Diflunisal targets the HMGB 1/ CXCL 12 heterocomplex and blocks immune cell recruitment

Abstract

Extracellular HMGB1 triggers inflammation following infection or injury and supports tumorigenesis in inflammation‐related malignancies. HMGB1 has several redox states: reduced HMGB1 recruits inflammatory cells to injured tissues forming a heterocomplex with CXCL12 and signaling via its receptor CXCR4; disulfide‐containing HMGB1 binds to TLR4 and promotes inflammatory responses. Here we show that diflunisal, an aspirin‐like nonsteroidal anti‐inflammatory drug (NSAID) that has been in clinical use for decades, specifically inhibits in vitro and in vivo the chemotactic activity of HMGB1 at nanomolar concentrations, at least in part by binding directly to both HMGB1 and CXCL12 and disrupting their heterocomplex. Importantly, diflunisal does not inhibit TLR4‐dependent responses. Our findings clarify the mode of action of diflunisal and open the way to the rational design of functionally specific anti‐inflammatory drugs.

Oxidation of HMGB1 Is a Dynamically Regulated Process in Physiological and Pathological Conditions

“…Extracellular HMGB1 has been identified as a drug-target protein in multiple diseases, in particular in inflammationassociated disorders, and as a target of aspirin (27), the most widely used drug worldwide, and of the salicylate diflunisal (28), demonstrating the importance of HMGB1 in clinic. A high level of serum HMGB1 appears to be a sensitive biomarker in diverse disorders, such as mesothelioma, but the different HMGB1 isoforms represent novel biomarker candidates that provide additional mechanistic information (29).…”

Discovery of 5,5′-Methylenedi-2,3-Cresotic Acid as a Potent Inhibitor of the Chemotactic Activity of the HMGB1·CXCL12 Heterocomplex Using Virtual Screening and NMR Validation

“…The structures of HMG boxes (BoxA, residues G3-Y77; BoxB, residues A93-G173) and CXCL12 (residues K1-K68) used for ligandability assessment, virtual screening (VS), and HADDOCK calculations were extracted from 2YRQ (first structure of the NMR bundle) and 4UAI, respectively (De Leo et al, 2019).…”

“…Water molecules were removed, hydrogen atoms were added, the protonation states were adjusted according to neutral pH, and finally the structures were minimized and optimized using OPLS2005 force field. For BoxA we generated a grid centered at 31.63, 27.3, 33.8 Å in correspondence to R23, the residue at the center of the experimentally validated binding pocket (De Leo et al, 2019). The size of the inner box, that is the ligand diameter midpoint box, was left at the default values of 10 Å edges; the outer box, that is the box within which all the ligand atoms must be contained, was enlarged to 46 Å edges to allow ligands to find unusual or asymmetric binding modes in the active site.…”

“…Interference with this heterophilic interaction is attractive but challenging for several reasons: (i) the three-dimensional structure of the complex is still unknown, and (ii) the interaction surface is expected to be large and dynamic, thus difficult to be targeted by small molecules (Schiraldi et al, 2012;De Leo et al, 2019;Fassi et al, 2019). Previous work has shown that it is possible to interfere with the pro-inflammatory properties of HMGB1 using small molecules including glycyrrhizin (Mollica et al, 2007), salicylic acid (SA), and its derivative amorfrutin (Choi et al, 2015).…”

“…Previous work has shown that it is possible to interfere with the pro-inflammatory properties of HMGB1 using small molecules including glycyrrhizin (Mollica et al, 2007), salicylic acid (SA), and its derivative amorfrutin (Choi et al, 2015). Moreover, we have recently shown that diflunisal, an aspirin-like nonsteroidal anti-inflammatory drug, is able to selectively interfere in vitro and in vivo with the HMGB1/CXCL12/CXCR4 inflammatory axis by disrupting the interaction between HMGB1 and CXCL12 (De Leo et al, 2019). This work has introduced the concept that the HMGB1•CXCL12 heterocomplex is a pharmacological target, herewith opening new perspectives for the rational design of novel inhibitors.…”