Inibidores de proteases de plantas como modelos estruturais para o desenho de peptídeos bioinspirados com potencial uso clínico sobre as proteases principais de SARS-COV-2, SARS-COV-1 e MERS-COV

Abstract

Human coronaviruses (HCoVs) associated with severe infections, such as MERS-CoV, SARS- CoV-1, and SARS-CoV-2, remain a global threat due to limited effective antivirals, reduced

efficacy of vaccines against new variants, and the risk of zoonotic outbreaks. The main viral protease (Mpro), essential for replication and highly conserved among HCoVs, represents a key therapeutic target. Given the potential of plant protease inhibitors (PIs) to modulate viral enzymes, this study aimed to identify plant-derived PIs targeting Mpro and evaluate their use as scaffolds for bioinspired antiviral peptides. A structural database of plant PIs was compiled from Protein Data Bank (PDB) entries, focusing on cysteine protease inhibitors. Molecular docking (FRODOCK 3.12 and ClusPro 2.0), molecular dynamics simulations (100 ns, GROMACS 2018.4), and quantum mechanical calculations (DMol3) revealed that phytocystatins from Ananas comosus and Sesamum indicum interact with allosteric sites of Mpro, with interaction energies of -225.7 and -301.9 kcal/mol, respectively. CASTp server analyses indicated that these interactions caused significant reductions in the active site area and volume of Mpro. Based on the sequences of these phytocystatins, two bioinspired peptides were designed — KFD-Pep1 (1276.31 Da) and MLL-Pep2 (1294.69 Da) — showing favorable in silico properties, including predicted cell penetration, positive net charges (+3 and +4), hydrophobicity (50% and 40%), Boman indices (2.1 and 1.34 kcal/mol), α-helical structures, simulated intestinal stability, and predictions of being non-hemolytic and non-allergenic. Notably, MLL-Pep2 also displayed an amphipathic nature. Molecular docking analyses showed

that both peptides interact with conserved catalytic regions of Mpro of SARS-CoV-2 (wild- type, naturally occurring mutants, and the E166R-induced mutant), SARS-CoV-1, and MERS- CoV. ADMET profiling predicted favorable pharmacokinetic properties and low toxicity,

comparable to the clinically approved inhibitor nirmatrelvir. These findings highlight KFD- Pep1 and MLL-Pep2 as promising candidates for further studies and support plant protease

inhibitors as valuable sources for broad-spectrum antiviral development against HCoVs.