Antiviral PROTACs

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Viral diseases caused a large number of deaths in the 20th century. For example, smallpox killed as many as 400 million people, the Spanish flu pandemic of 1918-1919 killed about 100 million people, and the human immunodeficiency virus (HIV) epidemic has killed 35 million cases. As of 25 January 2022, the number of confirmed cases of COVID-19 has reached 352 million, including 5.6 million deaths. Viral infections have become a serious concern for public health and safety. At present, the prevention and treatment of human virus infection mainly depends on the combination of drugs and vaccines. However, the growing number of resistant strains poses challenges to currently available antiviral treatment strategies, and vaccination often does not protect against mutated or novel viruses. Therefore, it is critical to find cutting-edge antiviral treatment strategies based on targeting or vaccination of these two novel drugs.

PROTAC technology has been widely studied for the targeted protein degradation treatment of POI, which relies on the use of heterotype bifunctional molecules to recruit the intracellular protein degradation mechanism to the vicinity of the target protein of interest, so that the target protein is ubiquitinated and thus degraded. Many advances have been made in the role of PROTACs as an antiviral therapy, and various PROTAC-based antiviral treatment strategies have been explored with improved resistance characteristics.

Antiviral strategy based on PROTAC

Depending on the characteristics of the virus and the propagation process into the host cell, some of these proteins can be used as target proteins for the design of PROTACs, including proteases, surface receptor proteins, host proteins, and cyclin-dependent kinases (CDKs). The following details the application of PROTACs designed based on these proteins in the field of antiviral.

Viral targets of different PROTAC-based approaches. (A) Illustration of putative antiviral PROTACs targeting different viral targets, including proteases, surface proteins, host proteins, and cyclin-dependent kinases. (B) Summary of viral targets and their putative antiviral drugs and E3 ligases that make up the PROTAC molecule. (Ahmad, H., 2023)

PROTAC virus, a novel vaccine strategy

Si et al. propose an innovative vaccine strategy that utilizes PROTAC technology to create artificially attenuated influenza strains, a strategy that promises to overcome the limitations of traditional vaccine preparation methods. By linking the proteasome-targeting domain (PTD) to a specific protein of the influenza virus, and using the host cell's ubiquitin-proteasome system, they successfully degraded the viral protein, significantly reducing the virus's ability to replicate.

PTD, a heptapeptide sequence (ALAPYIP) recognized by VHL E3 ubiquitin ligase, is linked to the viral protein via a TEV protease cleavage site (ENLYFQG), allowing the PTD to be conditionally excised in cells expressing TEV protease, avoiding degradation of the viral protein. This mechanism ensures that in certain cell lines, the virus replicates normally, while in others it is severely weakened, and the resulting attenuated virus can be used for vaccine production.

The research team performed PTD labeling tests on eight key proteins of influenza A virus and found that the M1 protein-labeled virus (M1-PTD) was more than 20,000 times less able to replicate in ordinary cells, but still replicated effectively in TEV protease-expressing cells. The M1-PTD virus elicits a significant immune response in both mouse and ferret models, including high levels of hemagglutinin inhibiting antibodies and neutralizing antibodies, as well as an enhanced T cell immune response.

The advantages of PROTAC virus as a vaccine candidate are that it can ensure safety while maintaining high immunogenicity, and activate the proteasome degradation pathway by degrading viral peptides, triggering a more effective immune response. Compared to traditional vaccine preparation techniques, PROTAC technology not only improves safety, but also reduces the loss of efficacy and productivity, and may reduce immune escape problems caused by rapid virus evolution. Therefore, PROTAC technology has become an important option for the production of safer and more effective vaccines. This breakthrough approach opens a new path to developing safer and more effective vaccines.

Ligand for VHL E3 ligase at BOC Sciences

PROTAC targeting proteases

Viruses encode one or more proteases as a typical technique for supporting compressed genome replication. To produce mature viral proteins, the viral genome encodes multiple proteins with integrated viral proteases that cut multiple proteins at several specific locations. Because of their necessity for reproduction, viral proteases are excellent therapeutic targets. Viral life cycle and replication depend on viral proteases. Proteases from different viral families differ from one another in terms of structure, catalytic mechanisms, and preferred substrates. Viral proteases have specific substrate preferences that can be used to design inhibitors to produce potent and selective drug-like compounds.

Yang et al reported a PROTAC compound targeting the non-structural 3/4A (NS3/4A) serine protease of hepatitis C virus (HCV). Terrapivir, a reversible covalent inhibitor, binds to the active site of HCV NS3/4A serine protease for the development of a PROTAC inhibitor for this protease.

The crystal structure of terrapivir with viral proteases shows that its pyrazine ring is solvent-exposed, allowing it to be coupled to the CRBN ligand via a linking chain. The resulting terapivir-based inhibitor DGY08-097 selectively inhibited and degraded HCV NS3/4A protease.

Ligand for CRBN E3 ligase at BOC Sciences

PROTACs targeting surface receptors

The viral envelope is a double layer of lipid that surrounds the capsid. It embeds a number of glycoproteins onto its surface, allowing the virus to attach to receptors in host cells. On the other hand, in addition to these glycoproteins present in the envelope, many coronavirus (CoVs) also have unique envelopes and membrane proteins that are necessary to maintain viral replication. Viral surface proteins, such as hemagglutinin and neuraminidase, are emerging as new targets for antiviral therapy.

Zhou et al report the design and synthesis of a novel PROTAC molecule based on the oseltamivir skeleton to combat severe annual influenza outbreaks. Oseltamivir is a neuraminidase inhibitor that prevents the emergence of newly synthesized virus particles by host cells. Oseltamivir based PROTACs degrade neuraminidase through the ubiquitin-proteasome pathway.

PROTAC targeting host proteins

After the host cell is infected with the virus, the virus hijacks the host cell to synthesize viral proteins and nucleic acids for rapid reproduction, in which many enzymes, such as polymerase, endonuclease and ligase, are essential for viral replication and proliferation. Antiviral strategies that target host proteins, rather than viral proteins, can avoid the problem of viral resistance because the virus relies on host mechanisms to replicate its genetic material. This makes host proteins an emerging target for antiviral therapy.

Indomethacin (INM), a non-steroidal anti-inflammatory drug, inhibits PGES-2 from participating in arachidase biosynthesis. Goracci et al. reported that INM combined with VHL E3 ligand pairs to form PROTACs via aliphatic or polyethylene glycol ligand chains. Because these PROTACs target the host protein PGES-2, they have significant antiviral activity.

CDK plays an important role in the viral life cycle, and its inhibitors have antiviral effects against a variety of viruses, including HSV, HIV, human cytomegalovirus (HCMV), and SARS-CoV-2. Hahn et al developed a CDK-based PROTAC, THAL-SNS032, that promotes the degradation of CDK9 as well as CDK1, 2 and 7 through the E3 ligase CRBN-mediated ubiquitination process, and also targets the pUL97 protein encoded by human cytomegalovirus, which plays a key role in viral replication.

Structure of THAL-SNS032. (Espinoza-Chávez, R. M., 2022)

Related ligand for target protein at BOC Sciences

PROTAC design for target proteins at BOC Sciences

Advantages and limitations of PROTACs antiviral strategy

PROTACs has great advantages over traditional antiviral drugs because of its specificity, high efficiency, catalytic capacity and other mechanisms of action.

Although PROTACs are more powerful than other small molecule inhibitors and completely deplete the required target proteins in cells, they may cause targeted toxicity in host cells and may impair normal cellular function. Some of the POIs targeted by PROTAC can have both enzymatic functions as well as other scaffold functions that may be important for normal cell function. Therefore, their complete elimination may be toxic to cells.

PROTAC degradation of the target protein may also degrade proteins directly or indirectly related to the target protein, resulting in off-target effects. If PROTAC binds to a new substrate, off-target effects will also cause new morphological interactions, so future studies should focus on further reducing the off-target toxicity of PROTACs.

References:

1. Ahmad, H., et al. Recent advances in PROTAC-based antiviral strategies. Vaccines. 2023, 11(2): 270.
2. Si, L., et al. Generation of a live attenuated influenza A vaccine by proteolysis targeting. Nature Biotechnology. 2022, 40(9): 1370-1377.
3. De Wispelaere, M., et al. Small molecule degraders of the hepatitis C virus protease reduce susceptibility to resistance mutations. Nature Communications. 2019, 10(1): 3468.
4. Xu, Z., et al. Discovery of oseltamivir-based novel PROTACs as degraders targeting neuraminidase to combat H1N1 influenza virus. Cell Insight. 2022, 1(3): 100030.
5. Espinoza-Chávez, R. M., et al. Targeted protein degradation for infectious diseases: from basic biology to drug discovery. ACS bio & med Chem Au. 2022, 3(1): 32-45.