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Effect of PEG Linker Length on Human Plasmin Inhibition by Benzamidine

Owing to its nontoxic, nonimmunogenic, non‐antigenic and amphiphilic properties, polyethylene glycol (PEG) is widely used for pharmaceutical applications. PEGylation is the covalent linkage of PEG to pharmaceutical agents. The enhanced performance of pharmaceutical agents due to PEGylation is attributed to the increase in hydrodynamic volume as well as its limited interactions with biomolecules in‐vivo effectively increasing drug circulation time by reducing clearance. While PEGylation typically refers to the modification of macromolecular drugs such as proteins and enzymes, PEG is also utilized as a common flexible linker with small molecules as well. In this study we sought to understand the effect of different PEG linker lengths on enzyme (plasmin) inhibition. Plasmin is an endogenous serine protease that is responsible for cleaving fibrin resulting in clot digestion. Benzamidine is a reversible, small molecule inhibitor of plasmin which competitively binds to its active site. Four bivalent molecules were synthesized with two benzamidines separated by 1.6, 2.6, 5.5 and 9.8 nm using 4‐aminomethyl benzamidine and monodisperse PEG linkers: PEG2, PEG5, PEG13 and PEG25, respectively. These inhibitors were purified using Reverse Phase – High Performance Liquid Chromatography (RP‐HPLC) and confirmed via mass spectrometry. Inhibition assays were carried out at a fixed concentration of human plasmin (42.5 nM) over a range of inhibitor concentrations (0–300 μM) and chromogenic substrate (S‐2251 100–500 μM) concentrations to determine inhibition constants (Ki values) via Dixon plot analysis. Lower the Ki, tighter is the inhibition. In addition to the four synthesized bivalent molecules, monovalent 4‐aminomethyl benzamidine and pentamidine were also tested. Pentamidine is a commercially available bivalent benzamidine molecule separated by 0.7 nm and is FDA approved for the treatment of infections. Ki values for synthesized bivalent molecules ranged from 58.9–326.3 μM and were linearly correlated (R2 = 0.99) to the PEG linker length. Pentamidine, the shortest bivalent inhibitor, had the smallest Ki of 1.7±2.0 μM exhibiting 192‐fold greater inhibition than the longest bivalent molecule tested. Moreover, even the longest bivalent molecule with the highest Ki is a 4‐fold better inhibitor than the monovalent benzamidine with a Ki of 1,395±165.8 μM molecule. This inhibition enhancement with the bivalent inhibitor molecules over the monovalent benzamidine can be attributed to a multivalent avidity affect. Avidity, or multivalent affinity, occurs at higher valency and is due to a higher probability of binding and rebinding that results in a tighter apparent inhibition constant (lower Ki). In addition, shorter PEG linkers minimize the distance between the two benzamidine inhibitor molecules and thus increase the chances of rebinding resulting in tighter inhibition. Further studies with higher order valency molecules (trivalent, tetravalent, etc.) and varying PEG lengths between benzamidine molecules will be carried out to expand on these inhibition correlations.