Chemoproteomics identifies a pyrimidopyrimidine analogue as a tubulin-tyrosine ligase binder

Abanti, R. R.; Georgiou, E. A.; Makarov, D.; Lechner, S.; Tsigara, A.; Küster, B.; Medard, G.; Kielkowski, P.; Persoons, L.; De Jonghe, S.; Kostakis, I. K.

doi:10.64898/2026.08.06.743276

bioRxiv · 10.64898/2026.08.06.743276

Chemoproteomics identifies a pyrimidopyrimidine analogue as a tubulin-tyrosine ligase binder

Abanti, R. R.·Georgiou, E. A.·Makarov, D.·Lechner, S.·Tsigara, A.·Küster, B.·Medard, G.·Kielkowski, P.·Persoons, L.·De Jonghe, S.·Kostakis, I. K.

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Abstract

Small-molecule drug discovery relies on identifying compounds that modulate specific protein targets, a process often hindered by cellular complexity. Through phenotypic screening of a kinase-focused diazaquinazoline library, we serendipitously identified CEM198 as the first high-affinity ligand of tubulin-tyrosine ligase (TTL). Functional assays combining live-cell TTL inhibition, microtubule polymerization, cell cycle analysis, and proteomics revealed that CEM198 acts through a dual mechanism: directly binding to TTL and altering /{beta}-tubulin conformation. This interaction restricts -tubulin tyrosination and disrupts tubulin polymerization, leading to microtubule destabilization. The differential effects observed between SH-SY5Y and HEK293T cells indicate that effective TTL inhibition depends on both direct binding and structural modulation of the tubulin heterodimer. These findings introduce CEM198 as a chemical probe for investigating the tubulin tyrosination-detyrosination and demonstrate the potential of chemoproteomics to uncover novel modulators of microtubule dynamics.

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Abanti, R. R., Georgiou, E. A., Makarov, D., Lechner, S., Tsigara, A., Küster, B., Medard, G., Kielkowski, P., Persoons, L., De Jonghe, S., Kostakis, I. K.. 2026-08-07. Chemoproteomics identifies a pyrimidopyrimidine analogue as a tubulin-tyrosine ligase binder. https://doi.org/10.64898/2026.08.06.743276

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