proteasome inhibitors, the method comprising contacting the cells with a compound of formula (I) wherein: R 1 is A 1 -A 2 -A 3 -A 4 * (SEQ ID NO: 2) or A 1 -A 2 -A 3 -A 4 -A 5 -* (SEQ ID NO: 2); R 2 is: each R 3 is independently —N(CH 3 ) 2 or —OCH 3 ; A 1 is Leu or Ac; A 2 is Leu, Gly, or Arg; A 3 is Val, Pro, or Leu; A 4 is Tyr, Leu, or Arg; A 5 is Asp; each instance of n is independently 1 2, 3; and m is 1, 2, 3, and measuring proteasome activity by the accumulation of a fluorescent signal.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is U.S. National Stage Filing under 35 U.S.C. 371 of International Patent Application Serial No. PCT/US2021/021950, filed Mar. 11, 2021, and published as WO 2021/183789 A1 on Sep. 16, 2021, which claims priority to U.S. provisional patent application No. 62/987,919, which was filed on Mar. 11, 2020, both of which are hereby incorporated by reference in their entirety. STATEMENT OF GOVERNMENT SUPPORT This invention was made with government support under GM131206 awarded by the National Institutes of Health. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING A Sequence Listing is provided herewith as a text file, “2123125.txt” created on Mar. 9, 2021 and having a size of 4,096 bytes. The contents of the text file are incorporated by reference herein in their entirety. BACKGROUND Eukaryotic cells rely on the proteasome, a large enzyme complex with various catalytic, activities, to maintain healthy protein levels and control a wide range of cell cycle pathways. Specifically, the 26S proteasome, comprised of the 19S regulatory particle (19S RP) and the 20S core particle (20S CP), degrades proteins that are damaged or otherwise no longer required. Such proteins are tagged for degradation with a chain of ubiquitin (Ub) monomers, which is recognized and removed by the 19S RP before the protein is shuttled into the 20S CP to be hydrolyzed into small peptide units. The resulting peptide products can then be recycled for the synthesis of new proteins or other purposes as the cell needs. The improper regulation of the hydrolysis activity of the proteasome has been implicated in several different disease types. This includes numerous cancers, which require a higher level of protein degradation to keep up with their increased protein load, and protein aggregation disorders, which have been observed to have decreased proteasome activity for various reasons. As such, the proteasome has been recognized as a critical therapeutic target. For the past several decades, proteasome inhibitors have been studied and verified as a useful approach for the treatment of a variety of hematological cancers with the approval of bortezomib by the FDA for the treatment of multiple myeloma in 2003. In more recent years, small molecules which can stimulate the activity of the proteasome have also been discovered, providing a potential approach for the treatment of protein aggregation disorders, such as Parkinson's disease. Shortly after the discovery of the proteasome, a fluorescent substrate probe was described which can be used to monitor the protease-like activity of the 20S CP. This probe contains a modified four-mer peptide, with the sequence succinyl-Leu-Leu-Val-Tyr (Suc-LLVY; SEQ ID NO: 1), conjugated to a 4-amino-7-methylcoumarin (AMC) molecule (FIG. 1A). In this structure, the amidation of the AMC molecule quenches its fluorescence, which can be restored upon the recognition of the peptide and cleaving of the Tyr-AMC amide bond. Since the initial reporting of the Suc-LLVY-based probe, other peptide-AMC structures have been designed, and made commercially available, which are selective for the specific protease-like subunits of the 20S CP. Together, these AMC-based substrate probes have been vital tools for understanding a range of characteristics of the proteasome, and have been especially important in studying proteasome inhibitors. Although proven to be essential tools, these AMC-based probes suffer from several limitations. For example, the poor fluorescence properties of AMC require the use of high concentrations of the given probe in order to obtain suitable signals for in cell assays. At these concentrations, it is highly likely they are being cleaved by other cellular proteases and proteases in the cell media. To circumvent this issue, assays can be performed using cell lysate and generic proteases inhibitors, but it makes the evaluation of the effect of small molecule modulators of the proteasome confusing to assess. Although activity-based probes selective for different proteasome β-subunits have been described, they still are typically used with cell lysate and need to be repeated to include proteasome inhibitors to determine how much is attributed to non-proteasomal cleavage or lower molecular weight proteases must be removed from the lysate before assay preparation. To overcome some these concerns, Urru and co-workers developed a Förster resonance energy transfer (FRET)-based probe with improved selectivity. However, to obtain suitable cell permeability, a peptide sequence corresponding to residues 48-57 of TAT was included at the C-terminus, creating a probe which is 20 residues long and is cumbersome to synthesize making its application for a high throughput screen challenging. A more effective activity-based probe to monitor proteasome activity, with the goal of using it for various applications, including the evalu