Ginkgolic acids inhibit SARS-CoV-2 and its variants by blocking the spike protein/ACE2 interplay.

Targeting the interaction between the spike protein receptor binding domain (S-RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and angiotensin-converting enzyme 2 (ACE2) is a potential therapeutic strategy for treating coronavirus disease 2019 (COVID-19). However, we still lack small-molecule drug candidates for this target due to the missing knowledge in the hot spots for the protein-protein interaction. Here, we used NanoBiT technology to identify three Ginkgolic acids from an in-house traditional Chinese medicine (TCM) library, and they interfere with the S-RBD/ACE2 interplay. Our pseudovirus assay showed that one of the compounds, Ginkgolic acid C17:1 (GA171), significantly inhibits the entry of original SARS-CoV-2 and its variants into the ACE2-overexpressed HEK293T cells. We investigated and proposed the binding sites of GA171 on S-RBD by combining molecular docking and molecular dynamics simulations. Site-directed mutagenesis and surface plasmon resonance revealed that GA171 specifically binds to the pocket near R403 and Y505, critical residues of S-RBD for S-RBD interacting with ACE2. Thus, we provide structural insights into developing new small-molecule inhibitors and vaccines against the proposed S-RBD binding site.

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.

Structure-Based Virtual Screening and Identification of Potential Inhibitors of SARS-CoV-2 S-RBD and ACE2 Interaction.

The emergence and rapid spread of SARS-CoV-2 have caused a worldwide public health crisis. Designing small molecule inhibitors targeting SARS-CoV-2 S-RBD/ACE2 interaction is considered as a potential strategy for the prevention and treatment of SARS-CoV-2. But to date, only a few compounds have been reported as SARS-CoV-2 S-RBD/ACE2 interaction inhibitors. In this study, we described the virtual screening and experimental validation of two novel inhibitors (DC-RA016 and DC-RA052) against SARS-CoV-2 S-RBD/ACE2 interaction. The NanoBiT assays and surface plasmon resonance (SPR) assays demonstrated their capabilities of blocking SARS-CoV-2 S-RBD/ACE2 interaction and directly binding to both S-RBD and ACE2. Moreover, the pseudovirus assay revealed that these two compounds possessed significant antiviral activity (about 50% inhibition rate at maximum non-cytotoxic concentration). These results indicate that the compounds DC-RA016 and DC-RA052 are promising inhibitors against SARS-CoV-2 S-RBD/ACE2 interaction and deserve to be further developed.

Pseudoephedrine and its derivatives antagonize wild and mutated severe acute respiratory syndrome-CoV-2 viruses through blocking virus invasion and antiinflammatory effect.

The coronavirus disease 2019 has infected over 150 million people worldwide and led to over 3 million deaths. Severe acute respiratory syndrome (SARS)-CoV-2 lineages B.1.1.7, B.1.617, B.1.351, and P.1 were reported to have higher infection rates than that of wild one. These mutations were noticed to happen in the receptor-binding domain of spike protein (S-RBD), especially mutations N501Y, E484Q, E484K, K417N, K417T, and L452R. Currently, there is still no specific medicine against the virus; moreover, cytokine storm is also a dangerous factor for severe infected patients. In this study, potential S-RBD-targeted active monomers from traditional Chinese medicine Ephedra sinica Stapf (ephedra) were discovered by virtual screening. NanoBiT assay was performed to confirm blocking activities of the screened compounds against the interaction between SARS-CoV-2 S-RBD and angiotensin-converting enzyme 2 (ACE2). We further analyzed the blocking effect of the active compounds on the interactions of mutated S-RBD and ACE2 by computational studies. Moreover, antiinflammatory activities were evaluated using qRT-PCR, enzyme-linked immune sorbent assay, and Western blot analysis. As a result, pseudoephedrine (MHJ-17) and its derivative (MHJ-11) were found as efficient inhibitors disrupting the interactions between ACE2 and both wild and mutated S-RBDs. In addition, they also have antiinflammatory activities, which can be potential drug candidates or lead compounds for further study.

Potential therapeutic approaches for the early entry of SARS-CoV-2 by interrupting the interaction between the spike protein on SARS-CoV-2 and angiotensin-converting enzyme 2 (ACE2).

The COVID-19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has quickly spread around the globe. At present, there is no precise and effective treatment for the patients with COVID-19, so rapid development of drugs is urgently needed in order to contain the highly infectious disease. The virus spike protein (S protein) can recognize the angiotensin-converting enzyme 2 (ACE2) receptor on the host cell membrane and undergo a series of conformational changes, protease cleavage and membrane fusion to complete the virus entry, so S protein is an important target for vaccine and drug development. Here we provide a brief overview of molecular mechanisms of virus entry, as well as some potential antiviral agents that act on S/ACE2 protein-protein interaction. Specifically, we focused on experimentally validated and/or computational prediction identified inhibitors that target SARS-CoV-2 S protein, ACE2 and enzymes associated with viral infection. This review offers valuable information for the discovery and development of potential antiviral agents in combating SARS-CoV-2. In addition, with the deepening understanding of the mechanism of SARS-CoV-2 infection, more targeted prevention and treatment drugs will be explored with the aid of the advanced technology in the future.

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.

Glycyrrhizic acid exerts inhibitory activity against the spike protein of SARS-CoV-2.

Coronavirus causes a disease with high infectivity and pathogenicity, especially SARS in 2003, MERS in 2012, and COVID-2019 currently. The spike proteins of these coronaviruses are critical for host cell entry by receptors. Thus, searching for broad-spectrum anti-coronavirus candidates, such as spike protein inhibitors, is vital and desirable due to the mutations in the spike protein. In this study, a combination of computer-aided drug design and biological verification was used to discover active monomers from traditional Chinese medicine. Surface plasmon resonance (SPR) assays and NanoBit assays were used to verify the predicated compounds with their binding activities to spike proteins and inhibitory activities on the SARS-CoV-2 RBD/ACE2 interaction, respectively. Furthermore, an MTT assay was used to evaluate the cell toxicities of active compounds. As a result, glycyrrhizic acid (ZZY-44) was found to be the most efficient and nontoxic broad-spectrum anti-coronavirus molecule in vitro, especially, the significant effect on SARS-CoV-2, which provided a theoretical basis for the study of the pharmacodynamic material basis of traditional Chinese medicine against SARS-CoV-2 and offered a lead compound for further structural modification in order to obtain more effective candidate drugs against SARS-CoV-2.