Anti-platelet therapy plays an important role in the treatment of cardiovascular disease. In acute cases, αIIb/β3 integrin inhibitors are administered to block platelet integrin binding to fibrinogen – a key step in the thrombosis pathway. Pathological bleeding is commonly observed as a side effect of these drugs, possibly due to blocking of both integrin ‘outside-in’ and ‘inside-out’ signaling pathways. Talin is a cytoplasmic protein that has been shown to initiate the inside-out pathway, and thus presents an attractive target for selective pathway inhibition.
Previous NMR studies identified the structural basis of the talin/integrin interaction, revealing the mechanism of integrin activation. Using this structural information, we have designed a series of side-chain constrained peptides that mimic the integrin region involved in talin binding. Here, lactam constraints were incorporated at different points in the sequence, whilst ensuring key binding residues were left unchanged. Binding affinities, determined with NMR spectroscopy, were highly dependent on constraint location, with only one series member showing significantly increased binding affinity over the unconstrained parent peptide. Constraint at this location resulted in a five-fold improvement in affinity. Structural studies of these peptides, using CD and NMR spectroscopy, and proteolysis assays, suggest the optimised constraint site stabilises a region that is relatively unstructured in the parent peptide.
A second peptide series was produced, incorporating diverse hydrophobic linkers at the optimised site. These peptides were more helical than the original lactam series, and microscopy and flow cytometry studies showed a range of different cell types were able to take up the peptides.
Here we show that optimised covalent constraint of integrin mimicking peptides, increases peptide stability, cellular uptake and binding affinity for the target protein. Studies are underway to evaluate the effect of these peptides on integrin activation, and determine their signaling pathway specificity, and hence potential as novel anti-thrombotic therapeutics.