Identification of Aloe‐derived natural products as lead scaffolds for SARS‐CoV‐2 main protease (M^pro) 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 Mpro in 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.

The SARS‐CoV‐2 main protease (Mpro): Structure, function, and emerging therapies for COVID‐19

“…Further studies suggested that compound 382 was a reversible SARS‐CoV‐2 3CL pro inhibitor, while compound 415 might form a covalent bond with Cys145 of 3CL pro 232 . Similarly, aloesin was identified as a SARS‐CoV‐2 3CL pro inhibitor from a fluorescence resonance energy transfer (FRET)‐based THS assessment 233 …”

“…232 Similarly, aloesin was identified as a SARS-CoV-2 3CL pro inhibitor from a fluorescence resonance energy transfer (FRET)-based THS assessment. 233…”

A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors

“…Anacardic acid is the main component of extracts from Ginkgo biloba and Rhus verniciflua , which has a wide range of pharmacological activities such as antibacterial, anticancer, and antiviral ( Hundt et al, 2015 ; Park et al, 2018 ; Saedtler et al, 2020 ). Recent studies have confirmed that the EC 50 value of antiviral activity of anacardic acid is 9 μM in live SARS-CoV-2 virus by targeting Mpro ( Chen et al, 2021b ; Hicks et al, 2022 ; Li et al, 2022 ; Xiong et al, 2021 ; Yan et al, 2022b ). Our study demonstrated that anacardic acid is likely a novel competitive inhibitor targeting both PLpro and Mpro of SARS-CoV-2, and more in vivo experiments for its antiviral mechanism will be explored in the future.…”