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

Abstract

The binding of tumor necrosis factor alpha (TNF-alpha) to the type-1 TNF receptor (TNFRc1) plays an important role in inflammation. Despite the clinical success of biologics (antibodies, soluble receptors) for treating TNF-based autoimmune conditions, no potent small molecule antagonists have been developed. Our screening of chemical libraries revealed that N-alkyl 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones were antagonists of this protein-protein interaction. After chemical optimization, we discovered IW927, which potently disrupted the binding of TNF-alpha to TNFRc1 (IC(50) = 50 nM) and also blocked TNF-stimulated phosphorylation of Ikappa-B in Ramos cells (IC(50) = 600 nM). This compound did not bind detectably to the related cytokine receptors TNFRc2 or CD40, and did not display any cytotoxicity at concentrations as high as 100 microM. Detailed evaluation of this and related molecules revealed that compounds in this class are "photochemically enhanced" inhibitors, in that they bind reversibly to the TNFRc1 with weak affinity (ca. 40-100 microM) and then covalently modify the receptor via a photochemical reaction. 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 rings 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 the 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 nonphotoreactive inhibitors of the TNF-alpha-TNFRc1 interaction.

Figure 1

Chemical structures of some optimized TNF-α inhibitors.

Figure 2

Binding of IV703 to TNFRc1 under light and dark conditions. (A) IV703 was incubated with TNFRc1-coated plates (20 ng per well) at RT in the light for 30 min before adding Eu-TNF-α (2.4 nM) (squares). In the same experiment, IV703 was incubated with Eu-TNF-α (2.4 nM) at RT in the light for 30 min before adding the mixture to TNFRc1-coated plates (20 ng per well) (triangles). The standard binding assay was then performed in the dark. (B) TNFRc1-coated plates were preincubated with various concentrations of IV703 for 5 min at RT in the light or in the dark. The plates were then either washed extensively with PBS (triangles, light; diamonds, dark) or left untreated (squares, light; circles, dark). Eu-TNF-α was added and the standard binding assay performed in the dark. (C) TNFRc1-coated plates were preincubated with IV563, RQ989, 5B981, or IW927 for 5 min at RT at a concentration of 1 μM in the light or 100 μM in the dark. The plates were then either washed extensively with PBS (hatched bars) or left untreated (control, dark bars). Eu-TNF-α was added and the standard binding assay performed in the light or in the dark. In all cases, the data represent the average of triplicate wells.

Figure 3

The irreversible binding of IV703 with TNFRc1 is time-dependent. iTNFRc1-CHO membrane coated plates (10 ng per well) were preincubated with IV703 for various lengths of time at RT in the light. The plates were then washed extensively with PBS. Eu-TNF-α (1 nM) was added and the standard binding assay performed in the light. Data represent the average of triplicate wells.

Figure 4

Single crystal structure of covalent IV703–TNFRc1 complex. (A) Shown is the 2F_o − F_c electron density map (contoured to the 1.25 σ level) used to locate IV703. F_o values were from data of TNFRc1–IV703-soaked crystals, and F_c values were calculated from a TNFRc1 model without IV703. The structure shown is the TNFRc1–IV703 covalent structure to illustrate the fit between the final structural model (cf. B) and the initial electron density map. (B) A 2F_o − F_c electron density map (contoured at 1.5 σ) of the refined structure (2.9 Å resolution, R_factor = 27.5%; PDB ID code 1FT4) rotated 180° relative to A. Labeled receptor residues make van der Waals contacts with IV703.

Figure 5

A comparison of the general mechanistic schemes for a photochemically induced inhibitor (A → B → E) and a photochemically enhanced inhibitor (A → C → D → E). In the former case, the activated inhibitor (IV703*) scavenges the protein from solution; this would lead to nonselective reactivity. In contrast, in the case of the photochemically enhanced inhibitor, the mechanistic requirement for initial protein binding imposes an element of selectivity on its reactivity profile.

Figure 6

Superimposition of the crystal structures of IV703 bound to TNFRc1 (purple, this study) and that of TNF-β bound to TNFRc1 (green; ref. 11). Note that Tyr-108 (TNF-β) normally interacts with Ala-62 (TNFRc1) (11); this receptor residue is bound to IV703 in our structure.