Inhibiting the Interaction Between von Willebrand Factor and Collagen Using Novel Peptides to Prevent Thrombosis Initiation

Abstract

The field of peptide pharmaceuticals is gaining important prominence. Peptides have an attractive pharmacological profile and excellent safety, tolerability and efficacy in humans which makes them ideal candidates for new drugs. The macrocyclic arrangements of the amino acids in peptides can provide added stability, therefore the use of cyclic or looped peptides displaying unique secondary structure can aid in stability and efficacy. Heart attacks and strokes, many of which are attributed to arterial blood clots, remain major health concerns in America. Current therapies and preventatives target platelet interactions at the site of a clot and are rife with complications. We have previously targeted the interaction between von Willebrand Factor (vWF) and collagen, which initiates thrombosis, using our exceptionally stable heat‐to‐tail cyclized peptides to moderate potency. Due to the favorability of peptides as drug candidates, as well as the need to target thrombosis in a unique way that precludes platelets, our objective is to develop peptide inhibitors to clot initiation. Our current objective is broken into three key pieces: 1) the development of cyclic peptides with unnatural amino acid substitutions, 2) honing the structures and sequences of our cyclic peptides using rational design and 3) targeting the interaction using substituted lasso peptides displaying an epitope from vWF. Lasso peptides represent a special class of bacterial natural products containing a threaded macrocycle structure with a looped macrolactam ring, an isopeptide bond between the N‐terminus and an acidic side chain, and the C‐terminal tail threaded and trapped by steric hindrance. These peptides can behave as a macromolecular scaffold, retaining their structure when substitutions are made to the loop region. We hypothesize continued success in developing remarkably stable cyclic peptides that target the vWF‐collagen interface, as well as lasso peptides with unique function. A comparison between the cyclic and lasso peptides will provide relationships between stability, structure and efficacy. The potency of each peptide is tested using our novel fluorescently‐linked immunosorbent assay, using anti‐vWF antibodies to detect the presence or absence of vWF bound to collagen‐coated plates when treated with our peptides in a dose‐dependent manner. Additionally, protease stability assays testing our peptides with a panel of peptide‐degrading enzymes in cellular conditions provides a baseline of stability. In summation, we found that unnatural amino acid substitutions were well tolerated by the cyclic peptides and led to some improvements in efficacy and stability. The lasso peptides recombinantly express in abundance and are currently being tested for stability and inhibitor capabilities in comparison to the cyclic peptides. Therefore, all of these designs provide a springboard for future advances in exceptionally stable, active cyclic peptide drugs. Overall, our studies inform future peptidomimetic designs, especially in the development of short, structured peptides with biological function.