Chemoproteomics identifies a pyrimido[4,5- <i>d</i> ]pyrimidine analogue as a tubulin-tyrosine ligase binder

<jats:title>Abstract</jats:title> <jats:p> 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 <jats:bold>CEM198</jats:bold> 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 <jats:bold>CEM198</jats:bold> acts through a dual mechanism: directly binding to TTL and altering α/β-tubulin conformation. This interaction restricts α-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 <jats:bold>CEM198</jats:bold> as a chemical probe for investigating the tubulin tyrosination–detyrosination and demonstrate the potential of chemoproteomics to uncover novel modulators of microtubule dynamics. </jats:p>