Discovery and characterization of the covalent SARS-CoV-2 3CLpro inhibitors from Ginkgo biloba extract via integrating chemoproteomic and biochemical approaches.

BACKGROUND: The 3C-like proteases (3CLpros) are cysteine-rich homodimeric proteins and can be covalently modified by numerous natural and synthetic compounds, which in turn, block the proteolytic activity or the formation of enzymatically active dimeric forms. Although herbal medicines have been widely used to treat COVID-19, identification of the key herbal constituents that can covalently modify the 3CLpros in ß-coronaviruses (CoVs) remains a big challenge. AIMS: To construct a comprehensive approach for efficient discovering the covalent SARS-CoV-2 3CLpro inhibitors from herbal medicines. To decipher the key anti-SARS-CoV-2 3CLpro constituents in Ginkgo biloba extract 50 (GBE50) and to study their anti-SARS-CoV-2 3CLpro mechanisms. METHODS: SARS-CoV-2 3CLpro inhibition assay including time-dependent inhibition assays and inactivation kinetic analyses were conducted using a fluorescence-based biochemical assay. The constituents in GBE50 were analyzed by UHPLC-Q-Exactive Orbitrap HRMS. The peptides modified by herbal constituents were characterized by using nanoLC-MS/MS. RESULTS: Following testing the anti-SARS-CoV-2 3CLpro effects of 104 herbal medicines, it was found that Ginkgo biloba extract 50 (GBE50) potently inhibited SARS-CoV-2 3CLpro in dose- and time-dependent manners. A total of 38 constituents were identified from GBE50 by UHPLC-Q-Exactive Orbitrap HRMS, while 26 peptides modified by 18 constituents were identified by chemoproteomic profiling. The anti-SARS-CoV-2 3CLpro effects of 18 identified covalent inhibitors were then validated by performing time-dependent inhibition assays. The results clearly demonstrated that most tested constituents showed time-dependent inhibition on SARS-CoV-2 3CLpro, while gallocatechin and sciadopitysin displayed the most potent anti-SARS-CoV-2 3CLpro effects. CONCLUSION: Collectively, GBE50 and some constituents in this herbal product could strongly inhibit SARS-CoV-2 3CLpro in dose- and time-dependent manner. Gallocatechin and sciadopitysin were identified as potent SARS-CoV-2 3CLpro inhibitors, which offers promising lead compounds for the development of novel anti-SARS-CoV-2 drugs.

Discovery and characterization of the covalent SARS-CoV-2 3CLpro inhibitors from Ginkgo biloba extract via integrating chemoproteomic and biochemical approaches

Background : The 3C-like proteases (3CLpros) are cysteine-rich homodimeric proteins and can be covalently modified by numerous natural and synthetic compounds, which in turn, block the proteolytic activity or the formation of enzymatically active dimeric forms. Although herbal medicines have been widely used to treat COVID-19, identification of the key herbal constituents that can covalently modify the 3CLpros in β-coronaviruses (CoVs) remains a big challenge. Aims : To construct a comprehensive approach for efficient discovering the covalent SARS-CoV-2 3CLpro inhibitors from herbal medicines. To decipher the key anti-SARS-CoV-2 3CLpro constituents in Ginkgo biloba extract 50 (GBE50) and to study their anti-SARS-CoV-2 3CLpro mechanisms. Methods : SARS-CoV-2 3CLpro inhibition assay including time-dependent inhibition assays and inactivation kinetic analyses were conducted using a fluorescence-based biochemical assay. The constituents in GBE50 were analyzed by UHPLC-Q-Exactive Orbitrap HRMS. The peptides modified by herbal constituents were characterized by using nanoLC-MS/MS. Results : Following testing the anti-SARS-CoV-2 3CLpro effects of 104 herbal medicines, it was found that Ginkgo biloba extract 50 (GBE50) potently inhibited SARS-CoV-2 3CLpro in dose- and time-dependent manners. A total of 38 constituents were identified from GBE50 by UHPLC-Q-Exactive Orbitrap HRMS, while 26 peptides modified by 18 constituents were identified by chemoproteomic profiling. The anti-SARS-CoV-2 3CLpro effects of 18 identified covalent inhibitors were then validated by performing time-dependent inhibition assays. The results clearly demonstrated that most tested constituents showed time-dependent inhibition on SARS-CoV-2 3CLpro, while gallocatechin and sciadopitysin displayed the most potent anti-SARS-CoV-2 3CLpro effects. Conclusion : Collectively, GBE50 and some constituents in this herbal product could strongly inhibit SARS-CoV-2 3CLpro in dose- and time-dependent manner. Gallocatechin and sciadopitysin were identified as potent SARS-CoV-2 3CLpro inhibitors, which offers promising lead compounds for the development of novel anti-SARS-CoV-2 drugs. Graphical abstract Image, graphical abstract

Discovery of Isojacareubin as a covalent inhibitor of SARS-CoV-2 main protease using structural and experimental approaches.

The ongoing pandemic with the emergence of immune evasion potential and, particularly, the current omicron subvariants intensified the situation further. Although vaccines are available, the immune evasion capabilities of the recent variants demand further efficient therapeutic choices to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Hence, considering the necessity of the small molecule inhibitor, we target the main protease (3CLpro), which is an appealing target for the development of antiviral drugs against SARS-CoV-2. High-throughput molecular in silico screening of South African natural compounds database reported Isojacareubin and Glabranin as the potential inhibitors for the main protease. The calculated docking scores were reported to be -8.47 and -8.03 kcal/mol, respectively. Moreover, the structural dynamic assessment reported that Isojacareubin in complex with 3CLpro exhibit a more stable dynamic behavior than Glabranin. Inhibition assay indicated that Isojacareubin could inhibit SARS-CoV-2 3CLpro in a time- and dose-dependent manner, with half maximal inhibitory concentration values of 16.00 ± 1.35 µM (60 min incubation). Next, the covalent binding sites of Isojacareubin on SARS-CoV-2 3CLpro was identified by biomass spectrometry, which reported that Isojacareubin can covalently bind to thiols or Cysteine through Michael addition. To evaluate the inactivation potency of Isojacareubin, the inactivation kinetics was further investigated. The inactivation kinetic curves were plotted according to various concentrations with gradient-ascending incubation times. The KI value of Isojacareubin was determined as 30.71 µM, whereas the Kinact value was calculated as 0.054 min-1 . These results suggest that Isojacareubin is a covalent inhibitor of SARS-CoV-2 3CLpro .

Discovery and mechanism of action of Thonzonium bromide from an FDA-approved drug library with potent and broad-spectrum inhibitory activity against main proteases of human coronaviruses.

Although the effective drugs or vaccines have been developed to prevent the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), their efficacy may be limited for the viral evolution and immune escape. Thus, it is urgently needed to develop the novel broad-spectrum antiviral agents to control the coronavirus disease 2019 (COVID-19) global pandemic. The 3C-like protease (3CLpro) is a highly conserved cysteine proteinase that plays a pivotal role in processing the viral polyprotein to create non-structural proteins (nsps) for replication and transcription of SARS-CoV-2, making it an attractive antiviral target for developing broad-spectrum antiviral agents against SARS-CoV-2. In this study, we identified Thonzonium bromide as an inhibitor of SARS-CoV-2 3CLpro with an IC50 value of 2.04 ± 0.25 µM by fluorescence resonance energy transfer (FRET)-based enzymatic inhibition assay from the FDA-approved drug library. Next, we determined the inhibitory activity of Thonzonium bromide analogues against SARS-CoV-2 3CLpro and analyzed their structure-activity relationship (SAR). Interestingly, Thonzonium bromide showed better inhibitory activity than other analogues. Further fluorescence quenching assay, enzyme kinetics analysis, circular dichroism (CD) analysis and molecular docking studies showed that Thonzonium bromide inhibited SARS-CoV-2 3CLpro activity by firmly occupying the catalytic site and inducing conformational changes of the protease. In addition, Thonzonium bromide didn't exhibit inhibitory activity on human chymotrypsin C (CTRC) and Dipeptidyl peptidase IV (DPP-IV), indicating that it had a certain selectivity. Finally, we measured the inhibitory activities of Thonzonium bromide against 3CLpro of SARS-CoV, MERS-CoV and HCoV-229E and found that it had the broad-spectrum inhibitory activity against the proteases of human coronaviruses. These results provide the possible mechanism of action of Thonzonium bromide, highlighting its potential efficacy against multiple human coronaviruses.

Chemo- and Site-Selective Lysine Modification of Peptides and Proteins under Native Conditions Using the Water-Soluble Zolinium.

Site-selective lysine modification of peptides and proteins in aqueous solutions or in living cells is still a big challenge today. Here, we report a novel strategy to selectively quinolylate lysine residues of peptides and proteins under native conditions without any catalysts using our newly developed water-soluble zoliniums. The zoliniums could site-selectively quinolylate K350 of bovine serum albumin and inactivate SARS-CoV-2 3CLpro via covalently modifying two highly conserved lysine residues (K5 and K61). In living HepG2 cells, it was demonstrated that the simple zoliniums (5b and 5B) could quinolylate protein lysine residues mainly in the nucleus, cytosol, and cytoplasm, while the zolinium-fluorophore hybrid (8) showed specific lysosome-imaging ability. The specific chemoselectivity of the zoliniums for lysine was validated by a mixture of eight different amino acids, different peptides bearing potential reactive residues, and quantum chemistry calculations. This study offers a new way to design and develop lysine-targeted covalent ligands for specific application.

The SARS-CoV-2 main protease (Mpro): Structure, function, and emerging therapies for COVID-19.

The main proteases (Mpro), also termed 3-chymotrypsin-like proteases (3CLpro), are a class of highly conserved cysteine hydrolases in ß-coronaviruses. Increasing evidence has demonstrated that 3CLpros play an indispensable role in viral replication and have been recognized as key targets for preventing and treating coronavirus-caused infectious diseases, including COVID-19. This review is focused on the structural features and biological function of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease Mpro (also known as 3CLpro), as well as recent advances in discovering and developing SARS-CoV-2 3CLpro inhibitors. To better understand the characteristics of SARS-CoV-2 3CLpro inhibitors, the inhibition activities, inhibitory mechanisms, and key structural features of various 3CLpro inhibitors (including marketed drugs, peptidomimetic, and non-peptidomimetic synthetic compounds, as well as natural compounds and their derivatives) are summarized comprehensively. Meanwhile, the challenges in this field are highlighted, while future directions for designing and developing efficacious 3CLpro inhibitors as novel anti-coronavirus therapies are also proposed. Collectively, all information and knowledge presented here are very helpful for understanding the structural features and inhibitory mechanisms of SARS-CoV-2 3CLpro inhibitors, which offers new insights or inspiration to medicinal chemists for designing and developing more efficacious 3CLpro inhibitors as novel anti-coronavirus agents.

The SARS‐CoV‐2 main protease (Mpro): Structure, function, and emerging therapies for COVID‐19

The main proteases (Mpro), also termed 3‐chymotrypsin‐like proteases (3CLpro), are a class of highly conserved cysteine hydrolases in β‐coronaviruses. Increasing evidence has demonstrated that 3CLpros play an indispensable role in viral replication and have been recognized as key targets for preventing and treating coronavirus‐caused infectious diseases, including COVID‐19. This review is focused on the structural features and biological function of the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) main protease Mpro (also known as 3CLpro), as well as recent advances in discovering and developing SARS‐CoV‐2 3CLpro inhibitors. To better understand the characteristics of SARS‐CoV‐2 3CLpro inhibitors, the inhibition activities, inhibitory mechanisms, and key structural features of various 3CLpro inhibitors (including marketed drugs, peptidomimetic, and non‐peptidomimetic synthetic compounds, as well as natural compounds and their derivatives) are summarized comprehensively. Meanwhile, the challenges in this field are highlighted, while future directions for designing and developing efficacious 3CLpro inhibitors as novel anti‐coronavirus therapies are also proposed. Collectively, all information and knowledge presented here are very helpful for understanding the structural features and inhibitory mechanisms of SARS‐CoV‐2 3CLpro inhibitors, which offers new insights or inspiration to medicinal chemists for designing and developing more efficacious 3CLpro inhibitors as novel anti‐coronavirus agents. A comprehensive summary of recent advances in SARS‐CoV‐2 3CLpro inhibitors (including marketed drugs, peptidomimetic, and non‐peptidomimetic synthetic compounds, as well as natural compounds and their derivatives), including the inhibitory activities, inhibitory mechanisms, and key structural features, provides new insights for designing and developing more efficacious 3CLpro inhibitors as broad‐spectrum anti‐coronavirus agents.

Flavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CLpro: Inhibition potentials, covalent binding sites and inhibitory mechanisms.

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.

Identification of Vitamin K3 and its analogues as covalent inhibitors of SARS-CoV-2 3CLpro.

After the emergence of the pandemic, repurposed drugs have been considered as a quicker way of finding potential antiviral agents. SARS-CoV-2 3CLpro is essential for processing the viral polyproteins into mature non-structural proteins, making it an attractive target for developing antiviral agents. Here we show that Vitamin K3 screened from the FDA-Approved Drug Library containing an array of 1,018 compounds has potent inhibitory activity against SARS-CoV-2 3CLpro with the IC50 value of 4.78 ± 1.03 µM, rather than Vitamin K1, K2 and K4. Next, the time-dependent inhibitory experiment was carried out to confirm that Vitamin K3 could form the covalent bond with SARS-CoV-2 3CLpro. Then we analyzed the structure-activity relationship of Vitamin K3 analogues and identified 5,8-dihydroxy-1,4-naphthoquinone with 9.8 times higher inhibitory activity than Vitamin K3. Further mass spectrometric analysis and molecular docking study verified the covalent binding between Vitamin K3 or 5,8-dihydroxy-1,4-naphthoquinone and SARS-CoV-2 3CLpro. Thus, our findings provide valuable information for further optimization and design of novel inhibitors based on Vitamin K3 and its analogues, which may have the potential to fight against SARS-CoV-2.

Discovery of naturally occurring inhibitors against SARS-CoV-2 3CLpro from Ginkgo biloba leaves via large-scale screening.

3-Chymotrypsin-like protease (3CLpro) is a virally encoded main proteinase that is pivotal for the viral replication across a broad spectrum of coronaviruses. This study aims to discover the naturally occurring SARS-CoV-2 3CLpro inhibitors from herbal constituents, as well as to investigate the inhibitory mechanism of the newly identified efficacious SARS-CoV-2 3CLpro inhibitors. Following screening of the inhibitory potentials of eighty herbal products against SARS-CoV-2 3CLpro, Ginkgo biloba leaves extract (GBLE) was found with the most potent SARS-CoV-2 3CLpro inhibition activity (IC50 = 6.68 µg/mL). Inhibition assays demonstrated that the ginkgolic acids (GAs) and the bioflavones isolated from GBLE displayed relatively strong SARS-CoV-2 3CLpro inhibition activities (IC50 < 10 µM). Among all tested constituents, GA C15:0, GA C17:1 and sciadopitysin displayed potent 3CLpro inhibition activities, with IC50 values of less than 2 µM. Further inhibition kinetic studies and docking simulations clearly demonstrated that two GAs and sciadopitysin strongly inhibit SARS-CoV-2 3CLprovia a reversible and mixed inhibition manner. Collectively, this study found that both GBLE and the major constituents in this herbal product exhibit strong SARS-CoV-2 3CLpro inhibition activities, which offer several promising leading compounds for developing novel anti-COVID-19 medications via targeting on 3CLpro.