Novel chloroacetamido compound CWR-J02 is an anti-inflammatory glutaredoxin-1 inhibitor

• Published in: PloS one 2017-11, Vol.12 (11), p.e0187991-e0187991
• Main Authors: Gorelenkova Miller, Olga, Cole, Kyle S, Emerson, Corey C, Allimuthu, Dharmaraja, Golczak, Marcin, Stewart, Phoebe L, Weerapana, Eranthie, Adams, Drew J, Mieyal, John J
• Format: Article
• Language: English
• Subjects: Alkynes; Anti-inflammatory agents; Anti-inflammatory drugs; Beads; Binding sites; Biology and Life Sciences; Biotin; Chemical compounds; Chemical synthesis; Computational chemistry; Computer simulation; Cysteine; Cytokines; Diabetic retinopathy; Disease; Drug therapy; Gene expression; Genetic aspects; Glutaredoxin; Glutathione; Inflammation; Inhibition; Inhibitors; Kinases; Lipopolysaccharides; Medicine and Health Sciences; Microglia; Microglial cells; Molecular biology; Molecular dynamics; Oxidoreductase; Pharmaceutical sciences; Pharmacology; Physical Sciences; Physiological aspects; Protein disulfide reductase (glutathione); Proteins; Rodents; Signaling; Simulation; Spectrometry; Streptavidin; Substrates; Transcription
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0187991

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.