Publication Date

2012-05-02

Availability

Open access

Embargo Period

2012-05-02

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2012-04-13

First Committee Member

Peter Buchwald

Second Committee Member

Wasif N. Khan

Third Committee Member

Alberto Pugliese

Fourth Committee Member

Stephan C. Schürer

Fifth Committee Member

Vladlen Z. Slepak

Abstract

The CD40–CD154 protein-protein interaction (PPI) is an important tumor necrosis factor superfamily (TNFSF) interaction that mainly provides co-stimulatory signals to T-cells consequent to antigen presentation in adaptive immunity. Blockade of this PPI can cause immune unresponsiveness (anergy) and is, therefore, an important therapeutic target for immunosuppression. Following our recent discovery of the first small-molecule inhibitors of this PPI, the present work focuses on the identification of compounds with improved ligand efficiency and the confirmation of their activity in cell-based assays. Our best inhibitors identified to date are organic dyes that show low micro-molar activity in inhibiting CD40–CD154 binding and whose activity was also confirmed in several immune cell activation assays, for example, using THP-1 human monocytic leukemia cells or splenocytes from BDC2.5 / NOD mice activated with the corresponding antigen. Based on the analysis of the accumulated activity data, we have established clear structure-activity guidelines needed for CD154 inhibitory activity for this class of compounds. I have performed a series of computational docking experiments using available three-dimensional CD154 structures. Among our inhibitors investigated, docking scores (Glide, Schrödinger LLC) distinguished binders (specific and non-specific) from non-binders most at an allosteric site at the interior core of the CD154-trimer reiterating the established druggable nature of this site among TNFSF ligands. During this search for specific inhibitors, I have also identified a set of xanthene-based promiscuous inhibitors (erythrosine B, rose Bengal) that are the first promiscuous PPI inhibitors described in the literature. Erythrosine B (ErB) was a non-specific promiscuous inhibitor of a number of PPIs within the tumor necrosis factor superfamily (TNF-R–TNFα, CD40–CD154, BAFF-R–BAFF, RANK–RANKL, OX40–OX40L, 4-1BB–4-1BBL) as well as outside of it (EGF-R–EGF) with a remarkably consistent median inhibitory concentration in ~ 2-20 mg/L range. This was a novel, somewhat unexpected finding since small-molecule inhibition of PPIs is notoriously difficult due to the relatively large and flat nature of the interacting surfaces that do not have pockets suitable for the efficient binding of drug-like molecules. To identify the mechanisms of inhibition, extracellular CD154 was expressed with a histidine-tag in E. coli and purified using nickel affinity and size exclusion chromatography. The metachromatic shift exhibited by most of these inhibitors, which are organic dyes, upon protein binding (i.e., the shift in the absorption maxima) was exploited to determine the binding partner and derive binding constants. While the spectra of DR80, a specific inhibitor, shifted only upon incubation with CD154; that of ErB shifted upon incubation with both CD154 and CD40 indicating that it indeed bound both proteins. Stoichiometry analyses (Jobs plot) showed that proteins bind multiple ErB molecules. These results can now provide the basis for a more focused, structure-guided drug discovery effort to identify sufficiently efficient small-molecule CD40–CD154 inhibitors as well as to identify the mechanism of the unexpected promiscuous PPI inhibition we observed for the food colorant ErB.

Keywords

small molecules; protein–protein interactions; immunosuppression; food colorants; promiscuous inhibitors; erythrosine B

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