A Global Ligandability Map of Tryptoline Butynamide Stereoprobes Identifies Covalent Inhibitors of the Actin Maturation Protease ACTMAP.

Covalent chemistry coupled with activity-based protein profiling (ABPP) offers a versatile approach for small-molecule ligand discovery in native biological contexts. The covalent ligandability maps generated by ABPP that target cysteine have frequently leveraged the acrylamide as a reactive group due to its tempered electrophilicity and presence in many advanced tool compounds and therapeutics. More recently, alternative cysteine-directed reactive groups such as the butynamide have emerged as an additional source of covalent probes and drugs, but their global reactivity with the proteome remains largely unexplored. Here, we compare the ligandability maps of stereochemically defined acrylamide and butynamide compounds (stereoprobes) built from a common tryptoline core and find that the butynamides, despite exhibiting attenuated intrinsic and proteome-wide reactivity, preferentially engage a diverse set of proteins in human cancer cells. Among the butynamide-preferring proteins was C19orf54/ACTMAP, a cysteine protease required for the post-translational maturation of actin. We show that (1