COVID-19: MG-101 drug hinder the virus’s ability to infect cells by inhibiting protease processing of the spike protein

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Several FDA-approved drugs—including for type 2 diabetes, hepatitis C and HIV – significantly reduce the ability of the Delta variant of SARS-CoV-2 to replicate in human cells, according to new research led by scientists at Penn State.

Specifically, the team found that these drugs inhibit certain viral enzymes, called proteases, that are essential for SARS-CoV-2 replication in infected human cells.

“The SARS-CoV-2 vaccines target the spike protein, but this protein is under strong selection pressure and, as we have seen with Omicron, can undergo significant mutations,” said Joyce Jose, assistant professor of biochemistry and molecular biology, Penn State. “There remains an urgent need for SARS-CoV-2 therapeutic agents that target parts of the virus other than the spike protein that are not as likely to evolve.”

Previous research has demonstrated that two SARS-CoV-2 enzymes – proteases including Mpro and PLpro – are promising targets for antiviral drug development. Pfizer’s COVID-19 therapy Paxlovid, for example, targets Mpro.

According to Jose, these enzymes are relatively stable; therefore, they are unlikely to develop drug-resistant mutations rapidly.

Katsuhiko Murakami, professor of biochemistry and molecular biology, Penn State, noted that these virus proteases, because of their capabilities to cleave, or cut, proteins, are essential for SARS-CoV-2 replication in infected cells.

“SARS-CoV-2 produces long proteins, called polyproteins, from its RNA genome that must be cleaved into individual proteins by these proteases in an ordered fashion leading to the formation of functional virus enzymes and proteins to start virus replication once it enters a cell,” Murakami explained. “If you inhibit one of these proteases, further spread of SARS-CoV-2 in the infected person could be stopped.”

The findings published today (Feb. 25) in the journal Communications Biology.

The team designed an assay to rapidly identify inhibitors of the Mpro and PLpro proteases in live human cells.

“Although other assays are available, we designed our novel assay so it could be conducted in live cells, which enabled us to simultaneously measure the toxicity of the inhibitors to human cells,” said Jose.

The researchers used their assay to test a library of 64 compounds – including inhibitors of HIV and hepatitis C proteases; cysteine proteases, which occur in certain protozoan parasites; and dipeptidyl peptidase, a human enzyme involved in type 2 diabetes—for their ability to inhibit Mpro or PLpro.

From the 64 compounds, the team identified eleven that affected Mpro activity and five that affected PLpro activity based on a cut-off of 50% reduction in protease activity with 90% cell viability.

Anoop Narayanan, associate research professor of biochemistry and molecular biology, monitored the activity of the compounds using live confocal microscopy.

“We designed the experiment so that if the compound was affecting the proteases, you would see fluorescence in certain areas of the cell,” said Narayanan.

Next, the team evaluated the antiviral activity of the 16 PLpro and Mpro inhibitors against SARS-CoV-2 viruses in live human cells in a BSL-3 facility, the Eva J. Pell ABSL-3 Laboratory for Advanced Biological Research at Penn State, and discovered that eight of them had dose-dependent antiviral activities against SARS-CoV-2.

Specifically, they found that Sitagliptin and Daclatasvir inhibit PLpro, and MG-101, Lycorine HCl and Nelfinavir mesylate inhibit Mpro. Of these, the team found that MG-101 also hindered the virus’s ability to infect cells by inhibiting protease processing of the spike protein.

“We found that when the cells were pretreated with the selected inhibitors, only MG-101 affected the virus’s entry into cells,” said Narayanan.

In addition, the researchers found that treating cells with a combination of Mpro and PLpro inhibitors had an additive antiviral effect, providing even greater inhibition of SARS-CoV-2 replication.

“In cell culture, we showed that if you combine Mpro and PLpro inhibitors, you have a stronger effect on the virus without increasing toxicity,” said Jose. “This combination inhibition is highly potent.”

To investigate the mechanism by which MG-101 inhibits the activity of Mpro protease, the scientists, including Manju Narwal, postdoctoral scholar in biochemistry and molecular biology, used X-ray crystallography to obtain a high-resolution structure of MG-101 in complex with Mpro.

“We were able to see how MG-101 was interacting with the active site of Mpro,” said Narwal. “This inhibitor mimics the polyprotein and binds in a similar manner to the protease, thereby blocking the protease from binding to and cutting the polyprotein, which is an essential step in the virus’s replication.”

Murakami added, “By understanding how the MG-101 compound binds to the active site, we can design new compounds that may be even more effective.”

Indeed, the team is in the process of designing new compounds based on the structures they determined by X-ray crystallography. They also plan to test the combination drugs that they already demonstrated to be effective in vitro in mice.

Although the scientists studied the Delta variant of SARS-CoV-2, they said the drugs will likely be effective against Omicron and future variants because they target parts of the virus that are unlikely to mutate significantly.

“The development of broad-spectrum antiviral drugs against a wide range of coronaviruses is the ultimate treatment strategy for circulating and emerging coronavirus infections,” said Jose. “Our research shows that repurposing certain FDA-approved drugs that demonstrate effectiveness at inhibiting the activities of Mpro and PLpro may be a useful strategy in the fight against SARS-CoV-2.”

Other authors on the paper include Sydney A. Majowicz, graduate student, and Shay A. Toner, undergraduate student, Penn State; Carmine Varricchio, postdoctoral research associate, and Andrea Brancale, professor of medicinal chemistry, Cardiff University; and Carlo Ballatore, professor of medicinal chemistry, University of California, San Diego.


Currently there are few antivirals and no vaccines available for SARS-CoV-2. As such, it is imperative to identify drug targets that could lead to effective antivirals. Guided by research of the similar coronaviruses, SARS-CoV and MERS-CoV, several viral proteins have been prioritized as SARS-CoV-2 antiviral drug targets: the spike protein, the RNA-dependent RNA polymerase (RdRp), the main protease (Mpro), and the papain-like protease (PLpro).1,2

The SARS-CoV-2 RdRp inhibitor remdesivir was granted emergency use authorization from FDA on May 1st 2020. Remdesivir has broad-spectrum antiviral activity against SARS-CoV, SARS-CoV-2, and MERS-CoV in cell culture.3–5 The antiviral efficacy was further confirmed in MERS-CoV infection mouse and rhesus macaque models.6,7 Additional RdRp inhibitors under investigation for SARS-CoV-2 include EIDD-2801, favipiravir (T-705), ribavirin, and galidesivir.8,9

The fusion inhibitor EK1C4, which was designed based on the H2 peptide in the S2 domain of the HCoV-OC43 spike protein, showed promising broad-spectrum antiviral activity against SARS-CoV-2, SARS-CoV, MERS-CoV, as well as human coronaviruses HCoV-229E, HCoV-NL63, and HCoV-OC43.10,11 Meanwhile, the Mpro has been extensively explored as a drug target for not only SARS-CoV-2, but also SARS-CoV, MERS-CoV, as well as enteroviruses, rhinoviruses, and noroviruses.12

Mpro is a viral encoded cysteine protease that has a unique preference for a glutamine residue at the P1 site in the substrate, which was recently confirmed for SARS-CoV-2 by substrate profiling.13 Consequently, the majority of designed Mpro inhibitors contain either 2-pyrrolidone or 2-piperidinone at the P1 site as a mimetic of the glutamine residue in the substrate.14

Examples include compounds N3, 13b, 11a, 11b, and our recently identified GC-376,15–18 all of which have potent enzymatic inhibition in biochemical assay and antiviral activity in cell culture. Their mechanism of action and mode of inhibition were revealed by the drug-bound X-ray crystal structures.15–18

Interestingly, our previous study discovered two un-conventional SARS-CoV-2 Mpro inhibitors, calpain inhibitors II and XII, that are structurally dissimilar to the traditional Mpro inhibitors, such as GC-376.15 Specifically, calpain inhibitors II and XII incorporate the hydrophobic methionine and norvaline side chains in the P1 position.

This discovery challenges the idea that a hydrophilic glutamine mimetic is required at the P1 position. Furthermore, calpain inhibitor II is a potent inhibitor of human protease cathepsin L, with a Ki of 50 nM.19 Cathepsin L plays an important role in SARS-CoV-2 viral entry by activating the viral spike protein,20,21,22 and has a relatively broad substrate preference at the P1 position on the substrate.23,24 Studies have indicated that cathepsin L inhibitors can block or significantly decrease virus entry.20,25

To dissect the mechanism of action of these two promising drug candidates, we solved the high-resolution X-ray crystal structures of Mpro with calpain inhibitors II and XII (Fig. 1). We found that calpain inhibitor II is bound to Mpro in the canonical, extended conformation, but calpain inhibitor XII adopted an unexpected binding mode, where it assumes an inverted, semi-helical conformation in which the P1’ pyridine ring is placed in the S1 pocket instead of the P1 norvaline sidechain, as one would expect.

The complex structures of calpain inhibitors II and XII, together with the structure-activity relationship studies of calpain inhibitors II/XII, revealed the S1 pocket of Mpro can accommodate both hydrophilic and hydrophobic substitutions, paving the way for the design of dual inhibitors that target both the viral Mpro and host cathepsin L. Finally, guided by the X-ray crystal structure of SARSA-CoV-2 Mpro with GC-376 (PDB: 6WTT),15 three analogs UAWJ246, UAWJ247, and UAWJ248 were designed to profile the sidechain preferences of the S1’, S2, S3 and S4 pockets. The X-ray crystal structures and activity profile presented herein offer valuable insights into the substrate promiscuity of Mpro, as well as the design of new SARS-CoV-2 Mpro inhibitors.

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Fig. 1.
X-ray crystal structures of SARS-CoV-2 in complex with calpain inhibitors II (PDB: 6XA4) and XII (PDB: 6XFN).
Hydrogen bonds are shown as red dashed lines. SARS-CoV-2 Mpro in complex with (a) calpain inhibitor II (orange) and (b) calpain inhibitor XII (blue). Unbiased Fo-Fc electron density map, shown in grey, is contoured at 2 σ. c Comparison of calpain inhibitor XII (blue) and SARS-CoV-2 Mpro α-ketoamide inhibitor 13b (PDB ID 6Y2F) shown in purple. d Close-up comparison of calpain inhibitor XII (blue) and 13b (purple) in the S1 and S1’ sites. The P1 pyrrolidinone ring and P1’ benzene of 13b occupy the S1 and S1’ sites respectively. Conversely, the P1 norvaline and P1’ pyridine of calpain inhibitor XII adopt the S1’ and S1 sites. e Calpain inhibitor XII hydrogen bonding network in the catalytic core, accompanying a stereochemical inversion of the thiohemiketal adduct with Cys145, assuming the (R) configuration. f Hydrogen bonding network of 13b in the catalytic core. Like all other α-ketoamide inhibitors, the covalent adduct adopts an (S) conformation.

reference link: https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC7402059/


More information: Communications BiologyDOI: 10.1038/s42003-022-03090-9

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