c Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai, 201203, China.
*Corresponding author:
E-mail address: geguangbo@shutcm.edu.cn (G.-B. Ge)
Phone: +86-024-51323184
**Corresponding author:
E-mail address: xyzhuang59@foxmail.com (X.-Y. Z)
Abstract
The main proteases (Mpro) are highly conserved cysteine-rich proteins that can be covalently modified by numerous natural and synthetic compounds. Herein, we constructed an integrative approach to efficiently discover covalent inhibitors of Mpro from complex herbal matrices. This work begins with biological screening of sixty clinically used antiviral herbal medicines, among which Lonicera japonica (LJ) demonstrated the strongest anti-Mpro effect (IC50 = 37.82 μg/mL). Mass spectrometry-based chemical analysis and chemoproteomic profiling revealed that LJ extract contains at least 50 constituents, of which 22 exhibited the capability to covalently modify Mpro. We subsequently verified the anti-Mpro effects of these covalent binders. Gallic acid and quercetin were found to potently inhibit SARS-CoV-2 Mpro in dose- and time- dependent manners, with the IC50 values below 10 µM. The inactivation kinetics, binding affinity and binding mode of gallic acid and quercetin were further characterized by fluorescence resonance energy transfer, surface plasmon resonance, and covalent docking simulations. Overall, this study established a practical approach for efficiently discovering the covalent inhibitors of Mpro from herbal medicines by integrating target-based high-throughput screening and mass spectrometry-based assays, which would greatly facilitate the discovery of key anti-viral constituents from medicinal plants.
Keywords: SARS-CoV-2 Mpro, Lonicera japonica (LJ), anti-viral agents, covalent inhibitors, inhibitory mechanism
1. Introduction
The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has a significant impact on global health and the economy1. Emerging evidence highlights the potential value of herbal medicine in the treatment of viral infections including COVID-19, due to their potentials to block viral replication and relieve virus-induced systemic inflammation. For instance,Lonicera japonica, Pomegranate peel, and Bergenia ligulata have been shown to be capable of impede influenza virus replication2-4,Isatis tinctoria, Laggera pterodonta and Forsythia suspensa have been reported to both inhibit virus replication and suppress inflammatory responses5-7. Herbal medicines are undoubtedly a rich source of tool compounds and drug leads for developing novel antiviral drugs8,9. However, unlike Western medicines that commonly formulated with a single active ingredient, herbal medicines contain complex mixtures of chemically diverse natural compounds10. This complexity poses a challenge when it comes to identifying and isolating the specific active constituents responsible for their therapeutic effects. Furthermore, the availability of commercially purified compounds for certain constituents is often limited, further hindering in-depth characterization of their pharmacological effects and mechanisms of action.
Several virus-based and host-based targets have been validated for the discovery and development of antiviral agents to combat COVID-19. Among these therapeutic targets for β-coronaviruses (CoVs), the Mpro has garnered significant attentions from medicinal chemists due to its critical role in the viral replication and assembly11. There is increasing evidence indicating that inhibiting or disrupting Mprocan prevent the formation of replication-essential enzymes, thus impeding viral multiplication and replication12,13. The SARS-CoV-2 Mpro, also known as 3-chymotrypsin-like proteases (3CLpro), is a canonical cysteine protease that contains 12 cysteine residues per monomer14. Among these, Cys145 and Cys44, located in the catalytic pocket of the enzyme, are particularly important for its function15-17. They form the catalytic dyad that cleaves the viral polyproteins into smaller functional proteins necessary for viral replication. In addition, Cys156 and Cys300 play important roles in forming enzymatically active dimeric forms of the protein17-19. Targeting these cysteine residues is a promising avenue of research for developing effective inhibitors against SARS-CoV-2 Mpro. The scientific community has dedicated to discovering new covalent inhibitors for Mpro. Commonly employed approaches in this endeavor include high-throughput screening and structure-based virtual screening, both relying on known compound libraries or databases20,21. In this context, several natural compounds derived from traditional medicinal plants, such as myricetin, oridonin and isojacareubin, have been identified. These compounds possess catechol or Michael receptors that have the ability to covalently bind to the cysteine residues in SARS-CoV-2 Mpro16,17,22,23.
In this work, a practical strategy for rapidly discovering irreversible inhibitors of SARS-CoV-2 Mpro from complex components (such as herbal extracts or compound prescriptions) was demonstrated. Our approach integrates target-based high-throughput screening and mass spectrometry (MS)-based approaches, enabling efficient screening of the herbal medicines with potent time-dependent inhibition on SARS-CoV-2 Mpro and to rapidly identify the covalent Mpro inhibitors from crude herbal extract (Fig. 1 ). Firstly, fluorescence resonance energy transfer (FRET) technique was employed for screening the anti-Mpro effects of clinically used anti-viral herbal medicines and the results showed thatLonicera japonica (LJ) significantly inhibited SARS-CoV-2 Mpro in a time-dependent manner. After then, ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was used for global chemical profiling of the constituents in LJ extract, while the Mpro-compound conjugated were carefully characterized, which provided key information to decipher the key constituents in LJ extract with the capability to covalently modify Mpro, as well as the key modified site(s) of Mpro by small molecules. This strategy enables both multiplexed screening and direct identification of small molecule binders from complex herbal mixtures, eliminating the need for fractionation. Furthermore, inactivation kinetics, binding kinetics and covalent docking simulations were conducted to gain further insights into the interactions between the target protein and the newly identified Mproinhibitors from LJ extract.