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

Abstract

Costimulatory interactions are required for T cell activation and development of an effective immune response; hence, they are valuable therapeutic targets for immunomodulation. However, they, as all other protein-protein interactions, are difficult to target by small molecules. Here, we report the identification of novel small-molecule inhibitors of the CD40–CD40L interaction designed starting from the chemical space of organic dyes. For the most promising compounds such as DRI-C21045, activity (IC50) in the low micromolar range has been confirmed in cell assays including inhibition of CD40L-induced activation in NF-κB sensor cells, THP-1 myeloid cells, and primary human B cells as well as in murine allogeneic skin transplant and alloantigen-induced T cell expansion in draining lymph node experiments. Specificity versus other TNF-superfamily interactions (TNF-R1–TNF-α) and lack of cytotoxicity have also been confirmed at these concentrations. These novel compounds provide proof-of-principle evidence for the possibility of small-molecule inhibition of costimulatory protein-protein interactions, establish the structural requirements needed for efficient CD40–CD40L inhibition, and serve to guide the search for such immune therapeutics.

Therapeutic and Diagnostic Implications of T Cell Scarring in Celiac Disease and Beyond

“…thromboembolic complications have been developed. These reagents include PEG-conjugated antibodies [44,45] and immunomodulatory small molecules targeting the CD40L [46][47][48].…”

Methylene Blue Inhibits In Vitro the SARS-CoV-2 Spike – ACE2 Protein-Protein Interaction – A Mechanism That Can Contribute to Its Antiviral Activity Against COVID-19

“…Accordingly, it seemed logical to explore it for possible inhibitors of the SARS-CoV-2 S protein -ACE2 PPI that is an essential first step for the viral entry of this novel, highly infectious coronavirus. As a first step, we explored the feasibility of setting up a screening assays using a cell-free ELISA-type 96-well format similar to those used in our previous works with Fc-conjugated receptors coated on the plate and FLAG-or His-tagged ligands in the solution (Margolles-Clark et al, 2009b;Ganesan et al, 2011;Song et al, 2014;Chen et al, 2017). To establish assay conditions, we first performed concentration-response assessments using such a format with ACE2-Fc and SARS-CoV-2 S1 or RBD with His tag, and they indicated that both bindings follow classic sigmoid patterns with a slightly stronger binding for RBD than S1 (Figure 1).…”

“…Binding inhibition assays were performed in a 96-well cell-free format similar to the one described before (Margolles-Clark et al, 2009b;Ganesan et al, 2011;Song et al, 2014;Chen et al, 2017). Briefly, microtiter plates (Nunc F Maxisorp, 96-well; Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4 °C with 100 μL/well of Fc-conjugated ACE2 receptor diluted in PBS pH 7.2.…”

“…All binding inhibition and cell assays were tested in at least duplicate per plates, and assays were performed as at least two independent experiments. As before (Ganesan et al, 2011;Song et al, 2014;Chen et al, 2017), binding data were converted to percent inhibition and fitted with standard log inhibitor vs. normalized response models (Buchwald, 2020) using nonlinear regression in GraphPad Prism (GraphPad, La Jolla, CA, USA) to establish half-maximal (median) effective or inhibitory concentrations (EC 50 , IC 50 ).…”

“…Due to our interest in the chemical space of organic dyes to identify potential SMIs for PPIs (Margolles-Clark et al, 2009a;Margolles-Clark et al, 2009b;Ganesan et al, 2011;Song et al, 2014;Chen et al, 2017;, we initiated a screen of such compounds for their ability to inhibit the interaction between SARS-CoV-2 spike protein and its cognate receptor ACE2, which is the first critical step initiating the viral attachment and entry of this CoV. As part of this, we found that methylene blue, a tricyclic phenothiazine compound approved for the treatment of acquired methemoglobinemia and some other uses (Clifton and Leikin, 2003;Schirmer et al, 2011;Bistas and Sanghavi, 2020), inhibits this interaction, and we have confirmed that it does so in a concentrationdependent manner.…”