Small-molecule costimulatory blockade: organic dye inhibitors of the CD40–CD154 interaction

Abstract

Costimulatory blockade is one of the most promising therapeutic targets in autoimmune diseases as well as in transplant recipients, and inhibition of the cluster of differentiation (CD)40-CD154 interaction, which is required for T cell activation and development of an effective immune response, is particularly promising in islet transplant recipients. Here, we report the ability of several small-molecule organic dyes to concentration dependently inhibit this interaction with IC(50) values in the low-micromolar range. They were found to be considerably more active in inhibiting this interaction than the tumor necrosis factor (TNF)-R1-TNF-alpha or B cell-activating factor (BAFF)-R-BAFF interaction, which are members of the same family. They specifically inhibited CD154-induced cell responses in human B cells as well as in THP-1 myeloid cells, which can serve as surrogate dendritic cells, at concentrations well below their cytotoxic concentrations determined in the same cells. Flow cytometry experiments confirmed their ability to inhibit the CD154-induced, but not the Staphylococcus aureus Cowan I- or phorbol 12-myristate 13-acetate-induced increase in the surface expression of CD54, CD40, and major histocompatibility complex class II. Accordingly, these compounds can be useful not only for experimental investigations involving the inhibition of the CD40-CD154 costimulatory interaction but can also provide important structure-activity relationship information and can serve as the starting point of a targeted drug discovery program.

Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein–Protein Interaction

“…Because polymolecular conglomeration 76, 77 / aggregation 78, 79 is a frequent cause of promiscuous inhibition in screening assays 80 , we have checked for this by adding a non-ionic detergent (Triton-X 100, 0.01%) to the binding inhibitory assay, as recommended for the detection of such effects 81 . This already has been shown to have no effect for our original leads 68 , not even for the promiscuous PPI inhibitor erythrosine B (ErB) 71 . We have rechecked again here for the present compounds, and the presence of the detergent caused slight, but no significant shifts in the IC 50 s of all compounds, e.g., 10 (1.2×), 11 (1.9×), or ErB (1.04×) with the exception of 6 , our least soluble compound, which was more strongly shifted (8.0×).…”

“…The ability of compounds 5 – 11 (Figure 3; Table 1) to inhibit the CD40–CD40L interaction was quantified using a cell-free in vitro binding inhibition assay measuring the amount of soluble CD40L (CD154) bound to plate-coated CD40 in the presence of increasing concentrations of test compounds as described before 67, 68 . Organic dye 1 from our previous work was used as reference control.…”

A CD40-targeted peptide controls and reverses type 1 diabetes in NOD mice

“…That peptide demonstrated very low efficacy in blocking experimental autoimmune encephalomyelitis (EAE) [34]. Some small molecule organic compounds inhibited CD40–CD154 interaction but lost their activity in protein-rich medium [37], suggesting further non-specific effects. These compounds are structurally similar to suramin [38], which has toxic side-effects [39].…”

Porcine Islet Xenotransplantation for the Treatment of Type 1 Diabetes

“…Investigation is currently underway to search for compounds that can interfere with the CD154/CD40 interaction on the surface of T cells and APCs, respectively without the increase in thrombotic events. There is promise in the use of small inhibitory molecules which have demonstrated their ability to bind to CD154 in vitro and effectively block its binding to CD40 (Buchwald et al, 2009;MargollesClark et al, 2009;Margolles-Clark et al, 2010). These small inhibitory molecules have been identified as a number of organic dyes, including Direct Red 80 (DR80) and Mordant Brown 1 (MB1), compounds traditionally used in textile industry.…”

“…They have been found to bind to CD154 and block its interaction with CD40 in a dose dependent manner, with a much higher affinity than that of the anti-CD154 mAb itself. This inhibitory effect does translate to the inhibition of B and T cell proliferation in vitro (Buchwald et al, 2009;Margolles-Clark et al, 2009;Margolles-Clark et al, 2010;Mihalicz et al, 2011). Preliminary in vivo studies have demonstrated no effect as monotherapy in the prevention of NPI xenograft rejection in mice.…”