Small, But Mighty

In recent months, my research group has been involved in a large collaborative effort with the labs of David Ho, Alex Chavez, Tom Rovis, Farhad Forouhar, Brandon Fowler, John Hunt, Chuck Karan, and Waters. Together, we are aiming to develop small molecule inhibitors targeting the main protease of SARS-CoV-2. My lab normally focuses on the molecular mechanisms involved in cell death – specifically ferroptosis, which my lab discovered in 2012 – but we also conduct broader work related to drug discovery for cancer and degenerative diseases. When the pandemic hit, our focus turned to coming up with a viable therapeutic option for this enigmatic new virus.

While the media focus in on vaccine development, the most effective treatments for other devastating viral infections has historically been small-molecule protease inhibitors. For example, despite a massive effort over many years, there’s still no vaccine for HIV. What turned the virus from a death sentence into a treatable disease? Protease inhibitors. We decided to explore a similar approach for SARS-CoV-2.

After infecting a cell, SARS-CoV-2 genomic RNA is translated into proteins. Two large polyproteins are key to the virus’ lifecycle, but these massive polyproteins must be cleaved at more than 11 different sites in order to function as individual proteins. Inhibit this cleavage process, and you can block viral replication. Previous research on the original SARS-CoV virus told us that there are two proteases that do this cleaving. One of these, the main 3CL protease, has a substrate binding pocket that is similar across 12 different coronaviruses – a promising target for our research.

We’ve been applying chromatography and MS techniques to understand small-molecule inhibitor binding sites in the protease active site. As part of this effort, we’ve employed some creative digestion and modification experiments in conjunction with native size-exclusion chromatography (SEC) and reverse-phase separations.

These chromatography methods enable us to gather rich information on the binding of small-molecule candidates to the viral protease, while MS detection means we can determine where on the protease the small molecule has bound irreversibly. For example, using MALDI and LC-MS, we were able to observe the formation of a 1:1 complex when compound 18 (one of our most promising candidates) was mixed with the 3CL protease – confirmation that it is binding and inhibiting selectively as we’d hoped. Isothermal titration calorimetry has also added to the information we can gain from these MS analyses: by determining thermodynamic parameters, we can understand the specificity and energetics driving the interaction between drug candidate and protease.

The data gathered thus far have been incredibly useful in prioritizing candidates—time will tell if any of these have the potential to become a breakthrough medicine. After characterizing a number of different interactions, the compound GC376 seemed particularly promising. However, we still need to comprehensively evaluate ADME (absorption, distribution, metabolism, and excretion) properties of these compounds and their analogs.

The next hurdle will be getting an animal model set up for testing the optimized compounds. Mouse models for SARS-CoV-2 are only just emerging, and we’re setting up collaborations with other groups to test the first candidate in this way. We hope to do this by the end of summer.

The analytical community can make a big difference in the fight against COVID-19, in testing and beyond. Whether that difference will manifest itself in the form of small molecule inhibitors, viral proteins or biologics, we are yet to see. Despite the billions beings invested, we know that safe, long-lasting and effective vaccines are in many cases not feasible. While most of the world’s attention and funding is still focused on vaccine development, some of us predict that small-molecule inhibitors will ultimately be the breakthrough that brings back some degree of pre-pandemic normalcy to the world. In this sense, protease inhibitors for COVID-19 may perform the same task as penicillin in 1928 – turning an otherwise lethal infection into an inconvenience.