Suppressive effects of oroxylin A on intracellular proliferation ofToxoplasma gondiivia host cell ERK phosphorylation inhibition

30 Toxoplasma gondii poses a significant threat to immunocompromised patients, 31 resulting in high mortality rates. Considering the side effects of anti- Toxoplasma drugs, we 32 focused on a potential candidate, oroxylin A (OA), a common component extracted from 33 Astragalus membranaceus and Scutellaria baicalensis which suppressed the growth of T. 34 gondii in vitro and in vivo. Our result demonstrated that OA suppressed T. gondii intracellular 35 proliferation and downregulated phosphorylation of ERK1/2 of T. gondii-infected host cells 36 very similar to the MEK1 specific inhibitor PD98059. Our in silico analysis showed that OA 37 interacts sufficiently with the mammalian MEK1 region where established MEK1 inhibitors 38 like PD98059 and trametinib bind. Moreover, OA improved the survival rate in T. gondii -39 infected mice. This study proposes that host MEK1 is a novel potential target for anti-40 Toxoplasma drugs with a new mechanism of action. 41 42 43

44 Toxoplasma gondii , MAPK signaling pathway, ERK, in silico docking simulation, host-45 directed therapy 46 47 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 3 Toxoplasma gondii , a protozoan parasite known for its remarkable success as an 48 intracellular parasite, infects approximately one-third of the world’s population 1 and is 49 reported to infect nearly all warm-blood animals. In humans, toxoplasmosis caused by this 50 parasite typically remains asymptomatic in patients with a typical immune system. However, 51 in cases involving immunocompromised patients, such as those with human 52 immunodeficiency virus infection, organ transplantation, or pregnancy, T. gondii becomes 53 activated within the host’s organs, leading to severe diseases like toxoplasmic encephalitis, 54 ocular toxoplasmosis, and Toxoplasma pneumonia2–4. In general, pyrimethamine (PYR) and 55 sulfadiazine are clinically used to treat acute toxoplasmosis. However, their severe side 56 effects and the emergence of drug resistance pose serious issues that require continued 57 treatment5–7. Recently, considerable attention has been paid to developing novel drugs that 58 offer reduced side effects for combating toxoplasmosis. Hence, it becomes imperative to 59 identify various compounds capable of inhibiting the growth of T. gondii via diverse 60 mechanisms of action. 61 In the last decade, Traditional Chinese medicine (TCM) has gained increasing 62 recognition as an alternative treatment for infectious diseases due to its fewer side effects 8,9. 63 Artemisinin, extracted from Artemisia annua , has found widespread use in combating 64 malaria10. Similarly, Astragalus membranaceus (Am) and Scutellaria baicalensis (Sb) have 65 exhibited effectiveness in suppressing the proliferation of T. gondii proliferation in vivo11 and 66 in vitro12. These East Asian herbal medicines (e.g., TCM) have been trusted for their safety 67 and are used to treat various inflammatory and infectious diseases 12,13. However, the 68 properties of herbal products can considerably differ based on their sources, making quality 69 control and evaluation of efficacy based on active constituents essential. Therefore, the 70 identification of compounds within these plants suppresses T. gondii proliferation, and 71 elucidation of their mechanisms of action is paramount in developing novel drugs against 72 toxoplasmosis. Searching for potential active components within Am 14 and Sb 15, we 73 identified oroxylin A (OA) as a common constituent. 74 OA, a flavonoid, demonstrates a broad spectrum of bioactivities showcasing its 75 potential because of its anti-tumor, anti-virus, anti-protozoan parasite, anti-inflammation, 76 anti-oxidation and anti-allergy activities in addition to organ protection 16,17. Considering its 77 multi-bioactivities, OA holds considerable potency for clinical applications17. In addition, our 78 focus on OA led us to delve into the flavone backbone structure. Certain flavonoids, 79 including OA, can inhibit the MAPK signaling pathway 18. This pathway plays a critical role 80 in governing cellular processes such as cell proliferation, differentiation, stress response, and 81 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 4 apoptosis19,20. Interestingly, T. gondii infection triggers the activation of the host-cell MAPK 82 pathway, and studies indicate that inhibiting this pathway suppresses parasite proliferation21. 83 In this study, we hypothesized that OA could suppress T. gondii proliferation by 84 inhibiting the host-cell MAPK signaling pathway. To investigate this, we assessed the effects 85 of OA on T. gondii in vitro and in vivo, along with monitoring alterations in the host-cell 86 MAPK signaling pathway during parasite infection. Furthermore, utilizing in silico docking 87 simulation and in vitro assays, we inferred the target molecule and mechanism through which 88 OA operates. Our findings provide a novel strategy for the development of novel drugs 89 combating toxoplasmosis. 90 91 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 5

92 OA suppresses Toxoplasma proliferation in vitro. 93 Initially, we evaluated the effect of OA on the proliferation of host cells (Vero cells) 94 using the sulforhodamine B (SRB) assay22, 23. At concentrations of 50 µM and below, OA did 95 not demonstrate any significant effect (Figure 1). Next, we evaluated the effect of OA on 96 cytotoxicity induced by T. gondii proliferation using a monolayer disruption assay 24, with 97 PYR as the control. The results depicted in Figure 2A and 2B revealed a substantial decrease 98 in the percentage of bottom coverage due to T. gondii infection. However, OA exhibited a 99 dose-dependent increase in this coverage, indicating that OA suppressed T. gondii 100 proliferation. At a concentration of 50 µM, OA displayed a comparable effect to that of 2 µM 101 PYR. These results indicate that OA suppresses the proliferation of T. gondii. 102 103 OA suppresses the intracellular proliferation of Toxoplasma. 104 To evaluate the mechanism of OA effect on parasite proliferation, we employed the T. 105 gondii RH-GFP strain, engineered to express GFP constitutively, enabling the quantitative 106 assessment of parasite number and size through GFP signals. We confirmed that OA 107 suppressed the percentage of GFP signal density (Figure 3A and 3B). Thus, we utilized the 108 RH-GFP strain in subsequent experiments. 109 To determine whether OA affects intracellular parasites, we measured the size of the 110 parasitophorous vacuole (PV) within infected host cells. Results obtained at 30 h post-111 infection demonstrated a reduction in PV sizes due to the treatments (Figure 4A and 4B). In 112 addition, it was noted that the PV size of parasites treated with 50 µM OA was significantly 113 smaller compared to those treated with 10 µM OA (P < 0.001). These results suggest that OA 114 suppresses the intracellular development of T. gondii. 115 116 OA does not affect extracellular Toxoplasma viability. 117 Subsequently, we assessed the effect of OA on extracellular parasites. To assess this, 118 we pretreated the extracellular parasites for 1 h with OA before invasion. At 48 h post-119 infection, there was no difference in the number of parasites between the pretreated and 120 untreated conditions (Figure S1). Even upon extending the treatment duration to 4 h, 121 encompassing 1 h before invasion and 3 h for invasion, no difference was observed between 122 the conditions. These results suggest that OA does not influence the viability of extracellular 123 parasites, including their motility and host-cell invasion activities. 124 125 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 6 OA inhibits the host-cell MAPK signaling pathway induced by Toxoplasma infection. 126 OA, a flavonoid characterized by a flavone backbone structure (Figure 5A), has 127 demonstrated the ability to inhibit the MAPK pathway in osteoarthritis chondrocyte cells and 128 non-small-cell lung cancer cells 19,20. Furthermore, Han et al. 25 indicates that T. gondii 129 infection triggers the activation of the host-cell MAPK signaling pathway, and inhibiting this 130 pathway results in decreased parasite proliferation in Vero cells. In addition, T. gondii lacks 131 MAPK/ERK kinase (MEK) 1/2 homologs26. Therefore, we hypothesized that OA could target 132 on the host-cell MAPK signaling pathway, but not on the parasite. 133 To validate our hypothesis, we evaluated the activation of the MAPK signaling 134 pathway in T. gondii -infected cells with and without OA treatment. We assessed the 135 phosphorylation levels of extracellular signal-regulated kinase (ERK) 1 and ERK2 (Figure 136 5B), known as crucial endpoints within the MAPK pathway. We used PD98059 (Figure 5A), 137 a specific inhibitor for MEK1 27, as a positive control, which exhibited no cytotoxicity to the 138 host cell at concentrations of 50 μ M and below (Figure S2). We confirmed that host-cell 139 ERK1/2 phosphorylation was increased upon T. gondii infection using Vero cells (Figure 5B) 140 and human fibroblast cells (Figure S3) and the phosphorylation was decreased by PD98059 141 treatment (Figure 5B). We found that OA also inhibited infection-induced ERK1/2 142 phosphorylation (Figure 5B). These results demonstrated that OA counteracted the 143 phosphorylation of host-cell ERK1/2 within the MAPK signaling pathway activated by T. 144 gondii infection. 145 146 A combination of OA and PD98059 does not have an additive or synergistic inhibitory 147 effect on Toxoplasma proliferation. 148 To confirm that the host MAPK pathway is significant for T. gondi growth, we 149 examined the effect of PD98059 treatment on the parasite proliferation.We confirmed that 150 PD98059 exhibited a dose-dependent inhibition of T. gondii proliferation in Vero cells 151 (Figure 6A), as reported previously19,25. 152 To elucidate which step of MAPK pathway is inhibited by OA, we investigated the 153 impact of combining OA and PD98059 on the intracellular parasite proliferation. Our results 154 indicated no significant additive or synergistic effect when combining 10 or 25 µM PD98059 155 with 10 and 50 µM OA, as observed in the GFP signal distribution (Figure 6B) and the % of 156 GFP signal density (Figure 6C). Furthermore, the size of the PV housing the parasites did not 157 exhibit any additive or synergistic upon adding 10 or 25 µM PD98059 to 10 and 50 µM OA 158 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 7 (Figure 6D). These results suggest that OA and PD98059 share the same binding site within 159 MEK1. 160 161 In silico docking simulation supports that OA interacts with the host MEK1. 162 To provide comprehensive insights into our in vitro experiments, we conducted an 163 analysis to assess the physical and chemical affinity between OA and MEK1 using in silico 164 docking simulation techniques. Known MEK1-specific inhibitors, PD98059 27 and 165 trametinib28, have been identified to bind to the allosteric site of MEK1. We compared the 166 potential binding capacity of OA to MEK1 using PD98059 and trametinib as positive 167 controls for MEK1 allosteric site-binding molecules. Subsequently, we constructed the three-168 dimensional structures of the trametinib/MEK1, PD98059/MEK1, and OA/MEK1 complexes 169 closely evaluating the interaction between MEK1 and each ligand (Figure 7). Interaction 170 energies between MEK1 and each ligand were trametinib (-111.55 kcal/mol) > PD98059 (-171 60.79 kcal/mol, as a positive control) > OA (-54.21 kcal/mol), consistent with the results of 172 the in vitro experiments (Figure 6). Moreover, our analysis identified specific amino acid 173 residues within MEK1 commonly recognized by all ligands, D208, F209, V211, and S212 174 (Figure 7); these amino acids are conserved among mammals 29. This results strongly support 175 that OA binds to MEK1 in the same mode as the MEK1-specific inhibitors. 176 177 OA treatment increases the survival rates of Toxoplasma-infected mice. 178 Finally, we performed mouse infection experiments to assess the efficiency of OA in 179 suppressing T. gondii growth and the safety of OA in vivo. We used a cyst-forming Fukaya 180 strain (archetypal type II), closely resembling the clinical scenario observed in humans. We 181 confirmed that OA suppressed the growth of this strain in vitro (Figure S4). 182 We monitored the survival rates of infected mice treated with or without OA, PYR, 183 and sulfadiazine for 30 days (Figure 8A). In our experimental condition, all mice in the 184 challenge control group (infected DMSO treated; n = 8) succumbed by 11 days post-infection 185 (dpi.) (Figure 8B, black line). In contrast, all mice in the cure control group (infected P+S 186 treated) survived until 30 dpi (Figure 8B, magenta line). On the other hand, around 40% of 187 the mice in the infected OA-treated group (n = 8) survived at 11 dpi and they continued to 188 survive until 30 dpi (Figure 8B, green line) (P < 0.06 vs the challenge control). This suggests 189 that OA suppresses parasite growth also in vivo. 190 In addition, we monitored the effect of drug treatments on weight change (Figure 8C). 191 In the infected DMSO-treated group, weight loss continued from day 4 and resulted in death. 192 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 8 In contrast, the infected P+S-treated group stopped losing weight on day 7 and began 193 regaining weight from day 8. This suggests that P+S treatment eliminated T. gondii by day 7. 194 In the OA-treated survival group, weight loss stopped on day 8 and recovered weight from 195 day 10, and they continued to survive until 30 dpi. These suggest that T. gondii was 196 eliminated by about day 8 and that there were no fatal side effects of OA treatment on 197 survival, although the effects of OA were slower than those of P+S. These results indicate 198 that OA suppresses parasite growth in vivo with low side effects. 199 200 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 9

201 Developing new therapeutic drugs to combat toxoplasmosis requires identifying a 202 broader array of safe and efficient hit and lead compounds 31. The traditional East Asian 203 traditional herbal medicines Astragalus membranaceus (Am) and Scutellaria baicalensis (Sb) 204 inhibit T. gondii proliferation in vitro and in vivo11,12. This study focused on oroxylin A (OA), 205 a shared constituent found in Am and Sb. We evaluated its potential as a new medicine 206 against toxoplasmosis, where we demonstrated that OA suppressed Toxoplasma intracellular 207 proliferation by inhibiting the host-cell MAPK signaling pathway (Figure 9). Our findings 208 demonstrated that OA is one of the active components in Am and Sb for their anti- T. gondii 209 activity. Furthermore, our study proposes that host MEK1 is a novel potential target for anti-210 Toxoplasma drugs. 211 OA is a flavonoid compound known for its diverse pharmacological functions, 212 including anticancer and anti-inflammatory properties17. In general, many flavonoids seem to 213 cause protein kinase inhibition 32. In addition, it has been reported that inhibition of the 214 MAPK signaling pathway of host cells suppresses T. gondii proliferation 25. Therefore, we 215 hypothesized that OA inhibits the host MAPK signaling pathway, and inhibition of ERK 216 phosphorylation by OA leads to the suppression of T. gondii proliferation. In fact, we 217 demonstrated that OA inhibits the phosphorylation of host-cell ERK1/2 activated by T. gondii 218 infection. Also, we focused on the structural similarity between OA and PD98059, a specific 219 inhibitor of MEK1 possessing a flavone backbone. which provided further support for our 220 hypothesis. In fact, we demonstrated that OA inhibits the phosphorylation of host-cell 221 ERK1/2 activated by T. gondii infection, and its suppressive effect of OA on parasite 222 proliferation remains unaffected by PD98059. Finally, in silico docking simulations showed 223 that interacting energies of OA with MEK1 were comparable to those of PD98059. Taken 224 together, the target molecule of OA is likely to be MEK1. 225 This study highlights the host-cell MAPK signaling pathway as a novel target for 226 potential anti- Toxoplasma drug discovery. The MAPK signaling pathway demonstrates 227 activation in human cancers, where MEK1/2 plays critical roles 33. Therefore, inhibitors 228 specifically targeting MEK1/2 have been developed, with trametinib earning approval from 229 the US Food and Drug Administration (FDA) for clinical usage (FDA Reference ID: 230 4255758). The approval of trametinib demonstrates the feasibility of MEK1/2-targeted anti-231 toxoplasmosis drugs. However, the precise mechanisms underlying the inhibition of MAPK 232 signaling pathway activation by T. gondii infection, resulting in the suppression of parasite 233 proliferation, remain unclear. Our findings provide an impetus for further studies on the 234 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 10 relationship between T. gondii infection and the host-cell MAPK signaling pathway, leading 235 to drug development. 236 Recently, it has been established that intracellular pathogens, including viruses, 237 bacteria, and parasites, rely on host cell factors for their proliferation 36. Consequently, 238 inhibitors targeting these essential host factors crucial for pathogens are considered potential 239 drug candidates. This novel strategy, proposed as host-directed therapy (HDT), holds promise 240 in reducing the emergence of drug resistance 37. In parasitic diseases, this approach has been 241 applied in a study focused on cutaneous leishmaniasis38, showing that tofacitinib, an inhibitor 242 of the host cell Janus kinase 3, plays a critical role in the CD8 T cell IL-15 signaling pathway. 243 This approach was found to be a safe strategy, effectively blocking immunopathologic 244 responses locally while preserving protective responses. The mechanism of action identified 245 for OA aligns precisely with this HDT strategy that has been underexplored for 246 toxoplasmosis. Therefore, our findings could pave the way for HDT against Toxoplasma. 247 In conclusion, our findings revealed that suppressing host cell ERK phosphorylation 248 effectively suppressed the intracellular growth of Toxoplasma. Given that OA does not affect 249 the parasite directly, it is plausible that the emergence of drug resistance would be suppressed. 250 Kinase inhibitors like OA and PD98059 hold potential as initial candidates to advance HDT 251 studies in toxoplasmosis. Consequently, our study offers new insights into therapy against 252 toxoplasmosis. 253 254 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 11

255 Parasites. 256 The archetypal type I strains of T. gondii RH and RH-GFP (ATCC #50940), along with 257 the archetypal type II strain Fukaya, were cultured in Vero cells using high glucose 258 Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich Co. St. Louis, MO). The 259 medium contained 2% (v/v) fetal bovine serum (FBS) (Biowest, Nuaillé, France), 100 U/ml 260 penicillin, and 100 µg/ml streptomycin (FUJIFILM Wako Pure Chemical Co., Osaka, Japan). 261 All cells were maintained in a humidified incubator with 5% CO 2 at 37ºC. To harvest 262 Toxoplasma tachyzoites, T. gondii-infected Vero cells were gently scraped ten times using a 263 25-gauge needle and a 10-ml syringe. The resulting cell solution was centrifuged at 150 × g 264 for seven min to remove cell debris, and the supernatant, including tachyzoites, was collected. 265 The concentration of tachyzoites was assessed using a hemocytometer, and the solution was 266 diluted using DMEM with 2% (v/v) FBS. 267 Upon reaching confluency, T. gondii tachyzoites were placed onto Vero cells at a 268 density of 105/cm2 and then incubated with 5% CO 2 at 37 ºC. Following a 3-h incubation for 269 parasite invasion, we performed a single wash of the plate using phosphate-buffered saline 270 (PBS) (+) to eliminate any uninvaded tachyzoites. Subsequently, OA (MedChemExpress, NJ, 271 CAS# 480-11-5) or PYR (Tokyo Chemical Industry, Tokyo, Japan) was added to each well. 272 The culture medium, with or without these compounds, was replaced every two days. 273 274 SRB cytotoxicity assay. 275 Stock solutions of OA (50 mM) and PYR (2 mM) were prepared using DMSO and 276 stored at -80 ºC until needed. To assess the cytotoxicity of OA in host cells (Vero), we 277 examined the SRB (Tokyo Chemical Industry) cytotoxicity assay 22,23. Briefly, Vero cells 278 were seeded at a density of 10 5/cm2 in 24-well plates (Thermo Fisher Scientific, MA) and 279 maintained in DMEM supplemented with 2% FBS. After two days of cell seeding, we 280 confirmed the cell condition, i.e., confluence, before adding various concentrations (5 to 100 281 µM) of OA, PYR as an effective compound for anti-toxoplasmosis, or DMSO as a negative 282 control to each well. These cells were cultured for six days. The SRB colorimetric assay was 283 performed as described previously23. 284 285 Monolayer disruption assay. 286 Following the invasion of the T. gondii RH strain into Vero cells, as described in the 287 “Parasites” subsection, we cultured the parasites in 12-well culture plates for six days. This 288 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 12 culture was conducted with and without PYR (2 µM) or several concentrations of OA (5, 10, 289 25, 50, and 100 µM). Subsequently, the cultured cells were washed five times with 2 mL PBS 290 (+) to eliminate unattached cells. The cells that remained attached to the plate were fixed by 291 adding 1 ml of a 10% formalin neutral buffer solution (FUJIFILM Wako Pure Chemical Co.) 292 and left to stand for 24 h at room temperature. The fixed cells were stained with a 1% 293 aqueous solution of methylene blue (FUJIFILM Wako Pure Chemical Co.) for one hour. 294 Images of the stained cells in each well were captured from the bottom side of the culture 295 plate, and the coverage of stained cells in each well was analyzed using ImageJ software 296 (National Institutes of Health, Bethesda, Maryland). 297 298 Evaluation of the integrated density of Toxoplasma GFP signal and PV sizes in a host 299 cell. 300 The intracellular proliferation of the T. gondii RH-GFP strain was assessed in 12-well 301 culture plates. Confluent Vero cell Parasites were invaded by the parasites and treated with or 302 without PYR (2 µM) or different concentrations of OA (10 or 50 µM) for 48 h. Following 303 this treatment, nonadhesive parasites, cells, and cellular debris were removed by washing 304 twice with PBS (+). For each treatment group, images of five randomly selected fields were 305 captured, and the average GFP signal integrated density was measured and calculated using 306 ImageJ software. To determine percentages, the value of the GFP signal integrated density 307 obtained from each compound-treated infection group was divided by the value obtained 308 from the DMSO-treated infection group. 309 To analyze the PV sizes of T. gondii within individual host cells, Vero cells in 12-well 310 culture plates were infected with RH-GFP parasites. These infected cells were treated with or 311 without PYR (2 µM) or varying concentrations of OA (10 or 50 µM) for 30 h. The infected 312 cells were washed twice using PBS (+) to remove nonadhesive cells and extracellular 313 parasites. The cell nuclei were stained with a 0.05% Hoechst 33342 solution (Thermo Fisher 314 Scientific) in PBS (+) for 5 min. In each treatment group, fluorescence images of ten 315 randomly selected fields were captured, and the sizes of 100 PV were measured using ImageJ 316 software. 317 318 Evaluation of the effect of an ERK phosphorylation inhibitor on Toxoplasma 319 proliferation. 320 PD98059 was dissolved in DMSO to make a stock solution. The evaluation process of 321 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 13 PD98059’s effect on T. gondii proliferation was identical to the previously described method 322 but in a 6-well culture plate. The cells were harvested from culture plates by washing twice 323 with ice-cold PBS (-) on ice and centrifuged at 4 oC, 5,000 × g, for three min. Cell lysis was 324 achieved by incubating the cell pellet in 120 μ L of 1% Triton-X 100 buffer (1% Triton-X 100, 325 50 mM Tris-HCl (PH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulphonyl 326 fluoride, 1 × protease inhibitor) on ice for 15 min, followed by three 10-second cycles of 327 ultrasonic treatment. The total protein concentration was quantified using a bicinchoninic 328 acid assay kit (Thermo Fisher Scientific). The protein samples were combined with 3 × SDS 329 sample buffer containing 5% 2-mercaptoethanol and incubated at 95oC for 5 min. The protein 330 was loaded onto an SDS-PAGE gel using 5% stacking and 8% running gel. The proteins in 331 the gel were then transferred to a methanol-activated polyvinylidene difluoride membrane. 332 The membrane was treated with blocking buffer (1% nonfat milk in PBS (-) with 0.1% 333 Tween-20 solution (PBST)) for 10 min at room temperature. The membrane was incubated 334 with the primary antibodies (diluted in blocking buffer) for a minimum of one hour at room 335 temperature on a shaker. The following primary antibodies used were mouse monoclonal 336 antibody ERK 1/2 (1:1,000 dilution, sc-514302; Santa Cruz Biotechnology, Inc., CA), mouse 337 monoclonal antibody p-ERK 1/2 (1:1,000 dilution, sc-7383; Santa Cruz Biotechnology, Inc.), 338 and rabbit polyclonal antibody beta-actin (1:5,000 dilution, 5057S; Cell Signaling, Inc., MA). 339 After gently washing the membrane three times with PBST for 10 min, it was incubated with 340 secondary antibodies (1:1,000 dilution in blocking buffer) for 30 min at room temperature. 341 Goat anti–mouse IgG-horseradish peroxidase (HRP) (115-035-003; Jackson, Inc, PA) and 342 goat anti–rabbit IgG-HRP (111-035-003; Jackson, Inc) were used as secondary antibodies. 343 The membrane was then exposed to Immobilon Western Chemiluminescent HRP Substrate 344 (Merck, Darmstadt, Germany) for one min, and the results were visualized using a 345 luminescent image analyzer LAS4000 (FUJIFILM Wako Pure Chemical Co.). 346 347 In silico docking simulation. 348 The three-dimensional structure of the trametinib/MEK 1 complex was generated using 349 Molecular Operating Environment (MOE), version 2022.02 (CCG Inc, Montreal, Canada), 350 utilizing the Brookhaven Protein Databank 7JUR 28 as a reference. Docking simulations 351 within MOE were executed to mimic the trametinib-binding site of MEK 1 for PD98059 and 352 OA. Following this, the ligand interaction mode within MOE was used to assess the physical 353 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 14 and chemical parameters of trametinib, PD98059, and OA for MEK 1. 354 355 Mice. 356 Female C57BL/6J mice, weighing 20 ± 2 g and aged between 9 to 11 weeks, were 357 obtained from SLC (Hamamatsu, Japan). These mice were kept in controlled temperature and 358 humidity, under a 12-h day/night cycle, and provided with unlimited access to food and water. 359 All studies were conducted according to protocols approved by Chiba University. 360 361 Infection of Toxoplasma into mice. 362 To prepare the cysts, mice were orally infected with cysts of the archetypal type II T. 363 gondii strain Fukaya. Two months post-infection, the entire brain was extracted from the 364 infected mice and homogenized in 10 mL PBS (-) to create a brain suspension. The cyst 365 count was determined using a microscope. The suspension was then centrifuged at 440 × g 366 for 5 min, diluted to a concentration of 20 cysts in 500 µL suspension, and orally 367 administered to new mice. These newly infected mice were housed for four days prior to 368 treatment. 369 370 Compound treatment. 371 To prepare the compound solution, a 6.5% DMSO in water was used as the solvent. 372 Mice were orally administered with 500 µL of 6.5% DMSO as a negative control, 1 373 mg/kg/day of PYR, and 40 mg/kg/day of sulfadiazine in 6.5% DMSO as a positive control, or 374 50 mg/kg/day of OA in 6.5% DMSO orally once a day during 4-10 dpi. The body weight of 375 the mice was measured daily until 12 dpi. The survival curves were plotted by monitoring the 376 mouse health condition until 30 dpi. All animal treatments adhered to the guidelines 377 established set forth by the Chiba University Animal Ethics Committee. 378 379 Statistical analyses. 380 Statistical analyses were conducted using OriginPro, Version 2021 (OriginLab 381 Corporation, MA). All data except survival rate were analyzed using the ANOVA test. 382 Survival analysis was performed using the Kaplan–Meier method, and the log-rank test was 383 used for making comparisons. Data with P < 0.05 were considered statistically significant. 384 385 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 15 AUTHOR INFORMATION 386 Corresponding Author 387 Kenji Hikosaka 388 Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 389 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. 390 Email: [email protected] 391 392 Authors 393 Ziyue Z Zhang 394 Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 395 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan 396 Kazumi Norose 397 Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 398 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan; Department of Parasitology, Shinshu 399 University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan 400 Noriko Shinjyo 401 Laboratory of Immune Homeostasis, WPI Immunology Frontier Research Center, Osaka 402 University, Osaka 565-0871, Japan; School of Tropical Medicine and Global Health, 403 Nagasaki University, Nagasaki 852-8523, Japan; https://orcid.org/0000-0003-4501-4513 404 Xiaoxia X Lin 405 Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 406 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan 407 Akiko Suganami 408 Department of Bioinformatics, Graduate School of Medicine, Chiba University, 1-8-1 409 Inohana, Chuo-ku, Chiba 260-8670, Japan 410 Yutaka Tamura 411 Department of Bioinformatics, Graduate School of Medicine, Chiba University, 1-8-1 412 Inohana, Chuo-ku, Chiba 260-8670, Japan; https://orcid.org/0000-0001-5373-6909 413 Hirokazu Sakamoto 414 Department of Infection and Host Defense, Graduate School of Medicine, Chiba University, 415 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan; Department of Pathology, Stanford School 416 of Medicine, Stanford, California 94305, USA; https://orcid.org/0000-0001-9368-105X 417 418 419 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 16 Author Contributions 420 Z.Z.Z. and K.H. conceived the concept of this study. Z.Z.Z., K.N., N.S., X.X.L., H.S., and 421 K.H. designed the experiments. A.S. and Y.T. conducted the in silico docking simulation 422 analyses. Z.Z.Z. prepared first draft of the manuscript and the Figures. All authors edited the 423 manuscript and have approved the summitted manuscript. 424 425 Notes 426 The authors declare no competing financial interest. 427 428

429 This work was supported by JSPS KAKENHI (Grant Number JP19K07520, 430 JP19K16627, JP21KK0285, JP22H04633), AMED (Grant Number JP21fk0108569 and 431 JP22jm0610076), Ohyama Health Foundation, Hirose Foundation, Future Medicine 432 Education and Research Organization of Chiba University. 433 434 ABBREVIATIONS 435 Am, Astragalus membranaceus; FBS, fetal bovine serum; HDT, host-directed therapy; MEK, 436 mitogen-activated protein kinase/extracellular signal-regulated kinase; MOE, Molecular 437 Operating Environment; OA, oroxylin A; PV, parasitophorous vacuole; PYR, 438 pyrimethamine;Sb, Scutellaria baicalensis;TCM, Traditional Chinese medicine; Tg/ T. gondii, 439 Toxoplasma gondii; 440 441

442 (1) Flegr, J.; Prandota, J.; Sovi č ková, M.; Israili, Z. H. Toxoplasmosis--a Global Threat. 443 Correlation of Latent Toxoplasmosis with Specific Disease Burden in a Set of 88 Countries. PLoS 444 One 2014, 9 (3), e90203. https://doi.org/10.1371/journal.pone.0090203. 445 (2) Shen, C.; Zhang, G.; Song, X.; Xie, S.; Wang, C. Toxoplasma Gondii Pneumonia in an 446 Immunocompetent Individual. Am J Med Sci 2015, 350 (1), 70–71. 447 https://doi.org/10.1097/MAJ.0000000000000496. 448 (3) Lee, S.-B.; Lee, T.-G. Toxoplasmic Encephalitis in Patient with Acquired Immunodeficiency 449 Syndrome. Brain Tumor Res Treat 2017, 5 (1), 34–36. https://doi.org/10.14791/btrt.2017.5.1.34. 450 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 17 (4) Kalogeropoulos, D.; Sakkas, H.; Mohammed, B.; Vartholomatos, G.; Malamos, K.; 451 Sreekantam, S.; Kanavaros, P.; Kalogeropoulos, C. Ocular Toxoplasmosis: A Review of the Current 452 Diagnostic and Therapeutic Approaches. Int Ophthalmol 2022, 42 (1), 295–321. 453 https://doi.org/10.1007/s10792-021-01994-9. 454 (5) Caumes, E.; Bocquet, H.; Guermonprez, G.; Rogeaux, O.; Bricaire, F.; Katlama, C.; Gentilini, 455 M. Adverse Cutaneous Reactions to Pyrimethamine/Sulfadiazine and Pyrimethamine/Clindamycin in 456 Patients with AIDS and Toxoplasmic Encephalitis. Clin Infect Dis 1995, 21 (3), 656–658. 457 https://doi.org/10.1093/clinids/21.3.656. 458 (6) Montazeri, M.; Mehrzadi, S.; Sharif, M.; Sarvi, S.; Tanzifi, A.; Aghayan, S. A.; Daryani, A. 459 Drug Resistance in Toxoplasma Gondii . Front Microbiol 2018, 9, 2587. 460 https://doi.org/10.3389/fmicb.2018.02587. 461 (7) Ben-Harari, R. R.; Goodwin, E.; Casoy, J. Adverse Event Profile of Pyrimethamine-Based 462 Therapy in Toxoplasmosis: A Systematic Review. Drugs R D 2017, 17 (4), 523–544. 463 https://doi.org/10.1007/s40268-017-0206-8. 464 (8) Zhang, W.; Huai, Y.; Miao, Z.; Qian, A.; Wang, Y. Systems Pharmacology for Investigation 465 of the Mechanisms of Action of Traditional Chinese Medicine in Drug Discovery. Front Pharmacol 466 2019, 10, 743. https://doi.org/10.3389/fphar.2019.00743. 467 (9) Eigenschink, M.; Dearing, L.; Dablander, T. E.; Maier, J.; Sitte, H. H. A Critical Examination 468 of the Main Premises of Traditional Chinese Medicine. Wien Klin Wochenschr 2020, 132 (9–10), 469 260–273. https://doi.org/10.1007/s00508-020-01625-w. 470 (10) Maude, R. J.; Woodrow, C. J.; White, L. J. Artemisinin Antimalarials: Preserving the “Magic 471 Bullet”. Drug Dev Res 2010, 71 (1), 12–19. https://doi.org/10.1002/ddr.20344. 472 (11) Yang, X.; Huang, S.; Chen, J.; Song, N.; Wang, L.; Zhang, Z.; Deng, G.; Zheng, H.; Zhu, X.-473 Q.; Lu, F. Evaluation of the Adjuvant Properties of Astragalus Membranaceus and Scutellaria 474 Baicalensis GEORGI in the Immune Protection Induced by UV-Attenuated Toxoplasma gondii in 475 Mouse Models. Vaccine 2010, 28 (3), 737–743. https://doi.org/10.1016/j.vaccine.2009.10.065. 476 (12) Yang, X.; Huang, B.; Chen, J.; Huang, S.; Zheng, H.; Lun, Z.-R.; Shen, J.; Wang, Y.; Lu, F. 477 In Vitro Effects of Aqueous Extracts of Astragalus Membranaceus and Scutellaria Baicalensis 478 GEORGI on Toxoplasma gondii . Parasitol Res 2012, 110 (6), 2221–2227. 479 https://doi.org/10.1007/s00436-011-2752-2. 480 (13) Shi, H.; Ren, K.; Lv, B.; Zhang, W.; Zhao, Y.; Tan, R. X.; Li, E. Baicalin from Scutellaria 481 Baicalensis Blocks Respiratory Syncytial Virus (RSV) Infection and Reduces Inflammatory Cell 482 Infiltration and Lung Injury in Mice. Sci Rep 2016, 6, 35851. https://doi.org/10.1038/srep35851. 483 (14) Tan, X. X.; Zhu, H. M.; Tang, C.; Wang, X. HPLC-MS Identification on Flavonoids in 484 Astragali Radix Decoction. Drugs and Clinic 2021, 36 (2), 231–235. 485 https://doi.org/10.7501/j.issn.1674-5515.2021.02.004. 486 (15) Han, Y. K.; Kim, H.; Shin, H.; Song, J.; Lee, M. K.; Park, B.; Lee, K. Y. Characterization of 487 Anti-Inflammatory and Antioxidant Constituents from Scutellaria Baicalensis Using LC-MS Coupled 488 with a Bioassay Method. Molecules 2020, 25 (16). https://doi.org/10.3390/molecules25163617. 489 (16) Cho, P.; Shi, G.; Yap, M.; Boost, M. V. Effects of Lead Phytochemicals of Radix Scutellariae 490 on Acanthamoeba. Invest Ophthalmol Vis Sci 2016, 57 (15), 6591–6595. 491 https://doi.org/10.1167/iovs.16-20533. 492 (17) Lu, L.; Guo, Q.; Zhao, L. Overview of Oroxylin A: A Promising Flavonoid Compound. 493 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 18 Phytother Res 2016, 30 (11), 1765–1774. https://doi.org/10.1002/ptr.5694. 494 (18) Hong, G.-E.; Kim, J.-A.; Nagappan, A.; Yumnam, S.; Lee, H.-J.; Kim, E.-H.; Lee, W.-S.; 495 Shin, S.-C.; Park, H.-S.; Kim, G.-S. Flavonoids Identified from Korean Scutellaria Baicalensis Georgi 496 Inhibit Inflammatory Signaling by Suppressing Activation of NF- κ B and MAPK in RAW 264.7 497 Cells. Evid Based Complement Alternat Med 2013, 2013, 912031. 498 https://doi.org/10.1155/2013/912031. 499 (19) Zhang, Y.; Weng, Q.; Chen, J.; Li, M.; Han, J. Oroxylin A Attenuates IL-1 β -Induced 500 Inflammatory Reaction via Inhibiting the Activation of the ERK and PI3K/AKT Signaling Pathways 501 in Osteoarthritis Chondrocytes . Exp Ther Med 2021, 21 (4), 388. 502 https://doi.org/10.3892/etm.2021.9819. 503 (20) Wei, L.; Yao, Y.; Zhao, K.; Huang, Y.; Zhou, Y.; Zhao, L.; Guo, Q.; Lu, N. Oroxylin A 504 Inhibits Invasion and Migration through Suppressing ERK/GSK-3 β Signaling in Snail-Expressing 505 Non-Small-Cell Lung Cancer Cells. Mol Carcinog 2016, 55 (12), 2121–2134. 506 https://doi.org/10.1002/mc.22456. 507 (21) Milian, I. C. B.; Silva, R. J.; Manzan-Martins, C.; Barbosa, B. F.; Guirelli, P. M.; Ribeiro, M.; 508 de Oliveira Gomes, A.; Ietta, F.; Mineo, J. R.; Silva Franco, P.; Ferro, E. A. V. Increased Toxoplasma 509 Gondii Intracellular Proliferation in Human Extravillous Trophoblast Cells (HTR8/SVneo Line) Is 510 Sequentially Triggered by MIF, ERK1/2, and COX-2. Front Microbiol 2019, 10, 852. 511 https://doi.org/10.3389/fmicb.2019.00852. 512 (22) Vichai, V.; Kirtikara, K. Sulforhodamine B Colorimetric Assay for Cytotoxicity Screening. 513 Nat Protoc 2006, 1 (3), 1112–1116. https://doi.org/10.1038/nprot.2006.179. 514 (23) Shinjyo, N.; Nakayama, H.; Li, L.; Ishimaru, K.; Hikosaka, K.; Suzuki, N.; Yoshida, H.; 515 Norose, K. Hypericum Perforatum Extract and Hyperforin Inhibit the Growth of Neurotropic Parasite 516 Toxoplasma Gondii and Infection-Induced Inflammatory Responses of Glial Cells in Vitro. J 517 Ethnopharmacol 2021, 267. https://doi.org/10.1016/j.jep.2020.113525. 518 (24) Joyce, B. R.; Queener, S. F.; Wek, R. C.; Sullivan, W. J. Phosphorylation of Eukaryotic 519 Initiation Factor-2{alpha} Promotes the Extracellular Survival of Obligate Intracellular Parasite 520 Toxoplasma Gondii . Proc Natl Acad Sci U S A 2010, 107 (40), 17200–17205. 521 https://doi.org/10.1073/pnas.1007610107. 522 (25) Han, Y.; Adeyemi, O. S.; Kabir, M. H. Bin; Kato, K. Screening of Compound Libraries for 523 Inhibitors of Toxoplasma Growth and Invasion. Parasitol Res 2020, 119 (5), 1675–1681. 524 https://doi.org/10.1007/s00436-020-06673-9. 525 (26) O’Shaughnessy, W. J.; Dewangan, P. S.; Paiz, E. A.; Reese, M. L. Not Your Mother’s 526 MAPKs: Apicomplexan MAPK Function in Daughter Cell Budding. PLoS Pathog 2022, 18 (10), 527 e1010849. https://doi.org/10.1371/journal.ppat.1010849. 528 (27) Alessi, D. R.; Cuenda, A.; Cohen, P.; Dudley, D. T.; Saltiel, A. R. PD 098059 Is a Specific 529 Inhibitor of the Activation of Mitogen-Activated Protein Kinase Kinase in Vitro and in Vivo. J Biol 530 Chem 1995, 270 (46), 27489–27494. https://doi.org/10.1074/jbc.270.46.27489. 531 (28) Khan, Z. M.; Real, A. M.; Marsiglia, W. M.; Chow, A.; Duffy, M. E.; Yerabolu, J. R.; 532 Scopton, A. P.; Dar, A. C. Structural Basis for the Action of the Drug Trametinib at KSR-Bound 533 MEK. Nature 2020, 588 (7838), 509–514. https://doi.org/10.1038/s41586-020-2760-4. 534 (29) Scholl, F. A.; Dumesic, P. A.; Barragan, D. I.; Harada, K.; Bissonauth, V.; Charron, J.; 535 Khavari, P. A. Mek1/2 MAPK Kinases Are Essential for Mammalian Development, Homeostasis, and 536 Raf-Induced Hyperplasia. Dev Cell 2007, 12 (4), 615–629. 537 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 19 https://doi.org/10.1016/j.devcel.2007.03.009. 538 (30) Dubey, J. P.; Shen, S. K.; Kwok, O. C.; Frenkel, J. K. Infection and Immunity with the RH 539 Strain of Toxoplasma Gondii in Rats and Mice. J Parasitol 1999, 85 (4), 657–662. 540 (31) Alday, P. H.; Doggett, J. S. Drugs in Development for Toxoplasmosis: Advances, Challenges, 541 and Current Status. Drug Des Devel Ther 2017, 11, 273–293. https://doi.org/10.2147/DDDT.S60973. 542 (32) Hou, D.-X.; Kumamoto, T. Flavonoids as Protein Kinase Inhibitors for Cancer 543 Chemoprevention: Direct Binding and Molecular Modeling. Antioxid Redox Signal 2010, 13 (5), 691–544 719. https://doi.org/10.1089/ars.2009.2816. 545 (33) Zhao, Y.; Adjei, A. A. The Clinical Development of MEK Inhibitors. Nat Rev Clin Oncol 546 2014, 11 (7), 385–400. https://doi.org/10.1038/nrclinonc.2014.83. 547 (34) Abugri, D. A.; Witola, W. H. Interaction of Apigenin-7-O-Glucoside with Pyrimethamine 548 against Toxoplasma Gondii Growth. J Parasit Dis 2020, 44 (1), 221–229. 549 https://doi.org/10.1007/s12639-019-01185-5. 550 (35) Saito, H.; Murata, Y.; Nonaka, M.; Kato, K. Screening of a Library of Traditional Chinese 551 Medicines to Identify Compounds and Extracts Which Inhibit Toxoplasma Gondii Growth. J Vet Med 552 Sci 2020, 82 (2), 184–187. https://doi.org/10.1292/jvms.19-0241. 553 (36) Kaufmann, S. H. E.; Dorhoi, A.; Hotchkiss, R. S.; Bartenschlager, R. Host-Directed Therapies 554 for Bacterial and Viral Infections. Nat Rev Drug Discov 2018, 17 (1), 35–56. 555 https://doi.org/10.1038/nrd.2017.162. 556 (37) Zumla, A.; Rao, M.; Wallis, R. S.; Kaufmann, S. H. E.; Rustomjee, R.; Mwaba, P.; Vilaplana, 557 C.; Yeboah-Manu, D.; Chakaya, J.; Ippolito, G.; Azhar, E.; Hoelscher, M.; Maeurer, M.; Host-558 Directed Therapies Network consortium. Host-Directed Therapies for Infectious Diseases: Current 559 Status, Recent Progress, and Future Prospects. Lancet Infect Dis 2016, 16 (4), e47-63. 560 https://doi.org/10.1016/S1473-3099(16)00078-5. 561 (38) Novais, F. O.; Amorim, C. F.; Scott, P. Host-Directed Therapies for Cutaneous Leishmaniasis. 562 Front Immunol 2021, 12, 660183. https://doi.org/10.3389/fimmu.2021.660183. 563 564 565 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 20 Figure legends 566 Figure 1. OA did not exhibit cytotoxicity towards host cells at concentrations of 50 µM 567 and below. 568 Evaluation of OA toxicity to host cells (Vero cells) by SRB assay. The white and black bars 569 indicate the results of controls: no compound supplementation (DMSO-treated group) and 570 PYR supplementation, respectively. The gray bars indicate the results of various OA 571 concentration supplementations. The experiments were conducted three times independently 572 and in triplicates. Results are depicted as mean ± SEM. ***, P < 0.001 vs. the DMSO-treated 573 group. 574 575 Figure 2. OA suppresses T. gondii proliferation. 576 (A) Images of the monolayer disruption assay. Representative images for each condition are 577 shown. The experiments were independently repeated twice and triplicated within each. (B) 578 Comparison of the percentages of the bottom coverages for each condition. The white and 579 black bars indicate the results of controls: no compound supplementation (DMSO-treated 580 group) without and with T. gondii and PYR supplementation, respectively. The gray bars 581 indicate the results of various OA concentration supplementations. The experiments were 582 conducted three times independently and in triplicates. Data are presented as mean ± SD. 583 584 Figure 3. Assessing the effect of OA on T. gondii proliferation using the RH-GFP strain. 585 (A) Representative images of GFP signals from uninfected and RH-GFP-infected host cells 586 (Vero cells) at 48 h post-invasion. For the parasite-infected host cells, DMSO and PYR were 587 used as negative and positive controls, respectively. Scale bar: 100 µm. (B) Comparison of 588 the percentages of GFP signal-integrated density for each treatment. GFP signals were 589 calculated from six random fields using ImageJ software. Data are presented as the median ± 590 SD. ***, P < 0.001 vs. the infected DMSO-treated group. 591 592 Figure 4. OA suppresses the intracellular development of T. gondii. 593 (A) Representative images of GFP and Hoechst 33342 signals from uninfected and RH-GFP-594 infected host cells (Vero cells) at 36 h post-invasion. For the parasite-infected host cells, 595 DMSO and PYR were used as negative and positive controls, respectively. The host cells 596 were stained with Hoechst 33342. Scale bar: 100 µm. (B) The size of the area (µm 2) of the 597 parasitophorous vacuole (PV) across the indicated treatments. One hundred PVs in each 598 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 21 condition were quantified using ImageJ software. Data are presented as the median ± SD. ***, 599 P < 0.001 vs. the infected DMSO-treated group. ###, P < 0.001 vs. the infected 10 µM OA-600 treated group. 601 602 Figure 5. OA downregulates the host ERK1/2 phosphorylation in T. gondii-infected host 603 cells. 604 (A) The chemical structures of the flavone backbone, oroxylin A, and PD98059. (B) 605 Comparison of the phosphorylation levels of host ERK1/2 in each treatment by western 606 blotting. Phosphorylated ERK1/2 (p-ERK1/2) were detected using an anti-p-ERK1/2 607 antibody. Beta-actin was detected as a loading control. 608 609 Figure 6. The combination treatment of OA and PD98059 does not exhibit an additional 610 or synergistic inhibitory effect on T. gondii proliferation. 611 (A and B) Representative images of GFP signals from uninfected and RH-GFP-infected host 612 cells (Vero cells) treated with PD98059 (PD) (A) and single and combination treatments with 613 OA and PD (B) . Scale bar: 100 µm. (C) The percentages of GFP signal-integrated density 614 among the indicated treatments. GFP signals were calculated from six random fields using 615 ImageJ software. Data are presented as mean ± SD. (D) The size of the area (µm 2) of 616 parasitophorous vacuole (PV) among the indicated treatments. One hundred PVs in each 617 condition were quantified using ImageJ software. Data are presented as median ± SD. (A–D) 618 These experiments were repeated three times independently and conducted in triplicate. PD, 619 PD98059. 620 621 Figure 7. In silico docking simulation of ligand-bound complexes involving MEK1. 622 The results of the docking simulations between the MEK1 (gray) allosteric site and indicated 623 compounds (yellow). Trametinib and PD98059, known MEK1-specific inhibitors, are used as 624 controls for the simulation. The three-dimensional (upper panel) and two-dimensional 625 structures (lower panel) are shown. D208, F209, V211, and S212 in MEK1 are commonly 626 involved in the binding to the compounds. 627 628 Figure 8. OA increased the survival rate of T. gondii-infected mice. 629 (A) Experimental schedule of compound treatments. The T. gondii Fukaya strain was 630 infected at day 0 and the compounds were treated during day 4 to 11. The mice were then 631 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint 22 monitored for survival until day 30. (B) Survival curve depicting uninfected DMSO (n = 4), 632 infected DMSO (n = 8), infected P + S (n = 4), and infected OA (n = 8) treated mice for 30 633 days. P = 0.0638 between the infected DMSO-treated group and the infected OA-treated 634 group. (C) Changes in body weight of mice among the indicated conditions. Data are 635 presented as mean ± SD. ***, P < 0.001 vs. infected DMSO-treated group. DMSO, solvent; P 636 + S, PYR, and sulfadiazine. 637 638 Figure 9. Schematic illustration model of the mode-of-action of OA on T. gondii 639 proposed by this study. 640 T. gondii infection activates the MAPK signaling pathway in the host cell. Phosphorylation of 641 the host ERK1/2 promotes intracellular proliferation of T. gondii. Our findings suggest that 642 OA inhibits the host ERK1/2 phosphorylation via binding to the allosteric site of host MEK1. 643 This type of pathogen control by targeting host factors is known as host-directed therapy 644 (HDT). This model proposed by this study shows the potential for the development of a new 645 strategy, HDT, against Toxoplasma. PD: PD98059. 646 647 648 649 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint PYR (µM) OA (µM) - - 2 - - 5 - 10 - 25 - 50 - 100 0.0 0.5 1.0 1.5 2.0 2.5 *** Absorbance (510 nm) Fig 1._Zhang et al. Figure 1. OA did not exhibit cytotoxicity towards host cells at concentrations of 50 µM and below. Evaluation of OA toxicity to host cells (Vero cells) by SRB assay. The white and black bars indicate the results of controls: no compound supplementation (DMSO - treated group) and PYR supplementation, respectively. The gray bars indicate the

of various OA concentration supplementations. The experiments were conducted three times independently and in triplicates. Results are depicted as mean ± SEM. ***, P < 0.001 vs. the DMSO-treated group. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint A B PYR 2 µM Uninfected No treatment RH 5025105 Infected OA Bottom coverage in each well (%)0 20 40 60 80 100 PYR (µM) OA (µM) - - 2 - - 5 - 10 - 25 - 50 RH - - - + + + + + + Fig 2._Zhang et al. Figure 2. OA suppresses T. gondii proliferation. (A) Images of the monolayer disruption assay. Representative images for each condition are shown. The experiments were independently repeated twice and triplicated within each. (B) Comparison of the percentages of the bottom coverages for each condition. The white and black bars indicate the

of controls: no compound supplementation (DMSO-treated group) without and with T. gondii and PYR supplementation, respectively. The gray bars indicate the results of various OA concentration supplementations. The experiments were conducted three times independently and in triplicates. Data are presented as mean ± SD. (µM) (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint A B Uninfected Infected DMSO PYR 2 µM OA 10 µM OA 50 µM 100 80 60 40 20 0 GFP signal integrated density (%) PYR (µM) OA (µM) RH-GFP - - - - - + + + 2 - 10- + - 50 *** *** *** Fig 3._Zhang et al. Figure 3. Assessing the effect of OA on T. gondii proliferation using the RH-GFP strain. (A) Representative images of GFP signals from uninfected and RH-GFP-infected host cells (Vero cells) at 48 h post-invasion. For the parasite-infected host cells, DMSO and PYR were used as negative and positive controls, respectively. Scale bar: 100 µm. (B) Comparison of the percentages of GFP signal-integrated density for each treatment. GFP signals were calculated from six random fields using ImageJ software. Data are presented as the median ± SD. ***, P < 0.001 vs. the infected DMSO- treated group. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint RH-GFP Parasitophorous vacuole size (μm2) 200 150 50 0 PYR (µM) OA (µM) - - - - - + + + 2 - 10- 100 + - 50 *** *** ### *** A B Infected GFP Hoechst Merge UninfectedDMSOPYR 2 µMOA 10 µM OA 50 µM Fig 4._Zhang et al. Figure 4. OA suppresses the intracellular development of T. gondii. (A) Representative images of GFP and Hoechst 33342 signals from uninfected and RH -GFP-infected host cells (Vero cells) at 36 h post-invasion. For the parasite-infected host cells, DMSO and PYR were used as negative and positive controls, respectively. The host cells were stained with Hoechst 33342. Scale bar: 100 µm. (B) The size of the area (µm2) of the parasitophorous vacuole (PV) across the indicated treatments. One hundred PVs in each condition were quantified using ImageJ software. Data are presented as the median ± SD. ***, P < 0.001 vs. the infected DMSO-treated group. ###, P < 0.001 vs. the infected 10 µM OA -treated group. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint +T. gondii + + + OA (µM) - 10 -50 PD98059 (µM) - - - 10 - - - + - 25 WB: anti-p-ERK1/2 WB: anti-beta-actin – 37 – 50 p-ERK1 p-ERK2 A Oroxylin A PD98059 Flavone backbone (the basic structure of flavonoids) Fig 5._Zhang et al. Figure 5. OA downregulates the host ERK1/2 phosphorylation in T. gondii-infected host cells. (A) The chemical structures of the flavone backbone, oroxylin A, and PD98059. (B) Comparison of the phosphorylation levels of host ERK1/2 in each treatment by western blotting. Phosphorylated ERK1/2 (p-ERK1/2) were detected using an anti-p-ERK1/2 antibody. Beta-actin was detected as a loading control. B Beta-actin kDa – 37 – 50 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint A B C D PD 10 µM PD 25 µM PD 0 µM Infected Uninfected Infected OA 10 µMOA 50 µM PD 10 µM PD 25 µMPD 0 µM Parasitophorous vacuole size (μm2) 120 80 60 20 0 OA (µM) PD (µM) RH-GFP + 10 - + + + + 10 25 - - 10 25 10 10 + 50 - + + 10 25 50 50 40 100 100 80 60 40 20 0 GFP signal integrated density (%) OA (µM) RH-GFP + + + + + 10 - - 10 10 + 50 + 50 + 50 + - PD (µM) - 10 25 10 25- 10 25- - - - 120 Fig 6._Zhang et al. Figure 6. The combination treatment of OA and PD98059 does not exhibit an additional or synergistic inhibitory effect on T. gondii proliferation. (A and B) Representative images of GFP signals from uninfected and RH -GFP-infected host cells (Vero cells) treated with PD98059 (PD) (A) and single and combination treatments with OA and PD (B). Scale bar: 100 µm. (C) The percentages of GFP signal-integrated density among the indicated treatments. GFP signals were calculated from six random fields using ImageJ software. Data are presented as mean ± SD. (D) The size of the area (µm2) of parasitophorous vacuole (PV) among the indicated treatments. One hundred PVs in each condition were quantified using ImageJ software. Data are presented as median ± SD. (A–D) These experiments were repeated three times independently and conducted in triplicate. PD, PD98059. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint Fig 7._Zhang et al. Figure 7. In silico docking simulation of ligand-bound complexes involving MEK1. The results of the docking simulations between the MEK1 (gray) allosteric site and indicated compounds (yellow). Trametinib and PD98059, known MEK1-specific inhibitors, are used as controls for the simulation. The three-dimensional (upper panel) and two-dimensional structures (lower panel) are shown. D208, F209, V211, and S212 in MEK1 are commonly involved in the binding to the compounds. MEK1 Trametinib PD98059 OA (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint T. gondii cysts infection 0 4 11 30 Days Compounds treatment A B C Survival rate (%) Days post-infection Uninfected DMSO Infected P+S Infected OA (alive) Infected DMSO (dead)Infected OA (dead) Days post-infection Body weight (%) P = 0.0638 vs the infected DMSO group *** Fig 8._Zhang et al. Figure 8. OA increased the survival rate of T. gondii-infected mice. (A) Experimental schedule of compound treatments. The T. gondii Fukaya strain was infected at day 0 and the compounds were treated during day 4 to 11. The mice were then monitored for survival until day 30. (B) Survival curve depicting uninfected DMSO (n = 4), infected DMSO (n = 8), infected P + S (n = 4), and infected OA (n = 8) treated mice for 30 days. P = 0.0638 between the infected DMSO -treated group and the infected OA-treated group. (C) Changes in body weight of mice among the indicated conditions. Data are presented as mean ± SD. ***, P < 0.001 vs. infected DMSO-treated group. DMSO, solvent; P + S, PYR, and sulfadiazine. (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint MEK1 MAPKK MEK2 MEK1 MEK2 MAP3K Raf1 PD ERK1 ERK2 ERK2 ERK1 MAPK p p p p p Growth factor receptor T. gondii intracellular proliferation MAPK signaling pathway OA T. gondii infection Fig 9._Zhang et al. Figure 9. Schematic illustration model of the mode-of-action of OA on T. gondii proposed by this study. T. gondii infection activates the MAPK signaling pathway in the host cell. Phosphorylation of the host ERK1/2 promotes intracellular proliferation of T. gondii. Our findings suggest that OA inhibits the host ERK1/2 phosphorylation via binding to the allosteric site of host MEK1. This type of pathogen control by targeting host factors is known as host -directed therapy (HDT). This model proposed by this study shows the potential for the development of a new strategy, HDT, against Toxoplasma. PD: PD98059. Activate Activate Phosphorylation Phosphorylation (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 17, 2024. ; https://doi.org/10.1101/2024.03.17.585380doi: bioRxiv preprint