There are 4 effective drug to target the SARS-CoV-2 main protease (Mpro)

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As the death toll from the COVID-19 pandemic mounts, scientists worldwide continue their push to develop effective treatments and a vaccine for the highly contagious respiratory virus.

University of South Florida Health (USF Health) Morsani College of Medicine scientists recently worked with colleagues at the University of Arizona College of Pharmacy to identify several existing compounds that block replication of the COVID-19 virus (SARS-CoV-2) within human cells grown in the laboratory.

The inhibitors all demonstrated potent chemical and structural interactions with a viral protein critical to the virus’s ability to proliferate.

The research team’s drug discovery study appeared June 15 in Cell Research, a high-impact Nature journal.

The most promising drug candidates – including the FDA-approved hepatitis C medication boceprevir and an investigational veterinary antiviral drug known as GC-376 – target the SARS-CoV-2 main protease (Mpro), an enzyme that cuts out proteins from a long strand that the virus produces when it invades a human cell.

Without Mpro, the virus cannot replicate and infect new cells. This enzyme had already been validated as an antiviral drug target for the original SARS and MERS, both genetically similar to SARS-CoV-2.

“With a rapidly emerging infectious disease like COVID-19, we don’t have time to develop new antiviral drugs from scratch,” said Yu Chen, PhD, USF Health associate professor of molecular medicine and a coauthor of the Cell Research paper. “A lot of good drug candidates are already out there as a starting point.

But, with new information from studies like ours and current technology, we can help design even better (repurposed) drugs much faster.”

Before the pandemic, Dr. Chen applied his expertise in structure-based drug design to help develop inhibitors (drug compounds) that target bacterial enzymes causing resistance to certain commonly prescribed antibiotics such as penicillin.

Now his laboratory focuses its advanced techniques, including X-ray crystallography and molecular docking, on looking for ways to stop SARS-CoV-2.

Mpro represents an attractive target for drug development against COVID-19 because of the enzyme’s essential role in the life cycle of the coronavirus and the absence of a similar protease in humans, Dr. Chen said.

Since people do not have the enzyme, drugs targeting this protein are less likely to cause side effects, he explained.

The four leading drug candidates identified by the University of Arizona-USF Health team as the best (most potent and specific) for fighting COVID-19 are described below. These inhibitors rose to the top after screening more than 50 existing protease compounds for potential repurposing:

  • Boceprevir, a drug to treat Hepatitis C, is the only one of the four compounds already approved by the FDA. Its effective dose, safety profile, formulation and how the body processes the drug (pharmacokinetics) are already known, which would greatly speed up the steps needed to get boceprevir to clinical trials for COVID-19, Dr. Chen said.
  • GC-376, an investigational veterinary drug for a deadly strain of coronavirus in cats, which causes feline infectious peritonitis. This agent was the most potent inhibitor of the Mpro enzyme in biochemical tests, Dr. Chen said, but before human trials could begin it would need to be tested in animal models of SARS-CoV-2. Dr. Chen and his doctoral student Michael Sacco determined the X-ray crystal structure of GC-376 bound by Mpro, and characterized molecular interactions between the compound and viral enzyme using 3D computer modeling.
  • Calpain inhibitors II and XII, cysteine inhibitors investigated in the past for cancer, neurodegenerative diseases and other conditions, also showed strong antiviral activity. Their ability to dually inhibit both Mpro and calpain/cathepsin protease suggests these compounds may include the added benefit of suppressing drug resistance, the researchers report.

All four compounds were superior to other Mpro inhibitors previously identified as suitable to clinically evaluate for treating SARS-CoV-2, Dr. Chen said.

A promising drug candidate – one that kills or impairs the virus without destroying healthy cells – fits snugly, into the unique shape of viral protein receptor’s “binding pocket.” GC-376 worked particularly well at conforming to (complementing) the shape of targeted Mpro enzyme binding sites, Dr. Chen said.

Using a lock (binding pocket, or receptor) and key (drug) analogy, “GC-376 was by far the key with the best, or tightest, fit,” he added. “Our modeling shows how the inhibitor can mimic the original peptide substrate when it binds to the active site on the surface of the SARS-CoV-2 main protease.”

Instead of promoting the activity of viral enzyme, like the substrate normally does, the inhibitor significantly decreases the activity of the enzyme that helps SARS-CoV-2 make copies of itself.

Visualizing 3-D interactions between the antiviral compounds and the viral protein provides a clearer understanding of how the Mpro complex works and, in the long-term, can lead to the design of new COVID-19 drugs, Dr. Chen said.

In the meantime, he added, researchers focus on getting targeted antiviral treatments to the frontlines more quickly by tweaking existing coronavirus drug candidates to improve their stability and performance.

Dr. Chen worked with lead investigator Jun Wang, PhD, UA assistant professor of pharmacology and toxicology, on the study. The work was supported in part by grants from the National Institutes of Health.


Coronaviruses, a large family of viruses, causing upper-respiratory-tract infections in humans and other higher mammals (Bedford et al., 2020). Coronavirus outbreak has been reported three times in the 21st century namely SARS in 2002, MERS in 2012, and COVID-19 in November 2019.

The recent outbreak of COVID-19 caused by SARS-CoV-2 has emerged from China. Human-human transmission of this highly infectious zoonotic virus has led to exponential growth in the number of infected cases, which resulted in its pandemic outbreak worldwide (Fahmi, 2020).

Currently, more than 225 countries are affected due to COVID-19 with more than 2.5 million people to date which is increasing in thousands per day around the world and approximately 5 to 10% mortality rate (Fahmi, 2020).

Additionally, the lives of millions of people have been impacted due to mandatory lockdowns, isolations, and quarantines. Thus the severe effect of the COVID-19 outbreak has imposed major challenges for global health, society and economy (Fry, 2020). Currently, there are no specific antiviral drugs or vaccines available for the treatment and management of COVID-19, which is further making the situation difficult to handle.

At present preventive and supportive therapies are being implemented to prevent complications (Salata et al., 2020). Several efforts are going on to target key proteins of SARS-CoV-2 pathogenesis like the main protease (MPro), RNA-dependent RNA polymerase (RdRp), RNA binding N terminal domain (NTD) of Nucleocapsid protein (N protein), viral ion channel (E protein), 2′-O-RiboseMethyltransferase and human Angiotensin-converting-enzyme 2 receptor (hACE-2) (Gao et al., 2020; Gupta et al., 2020; Khan et al., 2020; Sarma et al., 2020; Shang et al., 2020; Zhang et al., 2020).

To propose or repurpose drug and/or lead molecules against SARS-CoV-2, it would be effective to target multiple virus pathogenesis specific proteins within a close network of interaction or having dependent functionality (Zhou et al., 2020).

For drug discovery purposes, the MPro of coronaviruses has been studied extensively. They are papain-like proteases involved in the self-maturation and processing of viral replicase enzymes (Zhang et al., 2020). MPro has 11 putative cleavage sites in 790 kD replicase lab indicating its predominance in the proteolytic processing of large polyprotein lab.

MPro from SARS-CoV-2 showed high sequence identity and structural similarity to that of the SARS-CoV MPro. It contains two catalytic domains, I (chymotrypsin) and II (picornavirus 3 C protease-like) each containing six-stranded antiparallel β-barrel containing active diad H41 and C145. Since they have a key role in virus replication, these proteases have emerged as important drug targets.

Furthermore, with their very low similarity with human proteases, inhibitors of MPro are found to be very less cytotoxic (Anand et al., 2003; Zhang et al., 2020). Preliminary studies have suggested the potential use of protease inhibitor lopinavir/ritonavir, commonly used drugs for human immunodeficiency virus (HIV), for the treatment of COVID-19 patients (Cao et al., 2020; Muralidharan et al., 2020).

Unfortunately, in the open-label randomized clinical study, these drugs are not found to be that impressive for COVID-19 treatment. Additionally, several other viral protease inhibitors like HCV Protease Inhibitor Danoprevir, HIV protease inhibitor Darunavir are under in vivo and clinical studies for the treatment of SARS-CoV-2 infection (Dong et al., 2020).

To find new or repositioning existing drug molecules, the understanding of sequence and structure of SARS-CoV- MPro in regards to various other coronavirus strains could be important. Their comparative understanding is enigmatic, which could be important to repurpose available antiviral protease inhibitors against SARS-CoV-2 MPro.

Coronavirus uses a multiprotein complex to replicate its RNA based genomes. Cleavage of viral polyproteins (ORF1a and ORF1b) produces a set of non-structural proteins (Nsp). Of these, RdRp or Nsp12 catalyzes the synthesis and is known to play an important role in the replication and transcription cycle of the virus. RdRp is a primary target for nucleotide analog antiviral inhibitors such as Remdesivir, a drug under evaluation for SARS-CoV2 infection in clinics (Gao et al., 2020; Imbert et al., 2006).

Remdesivir, a nucleotide analog, has shown broad-spectrum antiviral activities, and preclinical studies have also shown promising human safety data (Amirian & Levy, 2020; Dong et al., 2020). Recently, a report suggested that the significant inhibition of RdRp of SARS-CoV-2 by Remdesivir, and clinical studies for the same are ongoing to evaluate the efficacy of this molecule in the COVID-19 patients (www.clinicaltrials.gov; Study No: NCT04280705).

Apart from the maturation and replication, the entry of this virus by binding its surface spike protein to the hACE-2 is also a crucial process that can be targeted (Hasan et al., 2020; Kuhn et al., 2004; Shang et al., 2020).

Since this host cell receptor is essential for the virus entry; targeting hACE-2 has a promise for preventing SARS-Cov-2 infection. It has been shown that clinically approved serine protease inhibitor (TMPRSS2) can also bind to the SARS-CoV-2 receptor hACE-2 and inhibit viral entry (Hoffmann et al., 2020).

To date, the Chinese Clinical Trial Registry has recorded around 550 trials against SARS-CoV-2, mostly evaluating existing drug molecules. These trials include the application of antiviral (favipiravir, adalimumab, dihydroartemisininpiperaquine, leflunomide, lopinavir), antimalarial drugs (chloroquine or hydroxychloroquine), high‑dose vitamin C, etc (www.chictr.org.cn/index.aspx).

From the available data of experimental and clinical studies, it has been found that molecules targeting single protein will be ineffective antiviral lead. Thus, to devise an effective strategy, there is a need for molecules that can target multiple key proteins such as RdRp and hACE-2 along with the MPro as a major drug target (Wu et al., 2020).

In silico screening of the drugs is a very vital and useful tool for rapid screening. This enables to shortlist leads and thus meet the urgent demand for repurposing drugs for the treatment of SARS-CoV-2 infection.

Therefore, in this study, we have performed a comparative analysis of the major target of SARS-CoV-2, MPro for its structural characteristics to other viruses from the Coronoviridea family.

This information was further used for the virtual screening of the custom-made library of phytochemicals, active ingredients present in the commonly used ayurvedic anti-tussive medicines in India, and the synthetic anti-viral drugs against MPro. Top hit molecules from this screen were then docked against SARS-CoV-2 RdRP and hACE2 to find the multi-target-directed molecules.

We believe that this study will provide a new approach and a base for the discovery of multi-targeted antiviral molecules against SARS-CoV-2 pathogenesis. Thus these compounds could be potential candidates for in vitro and in vivo anti-viral studies followed by clinical treatment of SARS-CoV-2.

Anti-tussive molecules like δ-viniferin and myricitrin showed higher affinity for the active site of SARS-CoV-2 MPro and other targets

Screening of the custom made the library of ∼7100 molecules comprising of different types such as flavonoids, glucosinolates, anti-tussive, anti-influenza, anti-viral, terpenes, terpenoids, alkaloids and other know and/or predicated anti-SARS-CoV-2 molecules was done against SARS-CoV-2 MPro as the main target (Supplementary Data 2).

Sequence similarity search of MPro against human protein sequences was performed, which suggests that the viral proteases do not have any homolog in the human, thus targeting MPro will be specific to viral proteases only.

Docking analysis results demonstrated that several phytochemicals and anti-viral have strong binding in the active site of SARS-CoV-2 MPro. The selected molecules also displayed a high binding affinity to SARS-CoV-2 RdRp and hACE-2 (Supplementary Data 3). Likewise MPro, other viral targets considered in this study, RdRp, also showed that there is no human homolog present, thus could be a potential viral target with MPro.

Top molecules with the highest binding energy and with acceptable absorption, distribution, metabolism, and excretion (ADME) criteria were further selected for interaction analysis (Figure 4).

To validate our docking strategy and obtained outcomes we have used a set of known or predicted anti-SARS-CoV-2 molecules reported from the literature (Wu et al., 2020). For further interaction analysis, we have selected the molecules with a high binding score against all the targets. Also, we have taken into account of earlier reports that support the antiviral properties of these molecules against different viruses.

Figure 4. Heatmap of binding energies of top hits for different types of molecules used for screening, namely flavonoids, glucosinolates, anti-tussive, anti-influenza, synthetic anti-viral, terpenes, terpenoids and alkaloids.

In general, we noticed strong binding of ligands towards MPro and RdRp as compared to the hACE 2 receptor. Flavonoids showed promising results with better binding affinities than existing synthetic anti-viral drugs. For details of the docking score and names of the ligands check Supplementary Data 3.

Figure
Figure 4. Heatmap of binding energies of top hits for different types of molecules used for screening, namely flavonoids, glucosinolates, anti-tussive, anti-influenza, synthetic anti-viral, terpenes, terpenoids and alkaloids. In general, we noticed strong binding of ligands towards MPro and RdRp as compared to the hACE 2 receptor. Flavonoids showed promising results with better binding affinities than existing synthetic anti-viral drugs. For details of the docking score and names of the ligands check Supplementary Data 3.

As one of the symptoms of COVID-19 is cough, a library of ayurvedic anti-tussive molecules in Indian medicine (82 molecules) are screened against SARS-CoV-2 MPro. The resulting top 20 molecules were further screened against other targets- RdRp and hACE 2 receptors. Amongst these molecules, δ-viniferin, chrysanthemin, myritilin and myricitrin showed strong binding with SARS-CoV-2 MPro.

The δ-viniferin and myricitrin showed high solubility and bioavailability in computational ADME analysis; thus, analyzed for intermolecular interaction. With SARS-CoV-2 MPro, δ-viniferin exhibited binding energy of -8.4 Kcal/mol with several non-covalent interactions in the active site and substrate-binding site.

THR24 and HIS163 form polar contact with hydroxyl groups of δ-viniferin, while active site residue HIS41 shows Pi-Pi interaction with the central aromatic scaffold of a ligand. Apart from this, MET165 is involved in Pi-sulfur interaction with ligand and other residues from the substrate-binding pocket, which are involved in either attractive van der Waal interaction or carbon-hydrogen bond (Figure 5A).

In addition to MPro, δ-viniferin appeared to have a strong binding with the SARS-CoV-2 RdRp and hACE2 receptor with a score of -8.3 and -8.4 Kcal/mol, respectively. In the case of RdRp, binding pocket residues ALA762, TRP800, SER814 showed hydrogen bonds with the hydroxyl group of the ligand. While, active site and other key residues namely ARG553, ASP618, ASP623 and ASP761 display Pi interactions with the aromatic scaffolds of δ-viniferin.

Figure 5. δ-Viniferin with active site residues of (A) MPro (B) RdRp and (C) hACE-2. It is followed by interaction map of myricitrin with (D) MPro (E) RdRp and (F) hACE-2. The interaction type is distinguished by coloured circles (residues).

Dashed lines direct to the specific moiety in the ligand. Green residues symbolize van der Waals forces. Pink residues indicate those are involved in Pi-Pi stacking. Light pink indicates alkyl group interactions. Light orange colour indicates Pi-sulphur interactions.

Dark orange show pi-anion interactions. Aromatic rings are involved in Pi-Pi, Pi-anion and Pi-sulphur interactions. Intermolecular interaction between Taiwanhomoflavone A with active site residues of (G) MPro, (H) RdRp and (I) hACE-2. Next is the interaction map of Lactucopicrin 15-oxalate with (J) MPro, (K) RdRp and (L) hACE-2.

Figure
Figure 5. δ-Viniferin with active site residues of (A) MPro (B) RdRp and (C) hACE-2. It is followed by interaction map of myricitrin with (D) MPro (E) RdRp and (F) hACE-2. The interaction type is distinguished by coloured circles (residues). Dashed lines direct to the specific moiety in the ligand. Green residues symbolize van der Waals forces. Pink residues indicate those are involved in Pi-Pi stacking. Light pink indicates alkyl group interactions. Light orange colour indicates Pi-sulphur interactions. Dark orange show pi-anion interactions. Aromatic rings are involved in Pi-Pi, Pi-anion and Pi-sulphur interactions. Intermolecular interaction between Taiwanhomoflavone A with active site residues of (G) MPro, (H) RdRp and (I) hACE-2. Next is the interaction map of Lactucopicrin 15-oxalate with (J) MPro, (K) RdRp and (L) hACE-2.

The high density of these intermolecular non-covalent interactions is an indicator of strong and specific binding of δ-viniferin with RdRp active pocket. Furthermore, hydroxyl and aromatic groups of δ-viniferin also interacts through polar contacts and Pi-Alkyl interaction with the GLU35, LEU39, LYS68 and ALA71 residues of hACE-2 (Figure 5B and 6C).

The interaction of δ-viniferin with MPro, RdRp and hACE-2 suggests its high potential as a multi-target directed ligand against SARS-CoV-2. δ-Viniferin is a resveratrol dimer, a major stilbene produced by stressed grapevine leaves (Aresta et al., 2003). It is also one of the major stilbenes present along with resveratrol in the red wine (Vitrac et al., 2005).

Previous studies have also suggested that δ-viniferin shows potent antiviral activity against a variety of viruses viz. rotavirus (B. Yu et al., 2018), Human Immunodeficiency Virus (HIV) (Pflieger et al., 2013); hepatitis C virus (HCV) (Lee et al., 2019).

Figure
Figure 6. Schematic representing multi-target-directed drug ligands against SARS-CoV-2 infection.

Another anti-tussive molecule, myricitrin, found to have a strong binding with the active site residues of the MPro with a binding score of -8.9 Kcal/mol. Active site residues HIS41 and CYS145 form Pi-Alkyl and Pi-Sulfur interactions with the aromatic scaffold of ligand, respectively.

TYR54, PHE140, GLY143, HIS163 and GLU166 are involved in the formation of 6 hydrogen bonds with hydroxyl and carboxyl oxygen of ligand. Apart from this, there are other additional interactions like van der Waals forces that stabilize the binding of the ligand to the enzyme (Figure 5D).

Myricitrin exhibited equally strong interaction with RdRp and hACE-2 with a binding score of -7.9 and -7.5 Kcal/mol, respectively. For RdRp, the carboxyl group of myricitrin forms two hydrogen bonds with ARG553, ARG555 and THR556.

While the active site residue ASP623 is having Pi-Anion interaction with the aromatic rings of the ligand. Similarly, this ligand binds to hACE-2 by forming polar interaction with ALA348, PHE390, ARG393 and ASN394.

This interaction is then further stabilized by Pi-stacking of PHE40 and Pi-anion interactions of ASP350, ASP382 with aromatic rings of the ligand (Figure 5E and F).

Myricitrin is a glycosylated analog of myricetin, present in the Myrica esculenta. Although in our docking study, myricetin has shown relatively lesser binding affinity when compared to myricitrin, previous studies have shown that it possesses potent antiviral activity.

Myricetin inhibited VP35 protein with double-stranded RNA interaction in Ebola virus (Daino et al., 2018). The glycosylated myricetin, i.e. myricitrin has been found to inhibit HIV (Ortega et al., 2017), herpes simplex virus (Li et al., 2020), SARS coronavirus (Keum & Jeong, 2012; Yu et al., 2012).

Together, our results suggest that δ-viniferin and myricitrin could be potent molecules for the mitigation of SARS-CoV-2. Interestingly, top anti-tussive molecules in the present study mainly δ-viniferin, chrysanthemin, myritilin and myricitrin are present in the extract of black grapes, which is one of the key ingredients of ayurvedic antitussive medicines, and energy and immune booster, Chyawanprash (Georgiev et al., 2014).

Thus, these medicines could be helpful in the management and mitigation of COVID-19. Particularly, the herbal formulation and beverages containing Vitis vinifera might be helpful in the management of COVID-19.


More information: Chunlong Ma et al, Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease, Cell Research (2020). DOI: 10.1038/s41422-020-0356-z

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