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Deregulation of the TNF-like weak inducer of apoptosis (TWEAK)-fibroblast growth factor-inducible 14 (Fn14) signaling pathway is observed in many diseases, including inflammation, autoimmune diseases, and cancer. Activation of Fn14 signaling by TWEAK binding triggers cell invasion and survival and therefore represents an attractive pathway for therapeutic intervention. Based on structural studies of the TWEAK-binding cysteine-rich domain of Fn14, several homology models of TWEAK were built to investigate plausible modes of TWEAK-Fn14 interaction. Two promising models, centered on different anchoring residues of TWEAK (tyrosine 176 and tryptophan 231), were prioritized using a data-driven strategy. Site-directed mutagenesis of TWEAK at Tyr(176), but not Trp(231), resulted in the loss of TWEAK binding to Fn14 substantiating Tyr(176) as the anchoring residue. Importantly, mutation of TWEAK at Tyr(176) did not disrupt TWEAK trimerization but failed to induce Fn14-mediated nuclear factor κ-light chain enhancer of activated B cell (NF-κB) signaling. The validated structural models were utilized in a virtual screen to design a targeted library of small molecules predicted to disrupt the TWEAK-Fn14 interaction. 129 small molecules were screened iteratively, with identification of molecules producing up to 37% inhibition of TWEAK-Fn14 binding. In summary, we present a data-driven in silico study revealing key structural elements of the TWEAK-Fn14 interaction, followed by experimental validation, serving as a guide for the design of small molecule inhibitors of the TWEAK-Fn14 ligand-receptor interaction. Our results validate the TWEAK-Fn14 interaction as a chemically tractable target and provide the foundation for further exploration utilizing chemical biology approaches focusing on validating this system as a therapeutic target in invasive cancers.

To perform highly sensitive cellular binding studies with TNF-like weak inducer of apoptosis (TWEAK), we developed a bioluminescent variant of soluble TWEAK (GpL-FLAG-TNC-TWEAK) by fusing it genetically to the C terminus of the luciferase of Gaussia princeps (GpL). Equilibrium binding studies on human (HT1080 and HT29) and murine (Renca and B16) cell lines at 37 °C revealed high affinities of human TWEAK from 53 to 112 pm. The dissociation rate constant of the TWEAK-Fn14 interaction was between 0.48×10(-3) s(-1) (HT29) and 0.58×10(-3) s(-1) (HT1080) for the human molecules, and the association rate constant obtained was 3.3×10(6) m(-1) s(-1) for both cell lines. It has been shown previously that oligomerization of soluble TWEAK trimers results in enhanced Fn14-mediated activation of the classical NFκB pathway. Binding studies with GpL-FLAG-TNC-TWEAK trimers oligomerized by help of a FLAG tag-specific antibody gave no evidence for a major increase in Fn14 occupancy by oligomerized ligand despite strongly enhanced induction of the NFκB target IL8. Thus, aggregated complexes of soluble TWEAK and Fn14 have a higher intrinsic activity to stimulate the classical NFκB pathway and qualitatively differ from isolated trimeric TWEAK-Fn14 complexes. Furthermore, determination of IL8 induction as a function of occupied activated receptors revealed that the intrinsic capability of TNFR1 to stimulate the classical NFκB pathway and IL8 production was ∼100-fold higher than Fn14. Thus, although ∼25 activated TNFR1 trimers were sufficient to trigger half-maximal IL8 production, more than 2500 cell-bound oligomerized TWEAK trimers were required to elicit a similar response.

The interaction between 4-1BB ligand (CD137L), a member of the tumor necrosis factor superfamily, and its receptor 4-1BB provides a co-stimulatory signal for T lymphocyte proliferation and survival. However, the structure of 4-1BBL has not been thoroughly investigated, and none of the human recombinant 4-1BBL molecules available have been described as capable of co-stimulating T cells. The present work provides a model of the three-dimensional structure of the tumor necrosis factor homology domain of 4-1BBL and describes the production of a recombinant human soluble 4-1BBL whose originality lies in that it contains the whole extracellular tail preceding the tumor necrosis factor homology domain and an AviTag peptide (AviTag-4-1BBL) allowing enzymatic biotinylation and multimerization via streptavidin. We provide evidence that this chimeric protein exists as a homotrimer, whereas commercial FLAG-tagged 4-1BBL does not. This resulted in a much higher affinity for 4-1BB (1.2 nM) as compared with FLAG-4-1BBL (55.2 nM). We demonstrate that the single extracellular cysteine residue in the tail (Cys-51) could form a disulfide bond, both in our recombinant protein and in physiologically expressed 4-1BBL. The mutation of this cysteine residue exerted no effect on trimerization but increased the dissociation rate of AviTag-4-1BBL from 4-1BB. In its soluble form, AviTag-4-1BBL did not stimulate purified T cells but dramatically inhibited proliferation of peripheral blood mononuclear cells stimulated with anti-CD3 mAb. In contrast, a very significant co-stimulatory effect was observed on purified T cells once AviTag-4-1BBL was immobilized onto streptavidin beads. In addition, we show that the cross-linking of two trimeric AviTag-4-1BBL molecules was the minimum step required to elicit significant costimulatory activity.