Refined USP25/28 inhibitors with improved selectivity towards c-Myc driven squamous lung cancer cells

Researchers developed refined USP25/28 inhibitors that eliminate off-target translation effects to selectively induce cell death in c-Myc-driven squamous lung cancer cells via the TP63/FBW7/c-MYC axis while sparing breast cancer cells.

The Gist

The Big Picture: A "Bad Boss" in the Cell Factory

Imagine your body is a massive city, and your cells are the factories running it. Inside these factories, there are managers called proteins that tell the factory when to grow, when to stop, and when to clean up.

One of these managers is a protein called c-MYC. In healthy cells, c-MYC is a good manager who keeps things running efficiently. But in some cancers (specifically a type of lung cancer called squamous cell carcinoma), c-MYC goes rogue. It becomes a "Bad Boss" that screams, "BUILD MORE! BUILD FASTER!" causing the factory to grow out of control and form a tumor.

Normally, the cell has a "Quality Control Inspector" named FBW7 who catches the Bad Boss and throws him in the trash (degradation) so the factory can calm down. However, there is another protein, USP28, that acts like a "Bodyguard" for the Bad Boss. USP28 grabs c-MYC and hides him from the Quality Control Inspector, keeping the cancer growing.

The First Attempt: The "Blunt Hammer"

Scientists wanted to stop the cancer by firing the Bodyguard (USP28). They developed a chemical "hammer" (a drug called FT224) designed to knock out USP28.

• The Plan: Hit the Bodyguard, expose the Bad Boss, and let the Quality Control Inspector do his job.
• The Problem: When they used this hammer on cancer cells, it worked, but it also smashed everything else in the factory. The cells died, but not just because the Bodyguard was gone. The hammer was too "noisy." It accidentally jammed the factory's assembly line (protein translation).

Think of it like trying to stop a specific worker in a car factory by throwing a wrench into the main conveyor belt. Sure, the worker stops, but now no one can build cars, and the whole factory shuts down. This is called an off-target effect. The drug was toxic to healthy cells (like breast cancer cells) because it stopped the assembly line everywhere, not just in the lung cancer cells.

The Investigation: Finding the Hidden Glitch

The scientists noticed something weird:

1. If they used a gene-editing tool (CRISPR) to simply delete the Bodyguard (USP28) from the cells, the cells didn't die. They were fine.
2. But if they used the chemical hammer (FT224), the cells died immediately.

This told them the hammer wasn't just hitting the Bodyguard; it was hitting something else. Using high-tech detective work (chemoproteomics), they found the hammer was also sticking to a part of the cell's ribosome (the machine that builds proteins). It was like the hammer got stuck in the "exit tunnel" of the assembly line, blocking new parts from coming out.

The Solution: The "Precision Laser"

The team realized they needed a better tool. They couldn't just use a hammer; they needed a laser.

They looked at the 3D structure of the Bodyguard (USP28) and saw exactly where the hammer was sticking. They realized the "handle" of the hammer was too long and was poking into the assembly line by accident.

So, they redesigned the drug. They took the same chemical core but trimmed the "handle" and reshaped the tip.

• The New Drug (Refined Inhibitors): These new molecules are like a precision laser. They still hit the Bodyguard (USP28) perfectly and stop him from protecting the Bad Boss.
• The Result: The laser does not jam the assembly line. It leaves the rest of the factory alone.

The Final Test: Who Gets Hit?

They tested these new "laser" drugs on two types of cells:

1. Breast Cancer Cells: These cells don't rely heavily on the Bodyguard. The new laser had almost no effect on them. They were safe.
2. Squamous Lung Cancer Cells: These cells are addicted to the Bodyguard. When the laser fired, the Bodyguard fell, the Bad Boss (c-MYC) was caught, and the cancer cells died.

The Takeaway

This paper is a story about precision medicine.

• Old Way: Use a sledgehammer to kill a specific bad guy, but accidentally destroy the whole house.
• New Way: Use a sniper rifle to take out the bad guy while leaving the house intact.

By understanding exactly how the drug was causing side effects (jamming the protein assembly line), the scientists were able to tweak the chemistry to remove the side effects. This means they now have a potential new treatment that could specifically target aggressive lung cancers without poisoning the patient's healthy cells. It's a major step toward making cancer treatments safer and more effective.

Technical Summary

1. Problem Statement

The ubiquitin-specific protease USP28 is a critical regulator of the oncogene c-MYC and the E3 ligase FBW7. Inhibiting USP28 is a promising therapeutic strategy for cancers dependent on c-MYC, particularly squamous lung cell carcinoma (LSCC), where USP28 stabilizes c-MYC and the transcription factor $\Delta$Np63.

However, previous generations of small-molecule USP25/28 inhibitors (specifically those based on the thienopyridine carboxamide scaffold, such as FT206, FT224, and FT811) exhibited a critical flaw:

• Phenotypic Discrepancy: While these inhibitors effectively killed cancer cells, genetic deletion (knockout) of USP28 did not replicate the same cytotoxic effects.
• Off-Target Toxicity: The inhibitors caused broad cellular toxicity and cell cycle arrest in cell lines (e.g., breast cancer) that were not dependent on USP28, suggesting the observed efficacy was driven by off-target mechanisms rather than specific USP28 inhibition.
• Lack of Selectivity: Existing inhibitors lacked the ability to distinguish between USP25 and USP28, and their off-target effects obscured the true therapeutic potential of targeting the USP28-c-MYC axis.

2. Methodology

The authors employed an integrated multi-omics and structural biology approach to deconvolute on-target vs. off-target effects:

• Chemical Biology & Profiling:
  • Used Activity-Based Protein Profiling (ABPP) with Ubiquitin-propargylamide (Ub-PA) probes to confirm target engagement of USP25/28 by compounds like FT224.
  • Generated USP28 Knockout (KO) cell lines (MCF-7, HCT116, HAP1) to compare genetic deletion vs. chemical inhibition.
• Chemoproteomics:
  • Created a biotinylated derivative of FT224 (FT639) to perform pull-down assays followed by quantitative mass spectrometry (MS/MS) to identify off-target binding partners.
• Functional Assays:
  • SUnSET and OPP Assays: Measured nascent protein synthesis to determine if inhibitors affected translation.
  • CRISPR-GOF (Gain of Function) & Genetic Screens: Validated the role of specific cellular components.
• Structural Biology:
  • Solved the X-ray crystal structure of the human USP28 catalytic domain in complex with a thienopyridine inhibitor (FT592).
  • Performed structural superposition with USP25, USP7, and USP8 to understand selectivity and binding modes.
• Structure-Activity Relationship (SAR):
  • Designed and synthesized a new panel of inhibitor derivatives with varied chemical scaffolds to retain USP25/28 potency while eliminating translation inhibition.
• In Vitro & In Vivo Modeling:
  • Tested refined compounds on MCF-7 (breast) and H-520 (squamous lung) cancer cells, comparing WT and USP28 KO backgrounds.

3. Key Contributions & Results

A. Identification of Off-Target Mechanism

• Translation Inhibition: The study revealed that thienopyridine carboxamide inhibitors (FT224, FT811) potently inhibit global protein translation at sub-micromolar concentrations, mimicking the effect of cycloheximide (CHX).
• Ribosome Binding: Chemoproteomics identified that these compounds bind to components of the translation machinery, specifically RPS27A and RPS27L (ribosomal proteins), near the polypeptide exit tunnel of the 60S ribosomal subunit.
• Mechanism of Toxicity: The toxicity observed in non-LSCC cell lines (like MCF-7) was driven by this off-target translation inhibition, not by USP28 inhibition. This was confirmed because USP28 KO cells remained sensitive to the translation-inhibiting effects of the drugs, and genetic deletion of USP28 alone did not reduce cell viability in these lines.

B. Structural Insights into USP28 Inhibition

• Binding Mode: The crystal structure of USP28-FT592 revealed the inhibitor wedges between the $\alpha$5 helix and the palm subdomain.
• Selectivity Basis: The binding pocket is formed by residues (e.g., T364, C644) that are less sterically demanding in USP25/28 compared to other DUBs like USP7 or USP8. This explains the high selectivity of this scaffold for the USP25/28 subfamily.
• Autoinhibition: The structure showed that the inhibitor occupies the same pocket as the Autoinhibitory Motif (AIM) in USP25, explaining why these compounds inhibit both USP25 and USP28.

C. Development of Refined Inhibitors

• Scaffold Optimization: Using the structural data, the authors synthesized new compounds (e.g., bin-07-07 and AV-24-34) that retained potent USP25/28 enzymatic inhibition but lacked the ability to inhibit protein translation.
• Decoupling Effects: These refined inhibitors showed no toxicity in breast cancer cells (MCF-7) regardless of USP28 status, confirming that the previous toxicity was off-target.

D. Selective Efficacy in Squamous Lung Cancer

• On-Target Activity: In H-520 squamous lung cancer cells (which are c-MYC and $\Delta$Np63 dependent), the refined inhibitors (bin-07-07, AV-24-34) significantly reduced cell viability.
• Specificity: This reduction in viability was dependent on USP28, as USP28 KO H-520 cells were resistant to the refined inhibitors.
• Conclusion: The refined compounds selectively suppress tumor growth in LSCC by targeting the TP63–FBW7–c-MYC signaling axis without the confounding cytotoxicity of translation inhibition.

4. Significance

• Resolving the "Genetic vs. Chemical" Gap: The study provides a definitive explanation for why previous USP28 inhibitors showed efficacy in cell lines where genetic deletion did not: the chemical inhibitors were "dirty" drugs acting via translation inhibition.
• Therapeutic Precision: By removing the off-target translation effect, the authors developed cleaner, more selective USP25/28 inhibitors. This allows for a clearer assessment of USP28 as a drug target and confirms its validity specifically in c-MYC-driven squamous lung carcinomas.
• Drug Design Strategy: The work highlights the importance of using chemoproteomics and structural biology to identify and eliminate off-target "hot spots" (in this case, the ribosome exit tunnel) during drug optimization.
• Clinical Implications: The refined inhibitors offer a promising therapeutic avenue for treating squamous lung cancers, a subtype with limited treatment options, while minimizing systemic toxicity associated with protein synthesis inhibition.

In summary, this paper successfully transitioned USP25/28 inhibition from a "dirty" chemical phenotype to a precise, mechanism-based therapeutic strategy by identifying and eliminating a critical off-target effect on the ribosome.