Study Offers Insight into Designing COVID-19 Drugs to Dually Inhibit SARS-CoV-2 Entry and Replication

Phoenix, AZ, USA) showed that the compounds can inhibit both the main protease (Mpro), 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 researchers 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 Mpro 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 Mpro 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. Calpain inhibitors can block other proteases, including cathepsin L, a critical human host protease involved in mediating SARS-CoV-2 entry into cells.

In this latest study, the researchers used advanced techniques, particularly X-ray crystallography, to visualize how calpain inhibitors II and XII interacted with viral protein Mpro. 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 Mpro 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. Besides the increased potency (desired drug effect at a lower dose) of targeting both viral protease Mpro and human protease cathepsin L, another benefit of dual inhibitors is their potential to suppress drug resistance.

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. 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 us say the same key can open two locks to help prevent COVID-19 infection; in this case the two locks are Mpro, the viral target protein, and cathepsin L, the human target protein. The researchers continue to fine-tune existing antiviral drug candidates to improve their stability and performance, and hope to apply what they have learned to help design new COVID-19 drugs. Their next steps will include solving how calpain inhibitors interact chemically and structurally with cathepsin L.

“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.”

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