Aspirin’s Active Metabolite Salicylic Acid Targets High Mobility Group Box 1 to Modulate Inflammatory Responses

Hyong Woo Choi, Miaoying Tian, Fei Song, Emilie Venereau, Alessandro Preti, Sang-Wook Park, Keith Hamilton, G. V. T. Swapna, Murli Manohar, Magali Moreau, Alessandra Agresti, Andrea Gorzanelli, Francesco De Marchis, Huang Wang, Marc Antonyak, Robert J. Micikas, Daniel R. Gentile, Richard A. Cerione, Frank C. Schroeder, Gaetano T. Montelione, Marco E. Bianchi, Daniel F. Klessig

Salicylic acid (SA) and its derivatives have been used for millennia to reduce pain, fever, and inflammation. In addition, prophylactic use of acetylsalicylic acid, commonly known as aspirin, reduces the risk of heart attack, stroke, and certain cancers. Since aspirin is rapidly de-acetylated by esterases in human plasma, much of aspirin's bioactivity can be attributed to its primary metabolite, SA. Here we demonstrate that human high mobility group box 1 (HMGB1) is a novel SA-binding protein. SA-binding sites on HMGB1 were identified in the HMG-box domains by NMR spectroscopic studies and confirmed by mutational analysis. Extracellular HMGB1 is a damage-associated molecular pattern molecule (DAMP), with multiple redox states. SA suppresses both the chemo-attractant activity of fully reduced HMGB1 and the increased expression of pro-inflammatory cytokine genes and COX-2 induced by disulfide HMGB1. Natural and synthetic SA derivatives with greater potency for inhibition of HMGB1 were identified, providing proof-of-concept that new molecules with high efficacy against sterile inflammation are attainable. An HMGB1 protein mutated in one of the SA-binding sites identified by NMR chemical shift perturbation studies retained chemo-attractant activity, but lost binding of and inhibition by SA and its derivatives, thereby firmly establishing that SA binding to HMGB1 directly suppresses its pro-inflammatory activities. Identification of HMGB1 as a pharmacological target of SA/aspirin provides new insights into the mechanisms of action of one of the world's longest and most used natural and synthetic drugs. It may also provide an explanation for the protective effects of low-dose aspirin usage.