Dual Inhibition of PB2 and JAK2 for Influenza: A Strategy Combining Antiviral and Host-Directed Immune Modulation

TL;DR

A new drug called PB05 can both fight influenza viruses and reduce harmful inflammation, offering a dual approach to treating severe flu.

Contribution

The development of PB05, a dual-target inhibitor that simultaneously targets viral PB2 and host JAK2 to combat influenza and inflammation.

Findings

PB05 showed strong antiviral activity against influenza A viruses with nanomolar EC50 values.

PB05 reduced pro-inflammatory cytokines like IL-6 and IL-1β by inhibiting JAK–STAT signaling.

In mice, PB05 lowered lung viral titers and reduced inflammation and tissue damage.

Abstract

Influenza virus infection remains a major global health burden, with severe disease outcomes driven not only by viral replication but also by excessive host inflammatory responses. Current antiviral therapies predominantly target viral components and fail to adequately control virus-induced hyperinflammation. In this study, we report a dual-target therapeutic strategy integrating direct antiviral activity with host-directed immunomodulation. Using a molecular hybridization approach, we designed and synthesized several dual-target inhibitors simultaneously targeting the influenza virus PB2 cap-binding subunit and host JAK2 kinase. Among them, PB05 emerged as the most promising candidate and was systematically evaluated in vitro and in vivo. PB05 exhibited potent broad-spectrum antiviral activity against influenza A viruses, with nanomolar EC50 values. Mechanistic studies demonstrated that PB05 directly binds to the PB2 cap-binding domain, thereby disrupting viral cap-snatching and RNA synthesis. In parallel, PB05 inhibited JAK–STAT signaling by suppressing STAT2 phosphorylation and downstream ISRE-mediated transcription, leading to a marked reduction in pro-inflammatory cytokine production, including IL-6, IL-1β, and IFN-β, in infected or stimulated immune cells. In a lethal influenza A/PR/8/34 (H1N1) mouse model, oral administration of PB05 at 100 mg/kg (twice daily) markedly decreased lung viral titers, attenuated pulmonary tissue damage and edema, and moderated excessive inflammatory responses. Collectively, these findings identify PB05 as a dual PB2/JAK2 inhibitor that effectively couples antiviral efficacy with immunomodulatory activity, promoting a therapeutic strategy for the treatment of severe influenza and other viral diseases associated with excessive inflammation.

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1. Introduction

Influenza is an acute respiratory infectious disease caused by influenza viruses, characterized by pronounced seasonality and rapid transmission. Epidemic outbreaks typically occur during the winter and early spring months each year [1]. According to the World Health Organization (WHO), seasonal influenza affects approximately 1 billion people worldwide each year, with up to 5 million cases developing into severe illness. Globally, around 650,000 deaths are attributed annually to respiratory complications associated with seasonal influenza, underscoring its substantial and ongoing burden on public health [2]. In addition to common symptoms such as fever, cough, dizziness, and headache, influenza infection can lead to severe and potentially life-threatening complications, including pneumonia and myocarditis. These complications are particularly prevalent among elderly individuals and those with compromised immune systems [3].

The influenza virus infection cycle includes viral binding and fusion with cells, viral genome replication and transcription/translation, and the assembly and release of progeny viral particles [4]. Several classes of antivirals have been developed to target different stages of the influenza virus life cycle, including M2 ion channel blockers, neuraminidase inhibitors, hemagglutinin inhibitors, and RNA polymerase inhibitors [5,6]. Among these, the RNA polymerase complex (PA, PB1, PB2) serves as a promising target with a high genetic barrier to resistance [7,8]. Favipiravir (PB1 inhibitor) and baloxavir marboxil (PA inhibitor) have demonstrated clinical efficacy [9,10], while PB2, a conserved and functionally essential subunit, represents an attractive target for next-generation influenza therapeutics. PB2 is primarily responsible for recognizing and binding the 5′-cap structure of host pre-mRNAs, thereby facilitating the endonuclease-mediated “cap-snatching” process required for viral mRNA synthesis [11]. Thus, disrupting this process represents a promising strategy for developing new antiviral agents, positioning PB2 as an attractive therapeutic target against influenza [12]. The PB2 inhibitor onradivir, derived from pimodivir and developed by Raynovent, exhibits potent antiviral activity against H1N1, with an EC_50_ of 0.03 nM [13,14,15]. In May 2025, onradivir was approved as the world’s first PB2 inhibitor for the treatment of uncomplicated influenza A in adults.

However, current antiviral therapies primarily act by inhibiting viral replication but fail to address the inflammatory and immunopathological consequences of infection [16]. During influenza infection, dysregulated immune activation and excessive cytokine production drive severe pulmonary inflammation and systemic injury, leading to ARDS and multi-organ failure even after viral clearance [17,18]. This hyperinflammatory state, rather than viral persistence itself, is a major contributor to severe and fatal outcomes. Moreover, most existing antivirals are only effective during the early phase of infection, offering limited benefit once systemic inflammation and immune dysregulation are established [19]. These limitations underscore the urgent need for therapeutic strategies that combine direct antiviral efficacy with modulation of host inflammatory responses.

Viral infection triggers the host inflammatory response. During influenza virus invasion, pattern recognition receptors (PRRs) activate downstream signaling pathways, inducing cytokines such as IFNs and IL-6, which in turn stimulate the JAK-STAT pathway. This cascade promotes the expression of pro-inflammatory cytokines and interferon-stimulated genes (ISGs), establishing an antiviral state that supports viral clearance when properly regulated [20,21,22]. An effective immune response requires not only efficient viral clearance but also the prevention of excessive inflammation and immune-mediated tissue damage [23]. In severe influenza virus infections, excessive production of inflammatory mediators—including IL-6, IFN-α/β/γ, and TNF-α—often triggers systemic hyperinflammatory cascades [24]. This excessive inflammatory condition, known as a cytokine storm, results in acute lung injury, multiple organ failure, and high mortality. It is now well established that such cytokine storms, arising from immune dysregulation, constitute a major pathological mechanism underlying the severe outcomes and elevated mortality associated with influenza virus infection [25]. Emerging evidence indicates that inhibition of the JAK signaling pathway effectively attenuates cytokine-mediated signaling cascades during the early stages of viral infection, thereby limiting hyperinflammatory responses. Accordingly, the JAK pathway has been identified as a critical regulatory axis governing cytokine-driven immune activation and inflammatory homeostasis [26].

The JAK-STAT signaling pathway comprises four Janus kinases (JAK1, JAK2, JAK3, and TYK2) and seven STAT transcription factors. Upon cytokine (e.g., interferon) binding to cell-surface receptors, paired JAKs undergo transphosphorylation and activate downstream STATs, which subsequently translocate to the nucleus to regulate target gene expression. This pathway plays a pivotal role in controlling cell proliferation, differentiation, apoptosis, and immune regulation [27]. Notably, JAK1 and JAK2 function as central signaling nodes within multiple cytokine-mediated pathways, serving as critical links between innate and adaptive immune responses [28]. Their dysregulation is associated with a spectrum of pathologies ranging from chronic inflammatory diseases to malignant tumors [29,30]. Beyond their immunoregulatory roles, JAK1 and JAK2 have been implicated in influenza A virus (IAV) replication, making them potential antiviral targets. Wang et al. reported that the active metabolite of leflunomide inhibits IAV replication by suppressing JAK2 activity, while simultaneously modulating the JAK-STAT pathway to reduce cytokine overproduction and mitigate the inflammatory storm associated with respiratory viral infections [31,32].

Initially, we screened a library of ~90 JAK inhibitors for anti-H1N1 activity and identified decernotinib, a non-selective JAK inhibitor previously developed for rheumatoid arthritis [33], with potential antiviral activity (EC_50_ = 8.88 μM) (Table S1). Structurally, decernotinib shares notable similarity with the PB2 inhibitor pimodivir. Based on this, we employed a molecular hybridization strategy to merge the two scaffolds, aiming to develop dual PB2/JAK2 inhibitors that combine direct antiviral and immunomodulatory effects. Among the synthesized derivatives, PB05 exhibited potent anti-influenza and anti-inflammatory activities. Mechanistic studies revealed that PB05 directly inhibits PB2 to block viral replication while suppressing the JAK-STAT pathway to reduce cytokine overproduction, thereby offering a dual-action therapeutic strategy against severe influenza infection.

2. Results

2.1. Design of the PB2/JAK2 Dual-Target Inhibitors

During the screening of a JAK inhibitor compound library for antiviral activity (Figure 1a), decernotinib was identified as a hit compound exhibiting moderate anti-influenza activity, achieving over 50% inhibition at concentrations of 10 and 20 μM. Notably, molecular docking analysis revealed that decernotinib shared a high degree of structural similarity in its core pharmacophore with pimodivir. Both compounds adopt comparable binding modes within the PB2 protein, forming key hydrogen-bond interactions with Val511, Lys376, and Glu361, which are critical residues for PB2 inhibition (Figure 1b).

Inspired by this observation, we designed a hybridization strategy to integrate the structural features of both compounds, aiming to develop a dual-target inhibitor capable of simultaneously inhibiting viral PB2 and host JAK2. Specifically, the azindole and pyrimidine moieties of pimodivir were fused with the side-chain fragment at the pyrimidine 4-position of decernotinib, followed by systematic side-chain modifications to fine-tune inhibitory activities against both targets (Figure 1c).

2.2. Chemistry

The synthesis of PB02 began from 5-fluoro-1H-pyrrolo [2,3-b]pyridine and proceeded through a regioselective bromination, N-protection, Miyaura–Suzuki cross-coupling, and controlled oxidation to afford a key sulfoxide intermediate. Subsequent nucleophilic substitution with D-(−)-isovaline, followed by amide coupling and final deprotection, furnished PB02 (Scheme 1). For PB03, intermediate 6 underwent nucleophilic substitution with L-(−)-isovaline to generate intermediate 9. Removal of the tosyl (Ts) protecting group using sodium methoxide, followed by lithium aluminum hydride reduction, afforded the target compound (Scheme 2). The synthesis of PB04 involved one-step aminolysis of intermediate 7 to give amide 11, which was reduced with 1 M borane–THF to produce primary amine 12. Subsequent Ts deprotection with sodium methoxide yielded PB04 (Scheme 3). The synthetic route to PB05 is outlined in Scheme 4. Nucleophilic substitution of intermediate 6 with α-amino alcohols afforded key intermediates 13A and 13B, which upon Ts deprotection in methanolic sodium methoxide provided the final target compounds.

2.3. Biological Evaluation of PB2/JAK Dual-Target Inhibitors

The in vitro anti-IAV activity of the resulting dual-target compounds was evaluated using cell-based cytopathic effect (CPE) assays, while PB2 and JAK2 enzymatic inhibition assays were conducted to assess target engagement. As shown in Table 1 and Figure S1, compounds PB01, PB03, PB04, and PB05 exhibited approximately 1000-fold enhanced antiviral activity compared with decernotinib, likely due to the introduction of PB2 inhibitory activity through subtle structural modulation. Structure–activity relationship analysis indicated that introduction of an amide side chain reduced antiviral activity, as exemplified by PB02. Reduction of the amide to an amine yielded PB04, which showed a marked improvement in anti-influenza activity (EC_50_ = 3.07 nM). Further substitution of the amino group with a hydroxyl group generated PB03, which retained potent antiviral activity (EC_50_ = 2.93 nM). Considering the sterically hindered side chain of pimodivir, a more rigid cyclohexanol moiety was introduced, affording two stereoisomers: trans-PB01 and cis-PB05. Both compounds displayed comparable PB2 inhibitory activity and thus similar antiviral potency. However, their JAK2 inhibitory activities differed, with the cis configuration favoring JAK2 binding. Specifically, PB05 exhibited a JAK2 IC_50_ of 74.56 nM, whereas PB01 showed weaker inhibition (IC_50_ = 327.20 nM). Further enzymatic analyses confirmed that PB05 possesses balanced and favorable inhibitory activity against both PB2 and JAK2, while maintaining low cytotoxicity (CC_50_ > 100 μM), highlighting its potential as a promising dual-target antiviral candidate.

2.4. Anti-Influenza Virus Activity of PB05 In Vitro

Subsequently, a CPE protection assay was performed to assess the antiviral activity of PB05 against influenza A virus subtypes H1N1 and H3N2 in MDCK cells. As summarized in Table 2, PB05 exhibited inhibitory activity against all tested strains, with EC_50_ values ranging from 2.60 to 38.37 nM, indicating broad-spectrum anti-influenza activity. Consistently, plaque reduction assays demonstrated that PB05 suppressed viral replication in a concentration-dependent manner, with pronounced inhibition observed at nanomolar concentrations (Figure S2). Immunofluorescence analysis further revealed that viral nucleoprotein (NP) was robustly expressed in virus-infected cells, whereas PB05 treatment led to a marked and dose-dependent reduction in NP fluorescence signals. Notably, treatment with 12.5 nM PB05 achieved a level of NP suppression comparable to that observed with 1 nM pimodivir (Figure 2a). Western blot analysis corroborated these findings, showing that PB05 significantly reduced the protein levels of both viral PB2 and NP in infected cells in a dose-dependent manner (Figure S3). Quantitative analysis confirmed a pronounced decrease in viral protein expression relative to virus-only controls (Figure 2b). At the transcriptional level, RT–qPCR analysis demonstrated that PB05 markedly suppressed viral NP and PB2 mRNA levels, suggesting inhibition of viral RNA synthesis or reduced RNA stability (Figure 2c). However, when evaluated solely from an antiviral potency perspective, PB05 remained less potent than the reference PB2 inhibitor pimodivir, exhibiting an approximately 10-fold lower activity.

RT–qPCR analysis was performed to evaluate the effects of PB05 on host innate immune responses in H1N1-infected cells (Figure 2d). Infection with A/WSN/1933 (H1N1) virus markedly induced the transcription of multiple proinflammatory and antiviral genes, including IFN-β, TNF-α, IFN-α, IL-6, CXCL10, and ISG15, compared with uninfected controls. Treatment with PB05 significantly modulated the expression of these immune-related genes in a concentration-dependent manner. Specifically, PB05 treatment resulted in a pronounced suppression of virus-induced IFN-β, TNF-α, IFN-α, and IL-6 mRNA levels, indicating effective attenuation of excessive inflammatory responses. In parallel, PB05 also significantly reduced the expression of downstream interferon-stimulated genes, such as CXCL10 and ISG15, compared with virus-only controls. Notably, PB05 exhibited a distinct immunomodulatory profile relative to decernotinib and pimodivir, with more balanced regulation of antiviral and inflammatory gene expression.

2.5. Mechanistic Characterization of PB05

These results confirm that PB05 displays potent cellular anti-influenza activity, motivating further investigation of its mechanism of action. Using a plasmid-based minireplicon assay, PB05 significantly inhibited viral RdRp-driven minigenome reporter activity in HEK-293T cells in a concentration-dependent manner (Figure 3a). Furthermore, surface plasmon resonance (SPR) analysis demonstrated that PB05 binds directly to the influenza virus PB2 cap-binding domain in a concentration-dependent manner (62.5~1000 nM), with no detectable binding observed for the PBS control, indicating a specific interaction. Kinetic analysis yielded an association rate constant (Ka) of 129 M^−1^·s^−1^ and a dissociation rate constant (Kd) of 1.06 × 10^−6^ s^−1^, corresponding to an equilibrium dissociation constant (K_D_) of 8.17 × 10^−8^ M, confirming that PB05 exhibits nanomolar affinity for PB2 and supporting PB2 as a direct molecular target of PB05 (Figure 3b).

Molecular docking of PB05 was performed using Maestro 13.6. PB05 showed favorable predicted binding to both targets, with docking scores of −7.665 kJ/mol for JAK2 (PDB: 4YTI) and −8.439 kJ/mol for PB2 (PDB: 6EUV). Docking analysis indicated that PB05 binds stably in the JAK2 ATP-binding pocket through a water-mediated hydrogen bond and hydrophobic interactions (Figure 3c). In the PB2 cap-binding domain, PB05 forms polar interactions with Glu361 and His357, as well as π–π and hydrophobic contacts with Phe323, Phe363, and Val311. PB05 adopts a binding mode highly similar to that of the co-crystallized ligand decernotinib in both proteins, occupying comparable key regions of the binding sites, supporting the reliability of the docking results.

2.6. PB05 Exerts Anti-Inflammatory Activity by Inhibiting JAK-STAT Signaling

The pathogenic effects of influenza virus infection are driven not only by viral replication but also by excessive host immune activation, often described as a cytokine storm, which contributes substantially to tissue injury and severe clinical outcomes. Therefore, therapeutic strategies that combine antiviral efficacy with regulation of dysregulated inflammatory responses may offer improved benefits. Based on the observed antiviral activity of PB05, its potential immunomodulatory properties were further investigated.

To assess the effects of PB05 on inflammatory signaling and interferon-responsive gene expression, multiple innate immune activation models were employed, with pimodivir and decernotinib included as comparator compounds (Figure 4). In RAW264.7 macrophages stimulated with lipopolysaccharide (LPS), PB05 modulated the mRNA expression levels of representative pro-inflammatory cytokines, including IL-6, IL-1β, IFN-α and IFN-β, in a concentration-dependent manner (Figure 4a). Compared with pimodivir, which showed minimal regulatory effects on cytokine transcription, PB05 exhibited a more pronounced influence, while its regulatory profile was generally comparable to, but distinct from, that of the JAK inhibitor decernotinib. These observations suggest that PB05 regulates inflammatory responses associated with TLR4-mediated signaling rather than acting solely through viral inhibition.

Similar trends were observed in RAW264.7 cells stimulated with poly(I:C), a synthetic analog of viral double-stranded RNA that activates RNA-sensing pathways. Under these conditions, PB05 influenced the expression of interferon-related and inflammatory genes, whereas pimodivir showed limited modulation, and decernotinib displayed broader effects consistent with JAK pathway interference (Figure 4b). These data indicate that PB05 modulates innate immune responses triggered by viral RNA–sensing mechanisms.

Because interferon signaling converges on interferon-stimulated response element (ISRE)-mediated transcription, the effect of PB05 on ISRE activity was further examined using luciferase reporter assays. PB05 influenced ISRE-driven reporter activity following stimulation with either influenza virus or exogenous IFN-β (Figure 4c,d). In contrast, pimodivir exerted minimal effects on ISRE activity, while decernotinib and tofacitinib showed stronger regulatory impact. Collectively, these findings suggest that PB05 influences inflammatory and interferon-responsive signaling at multiple levels, supporting further investigation into its effects on upstream regulatory pathways, including the JAK–STAT axis.

STAT2 phosphorylation was examined in virus-infected and IFN-β–stimulated THP1 cells to further assess the impact of PB05 on interferon-related signaling, with pimodivir and decernotinib included as reference compounds (Figure 5). In H1N1-infected THP1 cells, Western blot analysis showed that PB05 modulated the phosphorylation status of STAT2 in a concentration-dependent manner, while total STAT2 protein levels remained largely unchanged (Figure 5a). In contrast, pimodivir, a PB2-selective inhibitor, exhibited minimal effects on STAT2 phosphorylation, whereas decernotinib showed a more pronounced influence consistent with its JAK-inhibitory activity. Densitometric quantification of p-STAT2 normalized to total STAT2 is presented alongside the representative immunoblots. PB05 again influenced STAT2 phosphorylation without markedly affecting total STAT2 expression in IFN-β–stimulated THP1 cells (Figure 5b). As expected, decernotinib showed clear modulation of STAT2 activation, whereas pimodivir had limited impact. In parallel, RT–qPCR analysis indicated that PB05 regulated the transcriptional levels of STAT2-associated interferon-responsive genes following IFN-β stimulation. These results suggest that PB05 modulates interferon-driven STAT2 signaling at both the phosphorylation and downstream transcriptional levels, distinguishing it from a PB2-only inhibitor and aligning with its dual-target design.

2.7. Pharmacokinetic Properties, Anti-Viral and Anti-Inflammatory Effects of PB05 In Vivo

To evaluate the in vivo pharmacokinetic properties of PB05, we conducted single-dose administration experiments in mice (intravenous and oral routes). As shown in Table 3, after IV administration at a dose of 3 mg/kg, compound PB05 demonstrated moderate half-life (T_1/2_) and CL. When administered orally at a dose of 30 mg/kg, PB05 was rapidly and extensively absorbed, with an oral bioavailability of 44.9%, a half-life (T_1/2_) of 4.02 h, a maximum plasma concentration (C_max_) of 1090 ng/mL, and an area under the concentration–time curve (AUC_0–∞_) of 4183 h·ng/mL. These results suggest that compound PB05 confers favorable oral absorption potential, which may contribute to efficient pharmacological effects.

To further evaluate the in vivo efficacy of PB05, a lethal influenza A virus infection model was established in BALB/c mice (Figure 6). Mice were intranasally infected with influenza A/WSN/1933 virus (5 × LD_50_) and orally administered PB05 at different doses according to the experimental schedule shown in Figure 6a. Lung tissues and serum samples were collected at 6 days post-infection (dpi) for comprehensive analysis. Macroscopic examination of lung tissues revealed severe hemorrhage and consolidation in virus-infected control mice, whereas PB05 treatment markedly improved lung appearance in a dose-dependent manner, with higher doses showing more pronounced protection (Figure 6b). Consistently, PB05 treatment reduced lung weight, and significantly lowered the lung index compared with untreated infected mice, indicating alleviation of pulmonary edema and inflammation (Figure 6c). Viral burden analysis demonstrated that PB05 administration significantly reduced lung viral titers, as determined by plaque assays, with the higher dose achieving a more substantial decrease in viral replication (Figure 6d and Figure S4). At the molecular level, Western blot analysis of lung tissues showed that PB05 modulated the expression of representative viral proteins and host inflammatory signaling components relative to virus-infected controls (Figure 6e and Figure S5). In parallel, RT–qPCR analysis revealed that PB05 treatment reduced the transcriptional levels of viral genes as well as key pro-inflammatory cytokines in lung tissues in a dose-dependent manner (Figure 6f). Histopathological evaluation by hematoxylin and eosin (H&E) staining further supported these findings (Figure 7). Compared with extensive alveolar destruction, inflammatory infiltration, and septal thickening observed in virus-infected mice, lungs from PB05-treated animals—particularly at 100 mg/kg—exhibited markedly attenuated pathological alterations, including improved preservation of alveolar architecture, reduced inflammatory cell infiltration, and decreased edema. Collectively, these in vivo results demonstrate that PB05 confers dose-dependent protection against lethal influenza A virus infection, reflected by reduced viral replication, moderated inflammatory responses, and improved lung pathology.

3. Discussion

Influenza poses a major global health challenge, driven not only by viral cytopathic effects but also by dysregulated host immune responses. In severe cases, particularly among elderly individuals and patients with comorbidities, excessive cytokine release contributes to acute lung injury, ARDS, and multi-organ failure, representing a major cause of influenza-associated mortality [17,34]. Conventional antiviral agents, such as neuraminidase inhibitors (oseltamivir) and the PA inhibitor baloxavir marboxil, are designed primarily to halt viral propagation [9]. However, these agents are generally recommended for use during the early stage of viral infection, whereas most patients with influenza initiate treatment more than 48 h after symptom onset. As a result, they provide limited protection against immune-mediated tissue damage, which often plays a decisive role in patient outcomes [35].

For high-risk populations, including elderly or immunocompromised patients and those with chronic cardiopulmonary diseases, a therapeutic strategy that balances antiviral activity with modulation of excessive inflammation may provide added clinical benefit, even if its intrinsic antiviral potency is modest. By supporting broader host protection and attenuating cytokine-driven pathology, such an approach has the potential to reduce severe complications, lessen the need for intensive care, and improve overall outcomes [36]. This strategy aligns with insights gained from other severe respiratory infections, including COVID-19, where JAK inhibitors like baricitinib, used in combination with antivirals, have demonstrated survival benefits by blunting the hyperinflammatory response [37].

This study reports PB05, a small molecule developed through structural modification of the JAK inhibitor decernotinib to implement an integrated therapeutic strategy. PB05 concurrently targets the influenza virus RNA polymerase subunit PB2 and the host kinase JAK2. By inhibiting the PB2 cap-binding domain, PB05 interferes with the viral cap-snatching process, thereby limiting viral transcription and replication. In parallel, modulation of JAK2 signaling attenuates STAT2 phosphorylation and downstream interferon-stimulated gene expression, including pro-inflammatory cytokines such as IL-6 and IFN-β. PB05 exhibits distinct cytokine suppression characteristics compared to monotherapy. The PB2 inhibitor pimodivir primarily reduces cytokine production indirectly by lowering viral load, while the JAK inhibitor decernotinib broadly inhibits JAK-STAT signaling. PB05 combines both of these mechanisms of action. This dual mechanism enables PB05 to suppress not only the initial triggers of inflammation (viral PAMPs), but also directly attenuate the amplification effects of type I interferon (IFN) and interferon-stimulated genes (ISGs) by blocking the JAK-STAT pathway. Consequently, PB05 selectively and potently suppresses cytokines driven by the IFN-JAK-STAT pathway—such as IFN-β, ISGs (CXCL10, ISG15)—while substantially inhibiting IL-6. This synergistic suppression mechanism may mitigate immunopathological damage without completely compromising antiviral defenses, highlighting the therapeutic advantage of a dual-target strategy. This dual mode of action was supported in a lethal influenza mouse model, in which PB05 treatment was associated with reduced lung viral burden and alleviation of pulmonary inflammation, edema, and pathological injury. Although the direct antiviral potency of PB05 is lower than that of the PB2 inhibitor pimodivir, further structural optimization is ongoing to enhance PB2 inhibition and antiviral efficacy. Overall, this work supports a holistic therapeutic strategy for severe influenza based on PB2/JAK2 dual targeting, addressing both viral replication and host immune dysregulation.

In addition to its dual pharmacological actions, the pharmacokinetic profile of PB05 also merits discussion. Although this study primarily focuses on the biological effects of PB05 in JAK2 inhibition, we conducted a preliminary comparison of the pharmacokinetic characteristics of PB05 with the approved JAK inhibitor ruxolitinib through a literature review [38]. Based on available data, ruxolitinib exhibits a high C_max_ (9760 μg/L, 90 mg/kg) and AUC_0–∞_ (7250 h*μg/L, 90 mg/kg) in mice. In contrast, PB05 exhibits slightly lower oral absorption than ruxolitinib but remains at a high level, ensuring adequate drug uptake and utilization in vivo. Data indicate PB05’s half-life (T_1/2_) is approximately 4.02 h, about 2.7 times that of ruxolitinib, suggesting it may provide longer-lasting exposure in vivo, potentially facilitating sustained therapeutic effects. Additionally, PB05’s bioavailability (F) is approximately 44.9%, indicating moderate oral absorption efficiency. Ruxolitinib typically exhibits moderate to high oral bioavailability in comparable studies, though direct numerical comparisons require identical experimental conditions. In summary, compared to ruxolitinib, PB05 exhibits a longer half-life, lower systemic clearance, and moderate oral bioavailability, potentially attributable to differences in chemical structure and metabolic pathways. Future studies will systematically evaluate PB05’s metabolic stability, bioavailability, and direct pharmacokinetic comparisons with other JAK inhibitors to comprehensively assess its development potential.

4. Materials and Methods

4.1. General Chemistry

All chemicals and solvents were purchased from commercial suppliers and used without further purification unless otherwise stated. Flash chromatography was performed using silica gel (200–300 mesh). All reactions were monitored by thin-layer chromatography (TLC), using silica gel plates with fluorescence F_254_ and UV light visualization. Melting points were determined on an SGW X-4A micro melting point apparatus. ^1^H NMR and ^13^C NMR spectra were recorded on a Bruker AV-400 spectrometer and Bruker AV-600 spectrometer. Coupling constants (J) are expressed in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to an internal control (TMS). Low- resolution ESI-MS were recorded on an Agilent 1200 HPLC-MSD mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) and high-resolution ESI-MS on an Applied Biosystems Q-STAR Elite ESI-LC-MS/MS mass spectrometer (Thermo Fisher, Franklin, MA, USA). HPLC instrument for purity analysis was as follows: Dionex Summit HPLC (DIONEX, Ontario, CA, USA) (column: AD-H, 5.0 μM, 4.6 mm × 250 mm). The mobile phase consisting of hexane/isopropyl alcohol (80:20, v/v) was delivered at a flow rate of 1.0 mL/min.

4.1.1. (R)-2-((5-Fluoro-2-(5-fluoro-1H-pyrrolo [2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-N,N,2-trimethylbutanamide (PB02)

At room temperature, compound 8 (528 mg, 1.0 mmol) was dissolved in 5 mL of methanol, followed by the dropwise addition of 2 mL of a sodium methoxide solution in methanol (25% w/w). The reaction mixture was stirred at room temperature for 1 h, after which TLC analysis indicated complete consumption of the starting material. The pH was adjusted to 7 with a saturated NH_4_Cl solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 3) and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol = 80:1 to 40:1) to afford a white solid PB02 with a melting point of 205–207 °C (307 mg, 0.83 mmol, yield 83%). ^1^H NMR (600 MHz, DMSO-d6) δ 12.21 (s, 1H), 8.47 (dd, J = 9.8, 2.8 Hz, 1H), 8.24 (dd, J = 2.8, 1.4 Hz, 1H), 8.22 (d, J = 3.9 Hz, 1H), 8.16 (d, J = 2.8 Hz, 1H), 7.73 (s, 1H), 2.97 (s, 3H), 2.74 (s, 3H), 2.19 (dt, J = 15.9, 8.1 Hz, 1H), 1.95 (dq, J = 14.7, 7.4 Hz, 1H), 1.51 (s, 3H), 0.81 (t, J = 7.5 Hz, 3H). ^13^C NMR (151 MHz, DMSO-d6) δ 172.2, 157.3 (d, J = 6.6 Hz), 155.7 (d, J = 239.9 Hz), 149.6 (d, J = 10.8 Hz), 145.9, 143.3 (d, J = 255.8 Hz), 138.8 (d, J = 18.0 Hz), 131.5, 131.3 (d, J = 28.9 Hz), 117.8 (d, J = 7.9 Hz), 115.2 (d, J = 21.7 Hz), 113.9 (d, J = 4.2 Hz), 59.8, 37.1 (2C), 29.1, 22.1, 8.0. ESI-HRMS: m/z [M + H]^+^ calcd for C_18_H_21_F_2_N_6_O: 375.1739; found: 375.1737.

4.1.2. (S)-2-((5-Fluoro-2-(5-fluoro-1H-pyrrolo [2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)-2-methylbutan-1-ol (PB03)

At room temperature, compound 10 (90 mg, 0.26 mmol) was dissolved in 3 mL of tetrahydrofuran, followed by the dropwise addition of 0.52 mL of a 1 M LiAlH_4_ solution in THF (0.52 mmol). The reaction mixture was stirred at 45 °C for 12 h, after which TLC analysis confirmed complete consumption of the starting material. The mixture was cooled to room temperature and diluted with ethyl acetate. The reaction was quenched by the portionwise addition of sodium sulfate decahydrate. After filtration, the filtrate was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1 to 1:1) to afford a white solid PB03 (63 mg, 0.19 mmol, yield 73%) with a melting point of 214–216 °C. ^1^H NMR (600 MHz, DMSO-d6) δ 12.27 (s, 1H), 8.43 (dd, J = 9.8, 2.8 Hz, 1H), 8.28 (d, J = 1.5 Hz, 1H), 8.17 (d, J = 3.9 Hz, 1H), 8.11 (d, J = 2.8 Hz, 1H), 6.31 (s, 1H), 4.97 (s, 1H), 3.79 (d, J = 10.8 Hz, 1H), 3.60 (s, 1H), 2.13 (dd, J = 14.0, 7.3 Hz, 1H), 1.90 (dd, J = 14.0, 7.3 Hz, 1H), 1.42 (s, 3H), 0.83 (t, J = 7.5 Hz, 3H). ^13^C NMR (151 MHz, DMSO-d6) δ 157.5 (d, J = 6.7 Hz), 156.0 (d, J = 239.9 Hz), 152.0 (d, J = 9.4 Hz), 146.4, 144.2 (d, J = 255.6 Hz), 138.4 (d, J = 18.4 Hz), 131.9 (d, J = 28.8 Hz), 130.9, 118.4 (d, J = 7.6 Hz), 115.3 (d, J = 21.8 Hz), 114.4 (d, J = 3.8 Hz), 65.7, 58.8, 27.4, 21.7, 8.4. ESI-HRMS: m/z [M + H]^+^ calcd for C_16_H_18_F_2_N_5_O: 334.1474; found: 334.1477.

4.1.3. (R)-N2-(5-Fluoro-2-(5-fluoro-1H-pyrrolo [2,3-b]pyridin-3-yl)pyrimidin-4-yl)-2-methylbutane-1,2-diamine (PB04)

At room temperature, compound 12 (486 mg, 1.0 mmol) was dissolved in 5 mL of methanol, followed by the dropwise addition of 2 mL of a sodium methoxide solution in methanol (25% w/w). The reaction mixture was stirred at room temperature for 1 h, and TLC analysis confirmed complete consumption of the starting material. The pH was adjusted to 7 with saturated ammonium chloride solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 3) and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the crude product was purified by silica gel column chromatography (dichloromethane: methanol = 70:1 to 50:1) to afford a white solid PB04 (316 mg, 0.95 mmol, yield 95%) with a melting point of 187–189 °C. ^1^H NMR (600 MHz, DMSO-d6) δ 8.43 (dd, J = 9.8, 2.9 Hz, 1H), 8.30–8.27 (m, 1H), 8.16 (d, J = 3.9 Hz, 1H), 8.11 (s, 1H), 6.56 (s, 1H), 3.02 (d, J = 12.8 Hz, 1H), 2.72 (d, J = 12.8 Hz, 1H), 2.05 (dd, J = 14.0, 7.3 Hz, 1H), 1.97 (dd, J = 14.0, 7.2 Hz, 1H), 1.41 (s, 3H), 0.82 (t, J = 7.5 Hz, 3H).

4.1.4. (1R,2S)-2-((5-Fluoro-2-(5-fluoro-1H-pyrrolo [2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexan-1-ol (PB05)

At room temperature, compound 13A (675 mg, 1.35 mmol) was dissolved in 5 mL of methanol, followed by the dropwise addition of 2 mL of a sodium methoxide solution in methanol (25% w/w). The reaction mixture was stirred at room temperature for 1 h, after which TLC analysis indicated complete consumption of the starting material. The pH was adjusted to 7 with a saturated NH_4_Cl solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 3) and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 55:1 to 25:1) to afford a white solid PB05 (373 mg, 1.08 mmol, yield 80%) with a melting point of 219–221 °C. ^1^H NMR (400 MHz, DMSO-d6) δ 12.26 (s, 1H), 8.41 (dd, J = 9.9, 2.9 Hz, 1H), 8.28 (dd, J = 3.0, 1.5 Hz, 1H), 8.21 (s, 1H), 8.16 (d, J = 3.9 Hz, 1H), 6.82 (d, J = 7.3 Hz, 1H), 4.83 (d, J = 3.9 Hz, 1H), 4.14–4.04 (m, 2H), 1.85–1.70 (m, 4H), 1.60 (d, J = 9.8 Hz, 2H), 1.42 (d, J = 9.7 Hz, 2H). ^13^C NMR (151 MHz, DMSO-d6) δ 158.2 (d, J = 6.5 Hz), 156.0 (d, J = 239.9 Hz), 151.4 (d, J = 10.9 Hz), 146.4, 143.9 (d, J = 254.2 Hz), 138.8 (d, J = 17.7 Hz), 131.7 (d, J = 28.7 Hz), 130.9, 118.7 (d, J = 7.6 Hz), 115.3 (d, J = 21.7 Hz), 114.2 (d, J = 3.9 Hz), 66.3, 53.3, 32.5, 26.3, 24.9, 19.7. ESI-HRMS: m/z [M + H]^+^ calcd for C_17_H_18_F_2_N_5_O: 346.1474; found: 346.1472.

4.1.5. (1S,2S)-2-((5-Fluoro-2-(5-fluoro-1H-pyrrolo [2,3-b]pyridin-3-yl)pyrimidin-4-yl)amino)cyclohexan-1-ol (PB01)

At room temperature, compound 13B (500 mg, 1.0 mmol) was dissolved in 5 mL of methanol, followed by the dropwise addition of 2 mL of a sodium methoxide solution in methanol (25% w/w). The reaction mixture was stirred at room temperature for 1 h, and TLC analysis confirmed complete consumption of the starting material. The pH was adjusted to 7 with saturated ammonium chloride solution, and the mixture was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with saturated brine (20 mL × 3) and dried over anhydrous sodium sulfate. After filtration and concentration under reduced pressure, the crude product was purified by silica gel column chromatography (dichloromethane:methanol = 55:1 to 25:1) to afford a white solid PB01 (252 mg, 0.73 mmol, yield 73%) with a melting point of 207–209 °C. ^1^H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 8.48 (dd, J = 9.9, 2.9 Hz, 1H), 8.28 (dd, J = 2.9, 1.4 Hz, 1H), 8.20 (d, J = 2.5 Hz, 1H), 8.12 (d, J = 4.0 Hz, 1H), 7.32 (d, J = 7.6 Hz, 1H), 4.73 (d, J = 5.0 Hz, 1H), 4.01–3.89 (m, 1H), 3.55 (td, J = 9.8, 4.9 Hz, 1H), 2.12–1.94 (m, 2H), 1.79–1.65 (m, 2H), 1.38–1.24 (m, 4H). ^13^C NMR (151 MHz, DMSO-d6) δ 158.1 (d, J = 6.4 Hz), 156.1 (d, J = 239.8 Hz), 152.3 (d, J = 10.9 Hz), 146.4, 144.0 (d, J = 254.2 Hz), 138.5 (d, J = 18.0 Hz), 131.7 (d, J = 28.9 Hz), 130.8, 118.8 (d, J = 7.7 Hz), 115.5 (d, J = 21.8 Hz), 114.4 (d, J = 3.9 Hz), 71.5, 56.9, 35.2, 31.3, 25.0, 24.7. ESI-HRMS: m/z [M + H]^+^ calcd for C_17_H_18_F_2_N_5_O: 346.1474; found: 346.1472.

4.2. Biological Activity Assay

4.2.1. Molecular Docking Assay

The three-dimensional structure of the PB2 cap-binding protein was retrieved from the RCSB Protein Data Bank (PDB ID: 6EUV). The AMBER ff14SB force field and the AM1-BCC charge model were applied to the protein and the ligand, respectively. The docking procedure was performed using UCSF Dock 6.7. A grid-based scoring function was constructed by integrating the electrostatic and van der Waals interactions between PB05 and the binding site of the PB2 cap domain. Subsequently, cluster analysis was conducted with a root-mean-square deviation (RMSD) threshold of 2.0 Å to identify the optimal binding pose. A similar docking protocol was applied to PB05 with the JAK2 protein, in which the target protein was replaced by JAK2 (PDB ID: 4YTI).

4.2.2. Materials, Virus, Cell Lines and Culture Methods

Anti-NP (#GTX636247), anti-PB2 (GTX125926) and Anti-rabbit FITC-labeled secondary antibodies were purchased from GeneTex Inc. (Irvine, CA, USA). Anti-Stat2 (D9J7L) (#72604) and anti-phospho-Stat2 (Tyr690) (D3P2P) (#88410) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH (FD0063), β-actin (FD0060), horseradish peroxidase-labeled goat anti-mouse IgG (h + l) (FDM007), and horseradish peroxidase-labeled goat anti-rabbit IgG (h + l) (FDR007) antibodies were purchased from Fude Bio (Hangzhou, China).

Madin–Darby canine kidney (MDCK), 293T (a human embryonic kidney cell line), and mouse monocyte-macrophage leukemia cells (RAW264.7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. A549 (a human lung cancer cell line of alveolar epithelial cell origin) and THP1 (human monocyte leukemia cells) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. All of these cells were purchased from ATCC (Manassas, VA, USA) and frozen in liquid nitrogen after expansion culture in our laboratory.

IAV subtypes, including A/WSN/1933 (H1N1), A/FM-1/1/47 (H1N1), A/Aichi/2/68 (H3N2), A/Puetro Rico/8/34 (H1N1), A/PR/8/34 with NA-H274Y, and A/PR/8/34 (H1N1) murine lung-adapted strain virus, were propagated in the allantoic cavities of 9-day-old embryonated hen eggs at 37 °C for 48 h. The supernatant was centrifuged at 4 °C, 3000 rpm for 10 min and stored at −80 °C until use. Virus titers were measured via 10-fold serial dilutions (10^−1^ to 10^−9^) by infecting MDCK cells. The 50% tissue culture infecting dose (TCID_50_/mL) was calculated according to the Reed and Muench method.

Plasmids pHPHW2K-NP, pHW2K-PA, pHW2K-PB1, pHW2K-PB2, and pPolI-Fluc (firefly luciferase reporter plasmid) were generously provided by Professor Zheng Bojian (The University of Hong Kong, China). hRluc-TK (Renilla luciferase reporter plasmid) was purchased from Promega (Madison, WI, USA). pISRE-TA-luc (reporter gene plasmid) was purchased from Huayun Bio (Guangzhou, China).

BALB/c mice aged 6–8 weeks, with equal numbers of males and females (specific pathogen-free), were purchased from Guangzhou Southern Medical University Laboratory Animal Science Development Co., Ltd (Guangzhou, China). All animal research protocols were approved by the Animal Welfare Committee, and experimental procedures strictly adhered to the guidelines and policies of the Animal Care and Use Committees at each research institution.

4.2.3. Antiviral Activity, Cytotoxicity Assay, and Microscopic Observation

MDCK cells were seeded at a density of 1.5 × 10^5^ cells/mL in a 96-well plate. After the cells formed a confluent monolayer, they were infected with A/WSN/1933(H1N1) (MOI = 0.01) for 1 h. PB05 and pimodivir were diluted 2-fold in DMEM containing 1 μg/mL TPCK (Sigma-Aldrich, St. Louis, MO, USA) and added to wells after removing the viral solution. At 48 h post-infection (hpi), cytopathic effects (CPE) of virus-infected cells were observed microscopically, and antiviral activity was assessed using the CCK-8 (TargetMol, Shanghai, China) assay. For cytotoxicity assays, simply dilute the compound 2-fold in DMEM and add 200 μL per well to the cells. After 48 h, observe cell status under a microscope and assess compound cytotoxicity via CCK-8 assay.

4.2.4. Western Blot Analysis

Cells or lung tissue were thoroughly lysed at low temperature for 30 min using RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was quantified using the BCA protein assay kit. Samples were then resuspended in 5× Loading Buffer, vortexed thoroughly, and denatured by boiling at 105 °C to ensure complete protein denaturation. Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membrane was blocked at room temperature for 1 h in 5% (w/v) defatted milk dissolved in Tris buffer, then incubated overnight at 4 °C with the primary antibody. The following day, the membrane was washed and incubated with secondary antibody at room temperature for 1 h. The membrane was then exposed and developed using multifunctional imaging analysis system (Protein Simple, San Jose, CA, USA), with protein bands quantified and statistically analyzed using ImageJ 1.8.0.

4.2.5. Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted from virus-infected cells or lung tissue using an RNA extraction kit (Vazyme, Nanjing, China). The RNA was reverse-transcribed into cDNA. A qPCR reaction system was prepared containing 2 × concentrated real-time qPCR MasterMix, H_2_O, primers, and template cDNA. Three parallel replicates were set up. The prepared reaction mixture was loaded into a qPCR instrument (Roche LightCycler 480 II, Basel, Switzerland) for detection. Data processing and statistical analysis were performed. The primer sequences used are shown in Table S2.

4.2.6. Plaque Reduction Assay (PRA)

MDCK cells were seeded at 5 × 10^5^ cells/well density in 12-well plates for culture. After confluency was achieved, cell culture supernatant was collected per well from a virus-infected 12-well plate. After confluency, cells were incubated with virus-infected cell supernatant or lung tissue homogenate supernatant for 1~2 h. Cultures were then maintained at 37 °C, 5% CO_2_ in 2× DMEM supplemented with 1 μg/mL TPCK trypsin and 2% microcrystalline cellulose for 48 h. After removing the medium, cells were fixed and stained with crystal violet dissolved in 4% paraformaldehyde (Bio-Channel, Nanjing, China). The number of plaques formed was counted, and the viral titer was calculated based on the dilution factor.

4.2.7. Immunofluorescence Microscopy

MDCK cells in 48-well plates were infected with A/WSN/1933 (H1N1) (MOI = 0.01) for 1 h. The virus solution was removed and different dilutions of PB05 and the positive drug pimodivir were added. The cells were further cultured for 24 h. Then, the cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton-100 for 10 min. Afterward, the cells were blocked with 3% BSA at room temperature for 1 h. They were incubated with nucleoprotein (NP) antibody overnight at 4 °C. The next day, the cells were incubated with FITC-labeled secondary antibody at room temperature and in the dark for 1 h. The cells were stained with a mounting medium containing DAPI for 10 min and observed under a fluorescence microscope (Nikon, Tokyo, Japan).

4.2.8. Mini-Replicon Assay

The mini-replicon system is used to detect the RNA-dependent RNA polymerase activity of influenza viruses in cells. 50 ng of PB1, PB2, PA, and NP plasmids, along with 50 ng of pPolI-Fluc (firefly luciferase reporter plasmid) and 10 ng of hRluc-TK (Renilla luciferase plasmid), were transfected into 293T cells using PolyJet. Five hours post-transfection, the supernatant was replaced with fresh FBS-containing medium supplemented with PB05. After 48 h of incubation, cells were lysed for 20 min, and luciferase activity was measured using the luciferase reporter assay kit provided by Promega (Madison, WI, USA).

4.2.9. Fluorescence Polarization (FP) Assay

A 50 μL aliquot of the PB2 cap-binding domain sample was serially diluted using the assay buffer (100 mM KCl, 1 mM DTT, 0.5 mM EDTA, 50 mM HEPES, 1% DMSO, pH = 7.2). The diluted samples were transferred to a black 96-well plate. FITC-labeled m7GTP (EDA-m7GTP-ATTO 488; Jena Bioscience, Jena, Germany) was employed as the cap structure mimic probe. The 20 nM probe was incubated with the serially diluted PB2 cap-binding domain at room temperature for 30 min. Subsequently, gradient-diluted PB05 (at 2 × final concentration gradient) was added and the incubation was continued for another 30 min. The fluorescence polarization was ultimately measured using an Infinite M1000 Pro microplate reader (Tecan, Mannedorf, Switzerland).

4.2.10. JAK2 Kinase Inhibition Assay

A 10 μL kinase reaction containing 5 ng recombinant human JAK2, 1 μM ATP, 2 μM peptide substrate, and serially diluted PB05 in assay buffer (50 mM HEPES, pH 7.5, 10 mM MgCl_2_, 2 mM DTT, 0.01% Tween-20) was incubated for 60 min at 25 °C. The reaction was terminated by adding 10 μL EDTA-containing detection buffer with Europium cryptate-labeled anti-phosphotyrosine antibody and Streptavidin-XL665. After 60 min of incubation, HTRF signals were measured at 620 nm and 665 nm using a Spark microplate reader (Tecan, Mannedorf, Switzerland). Dose–response curves were fitted to calculate IC_50_ values.

4.2.11. Surface Plasmon Resonance (SPR)

Prior to the experiment, PB2 cap protein was immobilized onto the sensor surface. The DMSO stock solution of compound PB05 was serially diluted with 1‰ DMSO running buffer to concentrations of 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM working solutions, along with a running buffer (blank) without analyte as the zero concentration point. During the experiment, samples of varying concentrations were sequentially injected onto the chip from low to high concentration. Binding and dissociation processes were monitored in real time. Data were exported using Plexera DE software, adjusted with BIA Evolution software 4.1, and K_D_ values were calculated.

4.2.12. Influenza Virus Infection Mouse Model

BALB/c mice were purchased from Guangzhou Southern Medical University Laboratory Animal Science and Technology Development Co., Ltd. (Guangzhou, China). Six-week-old BALB/c mice (evenly distributed by gender) were randomly assigned to experimental groups. Under isoflurane inhalation anesthesia, the mice were intranasally inoculated with 50 μL of A/Puerto Rico/8/34 (H1N1) virus diluted in phosphate-buffered saline (PBS). At 24 h post-infection, the mice received oral gavage administration of the test compound (25, 50, or 100 mg/kg/day), a placebo as negative control, or pimodivir (20 mg/kg/day) as positive control. Both PB05 and pimodivir were administered orally via gavage in solution form. Compounds were dissolved using a formulation ratio of 10% DMSO + 40% PEG300 + 50% ddH_2_O. The mixture was thoroughly homogenized via ultrasonication and vortexing to form a uniform suspension. The suspension was shaken well prior to administration to ensure accurate dosing. Treatments were continued once daily for five consecutive days. On day 5 post-infection, all mice were euthanized by cervical dislocation, and lung tissues were collected for further analysis.

4.2.13. Statistical Analysis

All statistical analyses of data were performed using GraphPad Prism 10.0 software. Each experiment was conducted in triplicate, and results are expressed as mean ± standard deviation (SD). Statistical significance between groups was assessed using t-tests. p values < 0.05 were considered statistically significant.