Identification of Aloe-derived natural products as prospective lead scaffolds for SARS-CoV-2 main protease (M(pro)) inhibitors

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, a diverse range of antivirals is still needed to combat the inevitable resistance that will arise from new SARS-CoV-2 variants. The main protease of SARS-CoV-2 (M(pro)) 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, various 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 M(pro)in vitro, in order to identify structurally diverse compounds that could be further developed into antiviral leads. Our high-content screening efforts retrieved 27 hits each with > 50% inhibition in our M(pro) FRET assay. Of these, four of the top inhibitor compounds were chosen for follow-up due to their potency and drugability (Lipinski’s rules of five criteria): anacardic acid, aloesin, aloeresin D, and TCID. Further analysis via dose response curves revealed IC(50) values of 6.8 μM, 38.9 μM, 125.3 μM, and 138.0 μM for each compound, respectively. Molecular docking studies demonstrated that the four inhibitors bound at the catalytic active site of M(pro) with varying binding energies (-7.5 to −5.6 kcal/mol). Furthermore, M(pro) FRET assay kinetic studies demonstrated that M(pro) catalysis is better represented by a sigmoidal Hill model than the standard Michaelis-Menten hyperbola, indicating substantial cooperativity of the active enzyme dimer. This result suggests that the dimerization interface could be an attractive target for allosteric inhibitors. In conclusion, we identified two closely-related natural product compounds from the Aloe plant (aloesin and aloeresin D) that may serve as novel scaffolds for M(pro) inhibitor design and additionally confirmed the strongly cooperative kinetics of M(pro) proteolysis. These results further advance our knowledge of structure–function relationships in M(pro) and offer new molecular scaffolds for inhibitor design.