E-64

A cysteine protease, falcipain-2 (FP-2) belonging to papain family is an important haemoglobinase of erythrocytic parasite Plasmodium falciparum. FP2 inhibitors mainly functions by blocking haemoglobin hydrolysis thereby inhibiting parasite development, promoting FP2 as an applicable target to combat malaria. A library of MAAs was prepared and screened as potential FP2 inhibitors. E-64 was used as a reference inhibitor of FP2 for its comparability with the screened hits.

Molecular docking analysis was conducted on twenty-seven compounds to assess their potential interactions with FP2. Following the docking analysis, an in-silico exploration of ADME characteristics, along with an evaluation of oral toxicity, were performed for these compounds.

Out of twenty-seven screened compounds, seven showed favorable properties with no toxicity. The compounds exhibited favorable binding as evident from the docking score, and possess high stability as elucidated by the RMSD, RMSF and RoG plots. The top two hit compounds with maximum drug score, i.e., shinorine and mycosporine-hydroxyglutamicol were selected for simulation studies which determines the robustness of protein-ligand complex.

The results derived from these studies on FP2 inhibition by MAAs suggest that these metabolites possess a significant antimalarial activity by impeding the target enzyme's active site, thereby providing significant insights in playing crucial role in the emergence of novel drugs to combat malaria.

Previous studies have demonstrated that high-intensity ultrasound (HIU) could enhance the gel properties of salt-reduced (1.5 % NaCl) surimi gels. Understanding its mechanism, particularly the role of key endogenous enzymes, is essential for HIU-assisted modulation of salt-reduced surimi gels with satisfactory textural properties. In this study, enzyme-specific additives were employed to investigate the enhancement effect of HIU on the gelation properties of salt-reduced (1.5 % NaCl) surimi in terms of puncture properties, protein degradation and crosslinking, dynamic rheological properties, water distribution and microstructures. Results showed that CaCl₂ and E-64 significantly increased the breaking force of surimi gels. This improvement occurred because Ca²⁺ activated endogenous transglutaminase (eTGase), as evidenced by the increased formation of non-disulfide covalent bonds, while E-64 reduced protein degradation by inhibiting cysteine protease. The combination of HIU and CaCl₂ lowered the setting temperature during heating and facilitated the formation of non-disulfide covalent bonds, contributing to a three-dimensional gel network that effectively trapped more water. Consequently, the puncture properties were improved. The combination of HIU and E-64 effectively inhibited protein degradation, as evidenced by decreased TCA-soluble peptides and increased G' at the end of the modori stage. Notably, although the combination of HIU and NH₄Cl improved the gel properties, it still failed to form the typical three-dimensional gel network. Collectively, HIU enhanced the gelation properties of surimi through dual mechanisms of eTGase activation and cysteine protease inhibition, with eTGase activation contributing more substantially than protease inhibition, providing a theoretical foundation for HIU-assisted production of salt-reduced surimi gels.