PhD Candidate Purdue University Indianapolis, Indiana, United States
Tanmaye Nallan Chakravarthula (Indiana University School of Medicine, Indiana University School of Medicine, Indiana University School of Medicine)| Ziqian Zeng (Indiana University School of Medicine, Indiana University School of Medicine)| Nathan Alves (Indiana University School of Medicine, Indiana University School of Medicine)
Plasmin is a serine protease that plays a vital role in hemostasis, immune and inflammatory responses. It is the only in vivo enzyme that directly cleaves fibrin to achieve clot lysis. Direct plasmin derivatives or Plasminogen Activators (PAs) that convert plasminogen to plasmin are administered as thrombolytic agents for the digestion of pathological blood clots in diseases such as heart attack, ischemic stroke, and pulmonary embolism. There is emerging interest in applying the principles of multivalency to pathologically relevant enzymes such as glycosidases, carbonic anhydrases, and proteases to achieve highly controlled and strong inhibition. The aim of this study is to determine the effect of valency and linker length of multivalent benzamidine inhibitors on plasmin activity. Reversible monovalent, bivalent, and trivalent inhibitors of four different lengths each were synthesized. For monovalent and bivalent syntheses, 4-aminomethyl benzamidine (AMB) and monodisperse polyethylene glycol (PEG) molecules up to 10 nm (PEG25) were used. For trivalent syntheses, an additional trivalent TSAT core was used. These inhibitors were purified using RP-HPLC and were confirmed by mass spectrometry. Inhibition assays were performed for the synthesized inhibitors along with AMB and Pentamidine (an FDA approved bivalent benzamidine). A range of inhibitor (0-1200 µM) and chromogenic substrate S-2251 (100-500 µM) concentrations at a fixed human plasmin concentration (42.5 nM) were utilized to determine inhibition constants (Ki values) via Dixon plots. Parameters used to quantify multivalency such as relative potency (rp) and relative potency per unit benzamidine (rp/n) were computed. Ki values ranged between 259.4 - 521.1 µM, 44.3 - 290.4 µM, 3.9 – 241.9 µM for synthesized monovalent, bivalent, and trivalent inhibitors, respectively (lower the Ki, stronger the inhibition). Ki of free AMB was 1,395 µM, and pentamidine, the shortest bivalent inhibitor was the strongest with a Ki value of 1.7 µM. With AMB as the reference, pentamidine yielded an rp value of 820.5 and an rp/n value much greater than 1 (410.2) indicating a strong multivalent effect. All synthesized inhibitors demonstrated rp/n values > 1. The shortest trivalent inhibitor was comparable to pentamidine with a Ki of 3.9 µM. For similar inhibitor lengths, inhibition increased with valency owing to enhancement in statistical rebinding. Whereas, for a fixed valency, inhibition linearly decreased (R2=0.97) with increasing length. Since PEG is flexible, increasing its length increases conformational entropic penalties resulting in unfavorable free energy, therefore, increasing Ki and lowering the inhibition. Future studies with higher order valences with various linker lengths will be carried out to reaffirm these inhibition correlations.