Ziprasidone Suppresses Pancreatic Adenocarcinoma Cells Proliferation by Targeting GOT1 to Trigger Glutamine Metabolism Reprogramming


 Pancreatic ductal adenocarcinoma (PDAC) is a fatal malignant tumor that no effective treatment has been found. The redox state and proliferative activity of PDAC cells are maintained by the conversion of aspartic acid in the cytoplasm into oxaloacetate though aspartate aminotransferase 1 (GOT1). Therefore, GOT1 inhibitors as a potential approach for treating PDAC have attracted more attention of researchers. Ziprasidone effectively inhibited GOT1 in a non-competitive manner. The potential cytotoxicity and anti-proliferation effects of ziprasidone against PDAC cells in vitro and in vivo were evaluated. Ziprasidone can induce glutamine metabolism disorder and redox state imbalance of PDAC cells by targeting GOT1, thereby inhibiting proliferation, preventing migration and inducing apoptosis. Ziprasidone displayed significant in vivo antitumor efficacy in SW1990 cell-derived xenografts. What’s more, knockdown of GOT1 in SW1990 reduced the anti-proliferative effects of ziprasidone. As a novel GOT1 inhibitor, ziprasidone may be a lead compound for the treatment of PDAC.


Introduction
Pancreatic ductal carcinoma (PDAC), a deadly type of cancer, is listed as the second leading cause of cancer-related deaths in the United States by 2020 [1,2]. Because of the lack of early detection of biomarkers, the lethality of PDAC is largely due to the late onset of symptoms when the cancer has reached metastasis. The 5-year survival rate of pancreatic cancer is only 8% [3], prompting researchers to identify new targets for PDAC therapy.
Metabolic reprogramming is a distinctive feature of tumor cells. The entry of glutamine metabolites into the anabolic pathway is one of the distinctive features of tumor cells [4]. Emerging studies indicate that the KRAS-regulated non-classical glutamine (Gln) metabolic pathway could promote the proliferation and growth in partial PDAC cells [5]. As a nutrient for tumor cells, Gln provides a carbon source for the TCA cycle, a nitrogen source for nucleotide biosynthesis and hexosamine [6,7]. The formation of glutaminederived aspartate was promoted by the up-regulation of GOT1 in PDAC. GOT1 converted aspartate into oxaloacetate in the cytoplasm. Subsequently, this oxaloacetate is converted into malate and then pyruvate, which ultimately increasing the NADPH/NADP + ratio to maintain ROS balance [5]. Studies have shown that the proliferation of PDAC cells was inhibited by reducing the GOT1 expression without affecting the growth of normal tissue cells [6]. GOT1-targeted inhibitors have been reported to provide a much-needed treatment for pancreatic ductal carcinoma [8].
The GOT1 inhibitor iGOT1-01 discovered by high-throughput screening exhibits potential metabolic and growth inhibitory activities, but the speci c binding site and mechanism are not clear. [11].
Due to the important role of GOT1 in maintaining the PDAC cells redox homeostasis, the discovery of potential inhibitors of GOT1 may be a new strategy for the treatment of PDAC. We have long been committed to discovering GOT1 inhibitors with good anti-PDAC activity and exploring the mechanism in depth. Aspulvinone O, a natural GOT1 inhibitor, has been found to trigger glutamine metabolism and suppress PDAC cells proliferation in our previous study [12].
Drug repurposing, also referred to as drug repositioning or repro ling, is aiming at discovering new indications of existing drugs. Taking into account the malignancy of PDAC and the urgency of treatment, we tried to discover promising existing drugs for the treatment of PDAC through drug repurposing.
Ziprasidone, a selective monoamine antagonist, has strong binding a nity with dopamine (D2/3), serotonin (5-HT2A, 5-HT2C, 5-HT1A, 5-HT1D) and α1-adrenergic receptors, which is used for the treatment of acute agitated symptoms in patients with schizophrenia [13]. Ziprasidone induces glutamine metabolism disorders in SW1990 cells by inhibiting the catalytic activity of GOT1, which signi cantly suppresses the proliferation of SW1990 cell-derived xenografts. Brie y, the discovery of the new application of ziprasidone provides a novel strategy for PDAC treatment.

GOT1 protein expression and puri cation
Brie y, GOT1 protein was expressed by cloning human GOT1 ORF (GeneBank: NC_000010.11) into a pET28a vector containing 6 His-tag. The recombinant plasmids were transduced into E. coli strain BL21 and cultured in Luria-Bertani medium at 37 °C, and then 0.4 mM IPTG was added at 18 °C to induce GOT1 protein expression for 18 h. The details of GOT1 protein puri cation were as previously reported [14,15]. The puri ed GOT1 protein is quick-frozen and stored at -80°C for subsequent experiments.

GOT1 inhibition assay
An enzyme activity reaction system was constructed in vitro to evaluate the inhibitory activity of ziprasidone on recombinant GOT1 protein by monitoring the change in absorbance at 340 nm caused by the reduce of NADH on Microplate Reader (BioTek, USA). Total 100 μL reaction system, contained 0.1 mg/mL GOT1, 1 mM α-KG, 4 mM aspartic acid, 1 mM NADH and 1 unit/mL malate dehydrogenase [15].

Microscale Thermophoresis (MST) assay
The binding a nity between ziprasidone and GOT1 were determined by MST according to the instruction of Monolith™ NT.115 Protein Labeling Kit RED-NHS (Cat # MO-L011). GOT1 was dispersed into 20 mM HEPES (pH 7.5) solution with nal concentration of 10 μM and labelled by the RED-NHS dye. Ziprasidone was diluted to different concentrations based on gradient dilution and was incubated with the labeled GOT1 for 20 min. All samples were tested on the Monolith NT.115 instrument for their binding a nity to GOT1 (Nano Temper Technologies, Germany) [15].

Molecular Docking
The crystal structure of GOT1 (PDB ID: 3II0) was downloaded from the Protein Data Bank (http://www.rcsb.org/). The docking was executed through ICM 3.8.2 modeling software (MolSoft LLC, San Diego, CA). The ligand binding sites was selected though ICM software graphical tools for the molecular docking. The potential energy diagram of the receptor was calculated with default parameters. According to the Monte Carlo procedure 30, the numerous conformations of ligands were tested. Finally, the most promising conformations of the ligand were selected with the lowest-energy [16].

Cell viability
The MTT assay was used to evaluate the cell viability after compound treatment. 5 × 10 3 cells/well were seeded in 96-well plates until it converges to 80%, and then administrated with different concentrations of ziprasidone (0-100 μM). 100 μL of 5 mg/mL MTT was added after ziprasidone treatment for 24 h. After incubated for 4 h at 37 o C, DMSO (100 μL/well) was added to dissolve the formazan crystals. The absorbance was measured at 490 nm with a microplate reader.

Apoptosis analysis
After treating SW1990 cells with 20 μM and 40 μM ziprasidone for 24 h, the cells were collected and incubated with Annexin V-FITC/PI for staining. The speci c operation details referred to the Roche Annexin-V-FLUOS Staining Kit (Cat # 11858777001). Fluorescence was determined by FACS Verse ow cytometer.
2.8 Colony formation assay SW1990 cells were inoculated in a 6-well plate at 200/well and cultured for 24 h. Then, SW1990 cells were treated with different concentrations of ziprasidone. During the culture, the fresh medium containing ziprasidone was changed every three days until visible colonies were formed. SW1990 colonies were stained with 1% crystal violet, then washing with PBS for three times and counting colonies containing more than 50 cells.

Immuno uorescence staining
Brie y, cells were seeded into 96-well plates for 12 h and were treated with different concentrations of ziprasidone for 24 h, 4% paraformaldehyde for 20 min, 0.2% Triton X-100 for 10 min, and incubated with 50 μM EdU for 2 h. The EdU proliferation experiment was performed according to the kit instruction of the previous description [17].
For the Hoechst 33342 staining, SW1990 cells were seeded into 96-well plates with 1 × 10 4 /well and cultured for 12 h, then replaced with fresh DMEM containing DMSO or 10-40 μM ziprasidone, cultured for another 12 h, and dyed with 100 μL 20 μM Hoechst 33342 for 10 min. The morphology of nuclear chromosomes was observed under a uorescence microscope at 340 nm. (Nikon, Japan).

Transwell migration
First, 600 μL complete medium with ziprasidone were supplemented into the lower chamber, and then 5 × 2.11 Wound scratch assay SW1990 cells were cultured until the cell density reached about 85%, three straight scratches were made by 10 μL micro pipette tip per well. Wash the 6-well plates with PBS before treating the cells with different concentrations of ziprasidone. At 0 h and 24 h after ziprasidone treatment, microscopic pictures were taken to analyze the compound's ability to inhibit cell migration.

Western blot assays
SW1990 cells were seeded into a 6-well plate at 5 × 10 5 /well and cultured until the cell con uence was above 85%. SW1990 cells were treated with ziprasidone for 24 h and then collected. Cells were resuspended with RIPA lysis buffer (Beyotime Biotechnology) containing 0.1% PMSF to release total protein. The concentration of total protein was determined by BCA Protein Assay Kit (Beyotime Biotechnology). Total protein was separated by SDS-PAGE and transferred to PVDF membrane (Merck Millipore, 0.2 μm). The PVDF membrane was incubated with the primary antibody overnight at 4°C, and then with the secondary antibody for 2 h at room temperature, nally performed chemiluminescence imaging (Tanon 5200).

siRNA knockdown of GOT1
SW1990 cells were seeded into a 6-well plate at 5 × 10 5 /well and cultured until the cell con uence was 90%-95%. Mix 100 pM siRNA with 5 uL Hieff Trans™ Liposomal Transfection Reagent and incubate for 20 min at room temperature. siRNA-liposome complexes treated SW1990 cells for 6 h and then replaced them with fresh DMEM medium and continued to culture for 48 h. CETSA (cellular thermal shift assay) is usually used to evaluate the targeting effect between the compound and the protein based on the principle that the ligand can improve the thermal stability of the target protein. SW1990 cells were cultured to more than 80% con uence, replaced with fresh DMEM containing DMSO or 30 μM ziprasidone, and cultured for another 12 h. SW1990 cells were heat-treated from 42°C to 52°C for 3 min. Speci c experimental details referred to the previous description [18-20].

Measurement of ECAR and OCR
SW1990 cells were seeded in 24-well plates at a density of 5 × 10 4 and cultured for 12 h, and then treated with fresh DMEM medium containing 20 μM ziprasidone for 3 h. XF24 Extracellular Flux Analyzer (SeaHorse Bioscience) was used to determine the changes in oxygen consumption rate (OCR) and extracellular acidi cation rate (ECAR) of SW1990 cells after administration of ziprasidone. Experimental details were as previously reported [12].

Metabonomics experimental methods
The SW1990 cells were con uent to more than 80% and treated with 20 µM ziprasidone, and the control group was added with an equal volume of DMSO. After culturing for 24 h, we collected the cells and added 70% methanol aqueous solution to lyse on ice. The cells were lysed by repeated freezing and thawing of liquid nitrogen and the metabolites were dissolved in methanol aqueous solution. The supernatant is centrifuged to perform LC-MS/MS analysis. Parameter settings and operating mode for LC-ESI-MS/MS analysis were as previously described [17].

In vivo tumor xenograft study
CB-17/SCID mice (male, 4 weeks old) were s.c. inoculated with 3 × 10 6 SW1990 cells in the left abdomen. The tumors were allowed to grow for 6 d. The mice inoculated with tumor cells were randomly divided into three groups with 10 mice/group, including the control group, the low-dose ziprasidone group (100 mg/kg), and the high-dose ziprasidone group (200 mg/kg). Mice were given intragastric administration once a day. Tumor volume measurement and mouse body weight measurement were done as previously described [12].

Identi cation of ziprasidone as a novel GOT1 inhibitor
Over 500 compounds were selected for preliminary activity assessment against GOT1 from the approved drug library [10]. Ziprasidone exhibited the best inhibitory activity on GOT1 with an IC 50 value of 5.39 ± 1.13 μM (Fig. 1A). In order to explore the speci city of GOT1, malate dehydrogenase 1 (MDH1) was used as the control. The result indicated that the change of NADH content was related to the suppression of GOT1 enzyme activity, rather than MDH1. Ziprasidone exhibited potent GOT1 enzyme suppression activity and deserved more in-depth research.

Effects of ziprasidone on enzyme kinetics of GOT1
In order to explore the inhibitory mode of ziprasidone targeting GOT1, enzyme kinetics studies were applied to investigate the V max and K m of ziprasidone on GOT1. The results showed that ziprasidone had weak effect on the K m of α-KG and reduced V max dose-dependently, which is a typical non-competitive mode.

Ziprasidone speci cally binds with GOT1 in vitro
The MST evaluation was further applied to explore the direct interaction between ziprasidone and GOT1, which showed the K d value of 89.30 ± 5.35 μM (Fig. 1D). What's more, a DARTS experiment was carried out to detect the binding a nity between ziprasidone and GOT1. As shown in Fig. 1E-1F, ziprasidone enhanced GOT1 stability by binding to it without being degraded by pronase. These results indicated that ziprasidone could inhibit the activity of GOT1 by direct interaction.

Molecular docking shows the binding mode of ziprasidone and GOT1
To further clarify the binding conformation of ziprasidone with GOT1, molecular docking was performed [21]. The lowest-energy binding mode between ziprasidone and GOT1 was exhibited in Fig. 1G. Molecular docking results suggested that ziprasidone bound to an allosteric pocket of GOT1 (Fig. 1G). The binding domain of ziprasidone was located at the forefront of the catalytic active center, and some of the hydrophobic amino acid residues formed a hydrophobic pocket to hold ziprasidone, including Pro15, Val16, Phe19, Val38, and Tyr264 (Fig. 1H). The main hydrophobic interaction was formed between Arg42 and the carbonyl group of ziprasidone, and an apparent stacking was formed between Tyr264 and the indoline ring of ziprasidone. The predicted allosteric binding mode was consistent with the results that ziprasidone inhibited the enzyme activity of GOT1 in a non-competitive manner.
3.5 In vitro anti-proliferative activity of ziprasidone on SW1990 cells Pancreatic cancer (SW1990), breast cancer (HCC1806), liver cancer (HepG2), colorectal cancer (HCT116, SW620, SW480), non-small cell lung cancer (A549), brosarcoma cells (HT1080), normal cells (L929) were initially selected to determine GOT1 expression level by western blotting. GOT1 is highly expressed in all tested cell lines except L929 ( Fig. 2A). We attempted to evaluate whether ziprasidone could affect the cell viability of these cancer cells. The IC 50 values of ziprasidone were measured in a variety of tumor cell lines and normal cell lines ( Table 2). As shown in Fig. 2B, ziprasidone showed potent inhibitory activities on SW1990 cells growth.

Ziprasidone induces apoptosis in SW1990 cells
Based on the ability of ziprasidone inhibiting the viability of SW1990 cells, follow-up experiments were conducted to detect apoptosis-related indicators, including mitochondrial apoptosis pathway and death receptor apoptosis pathway. Ziprasidone could up-regulate the expression of cleaved caspase-3 and down-regulate the precursor of caspase-3 and caspase-9. Simultaneously inducing the cleavage of PARP, a substrate of caspase-3, indicated that ziprasidone may stimulate cell death by activating caspase. The Bcl-2 family proteins in the mitochondrial apoptotic pathway were also tested and found that the expression of anti-apoptotic protein Bcl-2 was down-regulated, and pro-apoptotic protein Bax was upregulated. It also proved that ziprasidone can promote the apoptosis of SW1990 cells (Fig. 2C,   2D). Hoechst 33342 staining was used to investigate the effect of ziprasidone on nuclear morphology. The SW1990 cells treated with ziprasidone showed signi cant chromatin condensation and fragmentation (Fig. 2E). Annexin-V/PI staining demonstrated that ziprasidone induced cell apoptosis in a dose-dependent manner (control: 0.27%, 20 μM: 10.94%, 40 μM: 70.07%) (Fig. 2F). In addition, ziprasidone induced cell cycle arrest at the G1 phase of SW1990 cells (Fig. 2G). The ability to form colonies of ziprasidone-treated SW1990 cells was down-regulated, indicating that ziprasidone can inhibit cell proliferation (Fig. 2H).

Ziprasidone suppresses SW1990 cells proliferation and migration
The results of the EdU proliferation experiment showed that the percentage of EdU + positive in SW1990 cells decreased after ziprasidone treatment, indicating that ziprasidone can inhibit the proliferation of SW1990 cells (Fig. 3A).
We performed the transwell experiment to investigate the inhibition of ziprasidone on the migration of SW1990 cells. As exhibited in Fig. 3B-3C, ziprasidone inhibited the migration of SW1990 cells in a dosedependent manner. Furthermore, the scratch wound assay exhibited that ziprasidone evidently decreased the migration ability of SW1990 cells (Fig. 3D), which was consistent with the above results in transwell.

Ziprasidone modulates Gln metabolism and ROS response
Aspartic acid (Asp) and oxaloacetate (OAA) are the substrate and product of the GOT1 enzymatic reaction, respectively. Therefore, to a certain extent, OAA can rescue cells lacking glutamine metabolism, but Asp cannot [22]. In our experiment, SW1990 cells were pretreated with Asp and OAA, and then treated with ziprasidone to investigate whether OAA can antagonize ziprasidone. The results exhibited that the sensitivity of SW1990 cells on ziprasidone was signi cantly reduced when pretreated with OAA, while Asp pretreatment was basically unchanged (Fig. 3E). It proved that ziprasidone indeed inhibited the enzyme activity of GOT1 to further affect the production of cell metabolites. As a metabolite of glutamine, OAA maintains the redox homeostasis of PDAC by increasing the ratio of NADPH/NADP + [22]. Ziprasidone down-regulated the NADPH/NADP + ratio in a dose-dependent manner to destroy the redox state of SW1990 cells (Fig. 3F). Furthermore, ziprasidone induced ROS production in SW1990 cells dosedependently (Fig. 3G). Therefore, we speculated that cells produced more reactive oxygen species (ROS) due to the inhibition of GOT1. These results indicated that the inhibition of ziprasidone can block the production of Gln-dependent NADPH and participate in the redox state of PDAC cells (Fig.  3G). Furthermore, ziprasidone induced ROS production in SW1990 cells in a dose-dependent manner. After ziprasidone treatment, the extracellular acidi cation rate (ECAR) of SW1990 cells was signi cantly reduced, the same as respiration (oxygen consumption rate, OCR) (Fig. 3H, 3I). The above results indicate that ziprasidone inhibits malate-aspartate shuttle and mitochondrial respiration by inhibiting GOT1.
We conducted an in-depth study on the ability of ziprasidone to inhibit GOT1. After knocking down the expression of GOT1, the inhibition of ziprasidone on the cell viability of SW1990 cells was signi cantly weakened (Fig. 4C, 4D). CETSA assay demonstrated that ziprasidone can signi cantly increase the thermal stability of GOT1 in the temperature range of 42-57°C (Fig. 4E). Both siRNA knockdown and CETSA prove that ziprasidone can directly target and bind with GOT1, and its biological function also depends on GOT1.

Ziprasidone treatment induces Gln metabolism and modulates mitochondria respiration
Metabolomics analysis of the metabolic regulation of ziprasidone on SW1990 was carried out by using UPLC-MS/MS. The fold change in metabolites content of ≥2 or ≤0.5 was de ned as the differential metabolites. Compared with the control group, the expression of 119 metabolites was signi cantly different in the ziprasidone group. The quantitative data of up-or down-regulated metabolites were identi ed in Table S1, Fig. S1. After ziprasidone-treatment, the GOT1 enzyme-related metabolites changed signi cantly (Fig. 5B).
Furthermore, we performed cluster analysis of 119 differential metabolites through KEGG analysis to discover the biological signi cance of ziprasidone in regulating metabolism. 119 differential metabolites are mainly concentrated in glucose metabolism, nucleotide biosynthesis, amino acid biosynthesis and other pathways. What's more, ziprasidone could down-regulate the endogenous intermediate metabolites in the amino acid biosynthetic pathway of SW1990 cells (Fig. 5C, 5D). These results indicate that ziprasidone disrupts the metabolic process of SW1990 cells by targeting inhibition of GOT1.
3.10 Ziprasidone suppresses tumor growth in SW1990 xenograft model GOT1 is commonly expressed in malignant pancreatic cancers. Based on the results of ziprasidone inhibiting the growth of SW1990 cells, we constructed a xenograft tumor model of SW1990 cells to investigate the in vivo inhibitory activity of ziprasidone. Three groups of SW1990 xenograft CB-17/Icr-scid mice were intragastric administrated with ziprasidone (200, 100 mg/kg) or vehicle once a day. The tumor volume of mice decreased signi cantly after two weeks of treatment with ziprasidone compared with the control group. The tumor weights of the 100 mg/kg and 200 mg/kg ziprasidone treatment groups decreased by 75.29% and 84.54%, respectively (Fig. 6B, 6D). As shown in Fig. 6A, after treated with 200 mg/kg ziprasidone, a decrease in mice body weight, and lethargy and anorexia were observed. H&E staining of the spleen, kidney and pancreas of mice showed no difference between the treated group and control group, indicating that ziprasidone did not damage the internal organs of mice (Fig. S2).
In order to further investigate the effect of ziprasidone on the metastasis and growth of solid tumors, H&E staining was performed on liver tissues and immunohistochemical analysis was performed on tumor tissues. H&E staining results showed that ziprasidone inhibited tumor cell metastasis to the liver. In addition, the tumor marker CD133 immunohistochemical results showed that ziprasidone can inhibit tumor growth (Fig. 6E).

Discussion
Glutamine-mediated metabolic reprogramming can not only maintain redox homeostasis, but also provide raw materials for biosynthesis and TCA cycle of PDAC cells proliferation. Targeting inhibition of GOT1 to disrupt tumor metabolic homeostasis, which is an important strategy for us to nd promising anti-tumor drug candidates. At present, the development of GOT1 inhibitors is still in the preliminary exploration stage. We hope that our long-term research can provide a strategy for the treatment of tumors that rely on glutamine metabolism.
Compared with the development of a brand-new drug for a certain indication, drug repurposing has obvious advantages, including reducing the risk and investment, and shortening the drug research and development time. Our research group is committed to the development of GOT1 inhibitors, and previous work has found lead compounds with good activity in natural products [12]. However, for PDAC, a fatal malignant tumor, no effective treatment has been found so far. There is an urgent need to develop drug candidates for treating PDAC. Based on the signi cance of GOT1 for PDAC to maintain redox homeostasis and the urgency of drug candidate discovery, we tried to nd potential drug candidates through the strategy of drug repurposing.
The antipsychotic drug ziprasidone displayed signi cant in vitro and in vivo antitumor e cacy by inhibiting GOT1. High-dose 200 mg/kg ziprasidone was administered to SW1990 cell xenograft tumor model, and no obvious adverse reactions were found. Regarding the safety of ziprasidone, P zer-Spain has recorded four cases of ziprasidone overdose, the highest of which was 4480 mg. QTc interval is within the normal range in all patients and there are no cardiac side effects, which suggests that overdose of ziprasidone may be relatively safe in patients without risk factors that contraindicate its use [31]. In this study, although our dosage of ziprasidone (100 mg/kg and 200 mg/kg) may be higher than its clinical dose (maximum recommended dose as 80 mg twice a day in particular cases), these doses are lower than the toxic dose suggested in the literature. Although, we observed a decrease in mice body weight in 200 mg/kg ziprasidone treated group, no apparent organ damage in spleen, kidney and pancreas were observed. Because we also observed lethargy and anorexia in this group of animals, we speculated that the major reason for the loss of body weight might be the reduced uptake of foods.
Although there was a little weight loss in the high-dose group, the anti-tumor effect of ziprasidone was not in uenced.

Conclusions
The enzyme activity inhibition experiment showed that ziprasidone had obvious inhibition effect on GOT1, and the inhibitory effects on related tumor cells were proved by the related pharmacological

Data availability statement
The data will be made available upon reasonable request.
Ethics approval and consent to participate All procedures in this study were performed in accordance with the ethical standards of the Animal Care and Use Committee of Shenyang Pharmaceutical University.     after ziprasidone treatment. Data are mean ± SD, (n = 5). * p <0.05, ** p < 0.01, *** p < 0.001 compared with the control by t-test.  The potential cytotoxicity and anti-proliferation effects of a novel GOT1 inhibitor (ziprasidone) against pancreatic adenocarcinoma cells in vitro and in vivo were summarized.