title: Small-molecule modulators of the OX40-OX40 ligand co-stimulatory protein-protein interaction.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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-κB 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.
  
  
  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 CD4(+) CD62L(+) Foxp3(-) cells to TH 9 phenotype in vitro.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Song
authors.forename: Emilio
authors.initials: E
authors.lastname: Margolles-Clark
authors.forename: Allison
authors.initials: A
authors.lastname: Bayer
authors.forename: Peter
authors.initials: P
authors.lastname: Buchwald
mesh_terms: D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D051858:Forkhead Transcription Factors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008822:Mice, Transgenic; D053278:OX40 Ligand / Q000235:genetics / Q000378:metabolism*; D053262:Receptors, OX40 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D053278:OX40 Ligand; D053262:Receptors, OX40