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Novel small molecule retrograde transport blocker confers post-exposure protection against ricin intoxication

Ricin is a highly toxic type 2 ribosome-inactivating protein (RIP) which is extracted from the seeds of castor beans. Ricin is considered a potential bioterror agent and no effective antidote for ricin exists so far. In this study, by structural modification of a retrograde transport blocker Retro-2cycl, a series of novel compounds were obtained. The primary screen revealed that compound 27 has an improved anti-ricin activity compare to positive control. In vitro pre-exposure evaluation in Madin–Darby Canine Kidney (MDCK) cells demonstrated that 27 is a powerful anti-ricin compound with an EC50 of 41.05 nmol/L against one LC (lethal concentration, 5.56 ng/mL) of ricin. Further studies surprisingly indicated that 27 confers post-exposure activity against ricin intoxication. An in vivo study showed that 1 h post-exposure administration of 27 can improve the survival rate as well as delay the death of ricin-intoxicated mice. A drug combination of 27 with monoclonal antibody mAb4C13 rescued mice from one LD (lethal dose) ricin challenge and the survival rate of tested animals is 100%. These results represent, for the first time, indication that small molecule retrograde transport blocker confers both in vitro and in vivo post-exposure protection against ricin and therefore provides a promising candidate for the development of anti-ricin medicines.


Introduction
Ricin is a type 2 ribosomal inactive protein 1 that is abundant in Ricinus communis seeds (castor beans) 2 . The LD 50 of ricin for human adults is approximately 5e25 mg/kg if the exposure is from injection or inhalation and the oral lethal dose (LD) is 1 mg/kg 3 . After injection of ricin, victim would develop multiple organ dysfunction syndrome (MODS) which eventually caused death within 3 days. The inhalation of ricin could cause acute respiratory distress syndrome at 18e24 h, death generally occurs three days after exposure 4 . The ingestion of ricin can cause toxin induced liver and kidney failure, which lead to death 3e5 days after exposure 5 . Owing to its high toxicity, high stability and relatively easy purification techniques, ricin is considered a potential bioterrorism agent 6 . To date, neither effective clinical treatment nor specific medicine for ricin intoxication exists. Supportive care remains the only measure when treating ricin-poisoned patients. Therefore, research and discovery of an effective ricin antidote is of great importance to public health.
Ricin consists of two subunits: a catalytically active A (RTA)-chain joined by one disulfide bond to a B (RTB) chain that acts as an N-acetylgalactosamine-specific lectin 7 . By virtue of RTB, ricin toxins can be internalized by endocytosis. Once inside the cell, ricin are transported from early endosomes to the endoplasmic reticulum (ER) via the Golgi apparatus 8 and this transport pathway has been termed retrograde transport 9,10 . Resorting to retrograde transport, the RTA chain is able to travel into the cytoplasm and depurinate a specific adenine residue in 28S ribosomal RNA, which disables the protein synthesis ability of ribosomes 11,12 and eventually causes cell death 13 .
Many attempts have been made to identify small molecule ricin inhibitors 14e16 . A retrograde transport blocker Retro-2 ( Fig. 1), discovered by Julien Barbier and coworkers 17 , proved to be a potential small molecule inhibitor of type 2 ribosomeinactivating proteins (RIPs) such as ricin and shiga toxins. In their experiments they reported that a 1 h pre-intoxication administration of 200 mg/kg Retro-2 can protect mice from a 2 mg/kg ricin exposure via the nasal route with a survival rate of 100%. Study of the mechanism revealed that Retro-2 does not inhibit the catalytically active site of type 2 RIPs; instead, Retro-2 blocks the retrograde transport of toxins from early endosomes to the ER through the trans-Golgi network (TGN) 18 . Later, Park et al. 19 reported that the bioactive compound was not Retro-2 but the cyclized product Retro-2 cycl (Fig. 1). Structuraleactivity relationship instructed structural modification of Retro-2 cycl produced a more powerful shiga toxin inhibitor compound 94 ( Fig. 1) with an in vitro EC 50 of 300 nmol/L in HeLa cells 20 . Anti-ricin data has not been reported for compound 94.
So far, all reported Retro-2 series compounds need to be administered pre-intoxication to achieve their anti-type 2 RIPs activity. Therefore, the compound can only be used as a prophylactic agent, which limits compounds' practicability. In this study, we modified the structure of Retro-2 cycl in order to obtain bioactivity improved and post-exposure effective compounds against ricin intoxication.

Structural modification of Retro-2 cycl
The optimization work of this study was focused on three main substitutable parts of Retro-2 cycl : the benzene ring of dihydroquinazolinone (R 1 ), the substituent group on 3-N (R 2 ) and the substituent group on 5-position of thiophene (R 3 ) (Fig. 2). The MadineDarby Canine Kidney (MDCK) cell line was used to assess the anti-ricin ability of new compounds at a concentration of 50 mmol/L. MDCK cell line is sensitive to ricin intoxication and a good correlation between cell survival rate and compound activity has been observed in the preliminary experiments, so in this section compounds activity were quantified by cell survival rate. Retro-2 cycl was used as the positive control in primary screen of new compound.
First, by replace 7-C or 8-C with N, we substitute the benzene ring with a more electrophilic pyridine ring. Then R 3 was subsequently substitute with various groups to explore the bioactivity of pyridine ring system, the results were shown in Table 1. Replace 8-C of Retro-2 cycl with N resulted in 1 and the compound retained the activity of Retro-2 cycl , which indicated the modification in 8-C is tolerable. Replacement of 7-C, however, resulted in a loss of activity. The substitution of R 3 with bromo retained the activity compare to Retro-2 cycl . Substitution with phenyl or heterocyclic 5-methylthiazole decreased the activity compare to Retro-2 cycl but still demonstrated a mild antiricin ability.
Then we probed the influence of substituent R 2 on activity, results were shown in Table 2. In the 8-N replaced series compounds, substitution of R 2 with o-chlorobenzene resulted in 9e12, they retained or slightly increased the anti-ricin activity. It was evidenced by 7 and 8 compare to 3 and 4 respectively that when replace benzene on R 2 with o-methoxybenzene, the activity increased. Certified that the o-substitution on 3-phenyl could benefit to the in vitro anti-ricin activity of compounds. The substitution of R 2 and R 3 did not improve the activity of 7-N series Figure 1 The structures of Retro-2, Retro-2 cycl and compound 94.
Novel molecule confers post-exposure protection against ricin intoxication.
compounds. Compounds 18e20 substitute benzene on R 2 with 5substituedthiazole, the modification showed little influence on activity compare to Retro-2 cycl .
Based on the results of 8-N series compounds we concluded that the increase of electrophilic on dihydroquinazolinone is a feasible modification strategy. Based on that we then explored the influence of a more electrophilic ring system on compounds' activity. A series of compounds with 6-F or 7-F dihydroquinazolinone structure were synthesized and evaluated, results shown in Table 3. Compounds with 7-F ring system demonstrated little difference in activity compare to Retro-2 cycl . But 6-F substituted compounds had a significant improvement of antiricin activity evidenced by activities of 25e27. Therefore, electrophilic substitute in 6-H increased compounds' activity. Additionally, substitute R 2 with o-methoxybenzene also improved the bioactivity of compounds as 24 possess the best activity among 7-F series compounds. The methylation on 1-N is a tolerable modification and may benefit to the pharmacokinetic quality of   compounds as it stabilized the secondary amine of dihydroquinazolinone. Compound 27, which combined the optimized structure discussed above, conferred a significant improvement in anti-ricin activity compare to Retro-2 cycl .

Chemistry
As outlined in Scheme 1, the general synthesize strategy of designed compounds was by condensation of amino intermediate 35a/35b with aldehyde intermediate 41/42. The required amino intermediate of each compound was synthised follow two routes: Route A produce the required nicotinamide intermediates for compounds 1e17 by condensation of nicotinic acid/isonicotinic acid 29 with amide 30, the condensation reagents were 1-(3dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt). Route B produce fluro substituted amide intermediates for compounds 21e27 from starting material F-substituted o-amide benzoic acid, by cyclization with thiposigene. F-substituted lsatoic anhydride 33 was produced, then 33 was retact with required amide in acid to form 35a or methylated by MeI to form 34, then 34 reacted with amide to form 35b. The required amino intermediates for 18e20 were also synthesized according to Route B by replace the starting material with o-amide benzolic acid. The aldehyde intermediates are mostly commercial available with one exception: 5-(2methylthiazol-4-yl)thiophene-2-carbaldehyde was synthesized from 5-acetylthiophene-2-carboxylic acid (36), the carboxylic group of starting material was protected by esterification with MeOH to form 37, then 37 was brominated by cupric bromide and cyclized with thiacettamide to form 39. Intermediate 39 was subjected to a reduction by LiAlH 4 and then oxidized by MnO 2 to form 41 (Route C).

In vitro pre-intoxication anti-ricin activity of 27
The anti-ricin ability of 27 against ricin intoxication in MDCK cells was evaluated to determine in vitro pre-intoxication anti-ricin activity of the compound. Compound 94, a powerful anti-shiga Retro-2 cycl series compound developed by Romain and coworkers 20 , demonstrated better anti-ricin ability compare to Retro-2 cycl in our pre-experiments. Therefore, compound 94 was used as a positive control in further evaluation experiments. In this assay, MDCK cells were pretreated with different concentrations of 27 (0.01, 0.03, 0.10, 0.30 or 0.90 mmol/L) or compound 94 (0.01, 0.15, 0.31, 0.62, 1.25, 2.50, 5.00, 10.00, 20.00, 30.00 or 40.00 mmol/L), incubated for 12 h and then challenged with 1 LC (5.56 ng/mL) of ricin. Then, cells were incubated for another 12 h, and then, the survival rate of cells in each group and the EC 50 values of tested compounds were calculated (results shown in Fig. 3). The results indicated that 27 was effective against ricin at a concentration higher than 0.01 mmol/L suggested by the improvement of cell survival rate, and 27 can protect cells against a ricin challenge with the maximum survival rate of 100.95AE6.72% at a concentration of 0.9 mmol/L, while compound 94 was effective at a concentration higher than 5 mmol/L and could convey a maximum cell survival rate of 66.21AE2.57% at the highest tested concentration of 40 mmol/L. The pre-exposure treatment EC 50 value of 27 was 41.05 nmol/L and the EC 50 value of compound 94 was 16.13 mmol/L. In summary, 27 have stronger anti-ricin activity compared to compound 94.  Then, the protection ability of 27 against ricin was assessed. In this assay, MDCK cells were pretreated with different concentrations of 27 (0.011, 0.033, 0.10, 0.30 or 0.90 mmol/L), incubated for 2 h and then challenged with different concentrations of ricin (0.06, 0.20, 0.60, 1.80, 5.50, 16.70, 50.00 or 100.00 ng/mL). The rest of the procedures were the same as in the above assay (results shown in Fig. 4). The results showed that 27 at a concentration as low as 0.033 mmol/L could protect cells from 1 LC (5.56 ng/mL) ricin challenge, with a cell survival rate of 102.68AE15.47%, the significant difference compare to ricin control group were determined by t-test with a result of P < 0.01. At a concentration of   .54% at the highest tested concentration of 10.00 mmol/L; therefore, the EC 50 for compound 94 cannot be calculated. The survival rate of 27 in all tested concentrations had a statistical significance compared to the survival rate of the ricin control group and compound 94 group (P < 0.001), therefore the therapeutic effect of 27 was significant.
The time-effect relationship of 27 was evaluated to determine the therapeutic window of the compound. In this assay, MDCK cells were intoxicated with one LC of ricin, and then, after 1, 2, 4 or 6 h, the cells were respectively treated with different concentrations of 27 (0.16, 0.31, 0.63, 1.25, 2.50, 5.00 or 10.00 mmol/L); the rest of the procedures were the same as in dose-effect assay (results shown in Fig. 6). Among the four sets of experiments, a 10 mmol/L administration of 27 in the 1 h post-exposure treatment groups conferred the best survival rate of the cells (100AE2.23%) and a lowest value of 0.16 mmol/L for 27 in 1 h group still conferred a survival rate of 64.40AE7.40%, which showed significant improvement compared to the survival rate of the ricin control group (12.57AE0.28%). The 2 h post-exposure treatment groups showed a milder yet still significant anti-ricin activity with the highest survival rate of 88.68 AE 2.28% and lowest rate of 40.43 AE 9.02%. In the 4 and 6 h groups, however, 27 demonstrated weaker anti-ricin activity. The highest survival rate in 4 h group was 58.64 AE 4.19% when given 5 mmol/L of 27, and an increasing of dose conferred no improvement (58.42 AE 2.31%), which indicated that the best survival rate that 27 could achieve when administered 4 h post-intoxication was approximately 58%. A similar result was observed in the 6 h group, with the best survival rate of approximately 50%. The results indicated that the therapeutic ability of 27 was correlated with the administration time and that an early treatment is crucial for 27 to achieve a better therapeutic effect.

In vivo pre-intoxication anti-ricin activity of 27
In vitro bioactivity evaluation results of 27 encouraged us to continue exploring the in vivo bioactivity of compound 27. Preexperiments were performed to determine the toxicity of 27 on mice. The results indicated that at a concentration of 50 mg/kg, 27 has no influence on the viability of female CD-1 mice. A model of ricin intoxication by i.p. administration was developed to mimic   exposure through injection. This is one of the most lethal approaches of ricin intoxication. A dose of 13 mg/kg for ricin was used in this model. Compound 94 was used as a positive control. Mice were randomly divided into the blank control group, ricin control group, positive control group and 27 group. The test groups of mice were respectively treated with 50 mg/kg of 27 or compound 94 1 h prior to intoxication with one LD (13 mg/kg) of ricin; then the number of surviving mice were recorded every day until 6 days after intoxication. The survival curve is shown in Fig. 7.
The results indicated that the death of mice started from day 3, with four deaths occurring in the ricin control group and six deaths in the compound 94 group, but only one mouse died in the 27 group. At day 4, only three mice were still alive in the ricin control group and the compound 94 group, while six survived in the 27 group. At the end of this experiment, all mice in the ricin control group and compound 94 group died, but one mouse survived in the 27 group and a regain of bodyweight was observed in that mouse. The survival curves of 27 group and ricin control group were analyzed by Log-rank (ManteleCox) Test, the result indicated that the survival curve of 27 is significant different compared with that of ricin control group (P Z 0.0374). The pre-treatment of mice with 27 could increase the survival rate of mice and prolong their survival time while no such delayed effect was observed in the positive control group.

In vivo post-intoxication anti-ricin activity of 27
The post-exposure anti-ricin activity of 27 in mice was also evaluated. The intoxicated animal model and ricin dose were the same as Section 2.5. Due to the poor activity of compound 94 in the pre-exposure assay, no positive control group was set in this assay. Mice were respectively treated with 50 mg/kg of 27 at 1, 2 or 6 h after intoxication. The behavior and survival rate of mice were observed every day for 12 days. The survival rate curve is shown in Fig. 8. Ricin control group mice started to die from day 2, three deaths occurred that day, and all the rest died the day after. The test group, however, revealed a delay of total death compared to that of the control group. Total death in the 1 and 4 h group occurred 4 days later than that of the control group. The same delaying effect was also observed in the 2 h group and one mouse survived the ricin challenge. The survival curve of 27 groups was significant different compared with that of ricin control group, tested by Log-rank (ManteleCox) Test (P < 0.05). Collectively, post-intoxication administration of 27 can prolong the survival time of mice.

Combined pharmacotherapy of 27 with mAb4C13 against ricin intoxication
It has been reported by Na Dong and co-workers 21 that monoclonal antibody mAb4C13 can recognize the specific epitope of ricin with very high affinity and the antibody demonstrated potent anti-ricin efficiency in both in vitro and in vivo experiments. Therefore, we explored the efficacy of a combined administration of 27 and mAb4C13 against ricin intoxication in mice.
The experiments were performed on the same intoxication model as in Section 2.5. The antibody group were treated with vehicle 0.5 h after intoxication and then treated with 300 mg/kg mAb4C13 2 h after intoxication. The combined group were treated with 50 mg/kg of 27 0.5 h after intoxication then treated with 300 mg/kg of mAb4C13 2 h after intoxication. Ricin control group were challenged with 1 LD (13 mg/kg) of ricin and then given vehicle at 0.5 and 2 h after intoxication. The results were shown in Fig. 9. The single use of mAb4C13 protected mice from ricin challenge to some extent and achieved a 53.85% survival rate 20 days after intoxication. The combination of 27 and mAb4C13 strongly protected mice from one LD ricin challenge with a survival rate of 100% at the end of the experiment. Log-Rank test confirmed that the combination curve was significantly different   from antibody curve (P < 0.05). Therefore, a combination of 27 with mAb4C13 is an effective therapeutic approach against ricin intoxication in mice.
Then the anti-ricin ability of this combination was also evaluated. In this experiment, each group of mice was challenged respectively with 2.5, 4 or 5 LD (32.5, 52 or 65 mg/kg) of ricin. Then each group was treated with 50 mg/kg of 27 0.5 h after intoxication and 300 mg/kg of mAb4C13 1.5 h after the administration of 27. Results were shown in Fig. 10. In the 2.5 LD group, only one mouse died 25 days after intoxication and the final survival rate is 90.90%, indicated that the combination is effective against 2.5 LD challenge of ricin. The survival curve of 4 and 5 LD group began decrease from day 3, and the final survival rate are 44.44% (4 LD) and 20% (5 LD), therefore the combination was less effective against a intoxication dose higher than 4 LD of ricin.
Former studies revealed that the inhibition bio-mechanism of Retro-2 cycl series compounds is to block the intracellular retrograde trafficking of toxins from early endosomes to the ER through the trans-Golgi network 17 , so intuitively, an advanced deliver of compounds is crucial for the anti-toxin activity of Retro-2 cycl series compounds. However, in our post-intoxication treatment experiments, Retro-2 cycl series compound 27 demonstrated specific anti-ricin activity when given after cell intoxication. These results of our study expanded the practicability of Retro-2 cycl series compounds against ricin. The reason of the improvement could be resulted from the stronger retrograde transport block ability of 27. The intracellular trafficking of ricin was proved to be sustained, so after intoxication, it took a period of time for ricin to transport inside the cell and accumulate RTA into cytoplasm to reach the required toxic concentration. Therefore, the administration of a transport blocker during that period can still protect the cell, however, a fairly effective blocker is required, as a considerable portion of RTA is already inside the cytoplasm, and the block of the rest should be timely and efficient. The structure modification promotes 27 to be a stronger retrograde transport blocker of ricin (which can be concluded from the in vitro experiments) and would be more efficient when blocking the intracellular trafficking of ricin.
Retro-2 was discovered by high throughput screen, it countered ricin intoxication by block the retrograde transport of the toxin from early endosome to ER. However, as the detailed molecular level mechanism of retrograde transport was not thoroughly explained, no specific protein could be used to explore the antiricin mechanism of 27. But as 27 possesses the same dihydroquinazoline structure of Retro-2 series compounds, the compound should share the same target protein as other Retro-2 series compounds. In the future when key proteins and the mechanism of retrograde transport route are studied, the mechanism study of 27 can be performed to interpret the specific target and binding sites of the compound to provide guidance for future developments of Retro-2 series compounds.

Conclusions
In this study, a novel compound 27 with a 12 h pre-exposure EC 50 of 41.05 nmol/L against ricin was discovered. Later experiments showed the compound had significant post-exposure therapeutic activity against ricin intoxication with a 1 h postexposure EC 50 of 144 nmol/L. This is the first time a retrograde transport blocker conferred post-exposure anti-ricin activity. The combined administration of 27 with mAb4C13 effectively rescued mice from LD ricin challenge and therefore provides a possible therapeutic approach for treatment of ricin intoxication.

Chemistry
Melting points were determined with an XRC-1 melting point apparatus (Beijing, China) and are reported as uncorrected. 1 H NMR spectra were measured on a Bruker AVANCE spectrometer (Bremen, Germany) at 400 MHz using tetramethylsilane (TMS) as an internal standard. Mass spectra (MS) were recorded by an HP 5989B spectrometer with an electrospray ionization (ESI) source (Palo Alto, CA, USA). High-resolution mass spectra were obtained with a Varian spectrometer (Palo Alto, CA, USA). All chemicals and solvents were of analytical or reagent grade. They were purchased commercially and used as received, unless otherwise stated.

Synthesis of 5-(2-methylthiazol-4-yl)thiophene-2-carbald ehyde (intermediate 41)
To a solution of 5-acetylthiophene-2-carboxylic acid (5 g, 29.4 mmol) in methanol (120 mL) and DMF (1.5 mL) was added dropwise SOCl 2 (12.24 g, 102.9 mmol) at room temperature, and the mixture was heated to reflux for 5 h. Next, the pH of the mixture was adjusted to 7 by a saturated NaHCO 3 solution. The mixture was filtered, and the precipitates were collected, washed with water and dried under a vacuum to produce 5acetylthiophene-2-methylacetate (intermediate 6) as a gray solid Methyl-5-acetylthiophene-2-carboxylate (4.75 g, 25.7 mmol), cupric bromide (11.48 g, 51.4 mmol) and ethyl acetate (150 mL) were added to a 250 mL round flask, and then, the mixture was stirred and heated to reflux for 8 h. Next, the mixture was filtered, and the filtrates were washed with water and dried over MgSO 4 . The solution was then evaporated to produce 5-(2-bromoacetyl) thiophene-2-methylacetate (intermediate 7) as a light green solid (6.2 g, Yield 91.2%). 1  To a 250 mL round flask were added 7 (6.2 g, 23.5 mmol), thioacetamide (1.95 g, 25.9 mmol) and DMF (100 mL), and the mixture was heated to reflux for 6 h. Then, the mixture was cooled to room temperature and quenched with saturated NH 4 Cl water solution. The mixture was filtered to collect the precipitate, and the filtered cakes were washed with water and dried to produce 5- To a suspension of LiAlH 4 (1.83 g, 48.3 mmol) in anhydrous THF (35 mL) at 0 C was added dropwise 8 (3.85 g, 16.1 mmol). The mixture was then stirred at 0 C for 90 min, the reaction quenched with water, and the solution filtered; the filtrate was dried over MgSO 4 . Next, the solution was evaporated to produce (5-(2-methylthiazol-4-yl)thiophen-2-yl)methanol (intermediate 9) as a yellow oil (3.28 g, Yield 97%), and the product was used without purification.
Intermediate 9 (3.28 g, 15.6 mmol) was dissolved in DCM (85 mL), and MnO 2 (27.1 g, 312.4 mmol) was added to the solution. The resulting mixture was stirred at room temperature for 2 h, and after completion, the mixture was filtered and the filtrate evaporated to produce 5-  13 1 mmol) was dissolved in 25 mL of THF and added dropwise to the system at 50 C. Then, the mixture was stirred at 60 C for 8 h. The solutions were evaporated and recrystallized in ethyl acetate (30 mL) to produce 33 as a yellow solid. 33 (18.9 mmol) was added dropwise to a suspension of 60% sodium hydride (w/w) in mineral oil (22.68 mmol) in DMF (35 mL) at 0 C. After stirring at 0 C for 30 min, the mixture was warmed to room temperature, and methyl iodide (22.68 mmol) was added. The mixture was allowed to stir overnight and then was quenched in an ice bath. The precipitates were collected by filtration to produce 34 as a white power (2.46 g, Yield 66.7%). Then 33 or 34 (12.6 mmol) and required amine (13.87 mmol) were dissolved in acetic acid (7 5 mL). The mixture was then stirred at 60 C for 1 h. Then, the reaction mixture was purified with silica gel chromatography. The solvent was evaporated to produce required intermediate 35a or 35b. Then 35a or 35b (1.4 mmol) and required aldehyde intermediate 41 or 42 (2 mmol) were disloved in AcOH (5 mL) and then stirred at room temperature for 24 h, the solution was evaporated under reduced pressure and the residue was purified with silica gel chromatography to produced pure products18e27.
Cell survival rate (%)Z(A 450 in the experiment group/A 450 in blank control group) Â 100 (1) The experiments in each group were repeated six times.

MDCK cells post-exposure treatment assay
The cell seeding, data retrieval and survival rate calculation methods were the same as those in Section 4.3.1. After removal of serum, cells were challenged with 5.56 ng/mL ricin, incubated for 1 h and then treated with gradient concentrations of 27 or compound 94 (0.16, 0.32, 0.63, 1.25, 2.50, 5.00 or 10.00 mmol/L and blank solution as ricin control), blank controls were also applied to cells. Next, cells were incubated for another 12 h, and data were collected the same as that in Section 4.3.1. The experiments in each group were repeated six times.

MDCK cells post-exposure time-effect relationship assay
The cell seeding, data retrieval and survival rate calculation methods were the same as those in Section 4.3.1. After the removal of serum, cells were challenged with 5.56 ng/mL ricin, incubated separately for 1, 2, 4 or 6 h, and then treated with gradient concentrations of 27 (0.16, 0.31, 0.63, 1.25, 2.50, 5.00 or 10.00 mmol/L and blank solution as ricin control); blank controls were also applied to cells. Each group of cells was incubated for another 12 h, and data were collected the same as that in Section 4.3.1. The experiments in each group were repeated six times.
4.3.6. Ricin-intoxicated mice pre-exposure treatment assay Animal experiments were approved by the Ethics Committee of Beijing Institute of Pharmacology and Toxicology. Female CD1 mice weighing from 20 to 22 g were randomly separated into four groups that were labeled the blank control group, ricin control group, 27 group and compound 94 group, with 10 mice each group. The 27 and compound 94 groups were injected i.p. with a 2 mg/mL solution of the tested compound, the final administration dose of compound was 50 mg/kg. The blank control and ricin control groups were given 25 mL/kg of blank solution. Then, after 1 h, the 27 group, compound 94 group and ricin control group were injected i.p. with a 1.3 mg/mL ricin normal saline (NS) solution; the final intoxication dose was 13 mg/kg. The blank control group was treated with 10 mL/kg NS. Survival was recorded daily until 6 days after intoxication.

Ricin-intoxicated mice post-exposure treatment assay
The separation of groups was the same as that in Section 4.3.6. The groups were labeled the ricin control group, 1, 2 and 6 h treatment groups. The compound solution, ricin solution and administration assay were the same as that in Section 4.3.6. All four groups of mice were challenged with ricin and given 27 separately at 1, 2 or 6 h after ricin intoxication, and the final dose of 27 was 50 mg/kg; the ricin control group was given 25 mL/kg blank solution 1 h after intoxication. Survival was recorded daily until 12 days after intoxication.

Ricin-intoxicated mice 27 and mAb4C13 combined theraputic assay
The separation of groups was the same as that in Section 4.3.6. The groups were labeled ricin control group, antibody group and combined group. The compound solution, ricin solution and administration assay were the same as that in Section 4.3.6. All groups of mice were challenged with 13 mg/kg of ricin and then combined group was given 50 mg/kg of 27 0.5 h after ricin intoxication while antibody group received corresponding amount of vehicle, then at 2 h both group were treated with 300 mg/kg of mAb4C13. The ricin control group was given vehicle at 0.5 and 2 h. Survival was recorded daily until 20 days after intoxication. When evaluating the anti-ricin ability of the combination, mice were divided into 2.5, 4 and 5 LD group and treated with 32.5, 52 or 65 mg/kg of ricin respectively, then all groups received 50 mg/kg of 27 at 0.5 h and 300 mg/kg of mAb4C13 at 2 h. Survival was recorded daily until 28 days after intoxication.