Abstract
Glutaredoxin (Grx1) is a ubiquitously expressed thiol-disulfide oxidoreductase that specifically catalyzes reduction of S-glutathionylated substrates. Grx1 is known to be a key regulator of pro-inflammatory signaling, and Grx1 silencing inhibits inflammation in inflammatory disease models. Therefore, we anticipate that inhibition of Grx1 could be an anti-inflammatory therapeutic strategy. We used a rapid screening approach to test 504 novel electrophilic compounds for inhibition of Grx1, which has a highly reactive active-site cysteine residue (pKa 3.5). From this chemical library a chloroacetamido compound, CWR-J02, was identified as a potential lead compound to be characterized. CWR-J02 inhibited isolated Grx1 with an IC50 value of 32 μM in the presence of 1 mM glutathione. Mass spectrometric analysis documented preferential adduction of CWR-J02 to the active site Cys-22 of Grx1, and molecular dynamics simulation identified a potential non-covalent binding site. Treatment of the BV2 microglial cell line with CWR-J02 led to inhibition of intracellular Grx1 activity with an IC50 value (37 μM). CWR-J02 treatment decreased lipopolysaccharide-induced inflammatory gene transcription in the microglial cells in a parallel concentration-dependent manner, documenting the anti-inflammatory potential of CWR-J02. Exploiting the alkyne moiety of CWR-J02, we used click chemistry to link biotin azide to CWR-J02-adducted proteins, isolating them with streptavidin beads. Tandem mass spectrometric analysis identified many CWR-J02-reactive proteins, including Grx1 and several mediators of inflammatory activation. Taken together, these data identify CWR-J02 as an intracellularly effective Grx1 inhibitor that may elicit its anti-inflammatory action in a synergistic manner by also disabling other pro-inflammatory mediators. The CWR-J02 molecule provides a starting point for developing more selective Grx1 inhibitors and anti-inflammatory agents for therapeutic development.
Highlights
Inflammation has long been recognized as a deleterious contributing factor in numerous disease conditions, prompting continuous pursuit of effective anti-inflammatory agents for therapy
To characterize J02 as a bona fide Grx1 inhibitor, we investigated the concentration-dependent effects of J02 on the activity of the isolated enzyme
Pre-incubation of isolated Grx1 for 30 min with various concentrations of J02 in the presence of 1 mM GSH resulted in a concentration-dependent loss of activity, with IC50 = 32 μM (Fig 1B)
Summary
Inflammation has long been recognized as a deleterious contributing factor in numerous disease conditions, prompting continuous pursuit of effective anti-inflammatory agents for therapy. In this context, many protein mediators of pro-inflammatory signaling are known to undergo reversible redox modifications on cysteine residues that can regulate their functions. Grx silencing inhibits pro-inflammatory cytokine release in both cell culture and animal models of inflammatory disease [4, 5, 10]. Grx-/- mice demonstrate blunted cytokine release from airway epithelial cells in response to both cigarette smoke and ovalbumin challenge used to simulate allergic airway response [4, 11]. We set out to discover an inhibitor of Grx that was effective intracellularly in model microglial cells and test whether such an inhibitor would display anti-inflammatory efficacy
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