Privileged Electrophile Sensors: A Resource for Covalent Drug Development

Abstract

Summary This Perspective delineates how redox signaling impacts activity of specific enzyme isoforms and how this property may be harnessed for rational drug design. Covalent drugs have resurged in recent years and several reports have extolled the general virtues of developing irreversible inhibitors. Indeed, many modern pharmaceuticals contain electrophilic appendages. Several invoke a warhead that hijacks active-site nucleophiles whereas others take advantage of spectator nucleophilic side-chains that do not participate in enzymatic chemistry, but are poised to bind/react with electrophiles. The latest data suggest that innate electrophile sensing—that enables rapid reaction with an endogenous signaling electrophile—is a quintessential resource for development of covalent drugs. For instance, based on recent work documenting isoform-specific electrophile sensing, isozyme non-specific drugs may be converted to isozyme-specific analogs by hijacking privileged first-responder electrophile-sensing cysteines. Because this approach targets functionally-relevant cysteines, we can simultaneously harness previously-untapped moonlighting roles of enzymes linked to redox sensing.

Single‐Protein‐Specific Redox Targeting in Live Mammalian Cells and C. elegans

“…Various powerful chemical biology toolsets comprising small-molecule- and protein-based fluorescent reporters and proteomics profiling methods have documented context-specific redox-sensor proteins and pathways, both in regulating physiology and supporting specific disease states (Bogdanova, Schultz, & Belousov; Brewer, Garcia, Onak, Carroll, & Chang, 2015; Jacobs & Marnett, 2010; Long, Poganik, et al, 2017; Weerapana et al, 2010). Despite these significant advances, we fall short in drawing a direct link between these modifications and function in the context of a living system.…”

“…Recent studies that have begun to address this knowledge gap indicate that redox modification of a single protein elicits signaling, underscoring the importance of precision in interrogating these individual chemical signaling events and how they drive function (Fang et al, 2013; Lin, Haegele, Disare, Lin, & Aye, 2015; Long & Aye, 2016, 2017; Long, Lin, et al, 2017; Long, Parvez, et al, 2017; Long, Poganik, & Aye, 2016; Long, Poganik, et al, 2017; Long, Urul, et al, 2017; Parvez et al, 2015; Parvez et al, 2016). This manuscript describes a detailed protocol to accomplish T-REX ( t argetable r eactive e lectrophiles and o x idants) in living human cells (Fang et al, 2013; Lin et al, 2015; Long, Lin, et al, 2017; Long, Parvez, et al, 2017; Parvez et al, 2015; Parvez et al, 2016; Zhao, Long, Wang, Zhang, & Aye, 2018) and a powerful model organism, C. elegans (Long, Urul, et al, 2017). With this protocol, the extent to which a protein is modified by r eactive e lectrophilic s pecies (RES) can be calculated and the RES-labeling site can be determined using proteomics.…”

“…With this protocol, the extent to which a protein is modified by r eactive e lectrophilic s pecies (RES) can be calculated and the RES-labeling site can be determined using proteomics. This protocol can be coupled to downstream assays, including various reporter assays (ARE luciferase and ARE GFP (Parvez et al, 2016)) and fluorescence resonance energy transfer (FRET (Long, Parvez, et al, 2017; Parvez et al, 2016)).…”

“…Emerging data indicate that LDEs can sub-stoichiometrically label specific cysteines on redox-sensor proteins, which in turn elicit phenotypically-dominant redox or other functional responses in a living cell or organism (Long & Aye, 2017). The ability to selectively perturb a specific protein of interest (POI) with a select reactive electrophilic signal (RES) in native settings at user-controlled time, locale, and POI/RES pair (Fang et al, 2013; Lin et al, 2015; Long & Aye, 2016, 2017; Long, Lin, et al, 2017; Long, Parvez, et al, 2017; Long et al, 2016; Long, Poganik, et al, 2017; Long, Urul, et al, 2017; Parvez et al, 2015; Parvez et al, 2016; Zhao et al, 2018) has altered the way that RES signaling pathways can be studied with surgical precision. The current conventional method to study RES biology in living models is bulk exposure of the organism to RES.…”

“…The HaloTag covalently reacts with hexyl chloride, a functional group that is inert to other biomolecules in living systems (fish (Long, Parvez, et al, 2017; Zhao et al, 2018), worms (Long, Urul, et al, 2017), cells (Fang et al, 2013; Lin et al, 2015; Long, Lin, et al, 2017; Long, Parvez, et al, 2017; Long et al, 2016; Parvez et al, 2015; Parvez et al, 2016; Zhao et al, 2018)). T-REX small-molecule probes integrate a hexyl chloride-tethered photocaged RES, termed Ht-PreLDE (Lin et al, 2015; Long, Lin, et al, 2017) for Halo-targetable precursor to lipid derived electrophiles (figure 1B). When this bioinert and cell-permeable T-REX probe is introduced to live cells or worms expressing the POI, and excess probe rinsed away, the HaloTag brings the photocaged RES into proximity of the POI.…”