Abstract
Semaphorin 4D (Sema4D) is expressed in the brain as a transmembrane protein and has been shown to regulate the development and maturation of GABAergic synapses via an unknown mechanism. Preliminary studies in the Paradis laboratory have shown that Sema4D is cleaved extracellularly in neurons with accumulation of an intracellular, C-terminal fragment. Using Western Blotting to monitor appearance of the Sema4D C-terminal cleavage product, we have demonstrated that Sema4D cleavage is metalloprotease-dependent in HEK293T cells using the metalloprotease activator, PMA, and broad spectrum metalloprotease inhibitor, GM6001. Additionally, we have used selective inhibitors for the metalloproteases ADAM10 and ADAM17, and our data suggest that ADAM10 cleaves Sema4D in HEK293T cells. We are currently exploring multivalent presentations of small molecule inhibitors to target Sema4D cleavage in neurons. To this end, we have synthesized a polymeric scaffold derived from the ring-opening metathesis polymerization (ROMP) amenable to subsequent appendage with amine-bearing small molecule inhibitors. Conjugation of the commercially available metalloprotease inhibitor, TAPI-2, to the polymeric scaffold generates a multivalent metalloprotease inhibitor. We have shown a 100-fold enhancement of TAPI-2 inhibition potency from the multivalent display and we propose that such a display can potentially improve inhibitor selectivity. To optimize multivalent inhibitor effectiveness, we will explore variations in the valency (polymer length), inhibitor density, and inhibitor ligand structures and gauge the effects of these structural variations using our Western blotting assay. We will then apply the most effective inhibitor on rat hippocampal neurons to test the role of metalloproteases on Sema4D cleavage in the brain.