Inhibition of Listeria Monocytogenes HtrA Protease with Camostat, Gabexate and Nafamostat Mesylates and the Binding Mode of the Inhibitors.

In many bacteria, the High Temperature requirement A (HtrA) protein functions as a chaperone and protease. HtrA is an important factor in stress tolerance and plays a significant role in the virulence of several pathogenic bacteria. Camostat, gabexate and nafamostat mesylates are serine protease inhibitors and have recently shown a great impact in the inhibition studies of SARS-CoV2. In this study, the inhibition of Listeria monocytogenes HtrA (LmHtrA) protease activity was analysed using these three inhibitors. The cleavage assay, using human fibrinogen and casein as substrates, revealed that the three inhibitors effectively inhibit the protease activity of LmHtrA. The agar plate assay and spectrophotometric analysis concluded that the inhibition of nafamostat (IC50 value of 6.6 ± 0.4 µM) is more effective compared to the other two inhibitors. Previous studies revealed that at the active site of the protease, these inhibitors are hydrolysed and one of the hydrolysates is covalently bound to the active site serine. To understand the mode of binding of these inhibitors at the active site of LmHtrA, docking of the inhibitors followed by molecular dynamics simulations were carried out. Analysis of the LmHtrA-inhibitor complex structures revealed that the covalently bound inhibitor is unable to occupy the S1 pocket of the LmHtrA which is in contrast to the previously determined camostat and nafamostat complex structures. This observation provides the first glimpse of the substrate specificity of LmHtrA which is not known. The obtained results also suggest that the development of novel inhibitors of LmHtrA and its homologs with active site architecture similar to LmHtrA can be pursued with suitable modification of these inhibitors. To date, only a very few studies have been carried out on identifying the inhibitors of HtrA proteolytic activity.

Inhibition of SARS-CoV-2 replication by zinc gluconate in combination with hinokitiol.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic is currently the major challenge to global public health. Two proteases, papain-like protease (PLpro) and the 3-chymotrypsin-like protease (3CLpro or Mpro), are indispensable for SARS-CoV-2 replication, making them attractive targets for antiviral therapy development. Here we screened a panel of essential metal ions using a proteolytic assay and identified that zinc gluconate, a widely-used zinc supplement, strongly inhibited the proteolytic activities of the two proteases in vitro. Biochemical and crystallographic data reveal that zinc gluconate exhibited the inhibitory function via binding to the protease catalytic site residues. We further show that treatment of zinc gluconate in combination with a small molecule ionophore hinokitiol, could lead to elevated intracellular Zn2+ level and thereby significantly impaired the two protease activities in cellulo. Particularly, this approach could also be applied to rescue SARS-CoV-2 infected mammalian cells, indicative of potential application to combat coronavirus infections. Our studies provide the direct experimental evidence that elevated intracellular zinc concentration directly inhibits SARS-CoV-2 replication and suggest the potential benefits to use the zinc supplements for coronavirus disease 2019 (COVID-19) treatment.

UHPLC-OrbiTrap MS Characterization of Phenolic Profiles in French Marigold Extracts and Analysis of Their Antifeedant Activity against Colorado Potato Beetle.

French marigold is an aromatic plant rich in polyphenolic secondary metabolites, which pesticidal potential was examined in this study. Ultra-high-performance liquid chromatography (UHPLC) connected with OrbiTrap mass spectrometer (MS) identified 113 phenolics and revealed the most detailed phytochemistry of French marigold published so far. Depending on plant material (flowers or leaves) and solvents used for extraction (water, methanol, dichloromethane), the phenolic composition varied. Methanol extract of flowers, with 89 identified phenolics and high antioxidant activity statistically comparable with positive control Trolox, was chosen for testing of antifeedant potential against the 3rd and 4th instars of Colorado potato beetle (CPB). A significant reduction in final body mass of 4th larval stage fed with potato leaves coated with methanol extract of flowers in the concentration of 10 mg/mL was observed (157.67 mg vs. 182.26 mg of controls fed with non-treated leaves). This caused delayed molting since treated larvae reached the maximal mass a day after controls and this delay persisted during the entire larval development. Continuous feeding caused a 25% decline in digestive proteolytic activity of the 4th instar in comparison to controls. The results suggest that French marigold methanol extract of flowers could be proposed as a promising antifeedant for CPB management, with an impact on the reduction in the environmental footprint associated with synthetic pesticide application.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.