Biophysical and Functional Characterization of a Bowman-Birk Antiviral Protease Inhibitor from Cleome viscosa Seeds

Plant protease inhibitors (PPIs) play a significant role against microbes, insects, and, to a considerable extent, against human pathogens. PPIs inactivate hydrolase enzymes or depolarize the plasma membrane of the pathogens, thereby inhibiting their growth, replication, and invasion. Here, an active serine protease inhibitor was isolated and purified from the seeds of Cleome viscosa. The purified inhibitor was homogenous and exhibited a molecular weight of around 12 kDa as a monomer. The secondary structure analysis indicated that the inhibitor is predominantly composed of α-helical content. The kinetics experiments demonstrated a non-competitive mode of inhibition towards serine protease when casein has been used as the substrate. The inhibitor formed a stable complex with serine protease having likely 1:1 stoichiometry as inferred from ITC, and the dissociation constant was examined to be Kd = 1.9 ×10-6 M with Gibb’s free energy ΔG = -8.079 (Kcal/mol). Further, in vitro preliminary studies revealed its inhibitory effects against HSV-2 function, evidence it may have a role in the treatment of viral infections.