Institute of Fundamental Technological Research

Plant pathogens pose a severe threat to global food security by compromising the availability, quality, and safety of crops for human and animal consumption. Given the urgent need for alternatives to chemical pesticides, natural inhibitors of phytopathogenic proteases represent promising biopesticides. Tarocystatin has been characterized in Colocasia esculenta as a defense protein against phytopathogenic nematodes and fungi. Despite its biotechnological potential, few studies describe its mechanical, structural, and energetic properties. In this study, we characterized the inhibitory mechanism of tarocystatin against papain using a computational biophysics approach. Through extensive molecular dynamics (MD) and steered molecular dynamics (SMD) simulations, we explored the dynamic, energetic, structural, and mechanical basis of tarocystatin and its specific binding to papain. Our results suggest that the stability of the complex is characterized by a lack of conformational rearrangements, showing invariability in its secondary structure across all MD replicas. Additionally, electrostatic analysis revealed a high complementarity of the tarocystatin-papain complex, which was later corroborated by the hydrogen-bond network established at the complex interface, explaining its strong inhibitory capacity. Moreover, we determined that the substrate-competitive inhibitory mechanism is due to the binding ability of conserved motifs in tarocystatin, which efficiently interact with the catalytic active site of papain. This was also confirmed through SMD, where we observed that the N-terminal region acts as a spring to prevent the dissociation of the complex under external pulling forces. Overall, our study is the first to provide a comprehensive exploration of the biophysical properties of the tarocystatin-papain complex, offering insights into the tarocystatin's inhibition mechanism. These results lay the foundation for future development of tarocystatin-based antifungal alternatives, as well as for exploring its inhibitory activity in other pathogens or enhancing its efficacy through molecular engineering.

Tarocystatin, Papain, Inhibition mechanism, Molecular dynamics, Computational biophysics

Context
SARS-CoV-2, responsible for COVID-19, has led to over 500 million infections and more than 6 million deaths globally. There have been limited effective treatments available. The study aims to find a drug that can prevent the virus from entering host cells by targeting specific sites on the virus’s spike protein.

Method
We examined 13,397 compounds from the Malaria Box library against two specific sites on the spike protein: the receptor-binding domain (RBD) and a predicted cryptic pocket. Using virtual screening, molecular docking, molecular dynamics, and MMPBSA techniques, they evaluated the stability of two compounds. TCMDC-124223 showed high stability and binding energy in the RBD, while TCMDC-133766 had better binding energy in the cryptic pocket. The study also identified that the interacting residues are conserved, which is crucial for addressing various virus variants. The findings provide insights into the potential of small molecules as drugs against the spike protein.

SARS-CoV-2, Molecular docking, Spike protein, Cryptic pocket, MMPBSA