S-Sulfocysteine (SSC), a bioavailable L-cysteine derivative (Cys), is known to be taken up and metabolized in Chinese hamster ovary (CHO) cells used to produce novel therapeutic biological entities. To gain a deeper mechanistic insight into the SSC biological activity and metabolization, a multi-omics study was performed on industrially relevant CHO-K1 GS cells throughout a fed-batch process, including metabolomic and proteomic profiling combined with multivariate data and pathway analyses. Multi-layered data and enzymatical assays revealed an intracellular SSC/glutathione mixed disulfide formation and glutaredoxin-mediated reduction, releasing Cys and sulfur species. Increased Cys availability was directed towards glutathione and taurine synthesis, while other Cys catabolic pathways were likewise affected, indicating that cells strive to maintain Cys homeostasis and cellular functions. In the biopharmaceutical industry, therapeutic proteins are produced in large-scale bioprocesses using Chinese hamster ovary (CHO) cells. As the demand for therapeutics is increasing, culture processes have to be constantly developed and optimized. This includes the development of cell culture media (CCM) and feeds (CCF) that support cell growth, viability, productivity , or drivKa value of the thiol group from 8.3–8.5 to 9.5–9.9 , characterized by an increase in the p 9.5–9.9 . Given t 9.5–9.9 ), NAC ex 9.5–9.9  that pro 9.5–9.9 . Based o 9.5–9.9 . Current 9.5–9.9 . Despite 9.5–9.9  and thus 9.5–9.9  and N-ac 9.5–9.9 , were in 9.5–9.9 . Upon ce 9.5–9.9 . Still,  9.5–9.9 .c−) in the uptake of SSC in CHO cells was studied. Fed-batch experiments using SSC in combination with inhibitors or activators of xc− showed a modulation of the SSC uptake rate, strongly suggesting the involvement of the antiporter in the cellular uptake  and the mixed disulfides L-cysteine glutathione (GS-Cys) and sulfo-glutathione (GS-SO3). These mixed disulfides were suggested to be reduced via unidentified intracellular enzymes, thereby maintaining and/or replenishing the intracellular Cys pool  collected during a two-week fed-batch process using either SSC or Cys feeding were analyzed. Multivariate data and pathway analyses were leveraged for the identification of intracellular effectors, which were validated by orthogonal methodologies. Overall, this study sheds light on the biological activity of SSC as well as mechanisms leading to the release of Cys and increase in intracellular GSH.In contrast to NAC and its derivatives, S-sulfocysteine (SSC) was developed through the addition of a sulfonic acid group to the thiol moiety, hence lowering its reactivity . SSC, wir uptake . Furtherr uptake , fungi , mouse anti-TFRC , rabbit anti-TST , rabbit anti-ETHE1  and rabbit anti-MPST .6 cells/mL in Cellvento® 4CHO medium and fed with Cellvento® ModiFeed Prime, with L-cysteine and S-sulfocysteine being added through a one- or two-feed system with the following feeding strategy (v/v): 2.75%, 5%, 6%, 6%, 6% and 5% on day 3, 5, 7, 9, 11 and 14 for the neutral pH feed, and one-tenth of the volume on the respective days for the separate alkaline 10 x concentrated L-cysteine feed. The pH in the bioreactor was controlled at pH 6.95 ± 0.15 from day 0–4, at pH 6.80 ± 0.05 from day 4–13, and at pH 7.00 ± 0.05 from day 13–17. The dissolved oxygen concentration was controlled at 50% air saturation by sparging with pure oxygen and air via an open pipe sparger. Temperature was set at 36.7°C and agitation was maintained at 280 rpm using a marine impeller. The redox potential probe was re-calibrated on day 3 to account for probe drift.Small-scale bioreactor experiments with CHO-K1 GS cells producing an IgG were performed in 1.2 L DasGip glass vessels . Cells were inoculated at 0.2.105 CHO cells were seeded in white clear flat-bottomed 96 well plates in Cellvento® 4CHO medium and mixed with the MT cell viability substrate and the NanoLuc® luciferase. Sulfur compounds were added to the cell preparation and the luminescence was monitored for 1 h at 37°C using an EnVision plate reader . For evaluation of the cell response upon peroxide insult, H2O2 was added at a concentration of 1.25 mM. Experiments were performed three times independently, using technical triplicates.The impact of sulfur containing substances on the intracellular redox potential was assessed using the RealTime-Glo™ MT Cell viability assay  according to the experiments of ® Ultra Reagent kit. Derivatization, ultra-performance liquid chromatography (UPLC), and data analysis were performed according to the supplier recommendations .The cell performance (viable cell density (VCD), viability) was assessed daily via the Vi-CELL™ XR 2.04 cell counter . Metabolite concentrations were monitored via the bioprocess analyzer Cedex BIO HT . Amino acid analysis was performed using an alkylation step with iodoacetamide and a pre-column derivatization using the AccQ TagVitamins and further small molecules were quantified in three different groups  using UPLC  coupled with a triple-quadrupole mass spectrometer .Chromatographic separation was performed on an Acquity UPLC HSS T3  for group 1 and 3 and an Acquity UPLC HILIC  for group 2. Column temperature was maintained at 40°C for all groups and 2, 2.5, and 3 µL were injected for group 1, 2, and 3, respectively. The mobile phase for group 1 and 3 consisted of 99% water/1% ammonium formate 1 M/0.1% formic acid (buffer A) and 99% methanol/1% ammonium formate 1 M/0.1% formic acid (buffer B). For group 2, the mobile phases were 99% water/1% ammonium acetate 1 M/0.1% acetic acid (buffer A) and 99% methanol/1% ammonium acetate 1 M/0.1% acetic acid (buffer B). The flow rate for group 1 was set to 0.4 mL/min from 0 to 3 min and to 0.6 mL/min from 3 to 5 min. The gradient elution was applied as follows (min/% B): 0/1, 2/1, 3/55, 3.1/99, 4/1, 5/1. The flow rate for group 2 was set to 0.6 mL/min and the gradient elution was applied as follows (min/% B): 0/95, 0.2/95, 0.8/55, 1/55, 1.01/20, 1.41/95, 1.9/95. The flow rate for group 3 was set to 0.5 mL/min and the gradient elution was applied as follows (min/% B): 0/1, 0.4/1, 1/55, 1.6/55, 1.61/99, 2/99, 2.01/99, 2.4/1.Analytes were detected in multiple reaction monitoring (MRM) mode and mass spectrometry analysis was performed with an API4000 mass spectrometer (Sciex) operating in the electrospray ionization (ESI) positive and negative mode. MS parameters were defined for each group as follows : collision gas 4 psi; curtain gas 25 psi (group 1 and 3), 20 psi in positive mode and 25 psi in negative mode (group 2); nebulizer and heater gas 50 psi; ion spray voltage 3,000 V (group 1 and 3), 3,000 V in positive mode and −3,000 V in negative mode (group 2); temperature 600°C; interface heater was turned on.2 weighting were used to generate standard curves, peak area was used for quantification.The analyst software  was used to operate the mass spectrometer and for data processing. Linear regression and 1/x6 and max 5.107 cells were treated with 2 mM N-ethylmaleimide (NEM) for 10 min at room temperature (RT). Cells were washed three times with cold PBS containing 0.5 mM NEM and the cell pellet was snap frozen in liquid nitrogen and stored at −80°C until extraction. After thawing, pellets were incubated 15 min in a thermomixer  and the intracellular content was extracted for 30 min  using pre-chilled acetonitrile/NEM extraction solution (5.2 mM NEM) containing isotopically labeled GSH and GSSG to compensate matrix effects. After centrifugation , supernatant was dried using a SpeedVac  and dry samples were kept at −20°C until LC-MS analysis.In order to prevent oxidation of GSH during sample preparation, at least 2.10Samples were reconstituted in 0.1% formic acid (buffer A) and analyzed using UPLC-MS . For GSSG, undiluted samples were analyzed and for GSH-NEM determination, samples were further diluted 1:1,000 with buffer A. 5 μL sample were loaded onto Acquity HSS T3 column  thermostated at 40°C with a flowrate of 400 μL/min. A 6 min gradient was applied using 0.1% formic acid in acetonitrile (buffer B) as follows (min/% B): 0.0/0.1, 2.0/2, 3.5/30, 4.5/95, 4.51/0.1, 6.0/0.1.MS detection in MRM mode was performed using a triple-quadrupole mass spectrometer equipped with an ESI source. MS acquisition was performed in positive mode using the following parameters: collision gas: 4, curtain gas: 20, gas 1: 50, gas 2: 60, ion spray voltage: 3,000 V. Data were evaluated using the software MultiQuant 3.0.3 . For quantification, the ratios of the analyte and internal standard peak areas (summed area of three transitions) were determined. For calibration, the peak area ratios obtained for the calibration solutions were plotted against the concentration of the analyte using a linear regressing with 1/x weighing. Finally, data was normalized to protein content, determined by colorimetric bicinchoninic acid assay.in vitro by incubating 300 µM of GS-SO3  in PBS with 60 ng of either GRX1 , GR , TRX1  or TRXR  and combination thereof. In selected conditions, 150 µM NADPH and 30 µM GSH were added as reducing equivalent. It is important to note that the GSH concentration was selected at 10% compared to the GS-SO3 concentration to avoid having more than 10% of reduction due to the chemical interaction between both molecules. After incubation at 37°C for 45 min, 500 rpm agitation, the reaction was stopped on ice for 5 min and the mixture was measured using LC-MS and the method described in 2.5.2. Data analysis was performed using Progenesis QI .The capability of oxidoreductases to reduce GS-SO3 was assessed The γ-glutamyltransferase (GGT) activity was assessed using the GGT colorimetric assay kit . The kit is based on the enzymatical cleavage of L-γ-Glutamyl-p-nitroanilide (γ-Glu-pNa) by GGT liberating the chromogen p-nitroanilide, whose formation can be monitored at 418 nm. To assess the effect of GS-SO3 on the GGT activity, increasing concentrations of GS-SO3 (between 130 μM and 2.5 mM) were added to the reaction and incubated for up to 1 h at 37°C with constant monitoring of the absorbance. Upon 1 h, the reaction was stopped by incubating on ice for 5 min and the mixture was measured using LC-MS and the method described in 2.5.2. Data analysis was performed using Progenesis QI .n = 42 or 48) were diluted 1:10 or were derivatized with either 2 mM NEM or 2 mM monobromobimane (MBBr) for the quantification of thiols or polysulfides respectively, prior to LC-MS measurement.For intracellular and extracellular metabolomics, samples (Cys and SSC conditions on day (0), 3, 5, 7, 10, 12, 14, 17, 7 cells/mL) and further incubated in a thermomixer . Samples were centrifuged at 18,000 g for 15 min at 0°C. The supernatant was dried using a SpeedVac using a temperature of 45°C for 60 min, 100 mTorr and a total run time of 90 min. Dry samples were kept at −80°C until LC-MS analysis.For intracellular metabolomics, the cell suspension (10 mL) was immediately quenched with 40 mL of pre-chilled 0.9% NaCl (0.5°C–1.5°C). Cells were centrifuged , the supernatant was discarded, and the pellet was snap frozen in liquid nitrogen and stored at −80°C. To extract intracellular metabolites, pellets were incubated 15 min on ice, extracted by vortexing 1 min in pre-chilled (−20°C) buffer consisting of 40% acetonitrile, 40% methanol (5.108 cells/mL) in pre-chilled water and analyzed using UHPLC  coupled with an ESI-Q-ToF mass spectrometer . 1 or 2 µL of sample were loaded in 99.9% buffer A (20 mM ammonium formate/0.1% formic acid) onto a XSelect HSS T3 column  thermostated at 40°C with a flowrate of 300 μL/min. An optimized 12 min linear gradient was applied using 100% methanol (buffer B) as follows (min/% B): 0/0, 2/0, 4/20, 6/30, 8/80, 8.5/100, 9.5/100, 9.6/0 12/0.Pellets were reconstituted . MS acquisition was performed in positive and negative modes with capillary voltages set at 4,500 V and 3,500 V, respectively, and the end plate offset set at 500 V. Nebulizer and dry gas (250°C) were set at 1.4 bar and 9.0 L/min, respectively. MS spectra were acquired over the m/z range 50–1,000 with a MS scan rate of 2 Hz in MS mode only for metabolite quantification. Additional LC-MS/MS acquisitions with a MS scan rate of 12 Hz using a fixed MS-MSMS cycle of 0.5 s and a dynamic exclusion list were performed for metabolite identification. Relative quantification of metabolites was carried out using Metaboscape 2021 . Data were processed using the T-ReX 3D workflow using the following parameters: an intensity threshold of 1,000 counts and a minimum peak length of 8 spectra were set for peak detection and peak area was used for feature quantification. Only features eluted between 0.8 and 9 min and exhibiting abundances >5,000 were considered. Injections of QC samples coupled to the “within batch-correction” processing were used to mitigate intensity drift effects occurring over the acquisition sequence.Various levels of confidence were assigned to the annotations based on the initial recommendation of ® 4CHO medium) was prepared at pH 7.0 ± 0.3 with or without the addition of 15 mM SSC, sulfite, sulfate, tetrathionate, or thiosulfate and frozen at −20°C until analysis with LC-MS.To identify interaction products between sulfur containing species and vitamin B12, 1.0x, 2.5x, and 5.0x concentrated cyanocobalamin  and sulfitocobalamin  was identified with a monoisotopic mass of 1,409.5282 Da and was eluted at 7.83 min.n = 42), at least 1.107 cells were collected, washed two-times with ice-cold PBS, snap frozen in liquid nitrogen and stored at −80°C until lysis. After thawing on ice, the cell pellets were resuspended in the respective amount of harsh RIPA lysis buffer   supplemented with 1X protease and phosphatase inhibitor cocktail (v/v)  to reach a final lysis buffer-to-cell ratio of 500 µL/1.107 cells. Samples were incubated for 20 min on ice with vigorous vortexing every 5 min. To reduce the viscosity of the samples, QIAshredders  were used and subjected to centrifugation for 2 min at maximum speed. Samples were centrifuged for 15 min at 14,000 g and 4°C to remove cell debris, and clarified supernatants were transferred into fresh tubes. Protein concentrations were measured using the Pierce™ Detergent Compatible Bradford Assay Kit . Reduction was conducted with 5 mM DTT  at 37°C for 30 min, and alkylation with 15 mM iodoacetamide  at RT for 30 min in the dark. Finally, an automatic clean-up  and on-bead digestion with trypsin  in a 1:5.75 trypsin-to-protein ratio (w/w) were carried out via the KingFisher Flex System .For each sample  reagents using the TMTpro 16plex Isobaric Label Reagent Set . Briefly, TMT label reagents were equilibrated to RT and dissolved in 30 µL of anhydrous acetonitrile, of which 10 µL were added to 25 µL of the respective sample (pH 8.0–8.5). Labeled samples were incubated for 1 h at RT and 600 rpm. To quench the excess of reactive TMT labeling reagents, 4 µL of 1 M TRIS at pH 8.0 were added and incubated for 15 min at RT and 600 rpm. Samples were equally mixed, 5% formic acid were added, dried in the SpeedVac at RT and high vacuum for about 2.5 h and then stored at −20°C until further use.Mixed samples were fractionated using the Pierce™ High pH Reversed-Phase Peptide Fractionation Kit  to reduce the sample complexity and then analyzed by nano LC-MS/MS. All nano LC-MS/MS experiments were performed using the nanoRSLC3000 system , directly coupled to the timsTOF Pro  with a nanospray ion source. 1 μL of each sample (400 ng of digested proteins) were separated on the Aurora UHPLC Emitter Column with nanoZero and Captive Spray Insert  at 50°C. Buffer A  and buffer B  were used, applying a gradient as follows (min/% B/µL/min): 0/2/0.15, 0.2/2/0.5, 6.5/2/0.15, 8/2/0.15, 9/2/0.2, 159/25/0.2, 169/35/0.2, 179/80/0.2, 187/80/0.5, 192/80/0.5, 197/2/0.5, 211/2/0.5, 212/2/0.15.2, with an accumulation and ramp time of 100 ms. The MS-MS/MS cycle time was set to 2.21 s, consisting of a single MS scan and 10 PASEF MS/MS scans, whereby only ions with an intensity of 2,500 and charge state of up to 5 were considered. The dynamic exclusion was activated with a defined time period of 0.4 min, enabling the acquisition of MS/MS spectra of less abundant ions. Excluded precursors were reconsidered, when exhibiting a 4-times higher intensity. The performance of the MS system was verified by running a Pierce™ HeLa Protein Digest Standard , leading to about > 5,200 protein groups.After nano LC separation, eluting peptides were ionized utilizing a CaptiveSpray source  in positive mode, setting the capillary voltage to 4.5 kV, the nebulizer to 0.4 bar, and the dry gas flowrate and temperature to 3 L/min and 180°C, respectively. The timsTOF was operated in the data-dependent acquisition (DDA) mode employing the parallel accumulation serial fragmentation (PASEF), providing an extreme improvement in speed and sensitivity and thereby extending the depth in the proteome coverage. The mass and ion mobility range were set to 100–1,700 m/z and 0.6–1.6 V Cricetulus griseus (Chinese hamster) from UniProt  and an in-house contaminant database . Database searching was carried out applying the following parameters: parent mass error tolerance of 10 ppm, fragment mass error tolerance of 0.05 Da and trypsin enzyme (specific) with a maximum of two missed cleavages. Carbamidomethylation of cysteine (+57.02 Da) and TMT16plex (+304.2072 Da) were defined as fixed post-translational modifications (PTM). A false discovery rate (FDR) of 1% was set, thus only peptides above the respective threshold and proteins exhibiting For the protein identification, the PEAKS Studio 10.6 software  was used. The generated peptide MS/MS spectra were searched against the reference proteome of To validate protein level changes, capillary-based Simple Western assays were performed in RePlex™ mode using the Jess device  according to the manufacturer’s protocol, employing an immunoassay followed by a total protein assay for normalization. Briefly, samples were mixed with fluorescently labeled molecular weight markers, reduced by 40 mM DTT and denatured at 95°C for 5 min. Prepared samples, primary antibodies specific for the target protein, horseradish peroxidase- or near-infrared-conjugated secondary antibodies, luminol-peroxide mix, RePlex™ reagent mix, blocking and rinsing reagents were added into proprietary 12–230 kDa microplates. For the immunoassay, the matrices (separation and stacking matrix) and the samples were loaded into the capillaries. The protein separation in the samples was conducted by applying a voltage of 375 V for 25 min, after which the separated proteins were immobilized to the capillary wall upon UV light exposure and then probed with primary and secondary antibodies for 30 min each. Generated chemiluminescence or fluorescence signals were detected in the respective channel. Following the immunoassay, the capillaries were stripped with the RePlex™ reagent mix for 30 min and then reused for the detection of the total protein signals. Corresponding signals obtained were analyzed using the Compass software v 6.1.0 .For the differential expression analysis, the integral of the metabolite abundance/protein ratio over time, referred to as area under the curve (AUC), was used for the calculation of the fold change between the SSC and Cys condition. Metabolites with a minimum fold change of ± 1.5 were considered as differentially regulated, while the threshold for proteins was set to ± 1.1.For multivariate data analysis (MVDA), a supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was performed on metabolomic and proteomic data using SIMCA v 17.0 , allowing a clear differentiation of the Cys and SSC conditions and the identification of biomarkers with the largest discriminatory power. The OPLS-DA model was built with 36 observations  and 4,889 intracellular features/5,506 extracellular features or 5,077 proteins used as variables, which were subjected to either pareto (Par) scaling or centering for the ratios. The model quality was assessed by the cumulative R2X and R2Y (goodness of fit) and cross-validated Q2 (goodness of prediction), while the model validity was evaluated by a 200-times permutation test. Features or proteins, for which the loadings of the predictive component were greater than the standard deviation, were considered as statistically significant.Integrative metabolomic and proteomic data analysis was performed using the Ingenuity Pathway Analysis (IPA) software . Expression values (fold change) of metabolites and proteins were uploaded in IPA to execute the Core Analysis, enabling an insight into the biological roles, functions and canonical pathways being altered upon the cell adaptation to a new environment.To gain insights into the biological activity of S-sulfocysteine when used as a feed component in CHO-based culture, a fed-batch experiment in 1.2 L bioreactors was performed. An SSC containing feed was compared to a two-feed strategy using an equivalent concentration of Cys. The process used in this experiment was optimized to minimize differences in growth and productivity to focus on metabolic differences due to the usage of the modified amino acid.6 cells/mL, viabilities maintained above 70% over 14 days and a final titer of roughly 4 g/L on day 17 for both processes using either Cys or SSC containing feed. While the pH was maintained within the established deadband for both conditions, the SSC process revealed a lower redox potential when compared to the Cys process after day 3  model was used to focus on the differences between both feeding conditions. As shown in To validate the metabolomic dataset, some small molecules were quantified using an orthogonal method by prior quenching of thiol groups with NEM. The increase in reduced and oxidized glutathione in the SSC condition was confirmed . This quIn a next step, supernatants collected during the fed-batch experiment in the SSC vs. Cys conditions were analyzed using untargeted LC-MS. Similarly to the approach taken for intracellular metabolites, an OPLS-DA model was created to focus only on features that were differentially regulated in both feeding conditions. Results indicate that 4.3% of the variability in the dataset (predictive component) was attributed to features differentially regulated in both conditions, whereas 46.2% of the variability was due to the culture time  compounds were significantly impacted in the supernatants compared to the intracellular compartment, but similar behavior was detected for γ-glutamyl-peptides such as ophthalmic acid, γ-Glu-Val-Gly and γ-Glu-Leu-Gly with decreased quantities in the SSC condition compared to the control. In contrast, γ-glutamyl-AA such as γ-Glu-Cys or γ-Glu-Tyr or glutamyl-glutamate were increased in the SSC condition compared to the control. In the vitamin group, pterine and cyanocobalamin were decreased by a fold of 11 and 4 compared to the control, thus confirming the intracellular changes. Sulfitocobalamin was not detected in the dataset, but this may be due to a low concentration compared to the media components added. Indeed, when comparing both extracellular and intracellular workflows, the analysis of supernatant samples deals with a much wider dynamic range and thus the final sensitivity of the extracellular metabolomic method may be lower. Finally, whereas less CysS was confirmed in the SSC condition compared to the cysteine condition  and transferrin receptor protein 1 (TFRC) were exemplarily selected as representatives of the differentially expressed proteins for quantification using capillary-based Western blot. In agreement with the MS data, the differential expression of both proteins in the SSC condition compared to the Cys control condition was confirmed .Following the descriptive analysis of omics data, mechanistic studies were performed to understand the biological activity of SSC in CHO cells. As sulfur compounds are involved in many regulatory and redox pathways , the effNext, the link between the released sulfur containing molecules and the overall decreased redox potential shown in Since an increase in the thiosulfate sulfurtransferase (TST) expression was observed upon SSC treatment in the proteomic study , this ende novo synthesis. This mechanism was supported by the increase of molecules involved in GSH synthesis, such as intracellular cysteine  and γ-Glu-Cys  shown in c− expression was previously shown to be upregulated in presence of low intracellular Cys and GSH concentration , glutaredoxin 1 (GRX1), thioredoxin 1 (TRX1), thioredoxin reductase (TRXR) or combination thereof, together with NADPH and GSH  as reducing equivalent for the recycling reactions. The consumption of GS-SO3 as well as the production of GSH and NADP+ were followed using untargeted LC-MS. Results presented in Finally, as an important increase in GS-SO3 was detected in the cell culture supernatant, the impact of GS-SO3 on γ-glutamyltransferase (GGT) activity was studied. GGT is a membrane bound enzyme, known to cleave the γ-glutamyl bond of glutathione or glutathione conjugates, to release free glutamate (hydrolysis) or transfer the γ-glutamyl moiety to an acceptor amino acid or peptide to form new γ-glutamyl compounds (transpeptidation) . To evalIn the present study, industry relevant CHO cells producing a therapeutic antibody were cultured in a fed-batch process with either a Cys or SSC containing feed and subjected to a multi-omics approach. Metabolomic and proteomic profiling of CHO cells at multiple time points throughout the fed-batch, along with mechanistic studies, allowed the identification of key targets and interconnected biological pathways. The complementary information provides a more detailed picture of the SSC metabolization within the cell, as summarized in E. coli, calf thymus and humans  detected using metabolomic profiling of CHO cells. Albeit the SSC-derived Cys release kinetics were not determined herein, the elevated intracellular Cys and particularly GSH load in SSC-treated CHO cells point to the ability of this modified amino acid to support the de novo GSH synthesis more efficiently than Cys itself and other analogues.Another key observation upon SSC feeding is the increase in intracellular Cys. As already reported , SSC is Indeed, the accumulating literature surrounding NAC highlights a slow Cys delivery following its administration . RecentlUnexpectedly, the increase in the GSH synthesis pathway, following SSC feeding, was linked to another major observation of the metabolomic study. Indeed, the untargeted LC-MS workflow enabled the identification of several γ-glutamyl-AA  and γ-glutamyl dipeptides . Their abundance was drastically decreased upon SSC feeding compared to the control. Whereas several studies describe γ-glutamyl-AA as reservoirs for amino acids  or markeThe gathered multi-layered omics data further point towards an activation of other Cys catabolic pathways. As described in the literature, an intracellular Cys surplus is prevented through an elevated metabolic flux directed to the taurine pathway . Indeed,Finally, another hallmark of SSC treated cells is the increase of the intracellular sulfur species including sulfite, sulfate and thiosulfate that are exported, at least partly, to the extracellular space. Released sulfite was shown to interact with vitamin B12 leading to sulfitocobalamin through beta ligand exchange. Thus, the decrease in vitamin B12 most likely results from the chemical reaction between sulfite (released upon SSC treatment) and cyanocobalamin, and may not be related to the consumption of vitamin B12 for enzymatical reactions. Generated data further highlighted sulfite as a reducing molecule, able to decrease the cellular redox potential possibly contributing to the known antioxidative effect mediated by SSC. This increase in sulfur containing species was also accompanied by an elevated expression of the ETHE1 and TST enzymes. While both enzymes participate in persulfide or H2S catabolism mediated by the mitochondrial sulfide oxidation pathway , multiplConcluding, the multi-omics study on CHO cells treated with SSC offered deep insights into the cellular mechanism of the SSC/GSH mixed disulfide reduction mediated by GRX1, leading to an increased intracellular sulfur species and Cys availability. Increased GSH and taurine synthesis point out an enhanced antioxidant response. Several other Cys catabolism pathways in SSC-treated CHO cells were enriched to maintain Cys homeostasis and thereby sustain cellular functions.


The novel CBT-SiPc showed great potential in the application of lysosome-targeted and two-photon bioimaging-guided photodynamic cancer therapy.The development of efficient photosensitizers with high singlet oxygen quantum yield, strong fluorescent emission, excellent photostability, and specific organelle targeting is in great demand for the enhancement of PDT treatment efficiency. This study designed and synthesized a new two-photon photosensitizer chlorophenyl thiophene axially substituted silicon (IV) phthalocyanine (CBT-SiPc). CBT-SiPc showed specific targeting of lysosomes in living cells and good biocompatibility. Furthermore, high   Photodynamic therapy (PDT) has attracted attention for its low dark toxicity and reduced side effects, controllability, and high cure accuracy . PhthaloPDT efficacy has recently been shown to be associated with the amount of cancer cell uptake and subcellular localization of photosensitizers . BecauseMost PSs are preferentially localized in the plasma membrane , Golgi aBioactive molecules including benzothiophene derivatives have been widely used in agrochemical, medicinal agent, and chemical sensor applications . Moreovein vitro photodynamic efficacy of CBT-SiPc.This study prepared a novel di-((3-chlorophenylthiophene-2-ester)hexafluorophenoxy) axial substituted silicon phthalocyanine (CBT-SiPc). Thanks to the chlorophenyl thiophene groups, CBT-SiPc showed hindrance that reduced the aggregation of silicon phthalocyanines to some extent and showed specific lysosome targeting. The heavy atom S in the chlorophenyl thiophene groups also promoted intersystem crossing (ISC), which increased the ability to produce ROS for silicon phthalocyanine . We also2) and unsubstituted zinc (II) phthalocyanine (n-ZnPc) were prepared as we described previously , and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) were supplied by Energy Chemical Company . 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA), LysoTracker green, and the Apoptosis Detection Kit with Annexin V-FITC and propidium iodide were purchased from Beyotime Biotechnology . Cell Counting Kit-8 (CCK-8) was obtained from GlpBio Biotechnology Co. . Dichloride silicon phthalocyanine (SiPcCleviously . PhosphaA Perkin Elmer spectrometer was used to measure the infrared radiation (IR) spectra. 1 H nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity-400 NMR spectrometer. Mass spectra were recorded on a Bruker mass spectrometer. UV-Vis was recorded on a Cary 50 UV-Vis spectrophotometer, and fluorescence spectra were measured on an FL900/FS920 steady-state fluorescence spectrometer. High-performance liquid chromatography (HPLC) (SHIMADZU Essentia LC-16P) was used to analyze the CBT purity using acetonitrile: water (v/v = 40:60) as the mobile phase and a Zorbax SB-Aq C18  reversed-phase column as the solid phase at 25°C with a flow rate of 1 mL/min. High-resolution mass spectrometry was measured on an Agilent 6550 iFunnel Q-TOF LC/MS System (Japan).2Cl2) (40.00 mL) were refluxed at room temperature for 24 h with monitoring of the reaction by thin layer chromatography (TLC). The reacted mixture was washed with water. The collected organic phase was dried with anhydrous MgSO4 and then filtered. The organic phase was collected, and the organic solvent was evaporated under reduced pressure to obtain the crude solid product. The crude product was further purified on a silica gel chromatography column using CH2Cl2/ethanol (v/v = 5:1) as eluent twice. A white solid with a yield of 38.6% was obtained, with the following characteristics: IR/cm−1: 3446, 1610, 1499, 1192, 1059, 958, 926, 802, 744, 698, 586; 1H NMR : 5.21 , 12.16–12.18 , 12.47–12.49 , 12.71–12.80 , 12.87–12.96 , 13.28–13.37 . ESI-MS for C24H13ClF6O3S (m/z): 571. Found: 571.58 [M + K]+.3-Chloro-benzo[b]thiophene-2-carboxylic acid , hexafluoro bisphenol A , DMAP , EDC HCl , and dichloromethane , anhydrous K2CO3 , and toluene (40 mL) was added to a 100-mL flask and stirred at 140°C for 48 h. with monitoring by TLC. After being cooled to room temperature, the reaction mixture was filtrated, and the filtrate was evaporated under reduced pressure. The crude product obtained was separated and purified on a silica gel chromatography column using CH2Cl2 as an eluent, and a blue solid was collected. Yield: 30%, with the following characteristics: IR/cm−1: 1741, 1504, 1272, 1176, 1081, 1045, 928, 872, 738.1H NMR : 5.61–5.63 , 6.52–6.58 , 6.77–6.81 , 7.18–7.35 , 7.60–7.65 , 7.89–7.96 , 8.10–8.12 , 8.38–8.42 , 9.63–9.67 . ESI-MS for C80H44Cl2F12N8O6S2Si (m/z): 1599. Found: 1599.89 [M + H]+. Calculated percentages of C80H44Cl2F12N8O6S2Si: C 60; H 2.75; N 7; S 4; found: C 61.99; H 4.23; N 6.98; S 4.23.A mixture of CBT-OH , SiPcCl2. CBT-SiPc was dissolved in DMSO to make a stock solution (1.0 mM). The cells were seeded into 35 mm confocal dishes at a density of 1.0 × 104 cells per dish for 24 h and then treated with CBT-SiPc-containing medium (3.0 μM) for 10 h. Next, the medium was removed and the cells were washed twice with PBS. Fluorescence images of CBT-SiPc in MCF-7 breast cancer cells were obtained using a confocal laser scanning microscope (CLSM).MCF-7 breast cancer cells were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin at 37°C with 5% CO4 cells per dish for 24 h and then treated with CBT-SiPc-containing medium (3.0 μM) for 10 h. After incubation, the medium was removed and the cells were washed twice with PBS. LysoTracker Green (50 nm) was then added to dishes to stain the lysosomes of the cells for 30 min before rinsing three times with PBS. The experimental procedure was similar to that described previously  for 10 h. After that, the cells were irradiated with a 671 nm laser (100 mW/cm2) for 10 min and then treated with DCFH-DA for 1 h at 37°C. Next, the medium was removed and the cells were washed three times with PBS. Finally, the production ability of ROS was visualized by CLSM.DCFH-DA was used as the probe to determine the intracellular ROS generation ability in MCF-7 cells . MCF-7 c4 cells per dish for 24 h and then treated with CBT-SiPc-containing medium (3.0 μM) for 10 h. After incubation, the cells were exposed to laser irradiation  for 0, 5, 10, 15, and 20 min, respectively. Then, the cells were observed by CLSM.The MCF-7 breast cancer cells were seeded into 35 mm confocal dishes at the density of 1.0 × 104 cells/well, 24 h) and subsequently incubated with CBT-SiPc (3.0 μM) for 10 h. The blank group was directly cultured in fresh DMEM medium instead of the old medium for 10 h. After treatment, the cells were exposed to a 671 nm laser (100 mW/cm2) for 10 min and then incubated for another 4 h. The resulting cells were stained with the mixture of Annexin V-FITC (5 µL) and propidium iodide (PI) (10 µL) for 15 min, and the mixture was analyzed with flow cytometry to assess the number of apoptotic cells. All experiments were performed in triplicate.The apoptosis/necrosis mechanism of CBT-SiPc-mediated photodynamic therapy was detected by flow cytometry analysis with double staining using an Annexin V-FITC Apoptosis Detection Kit . MCF-7 c2. MCF-7 cells with a density of 1 × 104 cells per well were cultured in 96-well plates with 100 μL of culture medium for 24 h. The cells were then treated with various concentrations of CBT-SiPc  for 24 h. Next, the cells were exposed to the absence and presence of laser . Finally, the surviving cells were measured by CCK-8. The relative cell viability was calculated using Eq. 5 (The photodynamic therapy efficacy of CBT-SiPc against MCF-7 cells was determined by CCK-8 assay . MCF-7 cng Eq. 5 .Relativ2) via nucleophilic substitution to obtain CBT-SiPc. CBT-SiPc was soluble in common organic solvents such as dimethyl sulfoxide (DMSO), N, N-dimethylformamide (DMF), and tetrahydrofuran (THF). The structures of CBT-OH and CBT-SiPc were confirmed by 1H NMR, IR, ESI-MS, and HRMS.The scheme for the synthesis of chlorophenyl thiophene silicon phthalocyanine (CBT-SiPc) is shown in 1H NMR, HPLC, and HRMS spectra. Resonances at 9.63 and 8.38 ppm were assigned to signals of the phthalocyanine ring with 16 protons. The three resonances at 5.61–5.63, 7.89–7.96, and 8.10–8.12 were designated to the six aromatic protons of chlorophenyl thiophene, while the four sets of resonances at 6.52–6.58, 6.77–6.81, 7.18–7.35, and 7.60–7.65 were ascribed to the aromatic protons of the hexafluoro bisphenoxy groups , fluorescence lifetime (τs), and singlet oxygen quantum yield (ΦΔ) of CBT-SiPc were measured according to the literature (ΦΔ) was 0.30 , MitoTracker, and lipid droplet trackers BODIPY were incubated with CBT-SiPc, respectively, to assess organelles targeting by CBT-SiPc through two channels. As shown in 2) for 10 min, CBT-SiPc showed phototoxicity with an IC50 of 4.16 μM in MCF-7 cells. The cell viability also decreased to 33.14% when at a 5 μM concentration of CBT-SiPc after irradiation by a 671 nm laser (100 mW/cm2) for 10 min.The photodynamic efficacy and dark toxicity of CBT-SiPc against MCF-7 cells were evaluated using CCK-8 assays . CBT-SiPConfocal laser scanning microscopy was used to image the ROS generation of CBT-SiPc in MCF-7 cells using DCFH-DA as the probe. As shown in 2, 10 min), only 17% of cells were viable and 83% were apoptotic. As the percentage of necrotic cells was negligible, we concluded that apoptosis was the major cell death pathway for CBT-SiPc-mediated PDT.To understand the cell death mechanism induced by CBT-SiPc after PDT treatment, Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) co-staining was performed  and analyzed by flow cytometry. As shown in The morphological changes in MCF-7 cells were observed by CLSM . Before in situ, which showed impressive photodynamic activity by inducing cell apoptosis through lysosome dysfunction. These findings demonstrate that CBT-SiPc is a promising photosensitizer for photodynamic therapy.This study designed and developed chlorophenyl thiophene silicon (IV) phthalocyanine (CBT-SiPc). Due to the properties of the chlorophenyl thiophene groups, CBT-SiPc selectively targeted lysosomes, as tracked by its two-photon fluorescence. CBT-SiPc also generated ROS in the lysosomes of MCF-7 cells


In contrast, despite its presence in postnatal liver as a potential major RA catabolizing enzyme and its acute sensitivity to induction by RA, some data suggested that Cyp26a1 contributes only marginally to endogenous RA homeostasis postnatally. We report reevaluation of a conditional Cyp26a1 knockdown in the postnatal mouse. The current results show that Cyp26a1 mRNA in WT mouse liver increases 16-fold upon refeeding after a fast, accompanied by an increased rate of RA elimination and a 41% decrease in the RA concentration. In contrast, Cyp26a1 mRNA in the refed homozygotic knockdown reached only 2% of its extent in WT during refeeding, accompanied by a slower rate of RA catabolism and no decrease in liver RA, relative to fasting. Refed homozygous knockdown mice also had decreased Akt1 and 2 phosphorylation and pyruvate dehydrogenase kinase 4 (Pdk4) mRNA and increased glucokinase (Gck) mRNA, glycogen phosphorylase (Pygl) phosphorylation, and serum glucose, relative to WT. Fasted homozygous knockdown mice had increased glucagon/insulin relative to WT. These data indicate that Cyp26a1 participates prominently in moderating the postnatal liver concentration of endogenous RA and contributes essentially to glucoregulatory control.Considerable evidence confirms the importance of Cyp26a1 to all- Inhibiting catabolism with “conazoles” boosted RA potency and/or inhibited catabolism in cultured cells, providing early evidence that degradation tempers RA effects , 11, 12.ntiation . Ablatiotabolism . Also coolize RA . Even soogenesis . A condienotypes . Cyp26a1CYP26A1 mRNA, whereas refeeding increases its mRNA -inducible Cre mice were administered tam at 4-weeks-old for five consecutive days (A). Glucose tolerance (GTT) and insulin tolerance tests (ITT) were done at 7- and 8-weeks-old, respectively, on the same mice. Serum and liver tissues were harvested at 9-weeks-old following a 16 h fast or upon 6 h refeeding after a 16 h fast.The current experiments relied on a conditional knockdown (KD) of ive days A. GlucosCyp26a1 mRNA in WT mouse liver responded with a 16-fold increase to 6 h refeeding after an overnight fast (B). Tam-induced Cre-driven KD decreased Cyp26a1 mRNA in fasted heterozygous and homozygous mice to 19% and 1.6% of WT, respectively. After refeeding, Cyp26a1 mRNA increased to only ∼21 and 2% of WT in heterozygous and homozygous KD mice, respectively. Consistent with previous observations, refeeding prompted a 41% decrease in WT liver RA . Increased liver RA during fasting and decreased liver RA during refeeding serves its function as an anti-insulin autacoid . WT liver microsomes eliminated RA at half-lives of 89 and 65 min during fasting and refeeding, respectively. The t½ elimination rates increased to 141 and 110 min in homozygous fasted and refed KD mice, respectively. Neither retinyl esters nor retinol in liver varied by fasting versus refeeding or with genotypes (A and B). We also confirmed that serum RA does not reflect liver RA, as published previously (C) ..Cyp26a1 A and B). Although they were minimal to mild, the lesions accompanied yellow-green deposits of the iron-containing protein hemosiderin, distinguishable from spontaneous inflammation foci observed in WT liver sections (C–E).No apparent signs of gross liver damage presented in H&E-stained sections of any genotype, such as necrosis, steatosis, or fibrosis . Glycogesections , C–E.A). Upon an insulin challenge (ITT), glucose levels in each genotype decreased at similar rates (B). After 60 min, however, serum glucose levels began to diverge, eventually reaching a statistically significant increase in homozygous KD relative to WT. Serum glucose increased 46 mg/dl more in refed homozygous KD mice than WT (C). We hypothesized that the elevated glucose resulted from an increase in hepatic glucose production, indicating insulin resistance in liver. Western blots of Akt serine/threonine kinase 1 and 2 (Akt1 and Akt2) versus phosphorylated Akt1 and Akt2 showed that fasting increased phosphorylation of both Akt1 and 2 in WT mice, as expected, by >3-fold and 4.5-fold, respectively (D). The degrees of Akt1 and Akt2 phosphorylation between fasted and refed homozygous KD mice were blunted (∼2-fold each) and did not reach statistical significance (p ∼ 0.06). Liver triacylglycerol (TAG) did not differ significantly in WT fasting versus refed mice. TAG in livers of refed KD mice was significantly lower than in fasted mice, with homozygous KD mice attaining only 61% of the level in fasted WT mice (E). The glucagon/insulin ratio in fasted homozygous KD increased ∼2.3-fold more than WT (F), driven by the glucagon increase in the homozygote KD during fasting (G).GTTs did not differ among genotypes A. Upon aar rates B. After  than WT C. We hypectively D. The de WT mice E. The gl than WT F, driven fasting . Cortico fasting G.Figure Pck1) and glucose-6 phosphatase (G6pc) . G6pc expression increased ∼1.5-fold during fasting in homozygous KD relative to WT but did not attain statistical significance (p ∼ 0.08). (B). Gys2 mRNA, which encodes the rate-limiting enzyme of glycogen synthesis, did not change with genotype during fasting or refeeding , consistent with the increased serum glucose (C). Fasting induces Pdk4 expression, which inhibits the pyruvate dehydrogenase complex and increases pyruvate for gluconeogenesis . In contrast, Pdk4 mRNA in refed homozygous KD mice reached only 46% of the level in WT mice. This suggests a shift during refed homozygous KD mice from sparing pyruvate to generating acetyl-CoA. Glycogen phosphorylase (Pygl) mRNA, which encodes the rate-limiting enzyme of liver glycogenolysis, also did not change with fasting versus refeeding or with genotype (E). Insulin induced dephosphorylation and inactivation of phospho-Pygl in WT, but not in the homozygous KD mice (F).Phosphorylated Akt phosphorylates the FoxO1 transcription factor, which causes its nuclear export and proteolysis, resulting in decreased expression of phosphoenolpyruvate carboxykinase (e (G6pc) . Pck1 exefeeding A. G6pc e∼ 0.08). B. Gys2 mefeeding . Gck mRNve to WT C, consis glucose C. Fastinogenesis . Fastinggenotype D. In congenotype E. Insuli KD mice F.Figure Cyp26a1 mRNA in heterozygous and homozygous KD mice accompanied 56% increases in liver RA during refeeding relative to WT mice, consistent with a major contribution of liver Cyp26a1 to regulating RA. Changes in the RA elimination t½ provide additional insight. These values show that in addition to Cyp26a1 exerting a major impact during refeeding, it also does so during fasting. For example, in WT mice, the RA elimination t½ increased by 25 min during fasting relative to refeeding. In the homozygous KD, this value increased by 40 min during fasting relative to refeeding. Moreover, fasted homozygous KD mice had an extended RA elimination t½ of 52 min relative to fasted WT mice. Therefore, Cyp26a1 contributes to the catabolism of endogenous RA in liver during both fasting and refeeding. Lack of an increase in liver RA concentrations during fasting in KD versus WT mice likely indicates regulatory mechanisms other than Cyp26a1 activity. RA homeostasis reflects actions of cellular retinol-binding proteins, multiple retinol and retinal dehydrogenases, retinal reductases, retinol esterification enzymes, and retinyl ester hydrolases, besides catabolic enzymes , that is, inhibitors of Cyp26 isomers, have been evaluated for therapeutic use to increase endogenous RA concentrations , 51. ThiCyp26a1-deficient male mice were generated by crossing the tamoxifen-inducible Cre line, B6.129-Gt(ROSA)26Sortm1(cre/ERT2)Tyj/J (strain# 008463), purchased from The Jackson Laboratory, with Cyp26a1-floxed mice donated by Dr Martin Petkovich  were produced by F1 x F1 crossing (Cyp26a1 f/+ with tam Cre+/−). All mice were administered tam once daily by i.p. injection (75 mg/kg) at 4-weeks-old for five consecutive days, as reported previously . Mice were fasted 16 h (5 PM-9 AM) before the GTT and 4 h in the morning before the ITT and housed in individual cages without food, but with free access to water. Either glucose (2 g/kg body weight) or insulin (0.5 IU/kg body weight) was injected I.P. Blood glucose from tail tips was measured using an Ascensia CONTOUR NEXT glucometer.GTTs were done at 7-weeks-old. ITTs) were done at 8-weeks-old on the same mice A. Mice wSegments ∼1.5 cm long and 3 mm thick were cut from the middle of the left lobe and fixed in 10% neutral buffered formalin (Sigma HT501128) for 3 days. Formalin-fixed, paraffin-embedded liver segments were cut into 8-μm sections. Deparaffinized sections were stained with H&E. Tissue sections were imaged using a Zeiss Axio Imager M1 microscope equipped with QImaging 5MPix MicroPublisher.g to remove precipitates . RA catabolism was assessed at 37 °C. The 0.5 ml incubation mixture contained the assay buffer, 50 pmol RA, an NADPH regenerating system , and 50 μg microsomal protein. The reaction was terminated by adding 2 ml methanol.Liver samples (∼100 mg) were homogenized in buffer  using a glass pestle tissue grinder on ice. The homogenate was centrifuged 10 min at 10,000Proteins in liver samples were extracted with 1 ml RIPA buffer , which included protease and phosphatase inhibitors . The Bradford assay determined total protein concentrations. Thirty micrograms of protein were mixed 1:1 with Laemmli sample buffer , including 5% β-mercaptoethanol, heated 5 min at 95 °C, and cooled on ice. Protein samples were separated by 12% SDS-PAGE , transferred onto nitrocellulose membranes , which were blocked in Intercept (TBS) blocking buffer , and immunoblotted overnight at 4 °C with antibodies against Akt1 , phospho(S473)-Akt1 , Akt2 , phospho(S474)-Akt2 , phospho-(S15)-Pygl , and β-actin . Primary antibodies were diluted 1:2000. Near-infrared fluorescent dye-conjugated secondary antibodies were as follows: anti-rabbit  and anti-mouse  at 1:5000. Immunoblots were developed with a LICOR Odyssey Imaging System. Western blot band density was quantified by integrating areas under curves using ImageJ 1.53. Antibodies for phosphor-Akt1 and Akt2 were stripped by shake-incubating in Restore PLUS Western blot stripping buffer  at 37 °C for 10 min, followed by reblock and immunoblotting with the Akt1 and 2 antibodies. Signals were normalized to actin. For p-Pygl, actin-normalized signals were then normalized to the average of WT fasted set as 1. Data are provided in Gusb, and expressed as fold change relative to controls. Primers are listed in Total RNA was isolated using TRI Reagent , quantified using a ThermoScientific NanoDrop One, and reverse-transcribed using iScript cDNA Synthesis Kit . Quantitative PCR was performed with a Bio-Rad CFX Connect Real-Time Detection System using PrimeTime Gene Expression Master Mix (Integrated DNA Technologies (1055772). Gene expression was analyzed by the ΔΔ-Ct method, normalized to Insulin, glucagon, glycogen, and TAG were quantified using Ultra Sensitive Mouse Insulin ELISA Kit , Mouse Glucagon ELISA Kit , Glycogen Assay Kit , and Triglyceride Quantification Colorimetric/Fluorometric Kit , following manufacturers’ instructions.g and at 4 °C. Supernatants were transferred to round bottom glass tubes and methanol was evaporated under gentle N2 streams. Precipitates were resuspended in 2 ml of 0.2 M acetate buffer (pH 5.0) and vortexed 20 s. Ten milliliters of methyl t-butyl ether were added. Tubes were vortexed 20 s and centrifuged at 3 min 2000g. The upper organic phases were transferred to a new round bottom glass tube and evaporated under gentle N2 streams. Precipitates were resuspended in 40 μl methanol. One microliter was injected into HPLC-APCI/MS/MS.A mixture of 1.5 ml methanol and 40 μl corticosterone-d8 internal standard  was added into 2 ml Eppendorf tubes, each containing a metal bead. Liver (∼50 mg) samples were added and homogenized using a Qiagen TissueLyser II at 30/s for 30 s. Homogenates were centrifuged 10 min at 12,000via reverse-phase chromatography with an Agilent 1290 system equipped with a binary pump, column compartment, and autosampler. The column compartment was maintained at 40 °C. Samples were kept in the autosampler at 10 °C. Separation was achieved with an analytical Ascentis Express RP-Amide column  at a flow rate of 0.4 ml/min. Mobile phases were (A) 0.1% formic acid in water; (B) 0.1% formic acid in methanol. The following gradient was applied over 25 min: 0 to 2 min, 40% B; 2 to 15 min, 40 to 95% B; 15 to 20 min, holding at 95% B; 20 to 23.5 min, 95 to 40% B; 23.5 to 25 min, back to 40% B and reequilibrating for 1.5 min.Corticosterone was resolved Analytes were detected with a Sciex API-4000 triple-quadrupole mass spectrometer in positive atmospheric pressure chemical ionization mode. Analyst version 1.6 software controlled the instrument, which was operated in the multiple reaction monitoring mode. Mass transitions to produce optimum sensitivity were determined by injecting 1 pmol standards . Optimized MS variables were as follows: curtain gas, 10 psig; collision gas, 7 psig; ion source gas 1, 70 psig; nebulizer current, 3 μA; source temperature, 350 °C; declustering potential, 55 V; entrance potential, 10 V; collision exit potential, 5 V. Optimized collision energy value was 25 eV.p-values were reported for correcting multiple comparisons. Significance values are: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Western blot band density was measured by integrating areas under curves using ImageJ 1.53. Statistical testing was done using GraphPad Prism 9.5.0.Data are means ± SD. Two-tailed unpaired nonparametric Mann-Whitney tests compared data between two groups. False discovery rate (1%)-adjusted All data are contained within the manuscript.This article contains The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


First, the electrochemical performances of symmetric supercapacitors were characterized in an acidic aqueous electrolyte (0.5 M H2SO4). It was observed that a supercapacitor cell containing the 5 wt% AC-P2W18 hybrid symmetric displayed a noteworthy specific capacitance of 289 F g−1 and a remarkable energy density of 40 W h kg−1. Moreover, 5% AC-P2W18 symmetric supercapacitor cells showed 89% cyclic stability over 4000 cycles. Three LED lights were charged onto the electrode. The LEDs continued to illuminate continuously for red until 160 seconds, yellow until 20 seconds, and blue until 10 seconds after removing the electrode from the electrochemical workstation, demonstrating the device's power and energy density.The nanofabrication of electroactive hybrid materials for next-generation energy storage devices is becoming increasingly significant as supercapacitor (SC) technology develops rapidly. The present study utilizes activated carbon (AC) templates reinforced with Wells–Dawson polyoxotungstates (POMs) to produce nanohybrid electrodes for high-performance supercapacitors. This study analyzes Wells–Dawson polyoxotungstates (P 2W18) were impregnated on activated carbon (AC). In order to accomplish the real application as a device, the AC-P2W18 supercapacitor cell is lit up by a blue LED.In the fabrication of a novel three-symmetric supercapacitor cell, three different wt% of Wells–Dawson polyoxometalate (5%, 10% & 15% P Since supercapacitors can deliver higher power densities and longer cycle life than batteries with fast charge and discharge, they have gained significant attention.3,4 Supercapacitors were used in electronic devices, such as trains and buses, automobiles, cranes, and elevators.5 Double-layer capacitors (EDLC) and pseudocapacitors (PC) fall under the supercapacitor taxonomy. The electric doubler layer capacitor's energy storage and release mechanism rests entirely on the theory of charge separation at the electrode–electrolyte interface.6 As opposed to batteries, herein, there are no chemical oxidation-reduction reactions; thus, the nonfaradic nature of this physical charge transfer results in long cycling life.7,8 In EDLC, graphene-based,9–11 activated carbon,12–14 and carbon nanotube-based materials15,16 are typically used in the case of pseudocapacitors involved in the oxidation/reduction process. As a result, faradaic redox reactions have higher energy density.17,18 The materials used here include transition metal oxides,19,20 conducting polymers,21,22 metal sulfides,23 and polyoxometalates.24,25 The most usually used electrode material in commercial supercapacitors is activated carbon (AC) owing to its high surface area, low cost, high electrical conductivity, excellent corrosion resistance, high thermal stability, tuned pore structure, easy processability, and compatibility with other composite materials.26 However, because of its nonfaradic nature, the specific capacitance remains small. As a result, researchers began investigating new electrodes based on hybrid materials, the combination of an electric double layer, and a pseudocapacitor. It can boost the energy and power density while maintaining mechanical strength and the cell's long lifespan.The last few eras have seen rapid growth in several areas, such as industrialization and globalization. As a result, energy demands have increased exceptionally. In parallel, the human population has also grown, which accounts for much of the increase in energy consumption. Traditionally, fuels were used as energy sources, but fuel depletion led researchers to move toward electrochemical energy to replace them.27 The most unique property of POMs is that it can absorb and release electrons without changing its structural characteristics, which makes it an admirable supercapacitor electrode material. POMs comprise a multimetal oxide of early transition elements that can be altered in shape, size, and composition. It is nontoxic and nonvolatile, has a large molecular size, and relatively high molecular weight. Different types of POMs, such as Keggin,28 Dawson,29 Lindquist,30 Waugh,31 Silverton,32 and Anderson,33 and their multidirection applications are reported. POMs have been used in various applications, including sensors, catalysis, medicine, and energy storage. Among these, Keggin and Dawson types of POMs have been extensively investigated because of their thermal and redox stability and can engage reversible multielectron transfer reactions.34 This electron transfer metal-oxide cluster type suits electrochemical capacitors (EC). However, poor conductivity, low surface areas, and high solubility in an aqueous solvent prevent the direct application of POMs as an electrode material.35 To decrease the solubility, POMs are often deposited on a high surface area carbonaceous support to yield a nanocomposite for EC electrodes. Carbonaceous materials such as graphene,36 graphene oxide,9 single-walled carbon nanotubes (SWCNT),37,38 and multiwalled carbon nanotubes39,40 have been studied mainly as a support for the POM composite electrodes. Keggin POM clusters were mainly immobilized on the carbonaceous materials and investigated extensively in EC, resulting in reduced solubility and enhanced capacitance over soluble POMs. The following et al. successfully anchored molybdenum-containing Keggin POM [H3PMo12O40] (PMo12) onto the AC, and their electrochemical study was conducted in 1 M H2SO4, resulting in a specific capacitance value of 160 F g−1 at 2 A g−1 in a three-electrode system.41 In 2014, the same group developed an extensive method for impregnating [H3PW12O40] (PW12) on AC. A combination of double layer and redox activity was discovered to increase the specific capacitance by 254 F g−1 at 10 mV s−1.42 Hu C. et al. are interested in further exploring the same material PMo12-AC in an ionic liquid electrolyte (1 M [Bmim]HSO4), which attained a specific capacitance of 223 F g−1 at 1 mV s−1.43 In a follow-up study by Genovese et al., activated carbon from pinecone biomass was synthesized, and PMo12 was deposited onto the pinecone-derived AC surface. This resulted in a specific capacitance of 361 F g−1 at 10 mV s−1.44In this respect, inorganic metal–oxygen cluster polyoxometalates (POMs) have emerged as promising pseudocapacitive materials with diverse properties.et al. fabricated a symmetric cell using vanadopolymolybdates at the AC surface and achieved a capacitance of 430 F g−1 at 0.2 A g−1 in 0.25 M H2SO4 in a 2-electrode system.45 Next, Vannathan A. A. et al. developed manganopolyvandate into an AC matrix, and the electrochemical performance was measured on a symmetric device in 0.1 M H2SO4 electrolyte. The specific capacitance was observed at 479.7 F g−1 at 0.4 A g−1.46 The research has so far focused on Keggin-based polyoxometalates ranging from homopolyanions to heteropolyanions. In addition, Maity S. et al. synthesized Lindqvist polyoxometalate (NiV14) doped on AC surfaces and performed an electrochemical study in 0.5 M H2SO4, which showed a capacitance of 365 F g−1 at 0.2 A g−1 in a symmetric cell.27Maity S. et al. synthesized the first Dawson-based composite of octadecamolybdodiphosphate ((NH4)6[P2Mo18O62]·14.2H2O) doped on an activated carbon surface and investigated its electrochemical performance. It showed a specific capacitance of 275 F g−1 at a 6 A g−1 current density in a three-electrode system.47 Till today, there is no supercapacitor work reported using Wells–Dawson POM. Hence, it was interesting to investigate Well–Dawson-type POMs for supercapacitor applications. Herein, we explored activated carbon (AC)-supported K6[P2W18O62]. xH2O (P2W18)-based electrode for the electrochemical supercapacitor in a two-electrode system. In this paper, we doped P2W18 onto the AC matrix in different concentrations  to examine the maximum amount of P2W18 on AC porous surface, which can enhance its capacitive nature. A symmetric SC cell composed of 5 wt% AC-P2W18, 10 wt% AC-P2W18, and 15 wt% AC-P2W18 was synthesized, and its electrochemical performance was evaluated using a two-electrode system in 0.5 M H2SO4 electrolyte. It has been observed that 5 wt% AC-P2W18 exhibited an excellent specific capacitance of 289 F g−1 at 0.2 A g−1 with good energy and power density of 40 W h kg−1 and 1999 W kg−1.In this respect, Wells–Dawson-type POMs have been overlooked, which may be due to the larger size and higher charges than Keggin ions. In 2015, Mu A. 2.2.12W18. The characteristic chemical bands in all the three composite materials and pure P2W18 correlated well with those reported in the literature.48 The bands at 1108, 824, 934, and 996 cm−1 of P2W18 are induced by vibrations of the P–O, W–Oe–W, W–Oc–W, and terminal W PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O bonds  to determine the chemical structure of the materials.  Table S1. CC stre2.22W18 composites of different weight percentages are shown in 2W18 has been confirmed from the literature (48 and JCPDS (card no: 01-073-6183). AC-P2W18 exhibits amorphous and crystalline characteristics as a result of P2W18 being incorporated in AC. The sharp peaks at about 20.35°, 24.13°, 25.85°, and 26.63° are due to P2W18. Pure P2W18 shows more intense peaks due to its well crystalline nature , (1 0 0), and (2 0 0) correspond to 2θ of 24, 43.8, and 20.35, respectively, for AC and P2W18.The powder XRD patterns of AC-Pterature 48 and JCe nature . Convers2.32W18. The survey spectrum shows the peaks of the elements found as C 1s, O 1s, W 4f, and P 2p for 5% AC-P2W18 in Fig. S1.45 A Gaussian function centered at 531.2 eV, 532.1 eV, and 533.6 eV due to WO, O–W–O, and O–H, respectively, are satisfactorily fitted to the O 1s spectrum  was used to probe the electronic states of the nanohybrids. spectrum  of Well–spectrum , with pe2.42W18 nanohybrids, N2 adsorption/desorption isotherms data were collected using the Micromeritics physisorption analyzer . The AC possesses the highest surface area of 1340 m2 g−1 with the highest pore volume of 0.37 cm3 g−1  analysis was also carried out to identify the elements present in the composites. Fig. S2a2W18 of K, O, P, and W. The elemental compositions of the composites  are also confirmed by EDS results .  Fig. S2b. In addi2.5.146 The electrodes were tested in a cyclic  setup using 5% AC-P2W18, 10% AC-P2W18, and 15% AC-P2W18 composite materials in the potential window of 0–1 V using 0.5 M electrolyte solution at various scan rates. A CV graph measuring 5% AC-P2W18, 10% AC-P2W18, and 15% AC-P2W18 has been presented in −1. CV analysis indicates that AC-P2W18 oxidation-reduction peaks of 5% and 10% indicate that is P2W18 present over AC surfaces. It is noticeable that 15% AC-P2W18 shows a deformed curve compared to the other two, 5% AC-P2W18 and 10% AC-P2W18. To better understand the pseudo material's exact oxidation-reduction behavior, the bare P2W18 electrode was also used to perform CV  method was utilized to explore the electrochemical properties of the power cell. An experiment using cyclic voltammetry (CV) measurements was done to study the electrode material's chemical kinetics, degradation process, and specific capacitance.V Fig. S4 in the sity area . To eval2.5.22W18 composite electrode materials under a defined potential window.48 In a galvanostatic charge and discharge study, three symmetric SC cells were examined with current densities in the range from 0.2 to 5 A g−1 in the potential window of 0–1 V. The charge–discharge outline was reshaped by immobilizing POMs onto the AC surface, which differs from the EDLC behavior. The improper linear GCD curve was observed across lower current densities.46 At 0.2 A g−1 current density  at several current densities was examined to understand the electrochemical performance of the AC-P density , the 5% ensities  in the c plotted . It is o2.5.32W18 were deposited on AC and AC-P2W18 nanocomposites, and electrochemical impedance spectroscopy measurements were carried out using a dc potential of 0.01 V in the frequency range from 1 Hz to 100 kHz. Nyquist plots can be used to assess the internal resistance of the composites as well as their charge transfer kinetics and ion diffusion processes.51 The impedance spectroscopy results for the high-frequency region show the electrodes to be arranged in a semicircular arc. Low-frequency measurements of electrode–electrolyte impedance provide a visual representation of electron transfer kinetics of redox reactions due to limited mass transport.52,53 A partial semicircle was observed when the frequency increased, indicating the charge transfer resistance. As shown in Table S8,2W18 nanohybrids exhibit equivalent series and charge transfer resistance. In the higher frequency regime, the RCT value is calculated from the diameter of the semicircle in the Nyquist plot . All three different concentrations of Pist plot . The RCT at 3.35  ·xH2O was synthesized according to the published procedure.54 To achieve different concentrations of P2W18 on AC, the amount of P2W18 was varied. AC-P2W18 was prepared using the procedure outlined below by varying the amounts of P2W18 of 5 wt%, 10 wt%, and 15 wt%. 1 g AC in 0.010 L methanol was dispersed in a round bottom flask. The methanol solution was agitated for 10 minutes with a magnetic stirrer to ensure even distribution. To prepare the P2W18 solution, P2W18 was dissolved in a small quantity of water (<5 mL) and then added dropwise into the AC-methanol solution. The solution form was agitated for approximately 24 hours at room temperature. After drying under reduced pressure, the solution was rinsed with enough quantity of aqueous solution to remove the excess P2W18. The black residue was then collected and finally allowed to dry.The Wells-Dawson-type Polyoxometalate potassium octadecatungstate diphosphate K3.22W18, 10 wt% AC-P2W18, and 15 wt% AC-P2W18) mixed with 10% PVDF (as a binder). The mixture was taken in a mortar and pestle to blend the materials after it was combined with N-methyl-2-pyrrolidone  to make a uniform slurry. Next, a uniform layer of paste was coated on a flexible carbon cloth (each measuring 1 cm × 1 cm). The carbon cloth was weighed before and after modifications, which allowed us to determine the actual mass of the electrodes. We calculated only the active substance's mass . 0.5 M H2SO4 served as a proton-conducting electrolytes for SC cells, and Whatman filter paper was used as a separator, which was soaked in the electrolyte. Individually symmetric arrangements were constructed to conduct electrochemical studies. As part of the symmetric SC fabrication process, an electrolyte solution-soaked filter paper separated an equivalent number of identical components from the carbon cloth.Electrochemical performances were measured by taking 90% of the active material  impregnated on AC surface hybrid electrode material for supercapacitor applications. Furthermore, symmetric cells such as 5% AC-P2W18, 10% AC-P2W18, and 15% AC-P2W18 nanostructures were electrochemically tested in a 0.5 M H2SO4 electrolyte solution using the two-electrode configuration. The 5% AC-P2W18 symmetric cell shows an upgraded specific capacitance value of 289 F g−1 with a high energy density of 40 W h kg−1. 5% AC-P2W18 shows superior electrochemical faradaic charge storage performance compared to other symmetric cells, which indicates that P2W18 is firmly incorporated onto the AC surface. To determine a supercapacitor's application, the 5% AC-P2W18 electrochemical capacitors achieved cycle stability of 89% over 4000 cycles.The novel electrode materials were produced Madhusree J. E.: visualization, investigation, writing – original draft, software. Pranay R. Chandewar: investigation, data curation. Debaprasad Shee: writing – review and editing. Sib Sankar Mal: conceptualization, methodology, writing – review and editing, supervision.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.RA-013-D3RA04478E-s001


Introduction: Schizophrenia is a serious mental illness that requires effective treatment with minimal adverse effects. As preclinical and clinical research progresses, trace amine-associated receptor 1 (TAAR1) is becoming a potential new target for the treatment of schizophrenia.Methods: We used molecular docking and molecular dynamics (MD) simulations to discover TAAR1 agonists. The agonistic or inhibitory effects of compounds on TAAR1, 5-HT1A, 5-HT2A, and dopamine D2-like receptors were determined. We used an MK801-induced schizophrenia-like behavior model to assess the potential antipsychotic effects of compounds. We also performed a catalepsy assay to detect the adverse effects. To evaluate the druggability of the compounds, we conducted evaluations of permeability and transporter substrates, liver microsomal stability in vitro, human ether-à-go-go-related gene (hERG), pharmacokinetics, and tissue distribution.Results: We discovered two TAAR1 agonists: compounds 50A and 50B. The latter had high TAAR1 agonistic activity but no agonistic effect on dopamine D2-like receptors and demonstrated superior inhibition of MK801-induced schizophrenia-like behavior in mice. Interestingly, 50B had favorable druggability and the ability to penetrate the blood-brain barrier (BBB) without causing extrapyramidal symptoms (EPS), such as catalepsy in mice.Conclusion: These results demonstrate the potential beneficial role of TAAR1 agonists in the treatment of schizophrenia. The discovery of a structurally novel TAAR1 agonist (50B) may provide valuable assistance in the development of new treatments for schizophrenia. Schizophrenia is a complex and severe psychiatric disorder. The World Health Organization considers schizophrenia one of the top 10 disorders contributing to the global burden of disease, which affects >20 million people worldwide .The primary clinical symptoms of schizophrenia can be categorized into three groups: positive symptoms ; negative symptoms ; and cognitive deficits (impaired executive functioning and memory) . Due to 2-like receptors  receptors have been shown to produce fewer side effects associated with dyskinesia. However, they have not shown significant improvements in efficacy and have increased the prevalence of adverse metabolic effects and body weight gain (Since the discovery of the antipsychotic drug (APD) chlorpromazine in 1952, APDs have elicited clinical benefits in patients with psychosis, presumably through antagonist or partial-agonist effects at postsynaptic dopamine Deceptors . Howeverpairment . Atypicaght gain . Therefo2-like and 5-HT2A receptors, several other therapeutic targets have been studied extensively for their potential in treating schizophrenia, including receptors for α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, n-methyl-D-aspartic acid (NMDA), GlyT1, and muscarinic M1/M4   . An agon studies . Another1A and 5-HT2A and had no excitatory or inhibitory effect on dopamine D2-like receptors, indicating that 50B may be less likely to elicit adverse effects. Pharmacokinetic studies and studies on blood–brain barrier (BBB) permeability demonstrated that 50B entered the brain in sufficient amounts. An in vivo pharmacodynamic experiment and catalepsy assay revealed that 50A and 50B reduced MK801-induced schizophrenia-like behavior in mice significantly without causing excessive extrapyramidal symptoms (EPS). Druggability analyses suggested that 50B had good BBB permeability and stability without cardiotoxicity. Overall, 50B, as a novel TAAR1 agonist, has good druggability, efficacy, and safety in schizophrenia treatment and may become a novel and powerful APD.In the present study, two enantiomeric compounds were identified to have TAAR1 agonistic activity. Compound 50B activated TAAR1 better than SEP-856, as determined through molecular docking, molecular dynamics (MD) simulations, and functional assays on human TAAR1. Off-target assays revealed that 50B slightly activated 5-HT® cAMP Assay Kit (90-0075SM10) was obtained from Eurofins . FLIPR Calcium Assay Kit (R8194) was produced by Molecular Devices .Compounds 50A and 50B were supplied by Shanghai Wuxi Apptec Co., Ltd. (purity over 98.0%), and MS (ESI) m/z = 170.3 [M + H] +. SEP-856 (HY-136109A) and risperidone (HY-11018) were purchased from MedChemExpress (United States). Ham’s F-12K (Kaighn’s) Medium (11765070), Minimum Essential Medium , Dulbecco’s modified Eagle medium/Nutrient Mixture F-12 , and 0.25% Trypsin–EDTA solution (25200072) were purchased from Invitrogen . MK801 was purchased from Sigma-Aldrich (M107) . Fetal bovine serum (FBS) was purchased from Gibco (10099141C) . HitHunterAll procedures related to animal experimentation were in accordance with Chinese laws and regulations governing the use and care of laboratory animals and the regulations set by our institution. The number of animals used, experimental design, and procedures for animal handling were approved by the Experimental Animal Ethics Committees of Yantai University.Male Sprague–Dawley rats  obtained for studies on pharmacokinetics and tissue distribution and male C57BI/6J mice  used for the pharmacokinetics study were purchased from Pengyue Laboratory Animal Technology . All animals were kept in a standard environment .https://discover.3ds.com/discovery-studio/). As of 16 August 2021, the crystal structure of TAAR1 was unavailable in the RCSB Protein Data Bank (www.pdbus.org/). Hence, homology modeling was used to generate its three-dimensional structure using AlphaFold . The target was optimized for dehydration, hydrogenation, protonation, and energy minimization. The active site in TAAR1 was defined based on the verified contact residues Asp103 -K1 AGTRL1 Gi cells expressing human TAAR1 receptors on cell membranes were cultured with Ham’s F-12K (Kaighn’s) medium supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in an atmosphere of 95% air and 5% CO2L and D2S, recombinant Flp-In™-CHO cells expressing human dopamine D2L receptors or D2S receptors were cultured with Ham’s F-12K (Kaighn’s) medium supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and hygromycin B (600 μg/ml) at 37°C in an atmosphere of 5% CO2. The methods of cell inoculation and compound treatment were the same as those for TAAR1, and haloperidol was used as the MAX control. Percentage inhibition was calculated using the following equation:With regard to D1A, recombinant Gα15-CHO-Clone cells expressing human 5-HT1A were used in this test. Cells were cultured with MEM supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and hygromycin B (600 μg/ml) and were seeded in 384-well microplates. Cells were stimulated with different concentrations of compounds for 2 h at 37°C, and the agonistic effect was determined using the FLIPR Calcium Assay Kit. Serotonin was used as the MAX control. Percentage activity was calculated using the following equation:With respect to 5-HT2A, the agonistic or inhibitory activity of 5-HT2A of compounds was determined using the Flp-In™-CHO cell line expressing human 5-HT2A. Cells were cultured with Ham’s F-12K (Kaighn’s) medium supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 μg/ml), and hygromycin B (600 μg/ml) at 37°C in an atmosphere of 95% air and 5% CO2. Cells were stimulated with different concentrations of compounds for 2 h at 37°C after seeding in 384-well microplates. For the determination of agonistic activity, serotonin was used as the MAX control. In the determination of inhibitory activity, ketanserin was used as the MAX control.With regard to 5-HT2. Cells were seeded into 96-well cell plates and cultured continuously for 24 days for subsequent experiments. Compounds were administered with or without verapamil (2 µM). After incubation for 120 min, samples at the apical and basal ends were collected. The urea content in each sample was measured by liquid chromatography–tandem mass spectrometry (LC-MS/MS). The apparent permeability coefficient and the efflux ratio were calculated.Caco-2 cells were cultured with MEM supplemented with 20% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in an atmosphere of 95% air and 5% COCompounds were incubated with samples of liver microsomes from mice, rats, and humans and NADPH for 5, 10, 20, 30, or 60 min at 37°C. A cold acetonitrile solution containing tolbutamide was used to terminate the reaction. Testosterone and dextromethorphan were used as positive controls. LC-MS/MS was employed to determine the retention of compounds. The retention time, chromatogram acquisition, and chromatogram integration of compounds and positive control substances were analyzed using Analyst .2. They were passaged at a ratio of 1:5 every 48 h. The medium comprised 90% F12, 10% FBS, G418 (100 μg/ml), and hygromycin B (100 μg/ml). On the day of the assay, the cell culture medium was aspirated and washed with an extracellular solution. Then, 0.25% Trypsin–EDTA solution was added, and digestion was allowed for 3–5 min at room temperature. Digestive juice was aspirated, and cells were transferred to the experimental dish for electrophysiological recording after resuspension with the extracellular solution for future use.CHO cells stably expressing hERG cultured with DMEM/F12 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml) were cultured in cell culture dishes (diameter = 35 mm) in an incubator at 37°C in an atmosphere of 5% COCompound 50B was dissolved in physiologic (0.9%) saline. Male and female rats were fasted overnight before dosing to avoid the possible effects of food. Compound 50B was administered via intragastric (5 mg/kg) and intravenous (2 mg/kg) routes to female and male rats. Approximately 200 μL of whole blood was collected into heparin-coated tubes at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h after dosing. Plasma was collected immediately by centrifugation . Blood was placed in a heparinized Eppendorf™ tube. Plasma was centrifuged, and the concentration of compounds in the sample was determined by LC-MS/MS after pre-treatment. Drug-concentration data were processed in a non-atrioventricular model using WinNonlin™ 6.3 .Rats  were sacrificed at 0.25, 1, or 6 h after administration of 50B . Blood and brain tissues were harvested. The concentration of compounds in all tissue and blood samples was determined by LC-MS/MS. Drug concentration data were processed in a non-atrioventricular model using WinNonlin™ 6.3.Compounds 50A, 50B, and SEP-856 were dissolved in physiologic saline. Risperidone and aripiprazole were dissolved in a 0.5% sodium carboxymethyl cellulose (Na-CMC) solution.Ninety-nine male C57BI/6J mice were divided randomly into 11 groups. Mice were placed in a room 1 h before testing. They were administered (p.o.) vehicle (0.1 ml/10 g), 50A , or 50B . Mice were placed in open-field chambers  for 30 min to measure the baseline activity of compounds. Then, mice were administered (i.p.) MK801 . After 30 min, the distance that mice traveled was measured.Forty-six male C57BI/6J mice were divided randomly into four groups. The CN group contained four mice. Ten mice were in the vehicle group . Eight mice were in the SEP-856 group , 50B group , risperidone group  group, and aripiprazole group , respectively. The administration method described previously was employed.Twenty-four male C57BI/6J mice were divided randomly into four groups. Mice were placed in a room 1 h before testing. They were administered vehicle, risperidone , 50A , or 50B . The front paws of mice were placed on a horizontal metal bar raised 2 inches above a Plexiglas™ platform 30 min and 90 min after administration. The time the front paws of mice were placed on the platform was recorded at ≤30 s per trial. Each test was repeated three times.p-value of 0.05 or less was considered statistically significant.Statistical analysis was undertaken using Prism 8 . The results are the mean ± SD. For data with a single dose or time point, the differences between groups were analyzed by non-parametric ANOVA. A − of Asp103 in TAAR1 can form a salt-bridge bond with the positively charged N atom of SEP-856, which is beneficial for the antipsychotic activity. We undertook similarity searching to find compounds with similarity >0.7 using MACCS keys and the Tanimoto Index. Then, 50B and 50A with a high similarity score were modeled using molecular docking and MD simulations. The RMSD value by MD simulations showed that compound 50B reached a relatively stable position after achieving equilibrium in the MD trajectory , which weakened the potency of the agonist to the target markedly  of 50B on TAAR1 reached 0.405 μM, significantly lower than that of SEP-856  and 5-HT2A (EC50 = 61.23 μM and Emax = 54.88%) receptors (2A (IC50 > 100 μM and Emax = 31.49%), dopamine D2L (IC50 > 100 μM and Emax = 5.49%), and dopamine D2S (IC50 > 100 μM and Emax = 24.93%) receptors  was a weak partial agonism on 5-HTeceptors  but showeceptors .t was ∼18,929 h.nM, and oral bioavailability was ∼84.3%  = 24.39, p < 0.001)  = 32.35, p < 0.001) , the time-to-peak concentration was ∼0.833 h, the mean elimination half-life was ∼1.28 h, the concentration–time curve from 0 to time s ∼84.3% . Good pes ∼84.3% . Interes< 0.001) , the con< 0.001) . = 14.85, p < 0.001  = 9.037, p < 0.001  = 8.152, p < 0.001  = 6.692, p < 0.001 , wherea < 0.001 ; F. = 2.943, p = 0.033  = 28.22, p < 0.001 . Treatm < 0.001 . Compare < 0.001 . = 50.14, p < 0.001)  treatment increased the time mice spent holding bars significantly, whereas 50A and 50B at 100 mg/kg, respectively, did not increase this time. Hence, 50A and 50B did not cause EPS at doses ≥10 times the effective dose  . 50A andThe results stated previously suggested that as a novel agonist of TAAR1, 50B flowed to brain and had a role in the anti-positive symptoms of schizophrenia. Therefore, we further analyzed its druggability. Membrane permeability, microsomal metabolism, and cardiotoxicity of 50B were measured. 50B showed strong membrane permeability and good microsomal metabolic stability , 3, simi2-like receptors agonist APDs. Third-generation APDs are effective in relieving positive symptoms without causing hyperprolactinemia and have a lower EPS burden compared to first-generation APDs and lower weight gain and metabolic burden compared to second-generation APDs  group was −7.2 points after 4 weeks of treatment, which was significantly lower than the −9.7 points in the placebo group. With regard to safety, patients in the SEP-856 group experienced major adverse events . However, the prevalence of EPS and changes in blood levels of lipids, glycosylated hemoglobin, and prolactin were similar to those in the placebo group  . A clinibo group . These rMechanistic studies have shown that TAAR1 activation is involved in the antipsychotic effect of SEP-856. TAAR1 is activated by trace amounts (TAs) of amines . TAAR1 iSeveral studies have investigated the possible link between schizophrenia and TA disorders in urine, cerebrospinal fluid, and plasma. TAs of amines are significantly reduced in the brains of patients with various neurologic disorders, which may suggest that the downregulation of TAAR1 function is related to the pathogenesis of psychiatric disorders . HoweverStudies have shown that SEP-856 suppressed aberrant neuronal discharge in the ventral tegmental region of the midbrain and reduced ketamine-induced dopamine production in the striatum of mice  with the side-chain carbonyl O of Asp103. The thiophene moiety of 50B participated in π–π stacking interactions with Phe195, Phe268, and Trp264.50 = 0.405 μM and Emax = 80.078%). 50B had weak 5-HT1A and 5-HT2A receptor agonistic activity  and weak 5-HT2A antagonistic activity  and had no inhibitory effect on either isoform of dopamine D2-like receptors (D2S or D2L). The off-target results stated previously showed that 50B was a relatively pure TAAR1 activator and had weak agonistic or antagonistic effects on the 5-HT and dopamine systems.Subsequently, the functional effects of 50A and 50B on TAAR1 activation were investigated. 50B significantly promoted the cAMP accumulation of CHO-K1 AGTRL1 Gi cells expressing human TAAR1 receptors on cell membranes  reduced the distance traveled by mice after MK801 stimulation, and 50B  significantly reduced the distance traveled by mice after MK801 stimulation. Comparison of efficacy showed SEP-856 to have effects similar to those of 50B for suppressing MK801-induced schizophrenia-related symptoms. These data suggested that the superior role of 50B in resistance to the positive symptoms of schizophrenia is caused by its stronger activation of TAAR1. The agonistic activity of 50B on TAAR1 was superior to that of SEP-856. However, the plasma concentration of 50B was significantly lower than that of SEP-856 at the same time point after oral administration at 5 mg/kg. Thus, 50B and SEP-856 had similar effects in reducing the distance traveled by mice, which suggests that TAAR1 activation is involved in how 50B counteracts the positive symptoms of schizophrenia.To further analyze the role of TAAR1 agonists in improving schizophrenia-like behavior, the positive compounds, second-generation APD (risperidone), and third-generation APD (aripiprazole) were used. Our results show that 50B had a lower impact on reducing the distance mice traveled compared with second-generation APD but had similar effects compared with third-generation APD, which suggested that 50B plays a comparable role in suppressing positive symptoms of schizophrenia to third-generation APD. In terms of inducing EPS, 50B therapy did not increase the time mice spent holding bars in the catalepsy assay. Catalepsy symptoms were found at a risperidone dose of 1 and 5 mg/kg, whereas 50A and 50B did not cause catalepsy symptoms 10 times the effective dose (100 mg/kg). These data suggest that TAAR1 agonists are less likely to cause EPS compared with risperidone. Further analysis of the druggability of 50B revealed strong permeability through the BBB, good microsomal metabolic stability, and no cardiotoxicity in the hERG assay.Our study demonstrated the role of TAAR1 activation in schizophrenia treatment. The TAAR1 agonist 50B significantly improved the positive symptoms of schizophrenia induced by MK801 in mice. 50B had favorable safety and efficacy. 50B may become a candidate compound for schizophrenia treatment.