Electrophilic Multi-Kinase Probes Identify Degradation and Aspartate Conjugation of ULK1

Kinases have proven to be one of the most fertile target classes for new drug approvals, however, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we established a systematic workflow to assess the cellular impact of minimal electrophilic modifications to ATP-site binding scaffolds. Amongst multiple potential starting points for monovalent kinase degradation that were revealed, we identified that one of our scout molecules was able to control the abundance of the master regulator of autophagy, Unc-51-like autophagy activating kinase 1 (ULK1) and that a scaffold-hop could be performed to generate a targeted monovalent ULK1 degrader chemical tool, we named ALP-1. Remarkably, further investigation of how ALP-1 engages ULK1 unveiled an atypical conjugation event between the chloroacetamide warhead of ALP1 and an aspartate residue on the lip of the ULK1 ATP-binding site. These discoveries reveal new understanding of the potential for non-cysteine reactivity of the commonly employed chloroacetamide warhead, as well as new opportunities for the targeting and covalent engagement of kinases.