Method Development towards Orthogonal Electrophile Delivery

Redox signaling events mediated by reactive electrophilic species (RES) are key players in several cellular processes. However, the studies on RES have been challenging due to their high reactivity towards numerous nucleophilic groups present in the cells and the toxicity caused by whole-cell flooding of RES at a high concentration. In recent years, T-REX (targetable reactive electrophiles and oxidants) delivery platform and G-REX (genome-wide profiling of sensors responsive to REX) profiling platform have been successfully applied in the identification and functional studies of sensor proteins of lipid-derived electrophiles in cultured mammalian cells, C. elegans and D. rerio. Here, we expanded the applicability of T-REX by introducing protein tags alternative to HaloTag. We demonstrated the compatibility of SNAP-tag and CLIP-tag with T-REX and proposed a novel method for orthogonal electrophile delivery using multiple tags. In parallel, based on T-REX guided discovery of kinase Akt3 as a sensor of HNE (4-hydroxynonenal), a lipid-derived electrophile, we showed that conjugating HNE or its derivative FNE to a known non-covalent Akt inhibitor, MK-2206, enabled the resulting bifunctional covalent inhibitor (MK-H(F)NE) to bind preferably to Akt3 instead of Akt1 and Akt2. To further understand the binding interactions of MK-H(F)NE and guide future design of potential drug leads, we isolated human Akt3 from mammalian expression system and conducted initial crystallization screens of apo Akt3 and Akt3–MK-FNE complex. Aside from original research aiming at better understanding the interactions between electrophiles and electrophile-sensor proteins, my thesis research period also provided me with an opportunity to contribute to a pedagogical review article which discussed the experimental evidence and potential contributing factors of the selectivity of electrophile sensors.