Publication Date

2015-04-29

Availability

Embargoed

Embargo Period

2017-04-29

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2014-12-04

First Committee Member

Peter Buchwald

Second Committee Member

Charles Luetje

Third Committee Member

R. Grace Zhai

Fourth Committee Member

Bonnie Blomberg

Fifth Committee Member

Michelle Arkin

Abstract

The activation and clonal expansion of T cells requires not just engagement of the T cell receptor (TCR), but also a functionally defined second co-stimulatory signal. Interactions between several co-stimulatory molecules within the tumor necrosis factor (TNF) superfamily (TNFSF) expressed by T cells and various other immune and non-immune cell types are central to T-cell function. Modulation of these interactions holds remarkable therapeutic promise as confirmed by various animal models of inflammatory and autoimmune diseases, transplant rejections, and cancer. Following the recent identification of the first small-molecule inhibitors of the CD40–CD40L co-stimulatory protein-protein interaction (PPI) in our group, the present work focuses on the identification of compounds with modulatory activity towards several other important ligand–receptor interactions within the TNF superfamily and, in particular, the OX40-OX40L interaction. Using a series of ELISA-type screening assays with recombinant human TNFSF proteins of interest, compounds capable of inhibiting the OX40–OX40L, RANK–RANKL, 4-1BB–4-1BBL, TNFα–TNF-R1, GITR–GITRL, and TRAIL-R1–TRAIL interactions have been identified. To follow up on the promising activity obtained in the OX40–OX40L assay, structure–activity relationships have been investigated in more detail, including computational screening experiments using available three-dimensional OX40 structures, and promising lead compounds have been identified. To elucidate the mechanism of the observed OX40–OX40L inhibition in this cell-free assay, the nature and mechanism of the binding were investigated, and these indicated a competitive and reversible binding with OX40 and not OX40L as the binding partner. To confirm and characterize activity and specificity, OX40-transfected sensor cells with NF-κB reporters were constructed and used. Interestingly, several compounds that inhibited OX40-OX40L binding in the cell-free assay, showed a concentration-dependent enhancement of the OX40L-induced NF-κB activation in these sensor cells, but not in the other TNFSF ligand sensors tested (i.e., TNFα and CD40L). Intriguingly enough, these compounds were able to cause NF-κB activation in concentration-dependent manner even without the presence of OX40L, but not to the same maximum activation level as can be achieved by OX40L. Hence, these cell assays indicated these compounds are partial agonists with low micromolar potency and adequate selectivity. Furthermore, the activity of our most promising compound (chlorazol violet N, CVN) has been confirmed in ex vivo T cell polarization assays, where it successfully mimicked the effects of an agonistic anti-OX40 antibody in suppressing regulatory T-cell generation and in diverting CD4+CD62L+Foxp3– cells to TH9 phenotype in vitro. CVN has also shown some activity in preventing hyperglycemia in an exploratory NOD mouse study, a widely used animal model for type 1 diabetes (T1D). Taken together, these results have validated our organic dye–based small molecule library as a reasonable starting point to identify small-molecule modulators for immunologically relevant protein-protein interactions within the TNFSF. Furthermore, the present discovery of the first small-molecule partial agonists for OX40 provides proof-of-principle evidence for the feasibility of small-molecule modulation of the OX40-OX40L co-stimulatory interaction, and should lead to new pharmacological tools to study OX40 mediated immune responses.

Keywords

Tpye 1 Diabetes; Tumor Necrosis Factor Superfamily (TNFSF); Cosimulation; Small Molecule Modulator

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