The Structural Basis of African Swine Fever Virus pS273R Protease Binding to E64 through Molecular Dynamics Simulations.

Identification of novel drugs for anti-African swine fever (ASF) applications is of utmost urgency, as it negatively affects pig farming and no effective vaccine or treatment is currently available. African swine fever virus (ASFV) encoded pS273R is a cysteine protease that plays an important role in virus replication. E64, acting as an inhibitor of cysteine protease, has been established as exerting an inhibitory effect on pS273R. In order to obtain a better understanding of the interaction between E64 and pS273R, common docking, restriction docking, and covalent docking were employed to analyze the optimal bonding position between pS273R-E64 and its bonding strength. Additionally, three sets of 100 ns molecular dynamics simulations were conducted to examine the conformational dynamics of pS273R and the dynamic interaction of pS273R-E64, based on a variety of analytical methods including root mean square deviation (RMSD), root mean square fluctuation (RMSF), free energy of ligand (FEL), principal component analysis (PCA), and molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) analysis. The results show that E64 and pS273R exhibited close binding degrees at the activity center of ASFV pS273R protease. The data of these simulations indicate that binding of E64 to pS273R results in a reduction in flexibility, particularly in the ARM region, and a change in the conformational space of pS273R. Additionally, the ability of E64 to interact with polar amino acids such as ASN158, SER192, and GLN229, as well as charged amino acids such as LYS167 and HIS168, seems to be an important factor in its inhibitory effect. Finally, Octet biostratigraphy confirmed the binding of E64 and pS273R with a KD value of 903 uM. Overall, these findings could potentially be utilized in the development of novel inhibitors of pS273R to address the challenges posed by ASFV.

Structural Analysis, Multi-Conformation Virtual Screening and Molecular Simulation to Identify Potential Inhibitors Targeting pS273R Proteases of African Swine Fever Virus.

The African Swine Fever virus (ASFV) causes an infectious viral disease in pigs of all ages. The development of antiviral drugs primarily aimed at inhibition of proteases required for the proteolysis of viral polyproteins. In this study, the conformation of the pS273R protease in physiological states were investigated, virtually screened the multi-protein conformation of pS273R target proteins, combined various molecular docking scoring functions, and identified five potential drugs from the Food and Drug Administration drug library that may inhibit pS273R. Subsequent validation of the dynamic interactions of pS273R with the five putative inhibitors was achieved using molecular dynamics simulations and binding free energy calculations using the molecular mechanics/Poison-Boltzmann (Generalized Born) (MM/PB(GB)SA) surface area. These findings demonstrate that the arm domain and Thr159-Lys167 loop region of pS273R are significantly more flexible compared to the core structural domain, and the Thr159-Lys167 loop region can serve as a "gatekeeper" in the substrate channel. Leucovorin, Carboprost, Protirelin, Flavin Mononucleotide, and Lovastatin Acid all have Gibbs binding free energies with pS273R that were less than -20 Kcal/mol according to the MM/PBSA analyses. In contrast to pS273R in the free energy landscape, the inhibitor and drug complexes of pS273R showed distinct structural group distributions. These five drugs may be used as potential inhibitors of pS273R and may serve as future drug candidates for treating ASFV.

Small molecule inhibitor E-64 exhibiting the activity against African swine fever virus pS273R.

African swine fever (ASF) is a viral disease in swine that results in high mortality in domestic pigs and causes considerable economic losses. Currently, there is no effective vaccine or drugs available for treatment. Identification of new anti-ASFV drugs is urgently needed. Here, the pS273R protein of the African swine fever virus (ASFV) is a specific SUMO-1-like cysteine protease that plays an important role in its replication process. To inhibit virus replication and improve treatment options, a set of small-molecule compounds, targeted inhibitors against the ASFV pS273R protease, were obtained through molecular screening by homology modeling and molecular docking based on structural information of pS273R. Our results clearly demonstrated that the 14th carbon atom of the cysteinase inhibitor E-64 could form one CS covalent bond with the Cys 232 amino acid of the pS273R protease and seven additional hydrogen bonds to maintain a stable binding state. Simultaneously, cell viability, immunophenotyping, and in vitro enzyme activity inhibition assays were performed to comprehensively evaluate E-64 characteristics. Our findings demonstrated that 4 mmol/L E-64 could effectively inhibit the enzyme activity center of the pS273R protease by preventing pS273R protease from lysing pp62, while promoting the upregulation of immune-related cytokines at the transcription level. Moreover, cell viability results revealed that 4 mmol/L E-64 was not cytotoxic. Taken together, we identified a novel strategy to potentially prevent ASFV infection in pigs by blocking the activity of pS273R protease with a small-molecule inhibitor.

One year of African swine fever outbreak in China.

African swine fever (ASF) is a major threat to domestic pigs and wild boars. Since 2018, ASF outbreaks have been ongoing in China. As of August 3, 2019, a total of 151 ASF clusters of outbreaks reported in China have caused severe economic losses for the industry, the pig farmers and pork producers, due to the lack of an efficacious vaccine. The present study aims to analyze the epidemiologic characteristics of ASF outbreak that occurred in several regions across China during the period August 2018- August 2019. Particular focus was on the epidemic distribution, main transmission routes, incidence/fatality, impact on pig production capacity, and the main preventive measures adopted to mitigate the risk of ASF spread in pig farming systems by Chinese government. Results show that anthropogenic factors, spatial distribution, efficient measures taken by China,and good response timely in implementation of preventive measures are important on the transmission of ASF and these suggest that effective ASF risk management in China will require a comprehensive and integrated approach linking science and implemented by all relevant stakeholders. This provides an empirical basis to optimize current interventions as well as develop new tools and strategies to reduce the risk transmission of African swine fever virus (ASFV) to domestic pigs and wild boars.