Identification of Aloe‐derived natural products as lead scaffolds for SARS‐CoV‐2 main protease (Mpro) inhibitors

ABSTRACT

In the past two years, the COVID‐19 pandemic has caused over 5 million deaths and 250 million infections worldwide. Despite successful vaccination efforts and emergency approval of small molecule therapies, diverse antivirals are still needed to combat the inevitable viral resistance that will arise. The main protease of SARS‐CoV‐2 (Mpro) is an attractive drug target due to the clinical success of protease inhibitors against other viruses, such as HIV and HCV. However, in order to combat resistance, diverse chemical scaffolds need to be identified that have the potential to be developed into potent inhibitors. To this end, we screened a high‐content protease inhibitor library against Mproin vitro, in order to identify structurally diverse compounds that may be further developed into antiviral leads. Our high‐content screening efforts retrieved 27 hits each with >50% inhibition in our Mpro FRET assay. Of these, three natural product compounds, anacardic acid, aloesin, and aloeresin D, were chosen for follow up IC50 screening due to their strong druglike properties (high bioavailability, low toxicity). Further analysis via dose response curves revealed IC50 values of 6.8 μM, 38.9 μM, and 125.3 μM respectively. Molecular docking studies demonstrated that the three inhibitors bound at the catalytic active site of Mpro with varying binding energies (‐7.8 to ‐5.6 kcal/mol). Furthermore, we determined that our kinetic data from the Mpro FRET assay fit better to a sigmoidal Hill model than the standard Michaelis‐Menten hyperbola, indicating substantial cooperativity between enzyme monomers, suggesting that the dimerization interface could be an attractive target for allosteric inhibitors. In conclusion, we identified two natural product compounds from the Aloe plant (aloesin and aloeresin D) as novel scaffolds for Mpro inhibitor design and confirmed the cooperative kinetics of Mpro. These results advance our knowledge of structure‐function relationships in Mpro and offer new molecular scaffolds for inhibitor design.