Potential Inhibitors of Monkeypox Virus Revealed by Molecular Modeling Approach to Viral DNA Topoisomerase I.

The monkeypox outbreak has become a global public health emergency. The lack of valid and safe medicine is a crucial obstacle hindering the extermination of orthopoxvirus infections. The identification of potential inhibitors from natural products, including Traditional Chinese Medicine (TCM), by molecular modeling could expand the arsenal of antiviral chemotherapeutic agents. Monkeypox DNA topoisomerase I (TOP1) is a highly conserved viral DNA repair enzyme with a small size and low homology to human proteins. The protein model of viral DNA TOP1 was obtained by homology modeling. The reliability of the TOP1 model was validated by analyzing its Ramachandran plot and by determining the compatibility of the 3D model with its sequence using the Verify 3D and PROCHECK services. In order to identify potential inhibitors of TOP1, an integrated library of 4103 natural products was screened via Glide docking. Surface Plasmon Resonance (SPR) was further implemented to assay the complex binding affinity. Molecular dynamics simulations (100 ns) were combined with molecular mechanics Poisson-Boltzmann surface area (MM/PBSA) computations to reveal the binding mechanisms of the complex. As a result, three natural compounds were highlighted as potential inhibitors via docking-based virtual screening. Rosmarinic acid, myricitrin, quercitrin, and ofloxacin can bind TOP1 with KD values of 2.16 µM, 3.54 µM, 4.77 µM, and 5.46 µM, respectively, indicating a good inhibitory effect against MPXV. The MM/PBSA calculations revealed that rosmarinic acid had the lowest binding free energy at -16.18 kcal/mol. Myricitrin had a binding free energy of -13.87 kcal/mol, quercitrin had a binding free energy of -9.40 kcal/mol, and ofloxacin had a binding free energy of -9.64 kcal/mol. The outputs (RMSD/RMSF/Rg/SASA) also indicated that the systems were well-behaved towards the complex. The selected compounds formed several key hydrogen bonds with TOP1 residues (TYR274, LYS167, GLY132, LYS133, etc.) via the binding mode analysis. TYR274 was predicted to be a pivotal residue for compound interactions in the binding pocket of TOP1. The results of the enrichment analyses illustrated the potential pharmacological networks of rosmarinic acid. The molecular modeling approach may be acceptable for the identification and design of novel poxvirus inhibitors; however, further studies are warranted to evaluate their therapeutic potential.

Deguelin inhibits HCV replication through suppressing cellular autophagy via down regulation of Beclin1 expression in human hepatoma cells.

AIMS: Deguelin, a natural compound derived from Mundulea sericea (Leguminosae) and some other plants exhibits an activity to inhibit autophagy, a cellular machinery required for hepatitis C virus (HCV) replication. This study aimed to illuminate the impact of deguelin on HCV replication and mechanism(s) involved. METHODS: HCV JFH-1-Huh7 infectious system was used for the investigation. Real time RT-PCR, Western blot, fluorescent microscopy assay were used to measure the expression levels of viral or cellular factors. Overexpression and silencing expression techniques were used to determine the role of key cellular factors. RESULTS: Deguelin treatment of Huh7 cells significantly inhibited HCV JFH-1 replication in a dose- and time-dependent manner. Deguelin treatment suppressed autophagy in Huh7 cells, evidenced by the decrease of LC3B-II levels, the conversion of LC3B-I to LC3B-II, and the formation of GFP-LC3 puncta as well as the increase of p62 level in deguelin-treated cells compared with control cells. HCV infection could induce autophagy which was also suppressed by deguelin treatment. Mechanism research reveals that deguelin inhibited expression of Beclin1, which is a key cellular factor for the initiation of the autophagosome formation in autophagy. Overexpression or silencing expression of Beclin1 in deguelin-treated Huh7 cells could weaken or enhance the inhibitory effect on autophagy by deguelin, respectively, and thus partially recover or further inhibit HCV replication correspondingly. CONCLUSIONS: Deguelin may serve as a novel anti-HCV compound via its inhibitory effect on autophagy, which warrants further investigation as a potential therapeutic agent for HCV infection.

Enhancement of γ-aminobutyric acid production in recombinant Corynebacterium glutamicum by co-expressing two glutamate decarboxylase genes from Lactobacillus brevis.

γ-Aminobutyric acid (GABA), a non-protein amino acid, is a bioactive component in the food, feed and pharmaceutical fields. To establish an effective single-step production system for GABA, a recombinant Corynebacterium glutamicum strain co-expressing two glutamate decarboxylase (GAD) genes (gadB1 and gadB2) derived from Lactobacillus brevis Lb85 was constructed. Compared with the GABA production of the gadB1 or gadB2 single-expressing strains, GABA production by the gadB1-gadB2 co-expressing strain increased more than twofold. By optimising urea supplementation, the total production of L-glutamate and GABA increased from 22.57 ± 1.24 to 30.18 ± 1.33 g L⁻¹, and GABA production increased from 4.02 ± 0.95 to 18.66 ± 2.11 g L⁻¹ after 84-h cultivation. Under optimal urea supplementation, L-glutamate continued to be consumed, GABA continued to accumulate after 36 h of fermentation, and the pH level fluctuated. GABA production increased to a maximum level of 27.13 ± 0.54 g L⁻¹ after 120-h flask cultivation and 26.32 g L⁻¹ after 60-h fed-batch fermentation. The conversion ratio of L-glutamate to GABA reached 0.60-0.74 mol mol⁻¹. By co-expressing gadB1 and gadB2 and optimising the urea addition method, C. glutamicum was genetically improved for de novo biosynthesis of GABA from its own accumulated L-glutamate.