Photochemically enhanced binding of small molecules to the tumor necrosis factor receptor-1 inhibits the binding of TNF-alpha

The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF re ceptor (TNFRc1) plays an important role in inflammation. Despite the clinic al success of biologics (antibodies, soluble receptors) for treating TNF-ba sed autoimmune conditions, no potent small molecule antagonists have been d eveloped. Our screening of chemical libraries revealed that N-alkyl 5-aryli dene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-prote in interaction. After chemical optimization, we discovered IW927, which pot ently disrupted the binding of TNF-alpha to TNFRc1 (IC50 = 50 nM) and also blocked TNF-stimulated phosphorylation of I kappa -B in Ramos cells (IC50 = 600 nM). This compound did not bind detectably to the related cytokine rec eptors TNFRc2 or CD40, and did not display any cytotoxicity at concentratio ns as high as 100 muM. Detailed evaluation of this and related molecules re vealed that compounds in this class are "photochemically enhanced" inhibito rs, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-1 00 muM) and then covalently modify the receptor via a photochemical reactio n. We obtained a crystal structure of IV703 (a close analog of IW927) bound to the TNFRc1. This structure clearly revealed that one of the aromatic ri ngs of the inhibitor was covalently linked to the receptor through the main -chain nitrogen of Ala-62, a residue that has already been implicated in th e binding of TNF-alpha to the TNFRc1. When combined with the fact that our inhibitors are reversible binders in light-excluded conditions, the results of the crystallography provide the basis for the rational design of nonpho toreactive inhibitors of the TNF-alpha -TNFRc1 interaction.