Immunomodulatory effects of the small molecule cyclotriazadisulfonamide (CADA) by down-modulation of the CD4 receptor and sortilin

The synthetic macrocycle compound, cyclotriazadisulfonamide (CADA), was identified as a potent inhibitor of human immunodeficiency virus (HIV) replication. It was shown that the antiviral potency of CADA is due to its down-modulating activity on cluster of differentiation 4 (CD4), the main receptor for HIV entry. CADA inhibits human CD4 protein translocation into the endoplasmic reticulum in a signal peptide-dependent way. Besides being the primary receptor for HIV infection, CD4 also plays a role in immunological processes such as T cell activation by enhancing T cell sensitivity to antigens. In addition, several non-depleting anti-CD4 monoclonal antibodies have been shown to exert potent immunosuppressive effects. Recently, the sorting receptor sortilin was identified as a secondary substrate of CADA, although to a lesser extent affected by our compound compared to CD4 (50% and 80% down-modulation for sortilin and CD4, respectively). Next to its role in trafficking of proteins to the cell surface, sortilin has been shown to interact with IL-6, IL-10, IL-12, IL-17A, interferon-α and interferon-γ. Therefore, sortilin is suggested to be involved in exocytic trafficking of certain cytokines. Due to the immunological relevance of both CADA substrates, we are interested in the immunomodulatory capacity of CADA and its potential use in the prevention of transplant rejection. In the mixed leukocyte reaction (MLR), an in vitro assay that reflects in vivo transplant rejection, CADA dose-dependently inhibits leukocyte proliferation in response to allogeneic major histocompatibility complex molecules. Inhibition of proliferation by CADA was consistently observed with leukocytes obtained from different donors, and could not be related to an overall cytotoxic effect of CADA, as was tested in the MTS-PES assay. In addition, CADA reduces the secretion of several cytokines in the supernatant of the MLR samples, as measured with the Bio-Plex 200 system (Bio-Rad). These data clearly point to an immunosuppressive potency of CADA. Non-depleting anti-CD4 monoclonal antibodies and inhibitors of sortilin will be used to further decipher the mechanism of action of CADA-induced immunosuppression.status: publishe