清華大學丁強團隊：布魯頓酪氨酸激酶抑制劑BTKi-2抑制猴痘病毒和痘苗病毒感染

作者

楊晨，趙潤初，霍恕婷，張亞青，馮婧熙，龔明麗，朱林，張從剛，張林琦，薛婧，張榮，王奇慧，丁強

單位

a清華大學基礎醫學院

b中國科學院微生物研究所，中國科學院病原微生物與免疫學重點實驗室

c中國醫學科學院醫學實驗動物研究所

d清華大學藥學院

e復旦大學上海醫學院基礎醫學院

摘要

近年來，全球猴痘病毒（mpox virus，MPXV）感染的暴發凸顯了開發正痘病毒抗病毒治療策略的迫切需求。本研究在生物安全二級條件下，建立了基于插入綠色熒光蛋白的改造型痘苗病毒（MVTT-GFP）的高內涵篩選平臺，并對 1,513 種激酶抑制劑進行了抗病毒活性篩選。結果顯示，布魯頓酪氨酸激酶抑制劑 BTKi-2 是一種具有顯著抗病毒活性的候選化合物，其在體外對痘苗病毒（vaccinia virus，VACV）和猴痘病毒的半數抑制濃度（IC50）分別為 0.535 μM 和 0.260 μM，且細胞毒性較低。在痘苗病毒感染的小鼠肺炎模型中，BTKi-2治療可使肺部病毒載量降低約 90%，并顯著提高小鼠存活率。進一步機制研究表明，BTKi-2的抗病毒作用不能完全歸因于對 BTK 或 EGFR/ErbB2 信號通路的抑制。上述結果提示，BTKi-2在體內外均具有抗正痘病毒活性，可作為未來開發抗正痘病毒療法的潛在候選藥物。

Fig. 1.Identification of BTK inhibitor-2 (BTKi-2) as a potent anti-VACV compound through high-content screening. A Representative images from high-content imaging depict MVTT-GFP-infected Huh7 cells treated with 10?μM ST-246 (positive control) or 0.1% (v/v) DMSO (negative control). Green fluorescence indicates infected cells, and blue fluorescence represents nuclei stained with DAPI. Scale bar: 200 μm. B Schematic of the high-content screening (HCS) assay workflow: Huh7 cells were infected with MVTT-GFP (MOI=0.02) and treated with compounds from the MCE kinase inhibitor library (1,513 compounds at 5?μM). ST-246 (5?μM) and DMSO (0.05%, v/v) served as positive and negative controls, respectively. After 24 hours, cells were fixed, stained with DAPI, and analyzed by HCS to quantify infection rates (GFP%) and cell counts (cytotoxicity). C Infection rates of positive (red) and negative (black) control wells treated with ST-246 (red, n=3 biological replicates) or DMSO (black, n=3 biological replicates). D Z’ factors of the screening plates. Plates with Z’ factors above 0.5 (dashed line) were considered qualified for further analysis. E Scatter plot of infection rates (% of DMSO) versus cell numbers (% of DMSO) for all tested compounds. DMSO and ST-246 are marked in blue and red, respectively. BTKi-2 (green) exhibited potent antiviral activity with minimal cytotoxicity.

Fig.2.BTKi-2 inhibits MVTT-GFP, ECTV, and MPXV infection in vitro. A Chemical structure of BTKi-2 (C24H25N5O3). B Dose-response curves for BTKi-2 (black, IC50; red, CC50) and ST-246 (blue). IC50 and CC50 values were calculated using GraphPad Prism software and represent one of three independent experiments. C Cytopathic effect protection assay in Huh7 and Vero cells treated with BTKi-2 or ST-246 at concentrations ranging from 0.02 to 5 μM. Cells were seeded in 24-well plates one day prior to infection with MVTT-GFP or ECTV at MOI = 0.02 or 0.2. At 72 hours (Huh7 cells) or 96 hours (Vero cells) post-infection, cells were fixed and stained with crystal violet. DMSO (0.05%, v/v) served as the negative control. D The IC50 value of BTKi-2 against ECTV was calculated using GraphPad Prism software based on CPE areas measured by ImageJ at different drug concentrations. E Schematic of the rdMPXVΔ96,158 cell culture model(Chen et al., 2025). Two genes indispensable for viral assembly, OPG96 and OPG158, were deleted and transcomplemented in CV-1-96,158 cells to ensure biosafety. Besides, the reporter expression cassette, mGreenLantern (mGreen)-P2A-Gaussia luciferase, under the control of the viral late promoter P11, was inserted into the thymidine kinase (tk) gene (OPG101) to visualize infection. F Dose-response curves for anti- rdMPXVΔ96,158 activity of BTKi-2 (black) and ST-246 (blue). IC50 values were calculated using GraphPad Prism software and represent one of three independent experiments.

Fig. 3.BTKi-2 disrupts the poxvirus replication cycle. A Time-of-Addition Assay Schematic. In Group 1, the drug (DMSO, ST-246, or BTKi-2) was added simultaneously with the virus (MOI=0.02) at 37°C for 1 h and then replaced with fresh medium. In Groups 2 and 3, drug treatment commenced 1-hour post-infection after virus attachment (4°C incubation, Group 2) or entry (37°C incubation, Group 3). Cells were fixed at 24 h p.i. and analyzed by flow cytometry for GFP positivity. B Infection rates of MVTT-GFP of each treatment group are shown. ST-246 (blue) served as a positive control, and DMSO (white) as a negative control. Statistical significance was determined by one-way ANOVA with Dunnett’s post hoc test; “ns”, no significance; ***P < 0.001. C Schematic representation of the VACV life cycle. Following entry, the virus initiates its replication cycle by transcribing and translating its genome to form immature virions (IVs), which mature into intracellular mature virions (MVs). MVs can be released via cell lysis or processed into wrapped virions (WVs) that are exocytosed as extracellular enveloped virions (EEVs). D Dose-dependent effects of BTKi-2 and ST-246 on MV and EEV yields at MOI=0.2. Huh7 cells infected with MVTT-GFP (MOI=0.2) were treated with DMSO (0.1%, v/v), 10 μM ST-246, or various concentrations of BTKi-2. At 24 h p.i., cell debris (for MVs) and supernatant (for EEVs) were collected and titrated. E Dose-dependent effects of BTKi-2 and ST-246 on MV and EEV yields at high MOI. Huh7 cells infected with MVTT-GFP (MOI=2) were treated with DMSO (0.1%, v/v), 10 μM ST-246, or various concentrations of BTKi-2. At 24 h p.i., cell debris (for MVs) and supernatant (for EEVs) were collected and titrated. F Relative quantification of viral gene mRNA and genomic DNA. Huh7 cells infected with MVTT-GFP (MOI = 0.2) were treated with DMSO, AraC (40 μg/mL), or BTKi-2 (10 μM). At 2 h p.i., E3L mRNA levels were quantified by RT-qPCR. At 8 h p.i., D13L and A3L mRNA levels were quantified by RT-qPCR, and viral genomic DNA levels were quantified by qPCR. Gene expression and viral DNA levels were normalized to GAPDH for comparison between groups (DMSO = 1). ns, not significant; ***P < 0.001.

Fig. 4.In vivo evaluation of BTKi-2 in a murine model of VACV-induced pneumonia. A Experimental design: BALB/c mice (n = 5 per group) received intraperitoneal (i.p.) injections of BTKi-2 (50 mg/kg), or vehicle (matching volume to BTKi-2 group). Three hours later, mice were intranasal (i.n.) challenged with VACV (1×106 PFU). From Day 1 to Day 5 post-infection (p.i.), BTKi-2 and vehicle groups received daily i.p. injections. B Kaplan-Meier survival analysis over 5 days p.i. for BTKi-2 (red), and vehicle (black) groups. Statistical significance was determined using a Log-rank (Mantel-Cox) test. *P < 0.05. C Body weight changes over 5 days p.i., presented as a percentage of initial weight (Day 0) for BTKi-2 (red), and vehicle (black) groups. Statistical significance was assessed by two-way ANOVA followed by Sidak's multiple comparisons test. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001. D Viral loads in lung were measured by quantitative real-time PCR (qPCR). Statistical significance was assessed by unpaired, two-tailed t-test. **P < 0.0.

Fig.5.The antiviral activity of BTKi-2 cannot be fully explained by BTK, EGFR, or ERBB2. A Total RNA from Huh7 cells was extracted, reverse transcribed, and the mRNA levels of the indicated kinases were quantified by RT-qPCR. Values were normalized to GAPDH mRNA levels. B Cells (2×105 per well in 24-well plates) were infected with 80 FFU of MVTT-GFP per well. After 72 h, cells were fixed with 4% paraformaldehyde (PFA), washed with PBS, and stained with crystal violet. “sgRNA1” and “sgRNA2” indicate different single-guide RNAs (sgRNAs) used for CRISPR/Cas9-mediated gene knockout. C Quantification of plaque diameters from (B). Each dot represents a single plaque, with the horizontal line indicating the mean plaque diameter. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001. D Cells with genes knockout as indicated were infected at an MOI of 0.02 and fixed for flow cytometry at 24 h post-infection (p.i.). E Viral yields (MV and EEV) in naive, NTC, EGFR KO, ERBB2 KO, and double KO cell lines. Cells were infected with MVTT-GFP at an MOI of 0.2. At 24 hours p.i., supernatants (for EEV) and cell debris (for MV) were collected and titrated. Data represent mean ± SD from three independent experiments. F Validation of BTK knockdown in HEK293T cells. HEK293T cells were transfected with 80 nM BTK-targeting siRNAs (siRNA-1, siRNA-2, and siRNA-3) or a negative control siRNA (siNC). At 48 h post-transfection, cells were harvested and lysed in 1× SDS loading buffer, followed by western blot analysis using anti-BTK and anti-β-actin antibodies. G Viral yields in BTK-knockdown HEK293T cells. HEK293T cells were transfected with 80 nM BTK-targeting siRNAs (siRNA-1, siRNA-2, and siRNA-3) or siNC for 48 h and then infected with MVTT-GFP at an MOI of 0.2 in the presence or absence of 2 μM BTKi-2. At 24 h p.i., cell debris were collected and titrated. Data represent mean ± SD from three independent experiments. Statistical significance was assessed by two-way ANOVA followed by Sidak’s multiple comparisons test. H Validation of BTK knockdown in HeLa cells. HeLa cells were transfected with 80 nM BTK-targeting siRNAs (siRNA-1, siRNA-2, and siRNA-3) or a negative control siRNA (siNC). At 48 h post-transfection, cells were harvested and lysed in 1 × SDS loading buffer, followed by western blot analysis using anti-BTK and anti-β-actin antibodies. I Viral yields in BTK-knockdown HeLa cells. HeLa cells were transfected with 80 nM BTK-targeting siRNAs (siRNA-1, siRNA-2, and siRNA-3) or siNC for 48 h and then infected with MVTT-GFP at an MOI of 0.2 in the presence or absence of 2 μM BTKi-2. At 24 h p.i., cell debris were collected and titrated. Data represent mean ± SD from three independent experiments. Statistical significance was assessed by two-way ANOVA followed by Sidak’s multiple comparisons test.

Supplementary Fig. S1.Dose-response determination and summary of positive hits from high-content screening. Kinase inhibitors with normalized infection < 20% and relative cell number > 80% were considered as potential antivirals, and their half-maximal inhibitory concentration (IC50, black) and half-maximal cytotoxic concentration (CC50, red) were determined. The table below summarizes IC50, CC50, and selectivity index (SI, CC50/IC50) values for each compound, as well as their reported molecular targets.

Supplementary Fig. S2.MVTT-GFP infection rates of cells treated with compounds annotated as BTK inhibitors in the HCS. Infection rates for all BTK-targeting compounds from HCS are shown as percentages relative to the DMSO control on the same plate. Detailed numerical values are listed in Table S1.

Supplementary Fig. S3.Dose-response determination of pirtobrutinib and zanubrutinib. The IC50 and CC50 values of another two BTK inhibitors were determined in Huh7 cells as previously described.

Supplementary Fig. S4.In vivo toxicity evaluation of BTKi-2 in BALB/c mice. A Experimental design. BALB/c mice (n = 3 per group) received intraperitoneal (i.p.) injections of BTKi-2 (10 μL/g body weight, equivalent to 50 mg BTKi-2/kg body weight) or vehicle (matched volume) from Day 0 to Day 5. B Body weight changes over 10 days, shown as a percentage of the initial body weight on Day 0, in the BTKi-2 (red) and vehicle (black) groups. Statistical significance was analyzed by two-way ANOVA followed by Sidak’s multiple-comparisons test. ns, not significant.

本文亮點

• BTKi-2 對痘苗病毒、猴痘病毒及鼠痘病毒均具有抗病毒活性

• BTKi-2 可破壞痘病毒 DNA 復制，并抑制病毒中期、晚期基因的后續表達

• BTKi-2 給藥能夠降低痘苗病毒肺炎模型小鼠的肺部病毒載量

• BTKi-2的抗病毒作用不能完全歸因于EGFR，ERBB2與BTK靶點

閱讀原文https://doi.org/10.1016/j.virs.2026.06.011

來源：中國病毒學英文版

編輯：吃一口小貓