Protein Degraders Targeting SARS-CoV-2 Main Protease
What is SARS-CoV-2?
The corona virus disease 2019 (COVID-19) that is sweeping the world is caused by a new single-stranded RNA coronavirus, The virus has been named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a coated plus-stranded single-stranded RNA virus, similar to severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and Middle East respiratory syndrome coronavirus (MERS-CoV).
The process of coronavirus entering host cells depends on the combination of spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of host cells, and then enters the cell through receptor-guided endocytosis to release the viral genome, complete the transcription of viral polymerase, and carry out genome replication and structural protein synthesis. Finally, it is assembled into complete virus particles, which are released into the cell through exocytosis to complete proliferation. When its genome enters the host cell, it encodes non-structural proteins through host cell mechanisms: Such as main protease (Mpro, also known as 3-chymotrypsin-like protease, 3CLpro), papain-like protease (PLpro), helicase, RNA-dependent RNA polymerase (RdRp) and structural proteins: Such as the spike protein and accessory protein for viral replication.
Potential therapeutic targets and drugs for SARS-CoV-2
The mechanism of SARS-CoV-2 infecting host cells is through a series of processes such as virus attachment, fusion, penetration, uncoating, transcription, translation and virion release. The main purpose of antiviral drugs is to block the infection and spread of the virus by inhibiting or interfering with the infection or replication process of the above different stages. Therefore, according to different drug targets, antiviral drugs can be divided into direct anti-SARS-CoV-2 drugs that target viral proteins such as spike protein, 3CLpro, PLpro and RdRp, and indirect anti-SARS-CoV-2 drugs that target host cell proteins such as ACE2 and TMPPSS2.
At present, the oral small-molecule drugs targeting SARS-CoV-2 have made breakthrough progress, and a variety of therapeutic drugs have entered phase III clinical trials. Gilead's Remdesivir and Pfizer's Paxlovid were the first marketed inhibitors of RdRp and 3CLpro, respectively.
Development of protein degraders targeting Mpro
Mpro is the main protease produced by novel coronavirus (SARS-CoV-2). Most functional proteins (non-structural proteins) of coronavirus are encoded by ORF1ab gene and translated into a polyproteome (7096aa). It is then cut by Mpro into multiple active proteins such as the viral replication protein RdRp.
In addition, the protein may cleave the intracellular protein NEMO (NF-κB essential regulator, also known as IKKγ), thereby inhibiting the activation of interferon signaling pathways. Therefore, antagonistic Mpro can effectively inhibit virus infection and replication. Therefore, Mpro is a key target for antiviral drug development. At present, the drug development research on Mpro targeting SARS-CoV-2 is mainly by inhibiting the activity of Mpro, thereby preventing the formation of non-structural proteins, and ultimately inhibiting the replication of SARS-CoV-2.
Mpro plays an important role in the viral life cycle, and as a result, Mpro has become one of the main drug discovery targets for SARS-CoV-2 infection, and protein degradation strategies have been shown to degrade proteins associated with viruses, such as hepatitis and influenza. However, its use in protein degradation for SARS-CoV-2 has not been reported.
First-in-class PROTAC degraders of SARS-CoV-2 main protease
A research team led by Benjamin Neuman, Wenshe Ray Liu, and Shiqing Xu from Texas A&M University published a preprint on bioRxiv titled "Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease." In this paper, a novel small molecule antiviral drug that can induce the degradation of SARS-CoV-2 Mpro was developed using PROTAC technology, which proved that MPD2 can effectively reduce the level of Mpro in 293T cells.
Chemical structure of MPD2. (Alugubelli, Y. R., 2024)
Starting from Mpro inhibitors MPI8 and MPI29 previously developed by the author team, a series of PROTAC molecules MPD1-10 were synthesized after certain modifications. In order to evaluate the inhibitory activity of these PROTAC molecules on Mpro, the author's team used a previously established experimental method, using a degradation substrate of Mpro - fluorescent peptide Sub3, by observing the fluorescence intensity in the system to evaluate the residual activity of Mpro in the system, and measured the IC50 value of these molecules.
Next, the authors constructed a 293T stable cell line of Mpro-eGFP to characterize the degradation activity of these PROTAC molecules. Meanwhile, MTT assay was performed to evaluate the cytotoxicity, and finally MPD2 with good degradation activity and low cytotoxicity was selected for further evaluation. It was found that MPD2 can effectively reduce the protein level of Mpro in 293T cells in a time-dependent, CRBN-mediated and proteasoma-driven manner. MPD2 not only significantly reduced the amount of Mpro protein in SARS-CoV-2-infected A549-ACE2 cells, but also showed strong antiviral activity against different SARS-CoV-2 variants including WA.1, BA.1 and XBB.1.5. We also observed a synergistic effect between MPD2 and Nirmatrelvir, the antiviral active ingredient in Paxlovid, suggesting potential synergistic use of Mpro inhibitors and Mpro degraders.
The mechanism of action of MPD2 relies on the binding of Mpro to CRBN, a process confirmed by the use of competing inhibitors MPI8 (Mpro ligand) and pomalidomide (CRBN ligand), and further confirmed by experiments in CRBN gene knockout cells. In addition, MPD2-induced Mpro degradation was effectively blocked when the cells were treated with the proteasome inhibitor MG132, suggesting that Mpro degradation is dependent on the activity of the proteasome.
Ligand for CRBN E3 ligase at BOC Sciences
Bifunctional molecule HP211206 degrading SARS-CoV-2 main protease
Ziwei Huang's team at the Faculty of Medicine of the Chinese University of Hong Kong reported the design, synthesis and biological characteristics of a novel heterobifunctional small molecule that can simultaneously bind E3 ubiquitin ligase and the viral protein Mpro, inducing Mpro degradation through the ubiquitin proteasome system, thus demonstrating a new alternative strategy to interfere with this important protein of the novel coronavirus.
The researchers used H117 as the target protein ligand and conjured the CRBN E3 ligand pomalidomide with linker to obtain a heterobifunctional small molecule HP211206, which was used as a small molecule degradation of SARS-CoV-2 virus protein. The molecule can effectively degrade SARS-CoV-2 Mpro and its drug-resistant mutants in HEK 293T cells. This provides a potential solution to the problem of resistance to traditional protein inhibitors, while bringing new candidate molecular entities to novel coronavirus drug development.
Chemical structure of heterobifunctional small molecule HP211206. (Sang, X., 2023)
Indomethacin-based PROTACs degrading SARS-CoV-2 main protease
In their 2024 study, Desantis et al. reported that they designed, synthesized, and evaluated a series of indomethacin (INM) -based PROTAC molecules that degrade the major protease of SARS-CoV-2 and exhibit broad-spectrum antiviral activity. By altering the linker part, the research team designed and synthesized a series of novel INM-based PROTACs capable of recruiting Von-Hippel Lindau (VHL) or cereblon (CRBN) E3 ligases. One piperazine-based compound (PROTAC 6) showed potent antiviral activity in SARS-CoV-2 infected with human lung cells. The results show that these INM-PROTACs do not work by degrading the human PGES-2 protein, but rather by inducing the degradation of SARS-CoV-2 Mpro in a concentration-dependent manner, both in Mpro transfected cells and in SARS-CoV-2-infected cells. In addition, compared with indomethacin, the antiviral activity of INM-based PROTACs was significantly iMproved, and the EC50 value reached the low micromolar/nanomolar range. To note, for the first time it has been confirmed that PROTAC technology applied to a weak affinity POI binder may result in potent antiviral degraders. The experimental elucidation of the ternary complex formation will be useful in the future to guide further optimization studies.
Ligand for VHL E3 ligase at BOC Sciences
References:
- Alugubelli, Y. R., et al. Discovery of first-in-class PROTAC degraders of SARS-CoV-2 main protease. Journal of Medicinal Chemistry. 2024, 67(8): 6495-6507.
- Sang, X., et al. A chemical strategy for the degradation of the main protease of SARS-CoV-2 in cells. Journal of the American Chemical Society. 2023, 145(50): 27248-27253.
- Desantis, J., et al. Design, synthesis, and biological evaluation of first-in-class indomethacin-based PROTACs degrading SARS-CoV-2 main protease and with broad-spectrum antiviral activity. European Journal of Medicinal Chemistry. 2024, 268: 116202.
