Small‐molecule modulators of the OX 40– OX40 ligand co‐stimulatory protein–protein interaction

Background and Purpose The OX40 – OX40L protein–protein interaction ( PPI ) is an important cell‐surface signalling co‐stimulatory regulator within the TNFR superfamily (TNFRSF) and a promising therapeutic target for immunomodulation. PPIs are difficult to modulate using small‐molecules. Here, we describe the identification of a small‐molecule OX40 modulator and confirm its partial agonist character. Experimental Approach Cell‐free screening assays were developed and used to identify OX40 – OX40L inhibitors. Modified versions of this assay were used to elucidate the binding partner and the binding nature of active compounds. OX40 ‐transfected sensor cells with NF ‐κ B reporters were constructed and used to confirm and characterize activity and specificity. Immunomodulatory activity and partial agonist nature were further confirmed by ex vivo   T ‐cell polarization assays. Key Results Several compounds that concentration‐dependently affected OX40 ‐ OX40L were identified. Cell assays indicated that they were partial agonists with low micromolar potency and adequate selectivity. Under polarizing conditions based on TGF ‐β, the most promising compound mimicked the effect of an agonistic anti‐ OX40 antibody in suppressing regulatory T ‐cell generation and diverting CD 4 + CD 62 L + F oxp3 − cells to T H 9 phenotype in vitro . Conclusions and Implications We identified, to our knowledge, the first small‐molecule compounds able to interfere with OX40 – OX40L binding and, more importantly, to act as partial agonists of OX40 . This is particularly interesting, as small‐molecule agonism or activation of PPIs is considered unusually challenging and there are only few known examples. These results provide proof‐of‐principle evidence for the feasibility of small‐molecule modulation of the OX40 – OX40L interaction and for the existence of partial agonists for TNFRSF ‐ PPIs .