M3F

Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control.

Hepatotoxicity, a critical liver damage often caused by drugs like paracetamol (PCM), involves oxidative stress, inflammation, and cellular injury. This study investigates the hepatoprotective potential of a benzimidazole-pyrazole derivative, 2-((1H-benzo[d]imidazol-2-yl) thio)-1-(3-(2-fluorophenyl)-5-phenyl-1H-pyrazol-1-yl) ethanone (M3F), using both in silico and in vivo approaches. Pharmacokinetic profiling was performed using SwissADME and ProTox to predict drug-likeness and toxicity. Molecular docking studies were conducted using AutoDock Vina integrated with PyRx to evaluate the binding affinity of M3F with key targets related to oxidative stress and inflammation. Subsequently, in vivo studies were conducted using a PCM-induced hepatotoxicity model in mice, wherein M3F was administered orally at doses of 100, 200, and 400 µg/kg for seven consecutive days, followed by a single dose of PCM (300 mg/kg, intraperitoneally) on the eighth day to induce liver injury. Serum markers of liver damage, lipid profiling, antioxidant assays, histopathological examinations and molecular markers analysis were performed to determine the therapeutic effects of M3F. In silico analysis revealed favorable pharmacokinetic properties and strong binding affinities of M3F to inflammatory and oxidative stress-related molecular targets. In vivo, M3F treatment led to a marked improvement in liver enzyme levels and lipid profiles, along with significant histological recovery of liver tissue. The study demonstrated a significant downregulation of IL-6, IL-1β, and TNF-α protein levels, as measured by ELISA, indicating reduced inflammatory responses following M3F treatment. Additionally, PCR analysis revealed a notable decrease in the mRNA expression levels of IL-17, NF-κB, and TLR4, alongside an increase in Nrf2 and HO-1 transcript levels. It is suggested that M3F demonstrated notable hepatoprotective effects in PCM-induced liver injury through dual modulation of inflammatory (NF-κB) and antioxidant (Nrf2) pathways. These findings support its potential as a novel hepatoprotective candidate for future studies.