Towards novel inhibitors of OX40L-OX40: A dominant negative approach
Proteins belonging to the tumor necrosis factor (TNF) superfamily show exceptional promise as targets for combating cancer, autoimmune and bone diseases, and other ailments. TNF proteins form homotrimers that interact with homotrimeric receptors on cell surfaces. Antibody-based inhibitors of these receptors have led to effective therapies in some cases, but an inhibitor built from the natural ligand should have advantages in terms of immunogenicity and tissue penetration. Other researchers have made a mutant TNF modified at two residues in the region of the receptor binding interface. The mutant forms heterotrimers that bind to but do not activate TNF receptors. This monomeric variant thus inhibits TNF signaling by diverting wild-type TNF subunits into inactive complexes.
We propose to test a novel approach to interrupt a different TNF-receptor interaction, specifically, the interaction of the receptor OX40 with its ligand OX40L (both of which interact as homotrimers). Such inhibitors could be useful for treating autoimmune diabetes, inflammatory bowel disease, and rheumatoid arthritis. Our approach will not rely on the rapid exchange of ligand subunits in the wild type binding interface. Instead, we propose to design and build heterotrimeric OX40L variants. This stoichiometry will be designed into the trimerization interface, thus ensuring that we form only inactive trimers that bind tightly to the receptor and block it.
We will be working with the COBRE Protein Production Group to express and purify each of these re-engineered variants, and assess their binding to OX40 in vitro by adapting an ELISA-based assay and by using surface plasmon resonance (Biacore).
We will also design and test OX40L variants which exist as obligate heterotrimers. In conjunction with the COBRE Protein Production Group, we will separately produce these OX40L variants and then determine in vitro whether these variants indeed form obligate heterotrimers via size-exclusion chromatography and NMR (working with the COBRE Protein Production and Bio-NMR Core Labs).
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