Synthesis and Testing of PB05, a Dual Inhibitor of IAV PB2 and Cellular JAK2

 Synthesis and Testing of PB05, a Dual Inhibitor of IAV PB2 and Cellular JAK2

Influenza A virus (IAV) is a member of the influenza family, which contains Influenza A-D. Influenza A and B are the subtypes primarily responsible for the seasonal influenza cases.  Influenza subtypes have further genetic diversity due to their hemagglutinin (HA) protein, which has 18 identified variants, and the neuraminidase (NA) protein that has 11 identified variants. The variants of these proteins present on the viral surface determine the nomenclature for a specific subtype of virus; for example, the influenza A H1N1 virus (Spanish Flu) would present variant 1 for both the HA and NA proteins (1).

Although vaccinations are available each year for the emerging strains, ~5 million people each year develop severe symptoms, with 650,000 yearly deaths. In addition to its well-known symptoms of fever, tiredness, dizziness, and headache, influenza infection can cause rarer, yet more dangerous symptoms such as pneumonia and myocarditis. A major source of the symptoms and major illnesses that come from influenza infection can be attributed to the immune response. This can be an issue when unregulated, pro-inflammatory molecules are produced out of control, leading to more severe symptoms. This process is heavily regulated by the JAK-STAT pathway, which is responsible for the activation of many pro-inflammatory molecules. Common viral signals are recognized by cells, which leads to signaling events that activate JAK proteins. Influenza antiviral drugs primarily target the viral replication process, while ignoring the burden the immune response can have on a patient. This highlights the need for antivirals that can target both the viral replication and immune response elements of IAV infection (2).

By nature of the atypical genome of IAV, it requires its own special enzyme that can produce its transcripts, called an RdRp. The IAV RdRp is also capable of stealing essential portions of cellular machinery in a mechanism called “cap snatching, ” which is essential for its replication. The PB2 subunit of RdRp contains the cap-binding region required to steal caps, and thus is a suitable target for drugs that seek to inhibit cap snatching, and by extension, viral replication (2).

The authors of this paper were able to screen for drugs that either inhibit JAK proteins or drugs that inhibit the PB2 domain of IAV RdRp. They identified two drugs with similar structures: decernotinib, which inhibits JAK, and pimodivir, which inhibits PB2. Their goal was to synthesize a drug that mimics both structures and is able to inhibit both JAK and PB2. The paper describes their process of synthesizing and testing this molecule, which they call PB05, against various influenza infection conditions (2).

Synthesis

Figure 1. Visual Representation of PB05. (b) Images of pimodivir and decernotinib in the cap binding domain of PB2. (c) Visual representation of the workflow to create a pimodivir and decernotinib-like molecule, with possible modifications to the green “R” group in the pink box

Both drugs shared a main skeleton of carbon and nitrogen-based rings, which interact with portions of PB2 (Figure 1b,c). Fluorine substituents present on pimdinovir were incorporated into the final molecule, while the modified carbon chain of decernotinib was incorporated as well. They synthesized 5 drugs with this structure, with slight structural variations named PB01, PB02, PB03, PB04, and PB05. These molecules were tested for IAV inhibition, JAK inhibition, and PB2 inhibition. From these tests, the authors were able to determine that the PB05 drug performed the best (2).

Antiviral Activity of PB05

Figure 2 PB05 and decernotinib binding visualization. (c) (Left) PB05 and decernotinib in the active region of JAK2. (Right) PB05 and decernotinib in the cap binding domain of PB2.

The effectiveness of PB05 was tested against the H1N1 and H3N2 strains of IAV, which demonstrated that PB05 was capable of reducing the levels of IAV proteins and genetic material, but about 10x less efficiently than pimodivir, the PB2 inhibitor. Their imaging technology revealed that PB05 was directly binding to PB2 (Figure 2c). As for JAK-STAT inhibition, PB05 was also able to decrease the levels of transcripts responsible for producing inflammatory molecules, and was also found to be interacting with JAK (Figure 2c). In mice, inflammation-induced damage was also severely decreased. From these experiments, they were able to prove that PB05 was an effective antiviral therapy against IAV, which worked by both inhibiting the immune response to reduce inflammation-based damage, but also by inhibiting a key IAV enzyme in viral replication (2). 

Pharmacokinetics

Aside from a drug's ability to act on its target, another key aspect of drug design is making a drug that can be taken into the body without interference from common pathways of degradation and removal. This means that digestion, excretion, and distribution of the drug can also become a problem. Thankfully, they determined that PB05 was able to be taken orally with a reasonable dosage that wasn't harmful to the mice they tested on, and stayed in the body long enough to be effective when taken twice daily (2).

Conclusion

This paper demonstrated the need for drugs that both inhibit the immune response to influenza infections to reduce the injuries caused by inflammation, and inhibit viral replication to assist the immune system in successfully clearing the infection. They were able to find two drugs that performed these actions with structural similarities, and synthesize a single drug that combined the properties of these two. They claim that PB05 is an important first step in developing better IAV treatments, but more studies are needed on its interactions with the body's metabolism and its ability to circulate. It functions by blocking the JAK pathway and the cap snatching enzyme of IAV, demonstrating the dual action ability of this drug.

Works Cited

1. Kyokha Ameen Y. Seasonal Influenza: A Narrative Review of Epidemiology, Clinical Features, and Preventive Strategies. Cureus. 2025 Oct 24;17(10):e95336. doi: 10.7759/cureus.95336. PMID: 41287674; PMCID: PMC12640676.

2. Rong B, Yang Y, Lu K, Zhou X, Zheng P, Lin X, Wen Y, Lin S, Deng X, Zhou Q, Liu S. Dual Inhibition of PB2 and JAK2 for Influenza: A Strategy Combining Antiviral and Host-Directed Immune Modulation. Molecules. 2026 Feb 17;31(4):696. doi: 10.3390/molecules31040696. PMID: 41752472; PMCID: PMC12943364.