O and 3-O carbamoylations. Our findings reveal a subclade of the CTase family and exemplify the potential of protein engineering for generating iterative enzymes.Iterative enzymes, which catalyze sequential reactions, have the potential to improve the atom economy and diversity of industrial enzymatic processes. Redesigning one-step enzymes to be iterative biocatalysts could further enhance these processes. Carbamoyltransferases (CTases) catalyze carbamoylation, an important modification for the bioactivity of many secondary metabolites with pharmaceutical applications. To generate an iterative CTase, we determine the X-ray structure of GdmN, a one-step CTase involved in ansamycin biosynthesis. GdmN forms a face-to-face homodimer through unusual C-terminal domains, a previously unknown functional form for CTases. Structural determination of GdmN complexed with multiple intermediates elucidates the carbamoylation process and identifies key binding residues within a spacious substrate-binding pocket. Further structural and computational analyses enable multi-site enzyme engineering, resulting in an iterative CTase with the capacity for successive 7- Carbamoyltransferases are a class of enzymes catalyzing carbamoylation of primary and secondary metabolites. Here, the authors show the molecular structure of the ansamycin-modifying carbamoyltransferase GdmN and repurpose the enzyme for the iterative carbamoylation of ansamitocins. Usually, enzymes offer distinctive advantages over chemical catalysts due to their chemo-, stereo- and/or regioselectivities, and enzyme-catalyzed reactions can be carried out under routine laboratory conditions, with high-performance productivity and convenient downstream purification of the target molecules. Moreover, compared with a series of canonical enzymes that can each only catalyze one step, iterative enzymes can provide catalytic versatility in synthetic biology, such as in the modification of therapeutic products. Therefore, repurposing of a canonical enzyme into an iterative biocatalyst is an attractive strategy for targeted generation of desired chemicals.Iterative enzymes catalyze two or more modification steps in natural product biosynthesis and have intriguing potential for the development of novel drugs and other chemicals with structural complexity and new biological activities. Known iterative enzymes include cytochrome P450 enzymes12. CTases play fundamentally important roles in cellular metabolism and also participate in the biosynthesis of many bioactive secondary metabolites with pharmaceutical potentials16. Carbamoylated bioactive secondary metabolites are often found in the biosynthetic assemblies containing nucleosides, polyketide synthases (PKSs), PKS-non-ribosomal-peptide synthetase hybrids (PKS-NRPSs), and aminocoumarins  to an amino group, hydroxyl group, or thiol group of the recipient substrates Fig. 10–12. CT17, and removal of the C7,9-cyclic carbinolamide group resulting from carbamoylation nearly abolished the antitumor biological activity of maytansinoids18. Maytansinoids are potent microtubule inhibitors and have served as the warhead molecules for antibody-drug conjugates for treating metastatic breast cancer19. Synthetic strategies for the antibody-drug conjugates developed so far include the initial preparation of maytansinol by chemical removal of the C-3 acyl group of ansamitocin under strict reaction conditions, which usually leads to low efficiency. Recently, 3-O-carbamoylation was reported to occur in ansacarbamitocins, with attachment by the CTase Asc21b from the asc cluster20. Dual carbamoylations at C-7 and C-3 of ansamitocins by engineered iterative CTases, instead of with a multi-step chemical approach, would offer a more efficient, economical, and eco-friendly process for obtaining warhead drugs. Although structure-guided protein engineering can broaden substrate capacity21, structural information on CTases, in terms of catalytic mechanisms and protein-substrate interactions, is limited.CTases are also important tailoring enzymes for generating chemical diversity and improving the pharmacological properties of targeted compounds. For example, carbamoylated albicidin showed a significantly higher inhibitory activity against bacterial gyrase than albicidin did (~8 vs 49 nM)2+, and then it transfers the carbamoyl group from carbamoyl-AMP to the thiol of the C-terminal cysteine of HypE15. CTases in class III, which modify secondary metabolites, adopt two nucleophilic half-reactions, the formation of carbamoyl-AMP derived from ATP, CP, and Mg2+ and the subsequent transfer of the carbamoyl group from carbamoyl-AMP to the secondary metabolites16. Further crystallographic investigations revealed the protein architectural differences of the different CTase classes  of geldanamycin and ansamitocins, respectively  pLysS  of Asm21, we reconstituted the carbamoylation reaction in vitro for GdmN. Similar to the reactions of Asm21, the incubation of GdmN with 20-O-methyl-19-chloroproansamitocin (1), CP, Mg2+, and ATP resulted in the specific appearance of N-demethyl-4,5-desepoxy-maytansinol (3)  Fig. . To dete(3) Fig. 27. Theseg2+ Fig. . Neverthg2+ Fig. .Fig. 2ChgdmN gene (M1-L575) in the CTase gene-disrupted mutant WJH10, did not accumulate AP-3  in each monomer built a four-stranded antiparallel β-sheet through the main chain hydrogen bonds, forming a vital geometry for the dimerization of GdmN , GdmN/ATP (2.00 Å resolution), and GdmN/carbamoyl-AMP (1.98 Å resolution) were determined. The electron density clearly revealed that 1, CP, carbamoyl-AMP, and the nucleotides were bound in different substrate-binding sites , loop 1 (G8-A30), loop 2 (D156-H162), and loop 4′ (H238-L246 from the other monomer), distant from the other side of the binding pocket consisting of helix 2 (L186-L196) and loop 3 (L197-D203), and leading to an extra space in the binding pocket when GdmN bound 1  oriented the binding pose of 1, with the C-3 hydroxy group pointing to H87 and the hydrophobic side towards the hydrophobic surface consisting of V24, F25, F26, and F201  did not affect the activity, whereas deletion of different amounts of residues in other mutants (mutants D3 and D7) greatly decreased and even abolished carbamoylation ability . In addition, the enzyme could not provide potentially sufficient interactions to bind 3 due to the large unoccupied space appearing in the vicinity of loop 1 (G8-A30) and helix 2 (L186-L196) in the active pocket of the GdmN/carbamoyl-AMP/1 complex.To generate a di-ses Fig. , severalO-carbamoylation, homology modelling of Asc21b based on GdmN was performed via the SWISS-MODEL server , albeit with relatively low catalytic efficiency  6545 MS with an electrospray ionization (ESI) source.Escherichia coli DH10B was used for all plasmids construction, and E. coli BL21(DE3)pLysS was used for protein expression. E. coli ET12567(pUZ8002) was used for conjugation. E. coli strains were cultivated in Luria-Bertani (LB) broth with corresponding antibiotics. Actinosynnema pretiosum subsp. pretiosum ATCC 31280 was obtained from the ATCC Global Bioresource Center. A. pretiosum ATCC 31280 and the mutants were cultivated in TSBY liquid medium or on ISP2 agar plates44. The TSBY liquid medium contained tryptone soya broth (30 g l−1), yeast extract (5 g l−1), sucrose (103 g l−1), pH 7.5. The ISP2 agar plate consisted of glucose (4 g l−1), malt extract (10 g l−1), yeast extract (4 g l−1), pH 7.5, and agar (15.0 g l−1). The IPS2 agar plates were used for the E. coli-Actinosynnema bi-parental conjugation.Strains and plasmids used in this study are listed in Supplementary Table A. pretiosum ATCC 31280 and the mutants were firstly cultivated in liquid TSBY medium at 30 °C, 220 r.p.m. for 24 h. 3.3% inoculum was added into liquid seed medium  and then cultivated at 30 °C, 220 r.p.m. for 24 h. Subsequently, 10% inoculum from the seed medium was added into liquid fermentation medium  at 25 °C, 220 r.p.m. for 7 days. Fermentation was conducted in baffled 250-ml flasks with 30 ml corresponding liquid medium.For fermentation of ansamitocins derivatives, A. pretiosum ATCC 31280 . The E. coli-Actinosynnema bi-parental conjugation was performed to introduce pLQ1051 into A. pretiosum ATCC 31280. After cultured for 5- day incubation at 30 °C, exconjugants grown on ISP2 plates with 20 mg ml−1 thiostrepton were cultivated in liquid TSBY medium and verified by PCR with ansa23-deletion-verify-FP/RP primers. Then, the correct single-crossover strains were inoculated into TSBY without antibiotics and incubated for 24 h at 30 °C, to eliminate the plasmid. Thiostrepton-sensitive strains were further verified by PCR with ansa23-deletion-verify-FP/RP primers to generate the mutant WJH10  by transformation and further into A. pretiosum ATCC 31280 by conjugation. After 5-day incubation at 30 °C, exconjugants grown on thiostrepton plates. Then, the exconjugants were cultivated in TSBY and verified by PCR using ansa21-deletion-verify-FP/RP primers. The correct single-crossover strains were inoculated into TSBY without antibiotics and incubated for 24 h at 30 °C. The correct double-crossover strains were selected by loss of thiostrepton resistance and PCR verification to generate the mutant WJH13  and then conjugated into WJH10. The correct recombinants were screened by apramycin resistance and PCR verification to generate the mutant WJH11  in WJH10, the truncated gdmN gene (M1-L575) was amplified with GdmNm -FP/RP primers  digested by NdeI and EcoRI, to obtain pLQ1055. After DNA sequencing, the plasmid pLQ1055 was introduced into E. coli BL21(DE3)pLysS for heterologous expression. The single colony was cultivated overnight at 37 °C in 50 ml of LB medium containing 50 μg ml−1 kanamycin and 34 μg ml−1 chloramphenicol. 50 ml overnight culture was transferred to 5 l LB medium containing 50 μg ml−1 kanamycin and 34 μg ml−1 chloramphenicol. Then, the culture was cultivated at 37 °C, 220 r.p.m. until the OD600 value reached 0.8. The culture was cooled to 4 °C and then induced with 0.4 mM IPTG. The culture was further cultivated at 16 °C and 220 r.p.m. for 18 h. The cells were harvested and resuspended in lysis buffer . The cells were centrifuged after ultrasonication, and the supernatant was purified using gravity chromatography columns with Ni Sepharose 6 Fast Flow beads (Cytiva) at 4 °C. The column was washed with washing buffer A , and the protein was eluted with elution buffer . Fractions containing GdmN were further purified by a fast protein liquid chromatography system (Bio-Rad) with a Superdex 200 Increase 10/300 GL gel filtration column (Cytiva) pre-equilibrated in buffer C . The fractions of the highest purity as evaluated by SDS-PAGE pLysS. Each mutation was verified by DNA sequencing. GdmN mutants were purified as described above for native protein purification.The primers listed in Supplementary Table PDB: 7VYO) and GdmN Y82F mutant (PDB: 7VZU), initial screenings were performed with crystallization screen kits (Hampton Research) by sitting-drop vapor-diffusion method at 20 °C. 1 μl drop consisted a 1:1 mixture of purified protein solutions (10 mg ml−1) and precipitant solutions. Needle crystals were observed in a precipitant solution  PEG3350 and 0.1 M Tris, pH 8.5) at 20 °C. After the optimization of pH and precipitant, rhomboid crystals of GdmN were obtained in a precipitant solution containing 0.2 M lithium sulfate monohydrate, 30% (w/v) PEG3350 and 0.1 M Tris, pH 9.0, and rhomboid crystals of GdmN Y82F mutant were obtained in a crystallization buffer containing 0.2 M lithium sulfate monohydrate, 27.5% (w/v) PEG3350 and 0.1 M Tris, pH 9.0.For GdmN (PDB: 7VX0), purified GdmN solution (10 mg ml−1) was incubated with 1.5 mM ATP for 1 h on ice. The 1 μl protein solution was then mixed with 1 μl precipitant solution. Rhomboid crystals were obtained in a precipitant solution containing 0.2 M lithium sulfate monohydrate, 28% (w/v) PEG3350 and 0.1 M Tris, pH 9.0.For the complex of GdmN with ATP (1 (PDB: 7VZY), purified GdmN solution (10 mg ml−1) was incubated with 1.5 mM CP, 1.5 mM AMP, and 1 mM 1 for 1 h on ice. The 1 μl protein solution was then mixed with 1 μl precipitant solution. Rhomboid crystals were obtained in a precipitant solution containing 0.2 M lithium sulfate monohydrate, 30% (w/v) PEG3350 and 0.1 M Tris, pH 9.0.For the complex of GdmN with AMP and PDB: 7VYJ), purified GdmN solution (10 mg ml−1) was incubated with 1.5 mM ATP, 1.5 mM MgCl2, and 1.5 mM CP for 1 h on ice. The 1 μl protein solution was then mixed with 1 μl precipitant solution. Rhomboid crystals were obtained in a precipitant solution containing 0.2 M lithium sulfate monohydrate, 32.5% (w/v) PEG3350 and 0.1 M Tris, pH 8.5.For the complex of GdmN with carbamoyl-AMP (PDB: 7VZQ), purified GdmN mutant solution (10 mg ml−1) was incubated with 1.5 mM ATP, 1.5 mM MgCl2, and 1.5 mM CP for 1 h on ice. The 1 μl protein solution was then mixed with 1 μl precipitant solution. Rhomboid crystals were obtained in a precipitant solution containing 0.2 M lithium sulfate monohydrate, 27.5% (w/v) PEG3350 and 0.1 M Tris, pH 9.0.For the complex of GdmN V24Y/G157A/R158A/G188R mutant with carbamoyl-AMP (1 (PDB: 7VZZ) and the complex of GdmN with natural tetrahedral intermediates, AMP, and 2 (PDB: 7VYP), purified GdmN solution (10 mg ml−1) was incubated with 1.5 mM ATP, 1.5 mM MgCl2, 1.5 mM CP and 1 mM 1 for 2 h on ice. 1 μl protein solution was then mixed with 1 μl precipitant solution. After 2 days, rhomboid crystals were obtained in a precipitant solution containing 0.2 M lithium sulfate monohydrate, 30.0% (w/v) PEG3350 and 0.1 M Tris, pH 9.0.For the complex of GdmN with natural tetrahedral intermediates, carbamoyl-AMP and 1 and carbamoyl-AMP (PDB: 7VZN), multiple crystals of GdmN with carbamoyl-AMP were soaked in a precipitant solution containing 0.2 M lithium sulfate monohydrate, 30.0% (w/v) PEG3350, 0.1 M Tris, pH 9.0, and different concentrations of 1.For the complex of GdmN with All crystals were soaked in a crystallization solution containing 20% (v/v) glycerol before being placed into liquid nitrogen.47. Structure of GdmN was determined by molecular replacement using the program Phaser 2.7.17 from Phenix suite48, whereby the structure of TobZ (PDB: 3ven) was used as a model. Iterative cycles of model rebuilding and refinement were performed using COOT49 and Phenix, to generate the final model of GdmN. Structures of other GdmN complexes were determined by molecular replacement using Phenix and the atomic coordinates of GdmN as a model. Refinement with Phenix48 along with model adjustment and ligand placement with COOT49 generated final models of GdmN complexes. MolProbity from Phenix suite was used to evaluate the quality of the structural models. The Refinement statistics of different structures are summarized in Supplementary Table All X-ray diffraction data of GdmN crystals were collected at the Beam Line 18U1 and 19U1 at the Shanghai Synchrotron Radiation Facility (SSRF). All datasets of different crystals were indexed, integrated, and scaled using the HKL2000 or HKL3000 package2, 1 mM carbamoyl phosphate, 2 mM ATP, 0.5 mM 1 or 3, 50 mM Tris, pH 8.5, 200 mM NaCl, and 40 μM proteins, was incubated at 30 °C for 12 h. Heat-inactivated GdmN (boiling at 100 °C for 15 min) was used as the negative control. The reactions were quenched with 100 μL methanol. Precipitated proteins were removed by centrifugation, and the supernatants were subjected to HPLC analysis. HPLC analyses were performed on the Agilent 1290 infinity instrument with an Agilent Eclipse Plus-C18 analytic column  equilibrated with 90% solvent A (water) and 10% solvent B (methanol). A gradient of 10–50% methanol was applied from 0 to 5 min, followed by the following gradients: 50%-60% methanol from 5 to 20 min, 60%-75% methanol from 20 to 28 min, 75%-95% methanol from 28 to 36 min, 95% methanol from 36 to 45 min, 95%-10% methanol from 45 to 46 min, and re-equilibration in 10% methanol from 46 to 55 min. The flow rate was 0.5 ml min−1, and absorbance was measured at 254 nm. The reactions were performed in triplicate (n = 3). The activity assays were analyzed using GraphPad Prism version 9.0.2.The reaction mixture (100 μl) for the biochemical assays of GdmN and mutants, consisting of 10 mM MgCl50. The alignment result was analyzed by the maximum likelihood method using the Molecular Evolutionary Genetics Analysis (MEGA 11) software51. The phylogenetic analysis used bootstrap test (1000 replicates) to check the robustness of result. The phylogenetic tree was further decorated by Interactive Tree of Life (iTOL)52.Amino acid sequences of different CTases were aligned by ClustalW multiple alignments1; (b) GdmN/carbamoyl-AMP/3; (c) GdmN V24Y/G157A/R158A/G188R mutant/carbamoyl-AMP/3. As GdmN formed a face-to-face homodimer through unusual C-terminal domains, the interactions between chain A and chain B should be taken into consideration. So, both chain A and chain B were retained in our MD simulations. The protonation states of titratable residues  were assigned on the basis of pKa values by PROPKA53 and visual inspection of local H-bonding networks. Histidine residues 27, 87, 133, 137, 238, 297, 308, and 422 were protonated at the δ position. Histidine residues 94, 134, 144, 162, 269, 286, 328, 332, 473, 495, 514, 606, and 608 were protonated at the ε position. All other Asp and Glu residues were deprotonated. The LEAP module in Amber 1854 was used to add the missing hydrogen atoms. The general AMBER force field (GAFF55) was used for the substrates, 1 and 3, while the RESP method56 was used to calculate the partial atomic charges at HF/6-31 G*. The force field for the iron-containing active site was parameterized using the MCPB57. To neutralize the total charge of the system, 46 Na+ ions were added to the surface of the protein, and the resulting protein was solvated with a 15 Å layer of TIP3P58 waters. We used the Amber ff14SB59 force field for the protein residues. For system (b) and (c), 3 were docked into the active site using Schrödinger Small-Molecule Drug Discovery Suite 2017-1 . Supplementary Fig. Three systems have been setup for the MD simulations: (a) GdmN/carbamoyl-AMP/60. All simulations were performed with the GPU version of the Amber 18 package54.After the setup, the energy of the system was minimized by 5000 steps of steepest descent and 5000 steps of conjugate gradient methods. Then the system was heated from 0 K to 300 K under a canonical ensemble for 0.5 ns. Subsequently, a 1 ns density equilibration was performed under the NPT ensemble at 300 K and 1.0 atm pressure to get a uniform density after the heating dynamics. Subsequently, we removed all restraints on the protein and further equilibrated the system for 4 ns under an NPT ensemble to obtain the well settled pressure and temperature. Finally, a productive MD run was conducted for 50 ns. During all MD simulations, the covalent bonds containing hydrogen were constrained using SHAKE61, combining Turbomole62 for the QM region and DL_POLY63 for the MM region. Residues within 12.0 Å of iron center were included into the active region. The electronic embedding64 scheme was employed to account for the polarizing effect of the enzyme environment on the QM region. Hydrogen link atoms65 with the charge-shift model66 were applied to treat the QM/MM boundary. B3LYP67 has been proved to be a successful functional for studying iron-based metalloenzymes68, and the oxidation state of +3 has been suggested for Fe cofactor in GdmN based on the study of HypF15. During QM/MM calculations, the QM region was studied by B3LYP at two levels of theory. The QM region consists of the truncated 1 and carbamoyl-AMP, and the side chains of His133, His137, Glu156, Glu333 as well as the Fe3+ ion as shown in Supplementary Fig. 69 method. The DL-FIND70 optimizer was used in the geometry optimization. The transition states were determined as the highest point on the potential energy surface along the reaction coordinates, for which a small increment of 0.02 Å was used for scanning near the transition states. All transition states (TSs) were located by relaxed potential energy surface (PES) scans followed by the full TS optimizations using the P-RFO optimizer.A representative structure of MD simulation for system (a) was chosen to do the following QM/MM calculations. All QM/MM calculations were performed using ChemShell34 were performed with the Gaussian 16 software71. The geometries of all of the TSs, reactants, and intermediates involved in the reaction were fully optimized in conjunction with the SMD continuum solvation model72 at the B3LYP/6-31 G(d) level. Harmonic frequency calculations were performed at the same level of theory as the optimizations in order to estimate the zero-point energies as well as the thermal and entropic corrections. The corrections between the stable structures and the transition states were ascertained by analyzing the corresponding imaginary frequency modes, as well as by limited intrinsic reaction coordinate (IRC) calculations. The energies were further refined at BMK/6-311 + +G73, which has been proven to be reliable for HCC model calculations76.All of the Hybrid cluster-continuum (HCC) model calculationsO-methyl-19-chloroproansamitocin (1), a 10-l scale fermentation of WJH10 was carried out. The fermentation broth was centrifuged, and the supernatant was extracted three times with ethyl acetate. Then, the crude extract was subjected to microporous resin XAD-16 (Amberlite), which was washed with 30%, 65%, and 100% ethanol. The fractions were analyzed by HPLC. Fractions containing compound 1 were concentrated and further purified by HPLC on a Thermo BDS HYPERSIL C18 column . For isolation of N-demethyl-4,5-desepoxy-maytansinol (3), a 10-l scale fermentation of WJH13 was carried out. The fermentation broth was centrifuged, and the supernatant was extracted three times with ethyl acetate. Then, the crude extract was subjected to MPLC over RP-18 silica gel  eluted with H2O, 50%, 75%, and 100% MeOH. The fractions were analyzed by HPLC. Fractions containing compound 3 were concentrated and further purified by HPLC on a Thermo BDS HYPERSIL C18 column . N-demethyl-3-carbamoyl-4,5-desepoxy-maytansinol (4) was obtained by scale-up of the conversion of 3 catalyzed by the GdmN-V24Y/G157A/R158A/G188R mutant. The large-scale reactions (20 ml) were performed in a reaction mixture containing 10 mM MgCl2, 1 mM carbamoyl phosphate, 2 mM ATP, 0.5 mM 3, 50 mM Tris, pH 8.5, 200 mM NaCl, and 40 μM proteins at 30 °C for 24 h. After incubation, the reaction mix was diluted by 1:5 in methanol. The sample was further purified by HPLC on a Thermo BDS HYPERSIL C18 column  with a flow rate of 2 ml min−1. The mobile phase consisted of 40% solvent A (water) and 60% solvent B (methanol). The resulting compounds were collected and dried for NMR analysis using MestReNova version 9.0.1 and high-resolution mass spectrometry (HRMS) analysis .For isolation of 20-1H NMR : δ 7.93 , 6.59-6.52 , 6.55 , 6.04 , 5.57 , 5.35 , 5.22 , 4.41-4.35 , 3.90 , 3.51 , 3.41 , 3.34 , 3.17 , 2.88 , 2.62–2.57 , 2.06-1.98 , 1.76 , 1.69 , 1.32-1.25 , 1.15 .13C NMR : δ 156.2 , 154.7 , 152.9 , 139.6 , 139.0 , 135.6 , 133.5 , 132.8 , 129.8 , 127.3 , 125.2 , 112.4 , 108.1 , 87.2 , 81.6 , 78.0 , 73.9 , 56.3 , 56.0 , 45.5 , 38.2 , 37.7 , 34.3 , 17.8 , 17.3 , 15.2 .4 were crystallized from a methanol solution by slow evaporation at room temperature. Data were collected on a Bruker D8 VENTURE CMOS Photon II diffractometer with helios mx multilayer monochromator Cu Kα radiation (λ = 1.54178 Å) at 173 K. Data collection, unit cell refinement and data reduction were performed using APEX3 v2019.11-0. The structure was solved by Intrinsic Phasing method using SHELXL-2018/3 program package77, and refined by full-matrix least-squares on F2 with anisotropic displacement parameters for the non-H atoms using SHELXL-2018/3 program package. The crystallographic data have been deposited at the Cambridge Crystallographic Data Centre with accession number 2122658. The detailed statistics are summarized in Supplementary Table Crystals of −1. Thyroglobulin (670.0 kDa), gamma globulin (158.0 kDa), ovalbumin (44.0 kDa), myoglobin (17.0 kDa) and vitamin B12 (1.3 kDa) from Gel Filtration Standard kits (Bio-Rad) were used for calibration. Blue Dextran was used to determine the column void volume. The calibration curve of Kav versus log(MW) was prepared using Eq.  with a Superdex 200 Increase 10/300 GL gel filtration column (Cytiva) pre-equilibrated in buffer C . The fractions of the highest purity as judged by SDS-PAGE were concentrated to 10 mg mlFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Plasma S100A1 protein is a novel inflammatory biomarker associated with acute myocardial infarction and neurodegenerative disease’s pathophysiological mechanisms. This study aimed to determine the levels of this protein in patients with acute ischemic stroke early in the disease progression and to investigate its role in the pathogenesis of acute ischemic stroke.κB p65, and IL-6 levels and cerebral infarction volume was detected by Pearson correlation analysis.A total of 192 participants from hospital stroke centers were collected for the study. Clinically pertinent data were recorded. The volume of the cerebral infarction was calculated according to the Pullicino formula. Multivariate logistic regression analysis was used to select independent influences. ROC curve was used to analyze the diagnostic value of AIS and TIA. The correlation between S100A1, NF-κB p65, and IL-6 among the AIS,TIA, and PE groups . Multivariate logistic regression analysis showed that S100A1 might be an independent predictive factor for the diagnosis of disease (P < 0.001). The AUC of S100A1 for diagnosis of AIS was 0.818 . The AUC of S100A1 for diagnosis of TIA was 0.720 . There were statistically significant differences in S100A1, NF-κB p65, and IL-6 among the SCI,MCI, and LCI groups . Plasma S100A1, NF-κB p65, and IL-6 were significantly different from cerebral infarction volume . There was a positive correlation between plasma S100A1 and IL-6 with statistical significance . There was no significant positive correlation between plasma S100A1 and NF-κB p65 (R < 0.3), but there was statistical significance . There was a positive correlation between IL-6 and NF-κB p65 with statistical significance .There were statistically significant differences in S100A1, NF-κB p65, and it was reasonable to speculate that this protein might mediate the inflammatory response through the NF-κB pathway during the pathophysiology of AIS.S100A1 might have a better diagnostic efficacy for AIS and TIA. S100A1 was associated with infarct volume in AIS, and its level reflected the severity of acute cerebral infarction to a certain extent. There was a correlation between S100A1 and IL-6 and NF- According to the latest global burden of disease data, acute ischemic stroke (AIS) was one of the main reasons for the increase in the number of years of life lost and all age disability-adjusted life years in China . The prein vivo , 20 patients with transient ischemic attack , and 31 physical examiners . AIS inclusion criteria were as follows: (1) patients with acute cerebral infarction of the first onset confirmed by cranial CT/MRI who meet the diagnostic criteria of the Chinese Guidelines for the Diagnosis and Treatment of Acute Ischemic Stroke 2018 ; (2) ageκB p65), and Interleukin-6 (IL-6). The ELISA kits for these three indicators were purchased from Beijing Green Source Boulder Biotechnology Corporation. The levels of plasma S100A1 protein, NF-κB p65, and IL-6 of the test individuals were assessed by skilled testers in accordance with the ELISA double antibody sandwich method’s usage guidelines. The fasting venous blood samples of the enrolled AIS and TIA patients were collected early on the second day of admission, and the PE group was completed in the hospital’s Health Screening Center. Other blood samples were tested by our experienced inspectors in our Clinical Research Laboratory.We gathered information about the research participants, such as their demographics, clinical information, risk factors, blood counts, biochemical indicators, and laboratory parameters. Age and sex were demographic traits. Admission NIHSS , and TOAST type (including large-artery atherosclerosis (LAA), cardioembolism (CE), small-artery occlusion (SAO), and others), admission diastolic blood pressure (BP), and admission systolic BP made up the clinical information. Risk factors included body mass index (BMI), alcohol drinking, smoking, triple-height, and heart disease. Blood counts contained blood glucose (BG), red blood cell (RBC) count, hematocrit count, leukocyte count, neutrophil count, lymphocyte count, monocyte count, red blood cell distribution width (RDW), and platelets (PLT) count. Biochemical indicators included total cholesterol (TC), triglycerides (TG), low-density lipoproteins (LDL), high-density lipoproteins (HDL), creatinine, Alanine transaminase (ALT), aspartate transaminase (AST), and homocysteine. Laboratory parameters contained plasma S100A1 calcium-binding protein, Nuclear factor kappa-B p65  group: total infarct volume 5–10 cm3; large-volume cerebral infarction (LCI) group: total infarct volume >10 cm3.The volume of cerebral infarction (according to the Pullicino formula ) = the lχ2 test were used to compare groups. The independent influencing factors connected to the illness diagnosis were chosen using multivariate logistic regression analysis. The Receiver Operating Characteristic curve (ROC) was used to evaluate the diagnostic efficacy of plasma S100A1 protein, a new inflammatory marker, for AIS and TIA. The area under the curve (AUC), the maximum Youden index (Jmax), the optimal cut-off value (cut-off), the sensitivity (Se), and the specificity (Sp) were calculated. Pearson or Spearman correlation analysis was used to test the correlation between the levels of plasma S100A1, NF-κB p65 and IL-6 and the volume of cerebral infarction and to explore the correlation among the three. A two-tailed value of P < 0.05 was considered significant.Statistical analysis was completed using Spss.26 software. The Kolmogorov–Smirnov test was used to assess the normal distribution. The mean and standard deviation (vs (130.90 ± 4.61) vs (130.45 ± 7.15), P < 0.001). Comparison between the groups suggested no significant difference between the TIA and PE groups (P > 0.05). The test indexes of blood samples include blood routine indexes, biochemical indexes, and laboratory parameters. Leukocytes and creatinine count were significantly increased in the AIS group  vs (5.45 ± 2.01) vs (5.41 ± 2.24), P = 0.015; Creatinine,  vs  vs , P = 0.009). Comparison between groups only suggested that leukocytes and creatinine count in the AIS group were significantly higher than in the PE group. At the same time, the peripheral blood monocytes, PLT, and ALT were significantly increased in the AIS group (P < 0.001). Comparison between groups also indicated that these indexes were significantly higher in the AIS group than in the other two groups  vs (0.34 ± 0.13) vs (0.33 ± 0.10), P < 0.001; PLT, (186.28 ± 59.29) vs (146.45 ± 48.99) vs (149.58 ± 42.32), P < 0.001; ALT,  vs  vs , P < 0.001). BG, RBC, hematocrit, neutrophils, lymphocytes, RDW, TC, TG, LDL, HDL, AST, and homocysteine were similar among the three groups. Laboratory parameters showed statistically significant differences in plasma S100A1, NF-κB P65, and IL-6 among the AIS group, TIA group, and PE group  vs (185.85 ± 43.24) vs (181.47 ± 27.39), P < 0.001; NF-κB p65, (3.99 ± 0.65) vs (3.58 ± 0.74) vs (3.51 ± 0.99), P = 0.001; IL-6, (13.32 ± 1.57) vs (11.61 ± 1.67) vs (11.42 ± 2.34), P < 0.001). Comparison between the groups suggested that the S100A1, NF-κB P65, and IL-6 in the AIS group were significantly higher than in the TIA group and PE group (P < 0.05). There was no significant difference between the TIA group and PE group (P > 0.05).Baseline characteristic data among the AIS, TIA, and PE groups were summarized in κB p65, and IL-6 were taken as independent variables. Multivariate logistic regression analysis showed that the values of systolic BP, ALT, and S100A1 were correlated with the diagnosis of disease, and their Chi-square values were 28.365 , 14.308 , 26.036 . The level of plasma S100A1 might be an independent predictive factor for disease diagnosis. Systolic BP and ALT might also be autogenous predictors of the diagnosis of different types of disease , when the optimum cutoff value (cut off) was 181.03, its maximum Youden index (Jmax) was 0.578, sensitivity (Se) was 95.0%, and specificity (Sp) was 62.7% , and when cut off was 150.14, its Jmax was 0.442, Se was 50.0%, and Sp was 94.2% (The diagnostic efficacy of AIS and TIA was assessed using the receiver operating characteristic curve (ROC) . The areas 62.7% . The AUCas 62.7%   vs , P < 0.001). Comparison between the groups suggested no significant difference between the SCI and MCI groups (P > 0.05). ALT and AST count were significantly increased in the LCI group  vs  vs , P = 0.002; AST,  vs  vs , P = 0.007). Comparison between groups only suggested that ALT and AST counts in the LCI group were significantly higher than in the SCI group. Systolic BP, diastolic BP, TOAST type, TC, TG, LDL, HDL, creatinine, and homocysteine were similar among the three groups. Laboratory parameters showed statistically significant differences in plasma S100A1, NF-κB P65, and IL-6 among the three groups. Comparison between the groups suggested that the levels of S100A1, NF-κB P65, and IL-6 in the LCI group were significantly higher than those in the MCI and SCI group  vs (225.42 ± 30.92) vs (223.98 ± 40.21), P = 0.001; NF-κB p65, (4.41 ± 0.45) vs (3.85 ± 0.64) vs (3.88 ± 0.66), P < 0.001; IL-6, (14.00 ± 1.40) vs (12.77 ± 1.31) vs (13.27 ± 1.65), P = 0.007) (P > 0.05).Baseline characteristic data for the different cerebral infarction volume groups were summarized in = 0.007) . There wκB P65, and IL-6 significantly differed from cerebral infarction volume. There was no significant positive correlation between plasma S100A1 and cerebral infarction volume (R < 0.3), but there was statistical significance   (R < 0.3), but there was statistical significance   . There w< 0.001) . There w= 0.036) .κB P65 and IL-6. There was no significant positive correlation between plasma S100A1 and NF-κB p65 (R < 0.3), but there was statistical significance     . There w< 0.001) . There w< 0.001) .κB p65 correlated with each other. It was reasonably speculated that this protein might mediate inflammatory responses through the NF-κB pathway in the pathophysiological process of AIS.In this study, we first assessed the diagnostic efficacy of plasma S100A1 protein for AIS and TIA diseases. The results showed that the diagnosis of AIS might be more favorable when S100A1 ≥181.03 pg/ml, while the diagnosis of TIA might be more advantageous when 150.14 pg/ml ≤S100A1 ≤181.03 pg/ml. At the same time, the correlation between plasma S100A1 protein and cerebral infarct volume was proposed in this study, and the results showed that its level could reflect the severity of AIS to some extent. Furthermore, this study also found that plasma S100A1 protein levels and IL-6 and NF-etc. Previous studies have shown that hypoxic cardiomyocytes released endogenous S100A1, which stimulated NF- κB p65 by binding to TLR4, thereby inducing an increase in pro-inflammatory cytokines; either endogenous or exogenous S100A1 significantly enhanced NF-κB phosphorylation in cardiomyocytes, thereby activating the expression of multiple pro-inflammatory cytokines involved in the inflammatory response   . This waκB p65, and IL-6 (P < 0.05). A two-by-two comparison revealed that the levels of the three in the AIS group were significantly higher than in the TIA and PE groups (P < 0.05). At the same time, no statistically significant differences were discovered between the TIA and PE groups. The results were more similar to the findings of In this study, clinical data were first compared among the AIS, TIA, and PE groups, and statistically significant differences were found in plasma S100A1, NF-P < 0.05). At the same time, no statistically significant difference was found between the MCI and SCI groups (P > 0.05). The levels of NF-κB p65 and IL-6 showed similar consequences among the three groups. Plasma S100A1 protein, NF-B p65, and IL-6 were well-established markers for the overall inflammatory status of the patient’s body, according to several earlier research , but the correlation coefficient was low. This might be due to the fact that plasma S100A1 protein was not released from brain tissue alone. Still, other systemic and local inflammatory responses, including the effects of certain underlying diseases, might also lead to changes in plasma S100A1 levels. Even nevertheless, plasma S100A1 levels in the LCI group were significantly higher than in the MCI and SCI groups with statistically significant differences, suggesting that S100A1 levels might in part reflect the severity of patients at the time of admission during the acute exacerbation. Meanwhile, the present study also compared the relationship between NF-κB p65, IL-6, and cerebral infarct volume, and the results also showed statistically significant but relatively low correlation coefficients. Previous studies have shown a moderate to a high positive correlation between these two factors and cerebral infarct volume (κB p65, and IL-6 were not statistically significantly different between the MCI and SCI groups, and it was conjectured that these inflammatory markers might need to reach a specific volume of cerebral infarction before their differences could be revealed, but the specific boundaries needed to be further investigated.In exploring the differences in plasma S100A1 levels in acute ischemic strokes of different severity, it was found that there was variability in S100A1 levels in patients with different volumes of cerebral infarction. The S100A1 levels in the LCI group were significantly higher than those in the MCI and SCI groups (research . Thus, pt volume . The limκB p65 and IL-6 was also compared. It turned out that there was some correlation between these three. All three were statistically significantly different from each other, with correlation coefficients preserved at around 0.3, showing a low correlation. This might be due to the complex pathophysiological mechanisms of cerebral infarction and the small number of studied samples. As previously indicated, S100A1 might regulate the inflammatory response of H9C2 cells through the TLR4 /NF-κB pathway. Necrosis of hypoxic cardiac tissue released endogenous S100A1 to activate TLR4, which in turn induced IL-6 production by binding to TLR4 and thus stimulating NF-κB p65 (κB pathway, thereby increasing the expression of inflammatory cytokines (κB-mediated expression of S100A protein levels through a negative feedback mechanism in PT (κB pathway. In summary, it could be hypothesized that the elevated levels of S100A1 in patients with acute cerebral infarction might enhance the expression of the inflammatory factor IL-6 and thus mediated the inflammatory response through the NF-κB pathway, which was also consistent with the conclusions obtained in this paper.Additionally, in this study, the correlation between plasma S100A1, NF-F-κB p65 . This saytokines . Along tsm in PT . In otheκB pathway in patients with acute cerebral infarction remained further confirmed by animal experiments. Finally, the possibility that other unknown confounders might influence the results could not be completely ruled out.There were several limitations to this report. First of all, an appropriate sample size and single-center study were important methodological defects, which required an expanded sample size and multi-center prospective study in the later period. Second, the time point at which plasma S100A1 protein levels were determined was single, which could not indicate the dynamic changes in this protein’s levels. Therefore, this protein’s dynamic monitoring and long-term follow-up should be strengthened in the future. Third, further grouping tests have to be conducted since it was possible that the experimental results would be impacted by including patients with anterior and posterior circulation cerebral infarction in the research sample. Fourth, whether plasma S100A1 protein actually enhanced the expression of the inflammatory factor IL-6 through the NF-κB p65, and it was reasonable to speculate that this protein might mediate the inflammatory response through the NF-κB pathway during the pathophysiology of acute cerebral infarction. Based on our findings and previous studies, plasma S100A1 protein has the potential to serve as an inclusion criterion for future clinical diagnostic trials in AIS, and lowering the level of this protein may be a novel therapeutic target to improve their severity.In summary, our study indicated that the plasma S100A1 protein was significantly correlated with the diagnostic value of AIS and the volume of cerebral infarction. This protein might have a better diagnostic effect for AIS and TIA. The diagnosis of AIS might be more favorable when S100A1 ≥181.03 pg/ml, while the diagnosis of TIA might be more advantageous when 150.14 pg/ml ≤S100A1 ≤181.03 pg/ml. Furthermore, plasma S100A1 protein was associated with infarct volume in AIS, and its level reflected the severity of acute cerebral infarction to a certain extent. Increased levels of this protein might mean more severe damage in AIS patients with larger brain infarct volumes, while patients with lower levels could benefit more. Concurrently, there was a correlation between plasma S100A1 protein levels and IL-6 and NF-10.7717/peerj.14440/supp-1Supplemental Information 1Click here for additional data file.


Kinesin family member 2A (KIF2A) induces gastric cancer (GC) growth and invasion, while its clinical relevance in GC patients is not reported. This study aimed to investigate the linkage of KIF2A with clinicopathological features, prognosis, and chemosensitivity of GC.A total of 160 surgical GC patients were reviewed, with their tumor and adjacent tissues acquired for immunohistochemical (IHC) assay to measure KIF2A expression, then scored by a semi‐quantitative method (IHC score: 0–12). KIF2A siRNA or nonsense‐siRNA were transfected into HGC‐27 and NCI‐N87 cells underwent various concentrations of capecitabine or oxaliplatin treatment followed by chemosensitivity assessment.p < 0.001). Besides, tumor KIF2A expression was related to larger tumor size (p = 0.014), higher N stage (p = 0.004) and TNM stage (p = 0.011); however, it was not linked with other clinicopathological features . Signally, tumor KIF2A high expression predicted poor overall survival (p = 0.037). After adjustment via multivariate Cox's regression, tumor KIF2A high expression independently linked with worse disease‐free survival (p = 0.033). Finally, KIF2A knockdown improved the oxaliplatin chemosensitivity vastly but only slightly affected capecitabine chemosensitivity in HGC‐27 and NCI‐N87 cells.Kinesin family member 2A expression was elevated in the tumor tissue compared to the adjacent tissue  independently associated with shorter DFS. Furthermore, KIF2A knockdown improved the chemosensitivity to oxaliplatin vastly and only increased that to capecitabine mildly in HGC‐27 and NCI‐N87 cells. A total of 160 GC patients were screened out and eligible for study analysis, based on the following criteria: (1) had a clinicopathological diagnosis of primary GC; (2) underwent surgical resection; (3) had complete clinicopathological data and survival data; (4) had accessible formalin‐fixed and paraffin‐embedded (FFPE) specimens of tumor and adjacent tissues; (5) had no history of other cancers or malignant diseases at diagnosis. The exclusion criteria were: (1) age less than 18 years old; (2) patients’ specimens were not available for immunohistochemistry assay; (3) lost to follow‐up within 6 months after surgery. Approval was acquired from the Institutional Review Board of our hospital with the written informed consent omitted.2.2H. pylori infection status, tumor location, tumor differentiation, tumor size, T stage, N stage as well as Tumor, Node and Metastasis (TNM) stage; (2) adjuvant treatment: adjuvant chemotherapy and radiotherapy  regimen, while some patients stopped the regimen due to intolerable toxicity; meanwhile, some patients with TNM stage III also received radiotherapy concurrently); (3) survival data: date of surgery, date of disease relapse, progression or death, and the date of last follow‐up  and overall survival (OS)). At the time of study analysis, the survival data were updated to March 2021. The median follow‐up duration was 46.5 months  with a range from 12.5 to 71.4 months.By reviewing the medical records, the following data were extracted: (1) clinical characteristics: age, gender, smoke and drink status, comorbidities, 2.3The FFPE specimens were processed by immunohistochemical staining for semiquantitative analysis of KIF2A expression. The IHC staining was implemented according to the standard procedures as described in a previous study.2.4Two human GC cell lines HGC‐27 and NCI‐N87 (Cell Blank of Chinese Academy of Sciences) were used for in vitro experiment. The RPMI1640 medium (Gibco) and 10% fetal bovine serum (Gibco) were applied for cell culture, which was performed in the following conditions: air 95%, carbon dioxide 5%, and 37°C.2.5The siRNAs for KIF2A and nonsense‐siRNA were purchased from the RIBOBIO Biotechnology Co., Ltd, which were respectively transfected into cells by Lipofectamine™ 2,000 Transfection Reagent (Invitrogen). According to the transfection, the cells were termed as follows: (1) KD‐KIF2A (knockdown KIF2A) group: the cells were transfected with siRNAs for KIF2A; (2) KD‐NC (negative control) group: the cells were transfected with nonsense‐siRNA; (3) blank group: the cells were not transfected. At 24 h after completion of transfection, all cells  were treated with capecitabine (Sigma‐Aldrich) and oxaliplatin (Sigma‐Aldrich), respectively. The concentrations of chemotherapeutic agents were set partly according to a previous study2.6t test and McNemar's test were applied for the paired sample comparison. The student's t test was used for independent sample comparison. Spearman rank correlation test was used for association analysis. Kaplan–Meier method and log‐rank test were used for DFS and OS analysis. Univariate and multivariate Cox's proportional hazards regression analyses were used to estimate the factors related to DFS and OS. Statistical analysis was completed using SPSS 22.0 (IBM Corp.), and graphs were constructed using GraphPad Prism 7.01 (GraphPad Software Inc.). p value < 0.05 indicated statistical significance.Paired 33.1H. pylori infection were 46 (28.7%), 61 (38.1%), 37 (23.1%), 45 (28.1%), 24 (15.0%), and 56 (35.0%), respectively. Regarding tumor location, 45 (28.1%), 19 (11.9%), and 96 (60.0%) patients’ gastric tumor located at the cardia, the gastric body and the gastric antrum, respectively. Besides, 20 (12.5%), 116 (72.5%), and 24 (15.0%) patients had well, moderate, and poor tumor differentiation, respectively. Additionally, 40 (25.0%), 68 (42.5%), and 52 (32.5%) patients presented with TNM stage I, II, and III, respectively. Moreover, there were 115 (71.9%) patients received adjuvant chemotherapy and 11 (6.9%) patients received adjuvant radiotherapy.A total of 160 GC patients were admitted into this study, of which detailed clinical characteristics were shown in Table 3.2p < 0.001)   Figure ; KIF2A h) Figure .3.3p = 0.014), more advanced N stage (p = 0.004) and TNM stage (p = 0.011). However, no association of KIF2A IHC score with tumor location, tumor differentiation or T stage was found  . However, no correlation was found in tumor KIF2A expression with accumulating DFS (p = 0.065) : 1.943 (1.057–3.573)), gender  : 3.418 (1.566–7.459)), tumor location of cardia (vs. location of antrum) : 2.253 (1.023–4.961)), poor tumor differentiation : 2.703 (1.490–4.904)), higher TNM stage : 3.712 (2.009–6.859)) were all independently associated with unsatisfying DFS, while adjuvant chemotherapy (yes vs. no) : 0.078 (0.022–0.270)) was independently associated with longer DFS  : 2.576 (1.029–6.447)), higher TNM stage : 7.159 (3.039–16.862)) were independently associated with shorter OS, while adjuvant chemotherapy (yes vs. no) : 0.051 (0.012–0.215)) was independently correlated with better OS      Figure . Additio) Figure  as well ) Figure .4According to previous studies, KIF2A is highly expressed in the tissues of several kinds of tumorsRegarding the correlation of KIF2A with prognosis, previous studies have been performed to investigate its association with prognosis in various tumors.Although a lot of findings were identified in this study, there were still some limitations. Firstly, the sample size could be expanded to improve the statistical power. Secondly, this study did not investigate the molecular mechanism of KIF2A involved in GC progression. Thus in vivo and in vitro experiments could be further conducted. Thirdly, some confounding factors might affect findings, such as treatment of adjuvant chemotherapy or neoadjuvant chemotherapy in these patients. Thus, we adjusted the findings in multivariate Cox's regression model to eradicate the effect of these confounding factors on the prognostication effect of KIF2A in GC patients. Fourthly, the effect of KIF2A on other commonly used chemotherapeutic agents for GC such as S1 and paclitaxel, as well as the in‐depth molecular mechanism, could be further investigated. Fifthly, since most of the patients were Chinese, it was unclear whether the findings of this study were valid in patients from other regions.In conclusion, KIF2A is highly expressed and its elevated expression associates with larger tumor size, advanced N stage, TNM stage as well as poor survival in GC patients; meanwhile, its knockdown enhances the chemosensitivity to oxaliplatin and capecitabine in GC cell lines. KIF2A might serve as a potential prognostic biomarker for improving the management of GC patients.The authors declare no competing interests.


Specifically, it had high brain penetration and specific binding that was demonstrated in both rodents and non-human primates. Here, we present the first automatic cGMP-level production of [18F]P10A-1910 and translational PET/MRI study in living human brains. Successful one-step radiolabeling of [18F]P10A-1910 on a GE TRACERlab FX2N synthesis module was realized via two different methods. First, formulated [18F]P10A-1910 was derived from heating spirocyclic iodonium ylide in a tetra-n-butyl ammonium methanesulfonate solution. At the end of synthesis, it was obtained in non-decay corrected radiochemical yields (n.d.c. RCYs) of 12.4 ± 1.3%, with molar activities (MAs) of 90.3 ± 12.6 μmol (n = 7) (Method I). The boronic pinacol ester combined with copper and oxygen also delivered the radioligand with 16.8 ± 1.0% n. d.c. RCYs and 77.3 ± 20.7 GBq/μmol (n = 7) MAs after formulation (Method II). The radiochemical purity, radionuclidic purity, solvent residue, sterility, endotoxin content and other parameters were all validated for human use. Consistent with the distribution of PDE10A in the brain, escalating uptake of [18F]P10A-1910 was observed in the order of cerebellum (reference region), substantial nigra, caudate and putamen. The non-displaceable binding potential (BPND) was estimated by simplified reference-tissue model (SRTM); linear regressions demonstrated that BPND was well correlated with the most widely used semiquantitative parameter SUV. The strongest correlation was observed with SUV(50–60 min) . Collectively, these results indicated that a static scan protocol could be easily performed for PET imaging of PDE10A. Most importantly, that [18F]P10A-1910 is a promising radioligand to clinically quantify PDE10A.Our previous work showed that [ Phosphodiesterases (PDEs) are widely present in various cells and catalyze the hydrolysis and inactivation of cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP). Moreover, they synergize with both ATP and GTP to regulate the content of these second messengers . Therefoin vivo information for exploring a host of physiological and pathological mechanisms. As a result, PET is useful in assessing the progression of related diseases, as well as help accelerate the development of targeted drugs  ranging from 3.0 to 5.0 in healthy people aged 29–47 years  can provide valuable, ed drugs . Given ted drugs . In part47 years . Clinica with HD . Howeverfication . To compd [18F]2  was moreimproved . Neverthimproved .18F]P10A-1910  2--4′,6′-dioxospiro 3 derived from these two methods was then performed to validate it for human use. Finally, a PET imaging study on healthy volunteers demonstrated that PDE10A expression in substantial nigra, caudate and putamen was sensitively quantified by [18F]3 via the static scan protocol. Taken together, this work will pave the way for routine radioligand production and widespread application, as well as facile clinical translation for further diagnosis of PDE10A-related neurological disorders.Our previous work revealed the successful radiosynthesis of -55 and BPE 6 were prepared from the common intermediate aryl iodide 4, which was obtained efficiently in gram scale according to our previously reported synthetic procedures (4 into the corresponding iodo (III) intermediate in a solution of TFA/CHCl3. After evaporation of the acid solvent, the crude mixture was directly dissolved in ethanol, then coupled with SPIAd under basic conditions to deliver the desired SCIDY 5 as a colorless powder in 69% yield. Additionally, BPE 6, a baby pink solid, was synthesized via Pd-catalyzed cross-coupling with bis(pinacolato)diboron at a comparable yield of 62%. When maintained at −78°C, both precursors remained stable radiolabeling efficiency for at least 6 months.As shown in ocedures . Particu5 according to our previously reported method  from 50% to 100%, n.d.c. RCYs were improved to 16.5%, while increasing the solvent volume decreased RCYs dramatically , 0.5 mg TEAB and 0.3 ml DMA at 120°C for 20 min would allow for the best automatic labeling of [18F]3 with the assistance of three-times air bubbling.Copper-induced nucleophilic erneur’s  and Scoterneur’s , is anoterneur’s , which werneur’s . Becauseerneur’s . Heatingerneur’s . By elevatically . Finally5 to 1:1 . Togethe18F]3 from either SCIDY 5 (Method I) or BPE 6 (Method II). The semipreparative HPLC purification chromatography of these two methods is shown in ®-GV filter and collected in a 25 ml sterile vial. The n.d.c. RCYs obtained by these two different labeling methods were 12.4 ± 1.3% and 16.8 ± 1.0%, respectively, both of which met our requirements for further clinical application.In summary, after conducting more than 100 tests in the FX2N module, we realized stable and highly efficient automatic radiolabeling of [18F]3, three of them prepared from Method I and all others obtained via Method II. The acceptance criteria and detection results are depicted in 18F]3 was determined to be 5.3 ± 0.2 min as measured by an analytical HPLC system equipped with a WondaSil C18-WR column , and eluted with CH3CN/H2O  with a flow rate of 1.0 ml/min. The tandem UV (λ = 254 nm) and Gamma detectors showed that radiochemical purities were greater than 95%, and the radioligand structure was confirmed by co-injection using standard reference (Method I) or 56.6–98.0 GBq/μmol (Method II) as calculated by a standardized curve derived from the reference compounds.According to the USP General Chapter <823> and ChP General Chapter <2321>, we conducted a comprehensive and meticulous validation and quality control for the six consecutive batches of 3. Demographic data and radioligand information for each study are summarized in 18F]3 was produced on the day of the PET/CT scan using the GE TRACERlab FX2N module in a well-shielded hot cell. The reconstructed data were shown with averaged standardized uptake values (SUV0–60min) and revealed an obvious, heterogenous radioactivity distribution pattern in the human brain. The highest accumulation was found in the caudate nucleus and putamen, followed by the substantia nigra. The lowest accumulation of tracer uptake was detected in the cerebellum, implying that this region had low PDE10A expression  aged 24–37 years  was estimated by simplified reference-tissue model (SRTM). As shown in BPND were observed in the putamen, followed by the caudate nucleus . Thus, the distribution of [18F]3 in the human brain closely matched the binding patterns that had been previously observed in both rodent and nonhuman primate (NHP) brain  brain . As expe studies . To the D and HD .10–20min, SUV20–30min, SUV30–40min, SUV40–50min, and SUV50–60min with specific binding . These results indicated that the widely used SUV PET signal derived from a 10-min static scan after 50 min post-injection can be used as a routine protocol for in vivo measurement of PDE10A density using [18F]3.In terms of clinical PET neuroimaging applications, either a dynamic or long time period scan would be challenging for patients due to physical or emotional causes and inescapable head movements. To verify a static scan with a reasonable duration, we then examined whether 10-min averaged SUV was practical for quantifying a target imaging signal. Comparisons between SUV binding  revealedAll commercial reagents were purchased from commercially available vendors and used as received. Unless otherwise noted, solvents were freshly dried and degassed according to the purification handbook Purification of Laboratory Chemicals before use. Analytical thin-layer chromatography (TLC) was performed on pre-coated glass-backed plates (EMD TLC Silica gel 60 F254) and visualized using either a UV lamp (254 nm) or potassium permanganate. Silica gel for flash chromatography was 300–400 mesh. NMR spectra were recorded on Bruker 400/500 MHz on Bruker spectrometers, and resonances chemical shifts (δ) were given in parts per million (ppm) relative residual solvent. Peak multiplicities are abbreviated by the following symbols: s, singlet; d, doublet; t, triplet; hept, heptahedron; m, multiplet; dd, doublet of doublets. HRMS spectra were measured on a Thermo Scientific LTQ Qrbitrap XL using ESI+.18F]Fluoride was produced by the GE Qilin MINITrace cyclotron via the 18O18F reaction. The automatic radiolabeling was conducted in an GE TRACERlab FX2N module. Radiochemical purity and molar activity of the radioligand were determined by the HPLC  with an analytical column . Radionuclide purity was tested on an energy disperse spectroscopy . The radioactivity of the product at the end of the synthesis was measured using a dose calibrator (Capintec CRC-55tR). The solvent and copper residue in the formulated product were tested by GC-FID (Agilent 6820) and ICP-MS (Agilent 7700x), respectively.dioxane-2,7'- methanoinden]-5-ylidene)-l3-iodaneyl)phenyl)-7-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)ethyl)-4-isopropoxyisoindoline-1,3-dione . According to our previous report (5 as obtained was a colorless powder (69% from aryl iodide 4). 1H NMR  δ: 7.95 , 7.70 , 7.61 , 7.43 , 7.33 , 4.79 , 3.88 , 2.63 , 2.43 , 2.35 , 1.94 , 1.77 , 1.72–1.57 , 1.28 , 1.27 .s report , SCIDY 54-isopropoxy-2-phenyl)-3,4-dihydroquinazolin-2-yl)ethyl)isoindoline-1,3-dione . To the solution of compound 4  in 5 ml of DMSO was added KOAc , bis(pinacolato)diboron , Pd (dppf)2Cl2 , and the reaction mixture was then stirred under N2 atmosphere at 80°C for 16 h. After cooling, the reaction mixture was diluted with EtOAc (50 ml) and washed with water (30 ml) and brine (30 ml). The organic layer was dried over Na2SO4 and concentrated to provide the crude residue which was purified by column chromatography (Hexane: EtOAc = 4:1) to deliver the desired product 6 as a baby pink solid in 62% yield. 1H NMR  δ 8.11 , 7.95 , 7.58–7.52 , 7.35–7.27 , 7.25–7.21 , 7.13 , 4.67 , 4.05 , 2.67 , 2.44 , 1.36 , 1.33   δ 169.4, 168.1, 163.6, 157.0, 154.7, 148.8, 146.7, 141.1, 137.9, 137.1, 136.1, 129.7, 129.2, 128.4, 128.3, 122.3, 120.0, 116.9, 85.6, 76.5, 74.2, 36.3, 35.1, 26.4, 23.4 (34H37BN3O6 (+) 594.2775, found 594.2768  ; 13C NMR.4, 23.4 ; HRMS  azeotropic drying of [18F]fluoride in REACTOR 2, 3) radiofluorination of SCIDY 5 (Method I) or BPE 6 (Method II) in REACTOR 2, and 4) HPLC purification followed by solid-phase formulation of the final product. Radiofluorination was performed using the sequential operations indicated in the schematic diagram (A schematic diagram of the GE TRACERlab FX2N radiosynthesis module used for the production of P10A-1910  was injected into the body from the cubital vein within 20 s, followed by dynamic brain PET collection in a 3D list mode for 60 min. Reconstruction of PET raw data was 32 frames . A specialist monitored the volunteers’ electrocardiograms (ECG) and blood pressure throughout the entire scan. After the scan, all HVs were advised to stay away from people, especially children and pregnant women, and to drink plenty of water to facilitate radioactivity excretion.HVs were placed in a supine position on a GE Discovery 690 PET/CT Elite scanner 3 (designated [18F]P10A-1910). Using the GE TRACERlab FX2N as our operation module, either the spirocyclic iodonium ylide tailing with an adamantyl auxiliary  or the boronic pinacol ester (6) coupled with copper salt and oxygen (Method II) delivered the product with comparable radiolabeling yields and molar activities. Moreover, the quality control of both was fully validated for human use. A subsequent imaging study in the living human brain revealed that the radioactivity accumulated in the caudate, putamen, as well as the substantia nigra. BPND was strongly correlated with the PET signal indicated by SUV50−60min. This easy, automatic radiolabeling, good in vivo stability and binding potential, as well as practical protocols for use in imaging scan in clinics would facilitate widespread use of [18F]3. Critically, its use would contribute to future exploration of PDE10A-related CNS pathologies in large-scale, multicenter trials.We have realized the first automatic radiolabeling of a novel PDE10A PET tracer [


Developing effective therapies and medicines to conquer nonalcoholic steatohepatitis (NASH) is of great significance for public health and is faced with a major challenge. The activation of the thyroid hormone receptor agonist THRβ could be regulated by target drugs that has brought huge potential to the treatment of NASH. In this work, pyridazinone compound YWS01125 was synthesized for the first time. In this study, an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method for YWS01125 determination was established, and the pharmacokinetics of YWS01125 was evaluated. The half-life values (t1/2)of three different doses of YWS01125 was 189.12 ± 95.27, 152.64 ± 37.98, and 181.95 ± 64.25 min, respectively, and the tissue distribution studies demonstrated that YWS01125 was quickly distributed to various tissues. With successful application in the pharmacokinetics study of YWS01125, the UPLC-MS/MS method has shown characteristics of high sensitivity, rapidity, and good selectivity. Nonalcoholic fatty liver disease (NAFLD) is characterized by heterotopic liver fat and steatosis . NAFLD iThe pathogenesis of NASH is very complicated. The “two-hit” theory is a widely accepted mechanism for NASH  in whichTreatments for NASH can be divided into two main categories: drug therapy and nondrug therapy. Nondrug treatment is used to make appropriate adjustments in diet, physical exercise, and psychological stress relief, while drug therapy may include antioxidant therapy, lipid-lowering drugs, and weight-loss drugs; however, no highly effective drugs are available in the treatment of NASH in the present.MGL-3196  includes two subtypes: THRα and THRβ. THRα is highly expressed in the skeletal muscle and heart, mainly regulating the heart rate, while THRβ is highly expressed in the liver, kidneys, and pituitary gland, mainly regulating the release of thyroid stimulating hormone . THRβ seMGL-3196 , a selecMGL-3196 .YWS01125 possesses the best selectivity and activity for THRβ with low cytotoxicity and was selected as the lead compound. In the pharmacodynamic study, it showed good in vivo efficacy in rat, such as hepatoprotective effect in an acute liver injury model, lipid-lowering function on acute hyperlipidemia mice, and alleviation of NASH. Therefore, we further conducted pharmacodynamics and pharmacokinetics research of YWS01125  was supplied by the Institute of Materia Medica, Shandong Academy of Medical Sciences ; MGL-3196  was purchased from Selleck Corporation . The structural equations of the two compounds are shown in Pyridazinones: The synthetic route of pyrazinone compound YWS01125 is as depicted in Synthesis of ethyl (2-cyano-3-methoxyacryloyl) carbamate (1).1 as a white solid . ESI m/z: C8H11N2O4 (M + H)+m/z = 199.1; molecular weight, 198.1780.Ethyl (2-cyanoacetyl) carbamate , trimethyl orthoformate , acetic anhydride (20 ml), and acetonitrile (40 ml) were added to a 100-ml flask. The solution was stirred at 80°C for 4 h, and then the reaction stopped. The solvent was evaporated. Diethyl ether (50 ml) was added to the mixture, and the resulting suspension was kept at 2–8°C overnight. The suspension was filtered and rinsed several times with ether (3 × 50 ml) to give target compound Synthesis of 3,5-dichloro-4-((6-chloro-5-cyclobutylpyridazin-3-yl) oxy) aniline (2).N, N-dimethylformamide (20 ml) was stirred at 120°C for 3 h. The reaction was stopped. An appropriate amount of diatomite was added into the sand core funnel, and the reaction solution was pumped and filtered to collect the filtrate. The filtrate was washed with 10% sodium chloride aqueous solution and saturated sodium chloride and was dried with anhydrous sodium sulfate. The solvent was evaporated to give a crude product which was purified by column chromatography (petroleum ether/ethyl acetate = 4/1) to obtain compound 2 as a light-brown solid . ESI m/z: C14H13Cl3N3O (M + H)+344.0; molecular weight, 344.6200.The solution of 6-dichloro-4-cyclobutylpyridine , 2, 6-dichloro-4-aminophenol , and potassium carbonate  in Synthesis of 6--4-cyclobutylpyridazin-3(2H)-one (3).2 , sodium acetate , and glacial acetic acid (20 ml) was stirred at 120°C for 24 h. The reaction was stopped and the mixture cooled to room temperature. Then, 10 ml of water was added. The pH was adjusted to 9–10 with 1N HCl. The aqueous phase was extracted with ethyl acetate (25 ml × 3). The organic phase was combined, washed with brine, and dried over anhydrous sodium sulfate. The solvent was evaporated to give a crude product which was used directly in the next step without further purification.In a 100-ml round-bottom flask, the solution of compound 3 as a gray solid . ESI m/z: C14H14Cl2N3O2 (M + H)+326.0; molecular weight, 326.1770.The above obtained solid was dissolved in methanol (24 ml), and NaOH solution  was added. The resulting mixture was stirred at 100°C for 24 h. The reaction was stopped. The solvent was evaporated. The suspension was extracted with ethyl acetate (25 ml × 3), and then the organic phase was combined, washed with brine, and dried over anhydrous sodium sulfate. The solution was concentrated under pressure and purified by column chromatography (petroleum ether/ethyl acetate = 2/1) to give compound Synthesis of ethyl  oxy) phenyl) amino) acryloyl) carbamate (4).1 , 3 , and chloroform (5 ml). The reaction mixture was stirred at reflux for 1 h and the reaction stopped. The mixture was then cooled to room temperature. The suspension was filtered and rinsed with ether. The filtrate cake was dried at 50°C. Compound 4 was obtained as a white solid . ESI m/z: C21H20Cl2N2O5 (M + H)+492.1; molecular weight, 492.2130.To a 10-ml reaction tube were added 1-oxy)phenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carbonitrile (YWS01125).4  was added into a 10-ml microwave reaction tube, followed by the addition of potassium acetate  and N, N-dimethylacetamide (2 ml). The mixture reacted at 140°C for 30 min, then stopped. The reaction solution was diluted with ethyl acetate (20 ml), transferred into a 150-ml separating funnel, and diluted with water (10 ml). The organic phase was separated, washed with brine, and dried over anhydrous sodium sulfate. The solvent was evaporated to give a crude mixture which was purified by column chromatography (methylene chloride/methanol = 30/1 to 10/1) to give the target compound YWS01125 as a light-yellow solid . 1H NMR  δ 12.31 , 12.22 , 8.90 , 7.88 , 7.50 , 3.59–3.53 , 2.30–2.24 , 2.19–2.10 , 2.07–1.97 , 1.85–1.80  ppm; 13C NMR  δ 160.9, 160.0, 154.7, 151.5, 151.4, 149.5, 145.9, 136.4, 128.9 (2C), 128.7 (2C), 120.0, 114.4, 89.2, 35.5, 27.3 (2C), 18.3 ppm; ESI-HRMS m/z calculated for C19H14Cl2N5O4 (M + H)+446.0423; molecular weight, 446.0420 . The permit number was SCXK (Lu) 20190003. The mice were allowed free laboratory sterile feed and sterile water and were housed in controlled environment conditions since at least 1 week before the experiment. The adjustable temperature was set at 20 ± 5°C and the moisture was 55 ± 15%, with a 12-h dark–light cycle. The tested mice were carried out according to the National Institute of Health Guideline for the Care and Use of Laboratory Animals, and performed by the Animal Ethics Committee of Institute of Materia Medica, Shandong Academy of Medical Sciences . Before the pharmacokinetic study, all experimental mice had fasted for 12 h with free drinking water.YWS01125 was prepared by accurately weighing a lot of the medicine, then dissolving it in methanol, yielding a final concentration of 1 mg/ml, and storing at 4°C. It was gradually diluted before use into a series of working solutions at concentrations of 4–400 ng/ml with acetonitrile. MGL-3196 powder was prepared as a 1 mg/mL internal standard stock solution and stored in refrigerator at 4°C. Immediately before use, use chromatographic acetonitrile to dilute step by step to prepare an internal standard solution with a concentration of 2 ng/ml MGL-3196, and placed in 4°C standby. These standards of the quality control (QC) samples were prepared by diluting the stock solution of YWS01125 to the blank plasma at low, medium, and high concentrations of 10, 50, and 300 ng/ml, respectively, and by depositing them at 4°C until analysis. All solutions were kept at 4°C before use.The stock solution of Hypersil GOLD C18 column . A 0.025% ammonia aqueous solution in water (solvent A) and acetonitrile (solvent B) were used as mobile phases with gradient elution. The gradient was as follows: 0.01 min, 15% B; 5 min, 20% B; 12 min, 80% B; 16 min, 80% B; 16.01 min, 15% B; 19 min, 15% B; and 19.01 min, stopped. The timing interval was 19 min, and the flow rate was set at 1 ml/min.® mass spectrometer  coupled to a Shimadzu Prominence UPLC  system. It was equipped with a binary pump, a degasser, an auto sampler, and an automatic thermostatic column compartment. The column was utilized from Agela Technologies UHP Innoval C18 . The whole process with a gradient elution program is as follows: 0.00–0.50 min, 5% B; 0.50–1.50 min, 90% B; 1.50–3.00 min, 90% B; 3.00–3.10 min, 15% B; and 3.10–5.00 min, 15% B. The mobile phase was made up of 0.10% formic acid in water (A) and acetonitrile (B), and the velocity of flow was 0.50 ml/min. The auto sampler and column temperature were set at 15 and 40°C, respectively. The injection volume was 1 μl. The Q-Trap mass spectrometer was operated with positive electrospray ionization source (ESI) and scanned using the multiple-reaction monitoring (MRM) mode. The Analyst® software version 1.6.3  was used to control the UPLC-MS/MS system. The best of the YWS01125 parameters was curtain gas, 35 psi; collision gas, medium; ion spray voltage, 5500 V; temperature, 500°C; ion source gas 1, 50 psi; and ion source gas 2, 50 psi. The quantitative analysis ion pairs of YWS01125 and MGL-3196 (IS) were m/z 446.2 → 375 and m/z 435.2 → 182, individually. The suitable collision energy (CE) of YWS01125 and IS was 42 and 50 eV, respectively, and the decluttering voltage (DP) was 160 and 180 V, respectively.Sorvall Biofuge Stratos high-speed refrigerated centrifuge was bought from the Thermo Fisher Scientific Co. Ltd. ; EL204 electronic balance was selected from Shanghai Mettler-Toledo Instruments Co. Ltd. ; IKA Vortex Genius 3 Vortex Mixer was purchased from Aika Instrument Equipment Co. Ltd. ; and the test was carried out using an AB SCIEX™ 5500 Q-TrapThe frozen mice plasma samples and tissue homogenates were thawed at room temperature, vortexed for 1 min, then taken out in 10-μl aliquots in a 1.50-ml centrifuge tube, and this was then mixed with 1 μl of standard solution and 50 μl of internal standard (IS) solution, which was then dissolved and diluted with acetonitrile. Subsequently, it was vortexed for 3 min and at 14,000 rpm centrifugation for 5 min. The adopted 1 μl of the supernatant was used for sample injection analysis.2) least-squares analysis was used to acquire the standard curve equations. The accuracy and precision were assessed by three different  QC samples by diluting with acetonitrile; each concentration had six samples and was continuously accessed for 3 days, and the accuracy, intra-day precision, and inter-day precision were obtained. Recovery was evaluated by comparing the peak area of protein precipitated with acetonitrile first and finally using it to calculate the recovery of the analyst. The matrix effect was measured by comparing the analyst peak area with the saline solution samples and QC samples at the three concentrations. The stability was assessed by defining the three QC levels in several different storage conditions: at room temperature for 6 h, injector stability at 15 °C for a whole night, and after three freeze–thaw cycles.The validation was carried out by the US Food and Drug Administration bioanalytical method guidance , namely The incubation system was carried out in 0.05 M Tris/KCl buffer (PH = 7.4) and included the following: pyrazinone compounds dissolved with dimethyl sulfoxide (DMSO), mouse liver microsomes and human liver microsomes at 0.50 mg/ml, 1.30 mM nicotinamide adenine dinucleotide sodium phosphate (NADP), 3.30 mM glucose-6-phosphate disodium salt (G-6-P), 0.40 units/mL glucose-6-phosphate dehydrogenase (G-6-PDH), and 3.30 mM magnesium chloride. The volume of the organic solvent did not exceed 1% of the reaction system. After preincubation for 3 min in an 850-rpm blender (37°C), liver microsomes were added to initiate the incubation reaction (37°C). The incubation time was 0, 30, 60, and 90 min. After incubation, an equal volume of cold acetonitrile was added to stop the reaction, and an internal standard (0.05 mg/ml acetaminophen solution) was added. The samples were centrifuged at 10,000 rpm for 10 min to precipitate proteins , and theYWS01125 by oral administration with a dose of 10, 20, and 40 mg/kg, respectively  with an independent-samples YWS01125 had no metabolites during the culture time in mice and human liver microsomes, suggesting that YWS01125 was not metabolized in the liver microsomal system  was 1.60 and 1.80 min, respectively. The production scan MS spectra of MGL-3196 and YWS01125 is shown in YWS01125 of 4 and 400 ng/ml, and IS of 2 ng/ml UPLC chromatographic images. During optimization of the mass spectrometric parameters, a higher response was observed in the positive mode than in the negative mode for all of the analysts. The deprotonated precursor molecular ions (M − H)− were chosen to be monitored. The quantitative analysis was carried out in the MRM mode at m/z 446.2 → 336 for YWS01125 and m/z 435.2 → 182 for MGL-3196 (IS). Meanwhile, the transitions were regarded as qualitative analysis. Finally, for YWS01125, the decluttering potential (DP), entrance potential (EP), cell exit potential (CXP), and collision energy (CE) were 160, 42, 15, and 28 V, respectively, while for MGL-3196, the DP, EP, CXP, and CE were 180, 50, 43, and 20 V, respectively.Under the above chromatographic conditions, the retention time for YWS01125 and IS were detected at 1.60 and 1.80 min, respectively  of 0.99 or better, and these were acquired on three consecutive days. The concentration of YWS01125 as abscissa and the peak area ratio of YWS01125 to IS as the ordinate implemented the regression equation with the weighted (1/x2) least squares method in acquiring the linearity of the calibration curve. The typical regression equations for the calibration curves were obtained as follows: Y = 0.0323 x + 0.00756 (r = 0.9994) for YWS01125. The The lower limit of quantification (LLOQ) with the relative standard deviation (RSD) ≤20% was found to be 4.00 ng/ml, with an RSD <9.24%  for 12 h of injector stability, room temperature for 6 h, and three freeze–thaw cycles. The results revealed RSD ≤ ± 15% and RE ≤ ± 15%, which has indicated that YWS01125 maintains stability in the above three environments. The measured results of YWS01125 are all within the acceptable limits during the entire process . YWS01125 was measured in the plasma until the last blood collection time (24 h). The plasma concentration–time profiles of YWS01125 following the single oral administration in C57/BL6J are presented in YWS01125 were processed by a no compartmental model using the DAS 2.0 software package, and the pharmacokinetic parameters are summarized in max) of each concentration of YWS01125 in the mice plasma is 188.50 ± 30.03 ng/ml at 55.00 ± 12.25 min for 10 mg/kg, 293.42 ± 60.43 ng/ml at 60.00 ± 0.01 min for 20 mg/kg, and 381.58 ± 30.55 ng/ml at 55 ± 12.25 min for 40 mg/kg. From a comparison of the maximum plasma concentration (Cmax), the time to reach Cmax (Tmax) and areas under the plasma drug concentration–time curve (AUC0–t and AUC0–∞) of YWS01125 in the C57BL/6J mice were markedly increased, and there were statistically significant differences in these parameters (p < 0.05).The validated method was applied to the pharmacokinetic study of YWS01125 were investigated in C57BL/6J mice at 0.25, 1, 6, and 24 h after oral administration. The examined organs or tissues included the heart, liver, spleen, lungs, kidneys, stomach, small intestine, colon, fat, muscle, testes, and brain. The results are presented in YWS01125 were not detected in the brain and muscle, suggesting that the blood–brain barrier may block the entry of the drug into the brain. The content of the compound in the stomach was much higher than that in any other organ. The highest levels were reached 0.25 h after oral administration, then decreased thereafter. For the heart, liver, kidneys, lungs, small intestine, colon, and fat, the highest level was reached 1 h after oral administration, and it reached the highest levels at 0.25 h in the spleen, stomach, and testes. These data show that YWS01125 is quickly distributed throughout the mice body after oral administration; however, long-term accumulation might not be observed.The tissue distributions of YWS01125 in mice. This study demonstrated that as a precursor drug, YWS01125 was absorbed very quickly and distributed extensively in C57BL/6J mice after oral administration. In addition, the UPLC-MS/MS method proved to be valuable in determining plasma YWS01125. In vitro experiments showed that YWS01125 was stable and may not be metabolized in the mouse and human liver microsomes. Subsequently, the retention of YWS01125 in the blood and its distribution in the various organs of mice were studied by in vivo experiments. It was found that YWS01125 reached its highest concentration in the blood of mice at 1 h, and no drug distribution was detected in the muscle and brain of the mice group. In summary, YWS01125, a pyridazinone compound which we newly designed and synthesized, as an effective target, exhibited excellent activity targeting THRβ, and we studied its pharmacokinetics in vivo and found that it is a potential drug for the treatment of nonalcoholic fatty liver disease. The results are beneficial to further investigate the mechanism of YWS01125 and provide useful information for its clinical application.In this study, a rapid, specific, and sensitive UPLC-MS/MS method for the determination of YWS01125 was developed and validated in the mouse plasma for the first time. In this method, carboxymethyl cellulose sodium suspension was used to dissolve drugs to maintain the homogeneity of the analytes. With the advantage of simple sample preparation procedure, short analysis time, and high sensitivity, the present method was successfully applied to the preliminary pharmacokinetic study of oral