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

Pinto-Fernandez, A.; Heride, C.; Turnbull, A. P.; Krajewski, W. W.; Bell, C.; Pedroso, D.; Smith, V.; Mullee, L.; Varca, A.; Charlton, T.; Jones, D. T.; McAllister, T.; Fischer, R.; Guerrero, E. N.; Ebner, D.; Kawamura, A.; Kim, S.; Guerin, D.; Hammonds, T. R.; Kearns, J.; Jones, N.; Buhrlage, S. J.; Urbe, S.; Komander, D.; Clague, M.; Kessler, B. M.

doi:10.64898/2026.03.17.712179

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bioRxiv · 10.64898/2026.03.17.712179

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

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Abstract

The ubiquitin specific protease 28 (USP28) is implicated in tumorigenesis by controlling the turnover of substrates including the oncogene c-MYC and the ubiquitin ligase FBW7. Here, we describe small molecule inhibitors of USP25 and USP28, leading to cancer cell cycle arrest and death. However, genetic deletion of USP25/28 does not replicate this effect. An integrated -omics approach revealed off-target effects for thienopyridine and thienopyrazine carboxamide compounds in protein translation. Chemoproteomics and biochemical analyses suggested binding of such compounds to a region near the ribosome complex polypeptide exit tunnel. Structural analysis of a USP28-inhibitor complex enabled the design of modified USP25/28 inhibitor molecules which minimized translation-related off-target effects. In distinction to earlier compounds, the optimized inhibitors were non-toxic to breast cancer cells yet retained potent anti-proliferative activity in squamous lung carcinoma cells, where USP28 is associated with disease progression. Together, our results demonstrate that refined USP25/28 inhibitors can selectively suppress tumor growth by targeting c-MYC driven pathways, offering a more precise therapeutic strategy for treating squamous lung cancers whilst minimizing undesired cytotoxicity.

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Pinto-Fernandez, A., Heride, C., Turnbull, A. P., Krajewski, W. W., Bell, C., Pedroso, D., Smith, V., Mullee, L., Varca, A., Charlton, T., Jones, D. T., McAllister, T., Fischer, R., Guerrero, E. N., Ebner, D., Kawamura, A., Kim, S., Guerin, D., Hammonds, T. R., Kearns, J., Jones, N., Buhrlage, S. J., Urbe, S., Komander, D., Clague, M., Kessler, B. M.. 2026-03-20. Refined USP25/28 inhibitors with improved selectivity towards c-Myc driven squamous lung cancer cells. https://doi.org/10.64898/2026.03.17.712179

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