A Review of the Current Landscape of SARS-CoV-2 Main Protease Inhibitors: Have We Hit the Bullseye Yet?

In this review, we collected 1765 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M-pro inhibitors from the bibliography and other sources, such as the COVID Moonshot project and the ChEMBL database. This set of inhibitors includes only those compounds whose inhibitory capacity, mainly expressed as the half-maximal inhibitory concentration (IC50) value, against M-pro from SARS-CoV-2 has been determined. Several covalent warheads are used to treat covalent and non-covalent inhibitors separately. Chemical space, the variation of the IC50 inhibitory activity when measured by different methods or laboratories, and the influence of 1,4-dithiothreitol (DTT) are discussed. When available, we have collected the values of inhibition of viral replication measured with a cellular antiviral assay and expressed as half maximal effective concentration (EC50) values, and their possible relationship to inhibitory potency against M-pro is analyzed. Finally, the most potent covalent and non-covalent inhibitors that simultaneously inhibit the SARS-CoV-2 M-pro and the virus replication in vitro are discussed.

In Vitro and In Silico Kinetic Studies of Patented 1,7-diEthyl and 1,7-diMethyl Aminoalkanol Derivatives as New Inhibitors of Acetylcholinesterase

Two aminoalkanol derivatives of 1,7-diEthyl-8,9-diphenyl-4azatricyclo (5.2.1.02,6) dec-8-ene-3,5,10-trione and two derivatives of 1,7-diMethyl-8,9-diphenyl-4-azatricyclo (5.2.1.02.6) dec-8-ene-3,5,10-trione were evaluated in vitro for their inhibition efficacy of acetylcholinesterase. The Km, Vmax, slope angles of Lineweaver–Burk plots, Ki and IC50 values showed that all four aminoalkanol derivatives are competitive inhibitors of acetylcholinesterase whose inhibitory potency depends, to a varying extent, on the nature of the four different substituents present in the main compound structure. Studies have shown that the most potent acetylcholinesterase inhibitors are derivatives containing isopropylamine and/or methyl substituents in their structure. In contrast, dimethylamine and/or ethyl substituents seem to have a weaker, albeit visible, effect on the inhibitory potency of acetylcholinesterase. Additionally, docking studies suggest that studied compounds binds with the peripheral anionic site and not enter into the catalytic pocket due to the presence of the sterically extended substituent.

Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants

SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron, as of December 2021. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19.

Molecular Docking Studies on Phenolic Constituents of Anethum graveolens L. Seed Extracts

Background: There are studies indicating that aqueous or hydroalcoholic dill extracts showed higher antioxidant activity compared to other fractions. Molecular docking studies would be relevant to get information on the mechanism of action of the phenolic constituents of Anethum graveolens seed extracts as bioactive compounds. Methodology: In order to perform the docking studies of antioxidant activity of phenolic constituents of Anethum graveolens seed extracts, BIOVIA Discovery Studio and AutoDock Vina software were used. Results: The orientation of flavonoids within Hck and CYP2C9 binding sites has been shown to be the main reason for their inhibitory potency. Conclusion: Molecular docking studies indicate that the compounds identified interact with the target enzymes Hck and CYP2C9 at molecular level through their condensed ring systems and hydroxyl substituents and therefore support the antioxidant capacity of the studied phenolic compounds.

Antibodies specific to SARS-CoV-2 proteins N, S and E in COVID-19 patients in the normal population and in historical samples

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally; recognition of immune responses to this virus will be crucial for coronavirus disease 2019 (COVID-19) control, prevention and treatment. We comprehensively analysed IgG and IgA antibody responses to the SARS-CoV-2 nucleocapsid protein (N), spike protein domain 1 (S1) and envelope protein (E) in: SARS-CoV-2-infected patient, healthy, historical and pre-epidemic samples, including patients’ medical, epidemiological and diagnostic data, virus-neutralizing capability and kinetics. N-specific IgG and IgA are the most reliable diagnostic targets for infection. Serum IgG levels correlate to IgA levels. Half a year after infection, anti-N and anti-S1 IgG decreased, but sera preserved virus-inhibitory potency; thus, testing for IgG may underestimate the protective potential of antibodies. Historical and pre-epidemic sera did not inhibit SARS-CoV-2, thus its circulation before the pandemic and a protective role from antibodies pre-induced by other coronaviruses cannot be confirmed by this study

Sulfate adenylyl transferase kinetics and mechanisms of metabolic inhibitors of microbial sulfate respiration

Sulfate analog oxyanions that function as selective metabolic inhibitors of dissimilatory sulfate reducing microorganisms (SRM) are widely used in ecological studies and industrial applications. As such, it is important to understand the mode of action and mechanisms of tolerance or adaptation to these compounds. Different oxyanions vary widely in their inhibitory potency and mechanism of inhibition, but current evidence suggests that the sulfate adenylyl transferase/ATP sulfurylase (Sat) enzyme is an important target. We heterologously expressed and purified the Sat from the model SRM, Desulfovibrio alaskensis G20. With this enzyme we determined the turnover kinetics (kcat, KM) for alternative substrates (molybdate, selenate, arsenate, monofluorophosphate, and chromate) and inhibition constants (KI) for competitive inhibitors (perchlorate, chlorate, and nitrate). These measurements enable the first quantitative comparisons of these compounds as substrates or inhibitors of a purified Sat from a respiratory sulfate reducer. We compare predicted half-maximal inhibitory concentrations (IC50) based on Sat kinetics with measured IC50 values against D. alaskensis G20 growth and discuss our results in light of known mechanisms of sensitivity or resistance to oxyanions. This analysis helps with the interpretation of recent adaptive laboratory evolution studies and illustrates the value of interpreting gene–microbe–environment interactions through the lens of enzyme kinetics.

Short tail stories: the hirudin-like factors HLF6 and HLF7 of the Asian medicinal leech, Hirudinaria manillensis

The leech-derived hirudins and hirudin-like factors (HLFs) share a common molecule structure: a short N-terminus, a central globular domain, and an elongated C-terminal tail. All parts are important for function. HLF6 and HLF7 were identified in the Asian medicinal leech, Hirudinaria manillensis. The genes of both factors encode putative splice variants that differ in length and composition of their respective C-terminal tails. In either case, the tails are considerably shorter compared to hirudins. Here we describe the functional analyses of the natural splice variants and of synthetic variants that comprise an altered N-terminus and/or a modified central globular domain. All natural splice variants of HLF6 and HLF7 display no detectable thrombin-inhibitory potency. In contrast, some synthetic variants effectively inhibit thrombin, even with tails as short as six amino acid residues in length. Our data indicate that size and composition of the C-terminal tail of hirudins and HLFs can vary in a great extent, yet the full protein may still retain the ability to inhibit thrombin.

Profil fitokimia dan aktivitas antibakteri fraksi etil asetat akar jarak pagar (Jatropha curcas Linn.)

Bacterial infections are a major cause of chronic infections and mortality that continues to threaten public health worldwide. Various medicinal plants used in folk medicine have demonstrated wound healing and antibacterial properties. The aims of the study are investigation phytochemical and antibacterial activity of ethyl acetate fraction from jarak pagar roots (Jatropha curcas Linn.). The roots were treated with maceration method using ethanol 70% and subsequently fractioned to ethyl acetate fraction. The fractions were used for phytochemical screening and antibacterial assay using Kirby and Bauer agar difussion method. The results showed that ethyl acetate fraction jarak pagar roots contained alkaloid, flavonoids, saponins, steroids, poliphenol dan tannins. The antibacterial activity of jarak pagar roots againts Staphylococcus aureus and Escherichia coli shows that the activity of this fraction with concentration 8 mg/mL has strong inhibitory potency against the growth S. aureus with inhibitory zone is 14,3 mm and has moderate inhibitory potency against E. coli with inhibitory zone is 9 mm. The increase concentrations of ethyl asetate fraction jarak pagar roots shows high inhibition diameter of bacterial growth.

Identification of α-Glucosidase Inhibitors from Leaf Extract of Pepper (Capsicum spp.) through Metabolomic Analysis

Metabolomics and in vitro α-glucosidase inhibitory (AGI) activities of pepper leaves were used to identify bioactive compounds and select genotypes for the management of type 2 diabetes mellitus (T2DM). Targeted metabolite analysis using UPLC-DAD-QToF-MS was employed and identified compounds that belong to flavone and hydroxycinnamic acid derivatives from extracts of pepper leaves. A total of 21 metabolites were detected from 155 samples and identified based on MS fragmentations, retention time, UV absorbance, and previous reports. Apigenin-O-(malonyl) hexoside, luteolin-O-(malonyl) hexoside, and chrysoeriol-O-(malonyl) hexoside were identified for the first time from pepper leaves. Pepper genotypes showed a huge variation in their inhibitory activity against α-glucosidase enzyme(AGE) ranging from 17% to 79%. Genotype GP38 with inhibitory activity of 79% was found to be more potent than the positive control acarbose (70.8%.). Orthogonal partial least square (OPLS) analyses were conducted for the prediction of the AGI activities of pepper leaves based on their metabolite composition. Compounds that contributed the most to the bioactivity prediction model (VIP >1.5), showed a strong inhibitory potency. Caffeoyl-putrescine was found to show a stronger inhibitory potency (IC50 = 145 µM) compared to acarbose (IC50 = 197 µM). The chemometric procedure combined with high-throughput AGI screening was effective in selecting polyphenols of pepper leaf for T2DM management.