Don't Lose Your (War)head: Structure-Activity Relationships of Covalent Warheads as Substrates for GST-Catalyzed Glutathione Conjugation

The resurgence of targeted covalent inhibitors (TCIs) has expanded the diversity of electrophilic warheads used in drug discovery. TCIs must balance efficient target engagement with resistance to rapid metabolic clearance. In drug development campaigns, intrinsic reactivity toward glutathione (GSH) is commonly used to estimate metabolic liability; however, in vivo GSH conjugation is primarily catalyzed by glutathione S-transferases (GSTs), a phase II metabolic pathway that is not captured by intrinsic reactivity measurements. Here, we establish a quantitative assay to determine GST k_cat and K_M values across a panel of structurally diverse warheads. We show that their intrinsic reactivities correlate poorly with GST-catalyzed conjugation rates, which are instead governed by warhead- and scaffold-dependent enzyme-substrate interactions. In contrast, GST k_cat/K_M values correlate closely with compound half-lives in human liver cytosol. Together, these findings establish GST susceptibility as a structurally tunable determinant of metabolic GSH conjugation and provide new principles for the optimization of TCIs.