Cys128 of AP1G1 is a new target for drug development in inhibiting viruses entry to host respiratory mucosal cell

\fancypagestyle firstpage\fancyhf \lhead \chead \rhead \cfoot فروردین ماه ۱۴۰۴ Background and Purpose：Itaconate has been proven to play a role in infectious diseases by interacting with target proteins through Michael addition. In this study, we reveal that itaconate can reduce virus invasion by inhibiting host endocytosis on virus particle, which greatly enhances host defense against respiratory infections. This study aims to give insight into the regulatory mechanism that itaconate achieves endocytosis inhibition. Experimental approach：Itaconate probe were applied for investigations in cells and animals to find out the target protein and binding site by which itaconate achieves endocytosis inhibition on multiple viruses. Key Results：It was found that itaconate targets to site Cys128 of host adapter protein AP1G1 to inhibit its membrane assemblage, thereby weaken the host endocytosis on virus particle, which indicating a general mechanism for antivirus. Logically, itaconate could be a promising choice for anti-virus drug/ prodrug molecule. However, itaconate is not clinically applicable. In order to bring the AP1G1-targetting strategy closer to clinical practice, we screened analogues of itaconate from the known antiviral herb Glycyrrhizae Radix et Rhizoma (licorice). The Licochalcone B was found to binds to Cys128 of AP1G1 and exhibits potent activity in reducing the invasion of multiple virus. Conclusions & Implications：This study proposed and demonstrated the feasibility of the AP1G1-tagetting strategy in inhibiting infections of wide-range of virus. Here we not only reveal a promising target for antivirus drug development, but also screen out the corresponding prodrug molecule. 2. Introduction Previous studies have shown that endogenous itaconate regulates inflammatory immune response and oxidative stress through transcriptional regulation 1-4 . However, Bidyadhar Sethy et al. conducted experiments and found that derivatives of itaconate can reduce viral load in the host during infectious diseases, and notably, this antiviral action does not rely on their anti-inflammatory properties 5 . This suggests that itaconate may play a significant role of inhibiting virus entry during viral infection processes.Respiratory viruses come into direct contact with the mucosal surface and result in illness wildly ranging from the common cold to severe pneumonia 6,7 . Host mucosal immunity is the first line of defense against viral infection 8-12 . Pathogens such as respiratory syncytial viruses (RSV), influenza viruses (AH1N1 influenza, H1N1), and coronaviruses (HCoV-229E, 229E) can break through the physico-chemical barrier of host innate immunity and launch invasion. Metabolic receptors mediate virus anchoring and following endocytosis decides the invasion rate of virus.Traditional antiviral methods rely on vaccines and virus-protein targeted drugs 13-17, but vaccine efficacy is limited and may fail against variants or in immunocompromised individuals, while virus-protein targeted drugs are prone to induce drug resistance. In contrast, intervening in virus endocytosis to reduce susceptibility can reduce the risk of drug resistance and achieve broader and more durable antiviral protection. Thus, this study aims to find out whether itaconate prevent respiratory virus invasion by regulating the endocytosis of host cells. Itaconate was found to covalently binds to Cys128 of AP1G1s, which is critical for cell endocytosis and a key host factor of virus infection 18 . Consistently, the binding of itaconate on AP1G1 weakens the cell endocytosis and inhibits entries of multiple virus. Thus, Cys128 of AP1G1s is a novel target site for modulating host defense with itaconate. Despite the comprehensive understanding of the mechanisms underlying itaconate, its translation into a viable therapeutic agent remains elusive, with a notable absence of human clinical trials. This gap underscores the urgency to delve into natural herbs in search of clinically applicable molecules. Therefore, in order to advance the clinical application of AP1G1 Cys128, we aim to screen small molecules from natural herbs that can serve as substitutes for itaconate. Finally, Licochalcone B was identified as the itaconate substitute which binds to AP1G1 at Cys128, and inhibits viruses from invading host mucosal cells. This study provides an approach for resisting broad-spectrum virus invasion and identifies relevant prodrug molecule for further development. 3. Methods 3.1. Materials and reagents Chinese herbal medicines (Arctii Fructus, Phragmitis Rhizoma, etc.) and standard substances (Licochalcone B, Glycyrrhizic acid, etc.) were purchased from China National Medicinal Materials Corporation and Chengdu Herbpurify Co., Ltd. Chemicals such as methanol, formic acid, and acetonitrile were from Fisher Scientific. Trypsin, Urea, BCA Protein Assay Kit, Streptavidin magnetic beads, and antibodies were obtained from various suppliers as indicated. \fancypagestyle firstpage\fancyhf \lhead \chead \rhead \cfoot فروردین ماه ۱۴۰۴ 3.2. Mice Male Kunming (KM) mice (20-30 g) were housed in a standard environment and randomly divided into groups. Mice were infected intranasally with H1N1, RSV, or 229E viruses, and treated with Licochalcone B (3 mg/kg) by tail vein injection. Control groups received saline. Blood and lung tissues were collected for analysis on the 5th day post-treatment. All animal experiments followed ethical guidelines approved by the China Academy of Chinese Medical Sciences. 3.3. Cell cultures and virus BEAS2B cells were cultured in DMEM with 10% FBS and 1% Penicillin-Streptomycin. Virus stocks of H1N1, RSV, and 229E were handled under biosafety level 2 conditions. 3.4. Itaconate identification and quantification Lung tissues were extracted with methanol/acetonitrile (3:1, v/v). UPLC analysis was performed on a Thermo Vanquish UHPLC system coupled to a Q Exactive Orbitrap MS. Data were acquired using Full Scan and Data-Dependent MS2 modes. The mobile phases contained 0.1% formic acid in water and acetonitrile. 3.5. Western blotting and RT-qPCR assay Proteins were measured using a BCA protein assay kit, separated by SDS-PAGE, and transferred to PVDF membranes for immunoblotting. For RNA detection, qPCR was performed using the 2−ΔΔCT method. 3.6. Immunoprecipitation and Western blot assay GST-tagged AP1G1 proteins were used for immunoprecipitation. Mutant constructs of AP1G1 were expressed in E. coli and purified with Glutathione beads. 3.7. Cloning and recombinant protein expression AP1G1 sequences were cloned into expression vectors and expressed in E. coli. Proteins were purified using Ni-IDA resin and dialyzed for further analysis. 3.8. Itaconate-probe synthesis and labeling Itaconate anhydride was reacted with 7-Octyn-1-ol to form the itaconate-probe, which was purified using silica chromatography. 3.9. Labeling and collection of itaconate-targeted proteins BEAS2B cells were incubated with itaconate-probe. Biotinylated proteins were pulled down using streptavidin beads and analyzed via western blotting. 3.10. In vitro itaconation identification and pull-down assays Recombinant proteins were incubated with itaconate-alkyne, followed by precipitation and analysis on SDS-PAGE. Competitive binding assays were performed with itaconate or Licochalcone B treatment prior to pull-down with streptavidin beads. 3.11. Molecular Docking Analysis The covalent docking of itaconate or Licochalcone B to proteins AF-O43747-F1 or AF-O15427-F1 was performed using the Covalent Dock program in Schrödinger. The top five docked poses were retained, with the best binding affinity being reported. 3.12. Cellular Fluorescence Imaging Images were captured using a Leica SP8 confocal microscope, and processed with LAS X software following the manufacturer’s protocol. 3.13. Subcellular Fractionation Cells were incubated in hypotonic buffer, homogenized, and subjected to centrifugation to separate nuclear and membrane fractions. Immunoblotting was performed on cytosolic and membrane fractions using tubulin as a control marker. 3.14. ShRNA and Lentivirus Infection Lentivirus expressing shRNAs targeting AP1G1 was produced by co-transfecting 293T cells. BEAS2B cells were infected with the viral supernatant, and positive cells were selected with puromycin. \fancypagestyle firstpage\fancyhf \lhead \chead \rhead \cfoot فروردین ماه ۱۴۰۴ 3.15. LiP-MS (Limited Proteolysis-Mass Spectrometry) Experiment AP1G1 protein was treated with licorice extract or water, followed by CNBr treatment for selective peptide fragmentation. The samples were processed using trypsin digestion and analyzed by LC-MS. DDA data was processed with Spectronaut, and spectral libraries were generated for DIA analysis to evaluate protein kinase site accessibility changes. 3.16. TPP (Thermal Proteome Profiling) Assay TPP-ELISA: Recombinant AP1G1 protein was incubated with licorice extracts or small molecules. Samples were heated at different temperatures, followed by ELISA detection of AP1G1 protein levels. TPP-WB: BEAS2B cell lysates were incubated with Licochalcone B or itaconate, followed by heat treatment at various temperatures. Samples were then analyzed by immunoblotting or mass spectrometry. 4. Results 4.1 Covalent addition of itaconate to the cysteine site of host proteins impairs the host cell s usceptibility to virus We found that compared with the control group (mice without virus infection), the content of itaconate in the lung tissue and serum of the model group (mice infected with H1N1) decreased(Fig. 1A). We further broaden the observation with the infection of H1N1, RSV, and 229E virus on BEAS2B cells (Figure 1B). The concentrations of itaconate in both BEAS2B cell body and the culture medium showed a decrease upon virus infections. Notably, the decline in itaconate levels was more pronounced within the cells compared to the culture medium(Figure 1C).This suggests that itaconate is consumed in host cells during viral infections. By labeling the virus with GFP, we trace the invasions and found that the content of H1N1, RSV, and 229E virus were all substantially reduced in itaconate treated cells (Fig. 1D). This finding suggests a close link between the consumption of itaconate and the susceptibilities of cells to virus. On the other hand, to locate the itaconate missing from the culture medium, we developed an itaconate probe ITAP (Fig.S1). Using ITAP, it was demonstrated that the level of bound itaconate on proteins increased(Fig. 1E). This binding is covalent since it’s uncleavable with the hydroxylamine (Fig. 1F). With its electrophilic group, the α, β-unsaturated carboxylic acid structure, itaconate is reported to adds to cysteine residues of host defense-related proteins with Michael addition, and contributes to the host immune response 19-21 . Here we found that the susceptibilities of cells to virus were significantly impaired upon itaconate treatment (Fig. 1K). By applying sulfhydryl blocking agent iodoacetic acid (IAA) (Fig. 1G, H), the itaconate bindings to proteins were significantly inhibited, which were indicated with the stable level of itaconate in culture medium as well as the weak itaconate-probe banding (Fig. 1I, J). Correspondingly, the susceptibilities of cells to virus, which is indicated with the viral load, were recovered when cells were IAA-pretreated (Fig. 1K). These results demonstrates that itaconate binds to the cysteine site of host proteins to lower the susceptibilities of cells to virus. 4.2 AP1G1 is an itaconate-targeted protein that contributes to reducing virus susceptibility of host In order to identify the target proteins of itaconate, we employed the itaconate probe (ITAP) in the copper(I)-alkyne/azide cycloaddition (CuAAC) chemical bioorthogonal experiment (Fig. 2A). AP1G1 has been identified as a host-dependency factor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection 22 . In this study, it was identified as a target protein of ITAP, ranking in the top 14.9% (Fig. 2B). In addition, AP1G1 exhibited significantly altered thermal stability following itaconate treatment (Fig. 2C, D). The immunoprecipitation assay revealed that itaconate interacts with AP1G1 (Fig. 2E). Moreover, excess itaconate can competitively bind to ITAP’s AP1G1-binding in situ (Fig. 2F). Thus, AP1G1 is an itaconate-targeted protein. The itaconation on recombinant AP1G1 (Fig. S2A, B) is found to be not cleavable by hydroxylamine, indicating that AP1G1 is modified by Michael addition instead of a thioester linkage (Fig. 2G). To investigate the functional relevance of AP1G1 as a target of ITAP, we assessed the impact of AP1G1-knockdown on H1N1, RSV, and 229E’s infection to BEAS2B cells. Compared to wild type (WT), sh-AP1G1 BEAS2B cells (Fig. S2C) exhibited higher cell viability(Fig. S3), suggesting significantly reduced viral invasion (Fig. 2H, I). Therefore, AP1G1 is a key target protein of itaconate that contributes to reducing virus susceptibility of host. \fancypagestyle firstpage\fancyhf \lhead \chead \rhead \cfoot فروردین ماه ۱۴۰۴ 4.3 Itaconate hampers the membrane location of AP1G1 by binding to Cys128 site Subsequently, we would like to clarify the specific interaction between itaconate and AP1G1. After administering itaconate, there was no effect on the expression level of AP1G1(Fig. 3A), therefore itaconate does not affect the function of AP1G1 by altering its expression level. Through the western blot assay, we found that itaconate administration leads to AP1G1 remains in the cytoplasm instead of translocating to the cytomembrane (Fig. 3B). In other words, itaconate weakens the ability of AP1G1 to enrich on the membrane, indicating that itaconate affects the cellular endocytosis function towards viruses by altering the subcellular localization of AP1G1. Further, to find out the binding site of itaconate on AP1G1, pull-down experiments with ITAP on GST-tagged sequences of AP1G1, including the N-domain (ND: 1-250 aa), Middle-domain (MD: 251-520 aa), and C-domain (CD: 521-822 aa) were carried out. The N-domain (ND: 1-250 aa) was found to be pulled down by ITAP (Fig. 3C), indicating that the itaconation occurs at cysteine residues within 1-250 aa of AP1G1. However, there are multiple cysteines in the N-domain sequence. In order to validate this conclusion, we mutated the cysteines among 1-250 aa area of AP1G1. When Cys106, Cys124, and Cys128 were mutated to Alanine in combinate, the interaction was attenuated (Fig. 3D). Further, we mutated Cys106, Cys124, and Cys128 separately. Mutation of Cys128 to Alanine (C128A) leads to reduced interaction between ITAP and AP1G1(Fig. 3E). Consistently, molecular docking indicated that the covalent interaction site may be Cys128 of AP1G1 (Fig. 3F), and the Lys156-Leu159 region is also affected, which aligns with the trend of reduced hydrolytic accessibility at these sites. This further implies that itaconate and AP1G1 possess mutually compatible spatial conformations at Cys128 and Lys156-Leu159. Taken together, Cys128 is the binding site of itaconate on AP1G1. In C128A BEAS2B cells, the membrane localization of AP1G1 has little change upon itaconate treatment (Fig. 3G). Correspondingly, the viral load of itaconate-treated cells remains as much as the control one (Fig. 3H), which indicating that itaconate lose its influence on cell susceptibility to virus. In summary, Cys128 of AP1G1 is the site that itaconate binds to, thus attenuates cell endocytosis to virus. 4.4 Exploring small molecule compounds interacting with AP1G1 through thermoproteomic analysis. We have confirmed the role of itaconate in reducing viral entry and identified its target site as cys128 on AP1G1. However, the clinical application of itaconate is currently limited to cellular and animal experiments, with no human trials conducted yet. To demonstrate the practical value of this research, we decided to search for an alternative to itaconate among antiviral traditional Chinese medicines with a long history of use. Using thermoproteomics technology for screening, we selected commonly used antiviral herbs such as Coptidis Rhizoma, Scutellariae Radix, and Glycyrrhizae Radix et Rhizoma . We found that after interaction with AP1G1, the thermal denaturation curve of AP1G1 showed a significant shift with the extract of Glycyrrhizae Radix et Rhizoma (Fig. 4A). Further, we selected dozens of small molecular compounds from Glycyrrhizae Radix et Rhizoma, including licochalcone B, glycyrrhizic acid, and glycyrrhizin. Ultimately, we determined that licochalcone B from Glycyrrhizae Radix et Rhizoma interacts significantly with AP1G1(Fig. 4B), suggesting that licochalcone B can serve as an alternative to itaconate. To validate this conclusion,we employed lip-ms technology, leveraging alterations in protease accessibility due to protein conformational changes to confirm the binding of licochalcone B to AP1G1. A total of 73 semi-specific peptides were identified, among which 27 exhibited higher accessibility as PK sites, while 46 displayed lower accessibility. The former indicates that the cleavage sites of AP1G1, upon treatment with itaconate, become exposed. Conversely, lower accessibility suggests that these cleavage sites are either bound to compounds or concealed due to conformational changes.In the higher accessibility PK sites, we observed five sites with an intensity increase ranging from 2 to 3 times, constituting 18.5% of the total and marked with *. Another five sites showed an intensity increase between 3 and 10 times, also accounting for 18.5% and denoted by **. Additionally, five sites exhibited an intensity increase of over 10 times, making up another 18.5% and labeled with ***. Notably, twelve sites were exclusively detected after binding or conformational changes, comprising 44.4% of the total and indicated by ****.(Fig. 4C).In further detail,within the higher accessibility PK sites, we observed five sites with an intensity increase ranging from 2 to 3 times, constituting 18.5% of the total and marked with *. Another five sites showed an intensity increase between 3 and 10 times, also accounting for 18.5% and denoted by **. Additionally, five sites exhibited an intensity increase of over 10 times, making up another 18.5% and labeled with ***. Notably, twelve sites were exclusively detected after binding or conformational changes, comprising 44.4% of the total and indicated by ****.On the other hand, within the lower-accessibility PK sites,nine sites displayed an intensity decrease ranging from 2 to 3 times, accounting for 20% of the total and marked with *. Thirteen sites showed a decrease in intensity between 3 and 10 times, representing 28% and denoted by **. Six sites experienced an intensity decrease of over 10 times, making up 13% and labeled with ***. Lastly, eighteen sites were solely detected before binding or conformational changes, comprising 39% of the total and indicated by ****(Fig. 4D).These changes in the accessibility of PK sites conclusively demonstrate the binding of licochalcone B to AP1G1.We have catalogued all the sites where protease hydrolysis accessibility has undergone changes. Notably, within the N domain (1-250aa), we observed a shift in the protease hydrolysis accessibility of Leu126, which is proximal to Cys128. This observation suggests that Cys128 may serve as a binding site(Fig. 4E). 4. 5 Licochalcone B is an itaconate substitute for \fancypagestyle firstpage\fancyhf \lhead \chead \rhead \cfoot فروردین ماه ۱۴۰۴ reducing host susceptibility to respiratory virus The molecular docking results showed that Licochalcone B could covalently bind to AP1G1 at the Cys128 residue(Fig. 5A). The labeling of Licochalcone B on AP1G1 was demonstrated by the altered thermal stability of AP1G1 (Fig. 5B, C). Importantly, Licochalcone B could compete with the binding of ITAP on AP1G1 in situ in BEAS2B cells (Fig. 5D). In addition, Licochalcone B competes with itaconation on ND (1-250aa) of AP1G1 (Fig. 5E). In BEAS2B cells pretreated with Licochalcone B and infected with respiratory tract viruses (Fig. 5F), Licochalcone B significantly restores the levels of itaconate (Fig. 5G). These research findings suggest that Licochalcone B can serve as an effective substitute for itaconate during respiratory tract virus infection. Further, Licochalcone B was found to be distributed in the blood and lung tissue of mice (Fig. S4), indicating its potential for treating respiratory tract infections. Viral genome copies are significantly downregulated by Licochalcone B administration in vivo (Fig. 5H, I). To sum up, Licochalcone B exerts the same effect as itaconate in reducing host susceptibility to respiratory virus by targeting Cys128 of AP1G1. 4.6 Licochalcone B reduces virus load of H1N1, RSV, and 229E in lungs and alleviates virus-induced lung injury in vivo To evaluate the effects of Licochalcone B on H1N1, RSV, and 229E viruses after their invasion into mice lungs (Figure 6A), we examined the levels of viral RNA, lung index, and inflammatory factors such as IL-1, IL-6, and TNF-α in mice lung tissues. Analysis of viral RNA levels in the lungs revealed that Licochalcone B significantly reduced the number of viral particles invading lung tissues compared to the control group, indicating its ability to lower the host susceptibility to respiratory virus (Figure 6B). Moreover, the lung weight/body weight ratio (lung index), an indicator of lung tissue inflammation and damage 23, was significantly lower in the Licochalcone B-treated group than in the control group, suggesting that Licochalcone B alleviates both virus-induced inflammation and lung damage (Figure 6C). Compared to the uninfected control group, the levels of pro-inflammatory cytokines in the lungs of virus-infected mice, including IL-1 (Figure 6D), IL-6 (Figure 6E), and TNF-α (Figure 6F), were significantly elevated. Treated with Licochalcone B, the expression levels of these cytokines were significantly reduced, which indicates that Licochalcone B can suppress the excessive inflammatory responses induced by viral infections. Taken together, Licochalcone B exhibits promising effect in alleviating virus-induced lung injury by reducing host susceptibility to respiratory virus. \fancypagestyle firstpage\fancyhf \lhead \chead \rhead \cfoot فروردین ماه ۱۴۰۴ 5. Discussion Endogenous metabolites, such as itaconate, play a role in various host-virus interactions and contribute to disease progression 2,3,24-26 .As an anti-inflammatory and antibacterial metabolite, itaconate exerts its actions by modifying proteins that are integral to inflammatory response and host defense 21 . It was reported that itaconate analogues, including dimethyl itaconate and 4-octyl-itaconate, are found to reduce pulmonary inflammation during respiratory virus infection 27,28 . The broad-spectrum anti-inflammatory, antiviral, and immunomodulatory effects of itaconate and its analogs are related to endocytosis mechanisms 29-33 . Endocytosis is not only a pathway for viruses to enter the host cell, but also essential for viral replication 34 .Clathrin adaptors are key factors in mediating endocytosis, facilitating the invasion of various viruses, including SARS-CoV-2, Ebola, HIV, and Hepatitis B 35-38 .Through bidirectional genome-wide CRISPR screening, AP1G1 was identified as a crucial host-dependent factor for the viral entry of SARS-CoV-2, MERS-CoV, and seasonal HCoVs 39 .This suggests that AP1G1 could be a potential target for therapeutic interventions. However, the approach of preventing virus entry by modulating AP1G1 remains undetermined.In this study, we reveal that the Cys128 of host proteins AP1G1 is a critical target of inhibiting the invasion of respiratory viruses. AP1G1 is a key player in virus endocytosis 39-41, it achieves this by participating in the subcellular localization of viral proteins to the so-called ”viral factory” 42 .Itaconate can target Cys128 of AP1G1,thereby disrupting its normal interactions and functions.Specifically, the occupation of Cys128 by itaconate disrupts the essential interactions that facilitate AP1G1’s membrane translocation.This inhibition, in turn, reduces the efficiency of virus endocytosis, thereby mitigating the invasion of respiratory viruses. Our findings underscore the strategic importance of Cys128 in AP1G1 as a critical target for inhibiting viral invasion, highlighting the intricate mechanisms by which itaconate modulates host-virus interactions at the molecular level.Currently, there are numerous reports on the antiviral effects of various natural drug molecules.For example, trans-δ-viniferin derivatives, pheophorbide a (Pba), Echinaforce®, and other natural drug molecules obtained from plants have shown broad-spectrum antiviral activity, which can effectively inhibit enveloped viruses such as influenza virus (IV), SARS-CoV-2, and herpes simplex virus 2 (HSV-2). With the discovery of the broad-spectrum antiviral target Cys128, screening and identifying promising drug precursor molecules from natural medicinal molecule libraries has become particularly important 43-45 .To enhance the practicality of the Cys128 targeting strategy in preventing pathogen invasion, this study integrated non-targeted metabolomics with multi-stage high-resolution native mass spectrometry. Through this approach, we identified an itaconate analogue, Licochalcone B, from the well-known antiviral traditional Chinese medicine, Glycyrrhizae Radix et Rhizoma (licorice). Further investigation revealed that Licochalcone B possesses the ability to specifically target the Cys128 site within the AP1G1 protein, effectively inhibiting viral replication and growth.This discovery not only strengthens the practical application of protein site-specific targeting strategies but also opens up novel avenues for antiviral drug screening. 6. Funding This research was funded by National Natural Science Foundation of China (grant number: 82141206), Natural Science Foundation of Beijing (grant number: 7244493), Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences (grant number: ZXKT22059, ZZ16-XRZ-071, ZZ17-YQ-023),Beijing University of Chinese Medicine Standardization Research Project on Experimental Techniques (Operating Procedures) (grant number: 2023-syjs-01). 7.Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 8. Reference 1. Shi, X., Zhou, H., Wei, J., Mo, W., Li, Q., and Lv, X. (2022). The signaling pathways and therapeutic potential of itaconate to alleviate inflammation and oxidative stress in inflammatory diseases. Redox Biol 58, 102553. 10.1016/j.redox.2022.102553.2. 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Meunier T, D.L., Bordage S, Bamba M, Hervouet K, Rouillé Y, François N, Decossas M, Sencio V, Trottein F, Tra Bi FH, Lambert O, Dubuisson J, Belouzard S, Sahpaz S, Séron K. (2022). . Antimicrob Agents Chemother., 66(62):e0158121. . 10.1128/AAC. 9.Figure legends Figure .1 (A) The level of itaconate in mice lung tissue and serum upon viral infection. Mice in the model group were exposed to H1N1 (100 TCID50) for 5 days. t-test, Lung tissue, p =0.0518, n=3. Serum, p =0.1525,n=3. (B) BEAS2B cells were exposed to virus infections of H1N1, RSV, and 229E. t-test, n=3. (C) The level of itaconate in BEAS2B cells and culture medium during viral infection. t-test, *p<0.05, **p<0.01, n=3. (D) BEAS2B cells were infected with GFP-tagged viruses of H1N1, RSV, and 229E (MOI=2, 24 hpi). The GFP signal was attenuated in the itaconate administration group compared with the WT group, indicating reduced virus invasion. Scale bar=100 μm. (E) The amount of itaconate in the bound state in cells. (F) Hydroxylamine can not cut off protein-bound itaconate. (G) The α,β-unsaturated carboxylic acid group in itaconate and its Michael addition. The carbon-carbon double bond in α,β-unsaturated carboxylic acid can undergo a Michael addition reaction with the exposed sulfhydryl group on cysteine to form a strong covalent bond. IAA can block sulfhydryl groups, preventing them from undergoing Michael addition reactions with itaconate. (H) BEAS2B cells were first pretreated with IAA and then exposed to virus infections of H1N1, RSV, and 229E. A t-test was performed, with n=3. (I) The level of itaconate in BEAS2B cells during viral infection. t-test, n=3. (J) Changes in bound itaconate content after IAA treatment. (K) Viral RNA level in infected cells. Cells were treated with itaconate, IAA, and itaconate+IAA. In the itaconate+IAA group, cells were pretreated with IAA (1 μM) for 24 h, and then subjected to itaconate administration (200 μM). t-test, **** p <0.0001, n=3. Figure.2 (A) Workflow of labeling and identifying the itaconate- targeted proteins with CuAAC-mediated click chemical reaction and Orbitrap MS.(B)Among the potential itaconate- targeted proteins, AP1G1 ranks in the top 14.9%. (C,D)In Cellular Thermal Shift Assay - Western Blot (CETSA-WB) assay, AP1G1 exhibited altered thermal stability when incubated with itaconate (50 mM). (E) ITAP pull-down assay indicates the interaction between itaconate and AP1G1 recombinant protein. (F) Itaconate competitively binding on AP1G1 and impede the pull-down efficacy of ITAP. (G) Hydroxylamine assay of AP1G1 recombinant protein. AP1G1 was incubated with 100 μM of ITAP for 1 h, followed by the click reaction with azide-Rho via CuAAC. The mixture was treated with 5% hydroxylamine. Itaconation on AP1G1 was found to be resistant to cleavage by hydroxylamine. (H) Viral genome copies in BEAS2B cells. Cells were infected with H1N1, RSV, and 229E viruses (MOI=2, 24 hpi). Cells were transfected with sh-AP1G1. t-test, **p<0.01, n=3. (I) BEAS2B cells were infected with GFP-tagged viruses of H1N1, RSV, and 229E (MOI=2, 24 hpi). The GFP signal was attenuated in the sh-AP1G1 group compared with the WT group, indicating reduced virus invasion. Scale bar=100μm. Figure.3(A) AP1G1 expression after the administration of itaconate. Itaconate administration does not alter the expression of AP1G1. The data represents three replicates. (B) Western blot assay showing the subcellular localization of AP1G1. In=input, C=cytoplasm, M=cytomembrane. (C) The direct interaction between AP1G1 and ITAP was measured by pull down experiment. The AP1G1 protein was synthesized in three GST-tagged fragments, including the N-domain (ND: 1-250 aa), Middle-domain (MD: 251-520 aa), and C-domain (CD: 521-822 aa). The N-domain (ND: 1-250 aa) was pulled down by ITAP.(D, E) Confirmation of cys128 as bin ding site by stepwise point mutation. (F)3D and 2D docking result illustrating interaction site between itaconate and AP1G1. (G) Membrane localisation levels of AP1G1 mutated at the cys128 locus after itaconate treatment and viral levels.(H) Viral RNA levels in BEAS2B cells. Cells were infected with H1N1, RSV, and 229E viruses (MOI=2). t-test, n=3. Figure.4 (A)The protein content change curves of the incubates of nine traditional Chinese medicines,A: Glycyrrhizae Radix et Rhizoma,B: Arctii Fructus,C: Phragmitis Rhizoma,D: Platycodonis Radix,E: Coptidis Rhizoma,F: Scutellariae Radix,G: Taraxaci Herba,H: Gardeniae Fructus,I: Forsythiae Fructus, with AP1G1 were observed. Compared with the control group, only the Glycyrrhizae Radix and Rhizoma-AP1G1 incubate exhibited a significant protein thermal shift.(B)The protein content change curves of the incubates of nine small molecule compounds—A: Licochalcone B,B: glycyrrhizic acid,C: glycyrrhizin,D: quercetin,E: glycyrrhetic acid, F: isoliquiritigenin, G: glabridin,H: licoricone J,I: licocoumarin—with AP1G1, when compared to the control group, showed a significant protein thermal shift only in the case of Licochalcone B-AP1G1.(C,D) Number, classification, proportion of higher and lower accessibility PK sites. (E)The sites in AP1G1 where the accessibility to protease K hydrolysis is upregulated or downregulated. Figure.5 (A)Molecular docking of Licochalcone B with the Cys128 site of AP1G1.(B, C) TPCA-WB experiments were performed with BEAS2B cell lysates. The melt curve of AP1G1 showed a thermal shift upon administration of Licochalcone B (100 μg/ml). n=3, mean ± SD. (D) Licochalcone B competes with ITAP for binding on the AP1G1 protein. (E) Competitive binding of Licochalcone B to ND (1-250 aa). BEAS2B cell lysates were incubated with 1 mM ITAP and then with 100 μg/ml of Licochalcone B for 24h.(F) BEAS2B cells were first pretreated with licochalcone B and then exposed to H1N1, RSV, and 229E virus infections. A t-test was performed with n=3. (G) Itaconate levels in BEAS2B cells after administration of Licochalcone B (50 μg/ml). Cells were infected with H1N1, RSV, and 229E viruses. t-test,*p < 0.05, **p < 0.01,n=3. (H)Viral RNA levels in BEAS2B cells following administration of Licochalcone B.Three experimental groups were included: the wild-type group infected with viruses (no Licochalcone B treatment), the wild-type group treated with Licochalcone B, and the C128A mutant group treated with Licochalcone B. BEAS2B cells were infected with H1N1, RSV, and 229E viruses (MOI=2). t-test, ****p<0.0001, n=6. (I) BEAS2B cells were infected with GFP-labeled H1N1, RSV, or 229E viruses (MOI=2) and observed at 24 hours post-infection (hpi). Scale bar=100 μm. The data represents three replicates. Figure 6. (A) Schematic of the experimental design. Mice were infected with viruses and treated with Licochalcone B (i.v. daily) for 5 days. Lung tissues were collected for analysis.(B) Effect of Licochalcone B on viral RNA levels in the lung tissues of virus-infected mice. t-test, ****p<0.0001, n=3.(C)Effect of Licochalcone B on lung weight/body weight ratio in virus-infected mice. t-test, ****p < 0.0001, n = 6.(D) Effect of Licochalcone B on IL-1 expression levels in the lung tissues of virus-infected mice. t-test, ****p < 0.0001, n = 6. (E) Effect of Licochalcone B on IL-6 expression levels in the lung tissues of virus-infected mice. t-test, ****p < 0.0001, n = 6. (F) Effect of Licochalcone B on TNF-α expression levels in the lung tissues of virus-infected mice. t-test, ****p < 0.0001, n = 6. 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Authors Metrics & Citations Metrics Article Usage 215views 140downloads Citations Download citation xinqi Deng, Qinling Rao, Zhixing Huang, et al. Cys128 of AP1G1 is a new target for drug development in inhibiting viruses entry to host respiratory mucosal cell. Authorea. 04 April 2025. DOI: https://doi.org/10.22541/au.174376458.84458625/v1 DOI: https://doi.org/10.22541/au.174376458.84458625/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.