University of South Florida Health-University of Arizona team reports compounds simultaneously targeting M^pro and cathepsin L may improve treatment of COVID-19 infection

Doctoral student Michael Sacco (sitting) with Yu Chen, PhD, associate professor of molecular medicine, in Dr. Chen’s laboratory at the USF Health Morsani College of Medicine | Photo by Allison Long, USF Health Communications

TAMPA, Fla (Nov. 6, 2020) — SARS-CoV-2, the respiratory virus that causes COVID-19, attacks the body in multiple steps. Gaining entry into cells deep within the lungs and hijacking the human host cell’s machinery to churn out copies of itself are two of the earliest steps — both essential for viral infection.

A new study offers insight into designing antiviral drugs against COVID-19 by showing that some existing compounds can inhibit both the main protease (M^pro), a key viral protein required for SARS-CoV-2 replication inside human cells, and the lysosomal protease cathepsin L, a human protein important for viral entry into host cells. The study, led by researchers at the University of South Florida Health (USF Health) Morsani College of Medicine and the University of Arizona College of Pharmacy, was published Nov. 6 in Science Advances.

“If we can develop compounds to shut down or significantly reduce both processes – viral entry and viral replication – such dual inhibition may enhance the potency of these compounds in treating the coronavirus infection,” said study co-principal investigator Yu Chen, PhD, a USF Health associate professor of molecular medicine with expertise in structure-based drug design. “Metaphorically, it’s like killing two birds with one stone.”

The USF Health-University of Arizona (UA) collaborators built upon their previous work, which identified and analyzed several promising, existing antiviral drugs as candidates to treat COVID-19.  All the candidates chosen to pursue target M^pro to block the replication of SARS-CoV-2 within human cells grown in the laboratory.

Two of the compounds, calpain inhibitors II and XII, did not show as much activity against M^pro as another drug candidate called GC-376 in biochemical tests. However, the calpain inhibitors, especially XII, actually worked better than GC-376 at killing SARS-CoV-2 in cell cultures, said lead author Michael Sacco, a doctoral student in Dr. Chen’s laboratory.

“We figured if these calpain inhibitors were less effective at inhibiting the virus’s main protease, they must be doing something else to explain their antiviral activity,” Sacco said. They learned from research done by other groups, including collaborator and study co-principal investigator Jun Wang, PhD, of UA, that calpain inhibitors can block other proteases, including cathepsin L, a critical human host protease involved in mediating SARS-CoV-2 entry into cells.

X-ray crystal structures of the SARS-CoV-2 protein Mpro interacting with calpain inhibitors II (depicted above in orange) and XII (below in blue). Calpain inhibitor XII adopts an atypical inverted binding pose. | Images courtesy of Michael Sacco, USF Health.

In this latest study, the USF Health researchers used advanced techniques, particularly X-ray crystallography, to visualize how calpain inhibitors II and XII interacted with viral protein M^pro. They observed that the calpain II inhibitor fit as expected into the targeted binding sites on the surface of the SARS-CoV-2 main protease. Unexpectedly, they also discovered that the calpain XII inhibitor adopted a unique configuration – referred to as “an inverted binding pose” — to tightly fit into M^pro active binding sites. (A snug fit optimizes the inhibitor’s interaction with the targeted viral protein, decreasing the enzyme activity that helps SARS-CoV-2 proliferate.)

“Our findings provide useful structural information on how we can design better inhibitors to target this key viral protein in the future,” Dr. Chen said.

Besides the increased potency (desired drug effect at a lower dose) of targeting both viral protease M^pro and human protease cathepsin L, another benefit of dual inhibitors is their potential to suppress drug resistance, Dr. Chen said.

SARS-CoV-2 can mutate, or change, its targeted genetic sequence. These viral mutations trick the human cell into allowing the virus to attach to the cell’s surface membrane and insert its genetic material and can alter the shape of viral proteins and how they interact with other molecules (including inhibitors) inside the cell.

Dr. Chen, the study’s co-principal investigator, is applying his expertise in structure-based drug design to look for new ways to stop COVID-19 viral infection. He collaborates with researchers at the University of Arizona College of Pharmacy. | Allison Long, USF Health Communications

When the virus mutates so it can continue reproducing, it can become resistant to a particular inhibitor, reducing that compound’s effectiveness. In other words, if the genetic sequence of the viral target (lock) changes, the key (inhibitor) no longer fits that specific lock. But let’s say the same key can open two locks to help prevent COVID-19 infection; in this case the two locks are M^pro, the viral target protein, and cathepsin L, the human target protein.

“It’s harder for the virus to change both locks (two drug targets) at the same time,” Dr. Chen said. “So a dual inhibitor makes it more difficult for antiviral drug resistance to develop, because even if the viral protein changes, this type of compound remains effective against the human host protein that has not changed.”

The USF Health-University of Arizona research team continues to fine-tune existing antiviral drug candidates to improve their stability and performance, and hopes to apply what they’ve learned to help design new COVID-19 drugs. Their next steps will include solving how calpain inhibitors interact chemically and structurally with cathepsin L.

Jun Wang, PhD, UA associate professor of pharmacology and toxicology, was the corresponding author for the Science Advances paper, along with Dr. Chen as co-corresponding author. The collaborative work was supported in part by grants from the National Institutes of Health.

Michael Sacco, lead author of the Science Advances paper, is a doctoral student in the USF Health Department of Molecular Medicine. | Allison Long, USF Health Communications

A University of Arizona-University of South Florida team  identified and analyzed four promising antiviral drug candidates in the preclinical study

TAMPA, Fla. (July 6, 2020) — 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.

Yu Chen, PhD, an associate professor of molecular medicine with expertise in structure-based drug design, has turned toward looking for new or existing drugs to stop SARS-CoV-2.

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 (M^pro), an enzyme that cuts out proteins from a long strand that the virus produces when it invades a human cell. Without M^pro, 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.

Using 3D computer modeling, Michael Sacco (left), a doctoral student in the Department of Molecular Medicine, worked with Dr. Chen to determine the interactions between antiviral drug candidate GC-376 and COVID-19’s main protease.

M^pro 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 M^pro 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 M^pro, 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 M^pro 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 M^pro inhibitors previously identified as suitable to clinically evaluate for treating SARS-CoV-2, Dr. Chen said.

Michael Sacco looks at COVID-19 viral protein crystals under a microscope.

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 M^pro 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 M^pro 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.

Two viral protein images generated by Yu Chen, University of South Florida Health, using X-ray crystallography. Above: The protein dimer (one molecule is blue and the other orange) shows the overall structure of the COVID-19 virus’s main protease (M^pro), the researchers’ drug target. Below: Three configurations of active sites where inhibitor GC-376 binds with the M^pro viral enzyme, as depicted by 3D computer modeling.

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.

-Photos by Torie Doll, USF Health Communications and Marketing

Yu Chen, PhD, an associate professor in the Morsani College of Medicine’s Department of Molecular Medicine, was among five USF faculty members who recently received the university’s Excellence in Innovation Awards for their exceptional research and innovation.

Each winner received a $2,000 award and plaque presented at the annual luncheon of the USF Chapter of the National Academy of Inventors on Aug. 31.

Yu Chen, PhD

The award recognizes Dr. Chen for his patented technology of novel beta-lactamase inhibitors licensed by Gordian Biotechnologies to tackle the growing problem of antibiotic resistance, for the development of collaborations with Achaogen Inc., and for his publications last year in the Journal of Medicinal Chemistry and Future Medicinal Chemistry.

Dr. Chen’s structure-based drug design approach has led to the development of novel small molecule inhibitors against multiple proteins involved in antibiotic resistance, metastatic cancer and Alzheimer’s disease. Using an interdisciplinary approach, he combines both computational and experimental techniques to investigate the function and inhibition of enzymes related to bacterial cell wall synthesis, the biological process targeted by antibiotics such as penicillin.

Dr. Chen has extensive experience in biochemistry, X-ray crystallography and molecular docking. He has characterized the catalytic mechanisms of three enzymes and determined about 40 crystal structures including protein complexes with DNA or small molecules.

The crystal of a protein used to help design better beta-lactamase inhibitors.