Wei Wei,Qingxuan Zeng,Yan Wang,Xixi Guo,Tianyun Fan,Yinghong Li,Hongbin Deng,Liping Zhao,Xintong Zhang,Yonghua Liu,Yulong Shi,Jingyang Zhu,Xican Ma,Yanxiang Wang,Jiandong Jiang,Danqing Song
Discovery and identification of EIF2AK2 as a direct key target of berberine for anti-inflammatory effects
Berberine,Anti-inflammatory,Target identification,Chemoproteomic technology,Photoaffinity probe,EIF2AK2
Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF-κB, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1β, IL-6, IL-18 and TNF-α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders.
Discovery and identification of EIF2AK2 as a direct key target of berberine for anti-inflammatory effects

Using chemoproteomic techniques, we first identified EIF2AK2, eEF1A1, PRDX3 and VPS4B as direct targets of berberine (BBR) for its synergistically anti-inflammatory effects. Of them, BBR has the strongest affinity with EIF2AK2 via two ionic bonds, and regulates several key inflammatory pathways through EIF2AK2, indicating the dominant role of EIF2AK2. Also, BBR could subtly inhibit the dimerization of EIF2AK2, rather than its enzyme activity, to selectively modulate its downstream pathways including JNK, NF-κB, AKT and NLRP3, with an advantage of good safety profile. In EIF2AK2 gene knockdown mice, the inhibitory IL-1β, IL-6, IL-18 and TNF-α secretion of BBR was obviously attenuated, confirming an EIF2AK2-dependent anti-inflammatory efficacy. The results highlight the BBR's network mechanism on anti-inflammatory effects in which EIF2AK2 is a key target, and inhibition of EIF2AK2 dimerization has a potential to be a therapeutic strategy against inflammation-related disorders.

During the past 20 years, more and more new biological effects of berberine (BBR, Fig. 1A), a natural antimalarial drug in China, have been gradually discovered and elucidated, such as type 2 diabetes, hyperlipidemia, atherosclerosis, and Alzheimer's disease1, 2, 3, 4. Though its several direct proteomic targets, including ubiquitin-like with PHD and ring finger domains 1 (UHRF1), retinoid X receptor alpha (RXRα) and ephrin-B2, as well as NIMA-related kinase 7 (NEK7) and mitogen-activated protein kinase 7 (MKK7) recently reported by our group, have been identified5, 6, 7, 8, 9, 10, 11, the complex and ingenious network mechanism of BBR has not been fully clarified. It is also unclear which proteins are the key targets that simultaneously regulate multiple signaling pathways, and how the key proteins play a more dominant role. If the key targets of BBR can be illuminated, it not only helps us understand the nature of biological evolution, but also provides the druggable targets for new drug discovery, worthy of further investigation. Based on our previous research, we continued to search for the key direct targets against inflammation through activity-based proteome profiling (ABPP) techniques,12, 13, 14, 15, using new BBR diphenyl-ketone photoaffinity probes. We first captured and identified the eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2, also known as PKR), eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), peroxiredoxin 3 (PRDX3) and vacuolar protein sorting 4 homolog B (VPS4B) as direct targets of BBR for its synergistically anti-inflammatory effects, through in vitro binding assays and orthogonal conﬁrmatory experiments. Among these four proteins, BBR strongly binds to EIF2AK2 via ionic bonds with the Kd value of 7.12 μmol/L, and simultaneously regulates multiple inflammatory pathways through EIF2AK2, suggesting that EIF2AK2 serves as a key target of BBR for anti-inflammatory potency. Furthermore, BBR could subtly inhibit the dimerization rather than enzymatic activity to selectively suppress multiple inflammatory signaling pathways, such as c-Jun N-terminal kinase (JNK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), protein kinase B (AKT) and nod-like receptor protein 3 (NLRP3), with an advantage of good safety profile. The in vivo study further confirms that BBR suppresses inflammation reactions in an EIF2AK2-dependent manner. Our data demonstrate that EIF2AK2 acts as a key target of BBR to regulate multiple signaling pathways, enabling BBR to synergistically perform anti-inflammatory efficacies.

A series of new BBR photoaffinity probes with diphenyl-ketone as a photo cross-linking group (7a–c, 11a–d, 15a–c, Supporting Information Fig. S4) were synthesized and evaluated for their activities against several inflammatory factors in PMA (phorbol 12-myristate-13-acetate)-differentiated THP-1 (human monocytic cell line-1) cells (PMA-THP-1) triggered by LPS plus nigericin16, 17, 18, 19, 20. As shown in Fig. 1A–C and Fig. S4, probes 7a and 11b exhibited better potencies on interleukin-1β (IL-1β) suppression in a time- or dose-dependent manner, better than that of BBR. The suppressive potencies were further confirmed (Supporting Information Figs. S5 and S6) on IL-6, IL-8, and monocyte chemoattractant protein-1 (MCP-1), and then both probes were used for the next step. The overall process of target capture and identification using ABPP assay is presented in Fig. 1D. Firstly, both 7a and 11b (10 μmol/L) were incubated with PMA-THP-1 cells to profile the direct target proteins of BBR in situ. Followed by 365 nm light exposure, the highly active diphenyl-ketone free radicals rapidly generated and covalently bond to the hydroxyl groups or α-hydrogen atoms of the captured target proteins,. After being clicked with Cy3-azide and resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), bound targets of 7a and 11b can be labelled and visualized with fluorescence scanning, which could be competitively inhibited by BBR (100 μmol/L, Fig. 1E), indicating a similar mechanism of action to BBR. More significant changes in fluorescence intensity were observed in probe 11b treatment group at the concentrations ranging from 2.5 to 20 μmol/L (Fig. 1F and G), and it was chosen as a functional probe for the next chemoproteomic profiling.

Next, PMA-THP-1 cells were incubated with 11b (10 μmol/L) for 6 h with or without BBR (100 μmol/L) pretreated for 3 h, taking DMSO as the control. Followed by 365 nm light exposure, the labeled proteome was linked with Cy3 and separated by SDS-PAGE. As shown in Fig. 2A, and fluorescence intensity of several different bands with the molecular weights (MW) between 25 and 80 kDa were detected. Compared with the 11b group, the fluorescent bands under the UV (365 nm) exposure were obviously enhanced, owing to covalently capturing the target proteins of 11b. The labeled bands were distinctly attenuated followed by an addition of BBR, which again verified the specific labeling capacity of 11b. Then the labeled proteins were coupled with biotin–azide, 11b–biotin complexes were formed and puriﬁed by streptavidin magnetic beads and identiﬁed with LC–MS/MS analysis. The quantitative ratio of proteome in group 11b/11b plus BBR reflects the extent of competition by the native BBR, with a larger ratio corresponding to greater competition. The DMSO control group was set to eliminate false-positive targets resulting from nonspecific binding to the beads. Three replicates were performed for each of the control and competition experiments with a cutoff of 2.0 for the quantified ratio analysis. We collectively identified 44 proteins between 20 and 80 kDa as potential targets, which appeared in the protein list twice or more (Fig. 2B and Supporting Information Table S2). Gene Ontology (GO) enrichment indicated that the 44 proteins were closely involved in various inflammatory signaling pathways (Supporting Information Figs. S7A and B), of which 13 (yellow dots in Fig. 2C) were inflammation-related proteins through protein interaction analysis. By analyzing the comprehensive function and involved pathways of these proteins, six proteins, including EIF2AK2, eEF1A1, PRDX3, DEAD-box helicase 3 X-linked (DDX3X), Vasodilator stimulated phosphoprotein (VASP) and VPS4B, were closely related to BBR's indications23, 24, 25, 26, 27. Also, these six proteins could regulate two or more inflammatory pathways and have the potential to play a key role in BBR's anti-inflammatory potency28, 29, 30, 31, 32, 33, 34, 35, 36. More recently, we have demonstrated the important role of VASP in BBR's antiplatelet aggregation, and thus other five proteins were selected for the further investigation. Of the five proteins, except for DDX3X, the EIF2AK2, eEF1A1, PRDX3 and VPS4B were successfully confirmed to be the potential direct target proteins of BBR by immunoblot assay using the active probe 11b pulling down (Fig. 2F). Obvious competitive inhibition could be detected while 100 μmol/L BBR was pre-treated, indicating the possible specific interactions between BBR and these four proteins. Then, CETSA experiment was performed in HEK-293 cells to verify the interaction between BBR and the four target proteins. The thermal stability of EIF2AK2, eEF1A1, PRDX3 and VPS4B was enhanced in different degrees with the addition of BBR (10 μmol/L) ranging from 35 to 75 °C (Fig. 3A), indicating possible direct interactions between BBR and these four proteins. Among them, BBR showed minor interactions with eEF1A1 and PRDX3, which may be due to the strong thermal stability in 55 to 65 °C temperature range of these two proteins. Subsequently, SPR analysis was conducted using the recombinant proteins for further confirmation, and the Kd values of BBR with EIF2AK2, eEF1A1, PRDX3 and VPS4B were 7.12, 13.4, 23.7 and 16.8 μmol/L (Fig. 3B–E), respectively. Furthermore, molecular docking between BBR and the four proteins was carried out using Discovery Studio 4.5 software, and docking scores were respectively 100.2, 88.8, 86.7 and 91.6, basically consistent with the affinity values (Fig. 3F, Supporting Information Figs. S8–S10). These results again verified the specific interactions between BBR and these four target proteins, in which BBR had the strongest affinity with EIF2AK2. Meanwhile, EIF2AK2 can modulate multiple signal transduction pathways, and is responsible for several inflammation-related diseases, such as virus-infections, glucose/lipid metabolism disorders and neuro-inflammations,,38, 39, 40. These results suggested that EIF2AK2 might play a more dominant role than other three proteins, and thus we conducted in-depth study on the interaction pattern with BBR and the anti-inflammation function of EIF2AK2.

Based on the strong affinity between BBR and EIF2AK2, it is speculated that there may be ionic bonds between them. Then, two cavities containing different ionic-bond interactions were selected to conduct molecular docking using Discovery Studio 4.5 software. Eight key amino acid residues, including D432, K296, D414, F278, K416 in Cavity I (Fig. 3F, upper row), and D316, E367, K291 in Cavity II (Fig. 3F, lower row), are potential to play the key roles. The single-point mutation of the eight residues was carried out one by one to identify the binding sites of EIF2AK2. All eight residues in sequence were respectively converted into alanine (A) that cannot easily interact with its substrate (Fig. 3F), and only the mutations of D316, E367 and K291 in Cavity II significantly abolished the binding affinity between BBR and EIF2AK2 (Fig. 3G). The results indicated that BBR could act on EIF2AK2 mainly through two ionic bonds with D316 and E367 and one Pi–cation bond with K291 in Cavity II. Of note, this cavity is precisely involved in the dimerization of EIF2AK2 (Fig. 3F), which is responsible for the downstream cascade inflammation-related reactions,, suggesting that BBR might directly target dimerization of EIF2AK2 to regulate the anti-inflammatory effects. Then, we used an immunoprecipitation approach to further verify if BBR interferes with EIF2AK2 dimerization. HEK-293 cells were cotransfected with plasmids expressing both HA-tagged EIF2AK2 and FLAG-tagged EIF2AK2 to conveniently observe dimerization behavior. The cells were treated with TNF-α for 15 min before lysed to trigger EIF2AK2 dimerization, and then subjected to immunoprecipitation using anti-FLAG magnetic beads. As shown in Fig. 4A, HA- and FLAG-tagged EIF2AK2 were dimerized in the control experiment, which was signiﬁcantly promoted after TNF-α treatment, and BBR dramatically decreased the level of dimerization between HA- and FLAG-tagged EIF2AK2, demonstrating that BBR inhibited dimerization interaction of EIF2AK2. Since autophosphorylation happens after EIF2AK2 dimerization,,, the effect of BBR on EIF2AK2 autophosphorylation was carried out. As depicted in Fig. 4B, followed by suppression of EIF2AK2 dimerization, BBR significantly inhibited the EIF2AK2 autophosphorylation on residues Thr446, as well as EIF2AK2 substrate eIF2α phosphorylation, further indicating that BBR affected the downstream functions by acting on EIF2AK2 dimerization. Directly targeting EIF2AK2 dimerization or its autophosphorylation might lead to different down-stream effect. To further understand this difference, EIF2AK2 autophosphorylation inhibitor C16 (Fig. 5C) was chosen for the determination of activities on EIF2AK2 inhibition. Half maximal inhibitory concentration (IC50) of BBR and C16 was respectively 50.0 and 1.01 μmol/L (Supporting Information Fig. S11). Meanwhile, 50% cytotoxity concentration (CC50) of both was also tested and calculated in THP-1 cells. BBR showed a moderate cytotoxicity with a CC50 value of 91.4 μmol/L, much higher than that of C16 with CC50 of 1.13 μmol/L (Fig. S11). These results suggested that BBR could avoid possible toxicities by targeting EIF2AK2 dimerization to selectively exert related activities, which provided a possible explanation for its good safety profile of BBR.

To further understand the functional effect of EIF2AK2, we first validated the function of NLRP3 inflammasome by measuring IL-1β release in PMA-THP-1 cells. BBR (10 μmol/L) inhibits the expression of NLRP3 and then blocks the downstream IL-1β release, while EIF2AK2 was pharmacologically inhibited by its inhibitor C16, such reductions in NLRP3 expression and IL-1β release were not observed (Fig. 5D and E),, which indicated that BBR might suppress NLRP3 expression and IL-1β release through acting on EIF2AK2. As EIF2AK2 is relatively high-expressed in brain tissue,,, we further verified the correlation between EIF2AK2 and the inflammatory pathways in human-derived microglia (HMC3) cells. As expected, BBR could down-regulate p-NF-κB p65 and p-JNK induced by LPS (Fig. 5F and G), while the effect of BBR on suppressing p-NF-κB p65 and p-JNK was lost partially or completely after EIF2AK2 was knocked down. Furthermore, when EIF2AK2 was overexpressed, the BBR-dependent inhibition of p-NF-κB p65 was reversed and p-JNK was attenuated. These results hinted that BBR could affect cerebral inflammatory reactions through targeting EIF2AK2, and provided a possible mechanism how BBR exerted anti-inflammatory related diseases in the brain, such as gliosis and cognitive impairment in Alzheimer's disease,,,. In addition, EIF2AK2 acts as a key signaling protein in fatty acid-induced ER stress to bridge the signals between lipid metabolism and inflammation through promoting JNK activation, followed by down-regulating NAD-dependent protein deacetylase sirtuin-1 (SIRT1) expression,, among which SIRT1 is an important biomarker closely related to lipid metabolism. As shown Fig. 5H and I, BBR could inhibit the level of p-JNK induced by palmitic acid (PA) and up-regulate the expression of SIRT1 protein in HepG2 cells. Knocking down or overexpression EIF2AK2 signiﬁcantly neutralized the decreasing p-JNK level and increasing expression of SIRT1 mediated by BBR, compared with wild type cells. The results further demonstrated that BBR may regulate inflammation-induced lipid metabolism disorders through binding to the key protein EIF2AK2.

Furthermore, we selected the classic inflammation-related pathways each protein involved for their functional verification. Overexpressing or knock-down models of the four proteins at cellular level were established to investigate whether BBR could regulate different classical inflammatory pathways in a dependent manner, such as JNK, NF-κB, mitogen-activated protein kinase (MAPK) and AKT signals. Firstly, in HEK-293 cells, BBR inhibits the degradation of IκBα protein, and the level of JNK phosphorylation (p-JNK) and p-AKT induced by tumor necrosis factor-α (TNF-α) (Fig. 5A and B). When EIF2AK2 was silenced by siRNA, the inhibitory effect of BBR on degradation of IκBα and p-AKT was abolished, while the BBR-dependent down-regulation of p-JNK was attenuated (Fig. 5A). Moreover, overexpression of EIF2AK2 also reversed the inhibitory effect of BBR (Fig. 5B) on IκBα degradation, p-JNK and p-AKT respectively. Similarly, the regulatory effects of BBR on p-JNK and p-NF-κB p65 were attenuated in eEF1A1 overexpressing or knock-down models (Fig. 5C and D),. Also, we have observed consistent results of BBR on p-NF-κB p65 and p-P38 MARK regulations, while PRDX3 was overexpressed or knocked down in HEK-293 cells (Fig. 5E and F). Finally, the decreased efficacy of BBR on p-P38 MARK signaling with VSP4B overexpressed or knocked down was observed (Fig. 5G and H). These results indicated that BBR modulates multiple signaling pathways including JNK, NF-κB, MAPK and AKT, through EIF2AK2, eEF1A1, PRDX3 and VPS4B dependent manners respectively, among which EIF2AK2 plays a dominant role, and then in vivo experiment was conducted for further confirmation.

It is known that IL-1β, IL-6, IL-18 and TNF-α secretions are closely linked to several factors, such as inflammasome NLRP3 expression, and activations of JNK and NF-κB pathways52, 53, 54, 55, and then we further demonstrated whether BBR affects IL-1β, IL-6, IL-18 and TNF-α release by targeting EIF2AK2 in vivo. Thus, an EIF2AK2 knockdown mouse model was successfully established by 35 days of intravenous injection of adeno-associated virus (AAV) shEIF2AK2 in mice to evaluate the effects of BBR on IL-1β, IL-6, IL-18 and TNF-α (Fig. 6A), the expression of EIF2AK2 was significantly decreased compared with the control group (Fig. 6B). Then, wild type mice and EIF2AK2 knockdown mice were respectively injected with BBR twice at 24 h intervals via intraperitoneal injection (i.p.) at a dose of 3 mg/kg, and then LPS was administrated. BBR significantly reduced the production of IL-1β, IL-6, IL-18 and TNF-α in the control group (Fig. 6B–F), while the effect of BBR was partly weakened in the EIF2AK2 gene knockdown group, indicating the EIF2AK2-relevant effect of BBR on IL-1β, IL-6, IL-18 and TNF-α. H&E staining of liver results (Fig. 6G) showed that the alleviation on inflammatory infiltration of liver by BBR was weakened in the EIF2AK2 gene knockdown mice. No significant lesions were found in other tissues (Supporting Information Fig. S12), consistent with the above conclusions. These results demonstrated that BBR might downregulate IL-1β, IL-6, IL-18 and TNF-α secretions via targeting EIF2AK2 with a good safety profile.

Since ionic bond is the strongest noncovalent bond, we consider that BBR owns the strongest binding force with EIF2AK2 via two ionic bonds. Meanwhile, EIF2AK2 modulates multiple signal transduction pathways, including AKT, JNK and NF-κB pathways in an EIF2AK2-dependent manner. Therefore, among the four newly identified direct targets of BBR for anti-inflammatory effects, including EIF2AK2, eEF1A1, PRDX3 and VPS4B, EIF2AK2 serves as a key target to treat several inflammation related diseases. Combining NEK7 and MKK7 recently reported by our group, BBR also directly targets EIF2AK2, eEF1A1, PRDX3 and VPS4B, to synergistically alleviate downstream inflammatory response and cytokines release, and the net mechanism of BBR's anti-inflammatory action was outlined as shown in Fig. 7. Since BBR exerts its diverse pharmacological activities through a multi-target network mechanism, it is necessary to further explore more direct targets and clarify the contribution of each target, in order to fully elucidate the complex and ingenious network mechanism, as well as the corresponding biological effects. In addition, the dimerization of EIF2AK2 followed by autophosphorylation leads to the full catalytic activation, which can trigger lots of typical inflammatory pathways and inflammation related diseases, such as virus-infections, glucose/lipid metabolism disorders and neuroinflammations,. Different from C16 targeting EIF2AK2 enzyme activity, BBR directly inhibits the dimerization of EIF2AK2 to exert synergistic anti-inflammatory effects with an advantage of good safety profile. Thus, inhibition of EIF2AK2 dimerization may be developed into an important therapeutic target for inflammation-related diseases.

Along with 10% FBS, DMEM was used for HEK-293 and HepG2 cells culture. RPMI with 10% FBS was applied for THP-1 cells culture. After 50 nmol/L PMA was added, THP-1 cells were differentiated for 1–2 days, before they were stimulated. Then, along with 10% FBS, HMC3 cells were cultured by MEM. For induction of inflammasome activation, PMA-THP-1 cells were plated in 6-well plates, and primed for 3 h with ultrapure lipopolysaccharide (LPS, 1 μg/mL) with or without BBR pre-treatment, then the cells were stimulated for 30 min with nigericin (5 μmol/L). The precipitated supernatants were analyzed by ELISA. For stimulated classical inflammatory pathways, HEK-293 cells were plated in 6-well plates, and transfected with EIF2AK2 siRNA or EIF2AK2 plasmid for 48 h, then cells were stimulated by TNF-α for 15 min with or without BBR pretreatment for 6 h. The cell extracts were analyzed by immunoblot. For stimulated neuro-inflammation, HMC3 cells were transfected with EIF2AK2 siRNA or EIF2AK2 plasmid for 48 h, then cells were stimulated by LPS for 4 h with or without and BBR pretreatment for 6 h. The cell extracts were analyzed by immunoblot. For stimulated hepatocyte steatosis, HepG2 cells were transfected with EIF2AK2 siRNA or EIF2AK2 plasmid for 24 h, then cells were stimulated by PA for 24 h with or without BBR pretreatment for 2 h. The cell extracts were analyzed by immunoblot.

The synthetic schemes and general procedures of 7a–c, 11a–d and 15a–c were shown in Supporting Information Figs. S1–S3 and synthesis section, respectively. Synthesis of 7a. (3-(4-Benzoylphenyl)-1-(ethynylamino)-1-oxopropan-2-yl)carbamic acid (1.1 eq), EDCI (1.2 eq), HOBT (1.2 eq) and triethylamine (2.8 eq) were mixed in 5 mL DMF. After stirring for 1 h, compound 6a was added and the mixture was stirred overnight. After the solvent was removed, the residue was purified through flash chromatography using CH2Cl2/CH3OH to obtain 7a as yellow solid, yield 41%; Mp: 89–91 °C; purity 96.7%; 1H NMR (500 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.92 (s, 1H), 8.37 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.96 (d, J = 2.0 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.80 (s, 1H), 7.74–7.72 (m, 2H), 7.70–7.69 (m, 2H), 7.68 (s, 1H), 7.67–7.66 (m, 2H), 7.65–7.63 (m, 2H), 7.10 (s, 1H), 6.18 (s, 2H), 4.94–4.92 (m, 2H), 4.55–4.53 (m, 1H), 4.49–4.47 (m, 2H), 4.11 (s, 2H), 3.19–3.16 (m, 6H), 2.89 (s, 1H), 2.76–2.74 (m, 4H), 1.93–1.91 (m, 2H); 13C NMR (126 MHz, DMSO-d6) δ 196.2, 173.6, 170.8, 150.5, 150.2, 148.4, 146.0, 144.4, 143.8, 143.5, 137.8, 137.7, 135.8, 135.7, 133.3, 132.2, 130.3, 130.2, 130.1, 129.3, 129.2, 129.2, 128.4, 124.3, 122.0, 121.1, 120.9, 119.4, 109.1, 106.1, 102.8, 84.3, 72.0, 67.6, 62.6, 54.5, 53.9, 38.6, 37.5, 34.6, 29.5, 27.0, 14.7. HRMS: calcd for C43H40N3O7Cl [M–Cl]+ 710.2863, found 710.2861. 11b. Yield: 39%; Mp: 90–92 °C; purity 97.9%; 1H NMR (500 MHz, DMSO-d6) δ 9.87 (s, 1H), 8.92 (s, 1H), 8.31 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H), 8.10 (t, J = 5.5 Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.5 Hz, 2H), 7.79 (s, 1H), 7.70 (d, J = 1.5 Hz, 2H), 7.68 (d, J = 3.5 Hz, 2H), 7.65 (d, J = 8.5 Hz, 2H), 7.62 (s, 1H), 7.09 (s, 1H), 6.18 (s, 2H), 4.92 (t, J = 6.5 Hz, 2H), 4.48 (d, J = 5.0 Hz, 1H), 4.25 (t, J = 6.5 Hz, 2H), 4.09 (s, 3H), 3.17 (m, 4H), 2.99 (m, 2H), 2.74 (d, J = 2.5 Hz, 1H), 2.28 (m, 4H), 1.80 (m, 2H), 1.43 (s, 2H), 1.23 (s, 2H); 13C NMR (126 MHz, DMSO-d6) δ 196.0, 170.7, 170.6, 150.3, 150.2, 148.2, 144.2, 143.7, 143.3, 137.9, 137.7, 135.6, 135.5, 133.4, 133.1, 131.1, 130.1, 130.0, 130.0, 129.9, 129.9, 129.9, 129.0, 129.0, 128.2, 124.1, 123.9, 119.2, 108.9, 105.9, 102.6, 84.1, 71.8, 69.9, 62.4, 55.6, 54.2, 38.8, 38.5, 34.4, 29.1, 28.9, 26.8, 23.2, 14.6. HRMS: calcd for C45H44N3O7Cl [M–Cl]+ 738.3176, found 738.3174.

HA-EIF2AK2 plasmid was purchased from Sino Biological and gene mutation was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent). The protein expression EIF2AK2 (258–551 residues, H412N, C551A) was ampliﬁed by gene synthesis and inserted into PGEX-6P-1 vector. All primer sequences used are listed in Supporting Information Table S1.

PMA-THP-1 cells were plated to 80%–90% confluence in 6-well plates, the medium was removed, and cells were washed twice with PBS and then treated with 2 mL growth medium containing probe with or without BBR (100 μmol/L). After incubation at 37 °C/5% CO2 for 3 h, the medium was aspirated, and cells were washed twice gently with PBS followed by UV irradiation (∼365 nm) for 30 min. The cells were then lysed by HEPES buffer (25 mmol/L HEPES, 150 mmol/L NaCl, 2 mmol/L MgCl2) containing 1% NP-40, and the protein concentration was determined by Bradford protein assay (Bio-Rad USA). Equal amount sample with different treatment was used for ﬂuorescent tagging or biotin tagging. For each click reaction, SDS (0.1%), TCEP (1 mmol/L), CuSO4 (1 mmol/L), TBTA (100 μmol/L) and Cy3-azide or biotin-azide (100 μmol/L) was sequentially added to the lysates, and samples were incubated for 2 h, then the labeled proteins were precipitated with acetone. For fluorescence detection, the samples were dissolved with 1 × SDS loading buffer and boiled at 95 °C for 10 min. For LC–MS/MS and immunoblot detection, the samples were dissolved in HEPES buffer with 1% SDS, and incubated with washed beads for 2 h. After washed 5 times with TBS-T, the beads were mixed with 1 × SDS loading buffer, followed by boiling at 95 °C for 10 min. Samples were detected by gel fluorescence or immunoblot blot assay (Tanon 5200 system, Tanon, Shanghai, China).

The proteome after 95 °C boiling to release from beads and identified with a label-free based LC–MS/MS approach (Beijing Qinglian Biotech Co., Ltd., China). MS was analyzed using Maxquant software (version 1.5.3.30) with the UniProtKB/Swiss-Prot human database.

HEK-293 cells were grown to 80%–90% confluence in 6-well plates under the above condition followed by BBR or DMSO treatment for 3 h. The cells were harvested and washed with PBS, and the suspension was incubated at each temperature point from 35 to 75 °C for 3 min. The samples were freeze-thaw 3 times and centrifuged at 20,000×g at 4 °C for 10 min. The 12 μL supernatant was mixed with 3 μL of 5 × loading buffer and then separated on a 10% SDS-PAGE for immunoblotting assay.

EIF2AK2 (258–551 residues, deletion 338–350 residues) with an N-terminal GST tag was cloned into PGEX-6P-1 vector and grown in E. coli Resetta2 (DE3) cells. The bacteria were grown at 37 °C for 4 h following induction with 0.5 mmol/L IPTG and continue cultured for at 18 °C 12 h. The protein was puriﬁed through GST beads and digested with HRV 3C enzyme. The protein was purified by size exclusion chromatography and the purity was checked via SDS-PAGE and Coomassie stain.

The SPR assay was performed on a BIAcore T200 biosensor system (GE Healthcare Life Sciences, Piscataway, NJ, USA) at 25 °C using a CM5 chip. The bindings of EIF2AK2, VPS4B, eEF1A1 and PRDX3 at different concentrations of BBR were performed in 1 × PBS-P + (GE Healthcare Life Sciences) at a flow rate of 20 μL/min for 120 s. After each binding reaction, a further dissociation time of 60 s was applied after each injection to allow the signal to return to the baseline.

An automated docking was carried out using the 3D structures of EIF2AK2 (PDB code: 2A19), eEF1A1 (PDB code: 6ZMO), PRDX3 (PDB code: 5UCX), VPS4B (PDB code: 7L9X) and Discovery Studio 4.5 software. The regularized protein was used in determination of the important amino acids in the predicted binding pocket. Interactive docking using Libdock protocol was carried out for all the conformers of BBR to the selected active site after energy minimization. The docked compound was assigned a score according to its binding mode onto the binding site.

The mice were transfected with adeno-associated virus (AAV-shEIF2KA2-GFP) to knock down EIF2AK2 (targeting sequence: 5′-GGAGTAGCCAT TACGTATAAA-3′) with adeno-associated virus expressing GFP (AAV-GFP) as a control. AAV-shEIF2KA2-GFP and AAV-GFP were purchased from Hanbio technology, China. Briefly, 100 μL of adeno-associated virus (1012 V·g/mL) were injected via tail vein.

Animal experiments were approved by the Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, with the approval number IBM20211102D201. Twelve-week-old male C57BL/6 mice were injected i.p. with BBR (3 mg/kg) or saline for twice every 24 h before i.p. injection of LPS (10 mg/kg). After 3 h, mice were sacrificed, and the blood and tissue were obtained and detected by ELISA or HE stain.

All values are expressed as the mean ± SD. Statistical analysis was performed with one-way ANOVA by SPSS 16.0 with all data points showing a normal distribution, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. No exclusion of data points was used. The researchers were not blinded to the distribution of treatment groups when performing experiments and data assessment. Sample sizes were selected to ensure an adequate power based on the preliminary results.