Design, Synthesis, and Evaluation of Novel Immunomodulatory Small Molecules Targeting the CD40⁻CD154 Costimulatory...

We report the design, synthesis, and testing of novel small-molecule compounds targeting the CD40⁻CD154 (CD40L) costimulatory interaction for immunomodulatory purposes. This protein-protein interaction (PPI) is a TNF-superfamily (TNFSF) costimulatory interaction that is an important therapeutic target since it plays crucial roles in the activation of T cell responses, and there is resurgent interest in its modulation with several biologics in development. However, this interaction, just as all other PPIs, is difficult to target by small molecules. Following up on our previous work, we have now identified novel compounds such as DRI-C21091 or DRI-C21095 that show activity (IC₅₀) in the high nanomolar to low micromolar range in the binding inhibition assay and more than thirty-fold selectivity versus other TNFSF PPIs including OX40⁻OX40L, BAFFR-BAFF, and TNF-R1-TNFα. Protein thermal shift (differential scanning fluorimetry) assays indicate CD154 and not CD40 as the binding partner. Activity has also been confirmed in cell assays and in a mouse model (alloantigen-induced T cell expansion in a draining lymph node). Our results expand the chemical space of identified small-molecule CD40⁻CD154 costimulatory inhibitors and provide lead structures that have the potential to be developed as orally bioavailable immunomodulatory therapeutics that are safer and less immunogenic than corresponding biologics.