Escherichia coli B strains but was inactive against E. coli K12 strain BW25113. These different activities could not be explained by the dissociation constants measured for 70S ribosome preparations from E. coli K12 and B strains. Mutations in the SbmA transporter that PrAMPs use to pass the inner membrane or proteolytic degradation of Api805 by lysate proteases could not explain this either. Interestingly, Api805 seems not to bind to the known binding sites of PrAMPs at the 70S ribosome and inhibited in vitro protein translation, independent of release factors, most likely using a “multimodal effect”. Interestingly, Api805 entered the E. coli B strain Rosetta faster and at larger quantities than the E. coli K-12 strain BW25113, which may be related to the different LPS core structure. In conclusion, slight structural changes in PrAMPs significantly altered their binding sites and mechanisms of action, allowing for the design of different antibiotic classes.In view of the global spread of multiresistant bacteria and the occurrence of panresistant bacteria, there is an urgent need for antimicrobials with novel modes of action. A promising class is antimicrobial peptides (AMPs), including them proline-rich AMPs (PrAMPs), which target the 70S ribosome to inhibit protein translation. Here, we present a new designer peptide, Api805, combining the N- and C-terminal sequences of PrAMPs Api137 and drosocin, respectively. Api805 was similarly active against two  Escherichiacoli, Salmonella typhimurium, Acinetobacter baumannii, and Klebsiella pneumonia  using the Levenberg–Marquardt algorithm, with dissociation constants (Kd) represented by the x0 coefficients .Kd plateau obtained for the cf-labeled peptide to be studied and added to each well (10 µL). After incubation , the cf-labeled peptide  was added and incubated . The fluorescence polarization was measured on a PARADIGMTM microplate reader in the top read position  in triplicate on at least two different days. Data were fitted to a nonlinear, dose–response logistical transition equation [y= y0 + a/(1 + (x/x0)b)] using the Levenberg–Marquardt algorithm, with the half-maximal inhibition constants (IC50) represented by the x0 coefficients . Inhibition constants, Ki, were calculated as described by Mathias and Jung [Ki was determined for ribosome extracts using cf-labeled and unlabeled peptides in ribosome preparation buffer. Black 384-well plates were prepared as described above. Unlabeled peptides or antibiotics  were added and 2-fold serially diluted in 23 wells in ribosome preparation buffer. A ribosome solution was prepared by adjusting the ribosome concentration to ~90% of the upper Kand Jung .v/v) in freshly prepared 1x pure system buffer , 1 mol/L potassium glutamate, 1 mol/L NH4Cl, 0.5 mol/L CaCl2, 1 mol/L spermidine, 0.1 mol/L putrescine, 0.1 mol/L DTT). GFP was expressed in a freshly prepared mixture of kit solution A (2 µL), kit solution B (1.5 µL), 50-fold diluted RF1 (0.5 µL), sfGFP template  or water as control (0.25 µL), PSB (0.25 µL), and PrAMP  dissolved in water (0.5 µL). The reaction mixture was transferred into a black 384-well plate , covered with a lid, and incubated in a microplate reader  at 37 °C for two hours. The fluorescence was recorded every 10 min .GFP was expressed using an NEB PureExpress Delta RF123 kit . The sfGFP DNA template coding for a UAA stop codon was amplified from the pY71sfGFP plasmid by PCR using sfGFP primers . Release600 of ~1. The cultures were adjusted to an OD600 of 1, and cf-labeled peptide was added to achieve a final concentration of 23 µmol/L (~64 µg/mL). After 0 min, 90 min, and 180 min, two aliquots (300 µL) of the culture were transferred to two tubes. One tube was immediately stored on ice in the dark. The second sample was centrifuged , and the supernatant was transferred to a fresh tube and stored on ice in the dark. The pellet was washed with ice cold PBS (300 µL) and centrifuged , and the supernatant was transferred into a fresh tube. Both the supernatant and the pellet resuspended in 300 µL ice cold PBS were stored on ice in the dark. After 180 min, an aliquot of the unprocessed sample (100 µL) was transferred to a translucent 96-well plate, and the absorbance was measured at 600 nm. Aliquots (100 µL) of the original cell culture, supernatants, wash solutions, and resuspended pellets were transferred into black 96-well plates to measure the fluorescence in a PARADIGMTM microplate reader in the top read position . Experiments were performed three times on different days. The relative fluorescence units were normalized to the measured OD600 values of the cell culture.Bacteria were grown in 25% MHBII medium overnight, diluted fourfold with fresh medium, and incubated to obtain an ODE. coli strain BL21(DE3) was randomly mutagenized by Tn10 insertion using the lambda phage lysate λNK1324 carrying a chloramphenicol resistance and grown on LB agar plates in the presence of chloramphenicol (15 µg/mL) at 30 °C overnight [® Genomic DNA purification kit  from the presumed double-resistant strains, and the sequences flanking the transposon insertion were determined by arbitrary primed PCR using arbitrary primer ARB6 and Tn10 transposon-specific primer IS10-R complementary to the IS10 element of Tn10 transposon [® Gel and a PCR cleanup kit  and sequenced using primer IS10-R , and the corresponding protein was identified in a sequence database .vernight ,38. The ansposon ,40. The E. coli Rosetta pLysS or E. coli BW25113 (protein concentration ~0.5 g/L) and incubated on an orbital shaker . After 0, 4, 8 and 24 h, aliquots were taken in duplicate or triplicate, and trichloroacetic acid was added to achieve a final concentration of 3% (v/v). Samples were incubated on ice (10 min) and centrifuged . The supernatant was neutralized with aqueous sodium hydroxide (1 mol/L) and stored at −20 °C. Samples were separated on an ACQUITY arc system  using a Jupiter C18 column (ID: 2 mm). Elution was achieved using a linear aqueous acetonitrile gradient containing 0.1% (v/v) formic acid. The absorbance was recorded on a 2489 UV/VIS detector at 214 nm (Waters GmbH), and mass spectra were acquired online with an ion trap mass spectrometer equipped with an electrospray ionization source . Peptide recoveries and the corresponding metabolites were calculated based on the peak areas (UV) of the initial peptide peak area at 0 min.Peptides (7.5 µg) were mixed with lysates of Api805 appears to follow a different mode of action than that of apidaecin-type PrAMPs exemplified by the well-studied Api137. We speculate that Api805 and Api88 use a new type of mechanism, which is not characterized by a well-defined single binding site in the exit tunnel of the 70S ribosome but relies on interactions with different binding sites at the ribosome or ribosome-attached proteins, i.e., a multimodal mechanism based on high intracellular PrAMP concentrations.


Fasting capillary blood glucose (FCG) and postprandial capillary blood glucose (PCG) both contribute to HbA1c in diabetes. Due to the collinearity between FCG and PCG, the HbA1c prediction model could not be developed with both FCG and PCG by linear regression. The study aimed to develop an HbA1c prediction model with both FCG and PCG to estimate HbA1c in type 2 diabetes.FCG) and PCG (MEANPCG) were calculated for each patient. The patients were randomized into exploratory and validation groups. The former was used for developing HbA1c prediction models and the latter for performance evaluation.A total of 1,642 patients with type 2 diabetes who had at least three FCG and three PCG measurements in the past 3 months were enrolled in the study. The mean of FCG (MEANFCG (mmol/L) + 0.187×MEANPCG (mmol/L) + 2.979, R2 = 0.668. Compared to linear regression models developed with FCG, PCG, fasting plasma glucose (FPG), and 2-hour postprandial plasma glucose (2-h PPG), respectively, the new HbA1c prediction model showed the smallest mean square error, root mean square error, mean absolute error. The concordance correlation coefficient of the new HbA1c prediction model and the linear regression models with MEANFCG, MEANPCG, FPG or 2-h PPG were 0.810,0.773,0.749,0.715,0.672.The new HbA1c prediction model using ridge regression expressed as HbA1c (%) = 0.320×MEANWe have developed a new HbA1c prediction model with both FCG and PCG, which showed better prediction ability and good agreement. Glycated hemoglobin HbA1c) is strongly correlated with mean blood glucose levels over 3 months. The American Diabetes Association (ADA) recommends that the goal of HbA1c is <7% in most non-pregnant adults with diabetes, which is directly associated with the reduction of diabetes complications c is stro. For thoStudies on the association between SMBG and HbA1c were limited in patients with type 2 diabetes. The A1c-Derived Average Glucose (ADAG) study provided the correlation between HbA1c and mean blood glucose from CGM and SMBG in patients with type 1 diabetes, type 2 diabetes and nondiabetic, and allowed the calculation of estimated mean blood glucose for a given HbA1c . HoweverThe multi-center, observational study enrolled patients with type 2 diabetes in endocrinology departments of eight hospitals in China from March 2018 to Jan 2020. The inclusion criteria included the following: 1) Type 2 diabetes aged ≥18 years referred to the endocrinology department; 2) being untreated, or receiving stable antidiabetic treatment including diet, exercise, or hypoglycemic agents for at least 3 months before the study; 3) having at least three FCG and three PCG measurements over a 3-month period before the study. The exclusion criteria included the following: 1) being pregnant or lactated; 2) using drugs that elevate blood glucose such as glucocorticoids, chemotherapy drugs within 3 months; 3) having conditions that may change blood glucose such as infection, myocardial infarction, tumor, inflammation, trauma, hyperthyroidism, hypothyroidism, Cushing’s syndrome or acromegaly within 3 months; 4) having a history of severe liver disease or alanine transaminase/aspartate transaminase ≥3 times the normal upper limit; 5) having a history of serious kidney disease or serum creatinine >133 μmol/L; 6) having known hematological disease or hemoglobin <90 g/L; 7) being hypoproteinemia with serum albumin <35g/L; 8) blood transfusion or blood donation within 3 months. The Ethics Committee of the Peking University People’s Hospital approved the study protocol. All patients signed informed consent before the interview and data collection. In this study, data were collected from 1737 patients with type 2 diabetes. After excluding 24 patients diagnosed with diabetes for less than 3 months, 5 patients who were found to have hemoglobin variants, 11 patients with hemoglobin <90 g/L, 16 patients with serum creatinine >133 umol/L, 22 patients with serum albumin <35 g/L, 9 patients with alanine transaminase/aspartate transaminase ≥3 times the normal upper limit, and 8 patients with glycated albumin (GA) information missing, data from 1642 patients were used for data analysis. The patients were randomized into an exploratory group including 819 patients and a validation group including 823 patients.2) was calculated. Blood pressure was measured using a mercury sphygmomanometer.Demographic characteristics and medical history of the patients were recorded. Patients’ glucose-lowering medications, records of 3 FCGs and 3 to 9 PCGs in SMBG, smoking, and alcohol consumption during the last 3 months before the interview were collected. Body weight and height were measured using a calibrated scale and body mass index , HbA1c, hemoglobin, serum creatinine, albumin, alanine transaminase, aspartate transaminase, total cholesterol, and triglycerides were measured in local laboratories. Patients had breakfast and take their daily hypoglycemic agents. The blood sample was collected 2 hours later to measure 2-hour postprandial plasma glucose (2-h PPG). Glycated hemoglobin HbA1c was tested using ion-exchange high-performance liquid chromatography, capillary electrophoresis, immunoassay, enzymatic assay or boronate affinity chromatography in local laboratories. Local laboratories were required to perform 10 samples comparison quarterly with the Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, which is an HbA1c Secondary Referral Laboratory certificated by the National Glycohemoglobin Standardization Program (NGSP) . At 1 yeFCG) and PCG (MEANPCG) were calculated for each patient. The MEANPCG was the mean PCG of each patient after three meals. The mean PCG of breakfast (MEANPCGB), lunch (MEANPCGL) and dinner (MEANPCGD) were also calculated for each patient.FCG referred to the capillary blood glucose before breakfast of a day. PCG referred to the capillary blood glucose 2 hours after breakfast, lunch or dinner. The mean of FCG  guidelines . The tarData analysis was performed using SPSS software . Continuous variables were presented as mean ± standard deviation or median (interquartile range). Categorical variables were expressed as number (percentage).FCG and MEANPCG was 0.827 (P <0.001). Correlation coefficient >0.8 was considered collinearity  was introduced to the regression equation to make the estimated value of the regression coefficient essentially stable. The trend of ridge trace became stable with the optimal ridge parameter k , root mean square error (RMSE), mean absolute error (MAE), and coefficient of determination (R2) in the validation group. The MSE, RMSE, MAE was calculated byThe patients were randomized into exploratory and validation groups by SPSS software. HbA1c prediction models were developed in the exploratory group and validated in the validation group. The correlation coefficient between MEANinearity . Due to ameter k . The optiX represents the predicted HbA1c, iY represents the actual measured HbA1c, m means the sum of participants. The smaller MSE, RMSE, and MAE demonstrated the better accuracy of the prediction model  were used to evaluate the agreement between actual HbA1c and predicted HbA1c in the validation group. The CCC >0.80 suggested a strong agreement between actual and predicted values. Receiver operating characteristic (ROC) curves were used to assess the sensitivity and specificity of the new HbA1c prediction model and simple linear regression models to detect patients with HbA1c <7% using MedCalc version 20.0. The areas under the ROC curves (AUC) of HbA1c prediction models were calculated and compared using the DeLong test. All FCG of 8.29 ± 2.64 mmol/L, average MEANPCG of 11.55 ± 3.73 mmol/L, and mean HbA1c of 7.80 ± 1.86%. The mean number of daily glucose tests was 7.51 ± 2.08. The percentage of patients experiencing hypoglycemia was 11.1% within 3 months. The clinical characteristics of the patients were not statistically different between the exploratory group and the validation group (P=0.784), but the mean number of daily glucose tests in non-insulin-treated patients was more than those in insulin treatment in the validation group .There were 1642 patients with a mean age of 59.3 ± 11.0 years in the study. Males accounted for 61.9%. The median duration of type 2 diabetes was 7.9 years. Patients had an average MEANon group Table 1.FCG (mmol/L) + 0.187×MEANPCG (mmol/L) + 2.979 when k = 0.03. The four simple linear regression models of HbA1c based on MEANFCG, MEANPCG, FPG, or 2-h PPG expressed as HbA1c (%) = 0.554×MEANFCG (mmol/L) +3.218, HbA1c (%) = 0.376×MEANPCG (mmol/L) +3.434, HbA1c (%) = 0.417×FPG (mmol/L) +4.251 and HbA1c (%) = 0.256×(2-h PPG) (mmol/L) +4.527, respectively. The R2 of the new HbA1c prediction model and the linear regression model with MEANFCG, MEANPCG, FPG or 2-h PPG was 0.668, 0.622, 0.599, 0.523 and 0.511, respectively, indicating that the predictors of the new HbA1c prediction model were more strongly correlated with HbA1c compared with the other linear regression models , the R2 of linear regression model of HbA1c based on MEANPCGB, MEANPCGL and MEANPCGD was 0.556, 0.504 and 0.506, respectively. And R2 of the linear regression model of 2-h PPG and HbA1c in 417 patients was 0.496.In the exploratory group, the trend of ridge trace became stable when ridge parameter k was 0.03 Figure 1FCG, MEANPCG, FPG, or 2-h PPG in the validation group , 0.06, -0.04, -0.02, respectively. Most of the differences between actual HbA1c and predicted HbA1c in different prediction models were within 95% limits of agreement. Of the new HbA1c prediction model and simple linear regression models, only the CCC of the new HbA1c prediction model was greater than 0.80, indicating that the predicted HbA1c with the new HbA1c prediction model had a stronger agreement with actual HbA1c , which was higher than simple linear regression models with MEANPCG , FPG  or 2-h PPG . But the new HbA1c prediction model did not show better predictive ability compared to the simple linear regression model with MEANFCG  (In the validation group, the new HbA1c prediction model for predicting HbA1c with MEANP=0.107] Figure 3FCG ≤7.2 and >7.2 mmol/L, MEANPCG <10.0 and ≥10.0 mmol/L, respectively. More than 80% of patients had the consistency of actual and predicted HbA1c for both <7% and both ≥7% in patients with both MEANFCG and MEANPCG within/outside the control target range (FCG within the control target range and MEANPCG ≥10.0 mmol/L, a proportion of 35.1% patients had actual HbA1c <7% but predicted HbA1c ≥7%. And two of these patients with actual HbA1c <7% but predicted HbA1c ≥7% had experienced hypoglycemia, one of them was given insulin treatment and the other one was given insulin secretagogues, compared with no one had experienced hypoglycemia in both actual and predicted HbA1c <7%. In patients who had MEANFCG >7.2 mmol/L and MEANPCG within the control target range, patients with actual HbA1c <7% but predicted HbA1c ≥7% accounted for 20.6%. Only one patient with insulin use had experienced hypoglycemia compared with no one who had experienced hypoglycemia in both actual and predicted HbA1c <7%.Patients were grouped with MEANet range Table 3.FCG ≤7.0 and >7.0 mmol/L, MEANPCG <10.0 and ≥10.0 mmol/L, respectively.Similar results were found when patients were grouped with MEANFCG and MEANPCG with HbA1c using ridge regression and developed a new model for predicting HbA1c by combining MEANFCG and MEANPCG. The new HbA1c prediction model predicted HbA1c with better performance than the HbA1c prediction model using MEANFCG, MEANPCG, FPG, or 2-h PPG alone. The new HbA1c prediction model had a better predictive ability to detect patients who had HbA1c <7% than simple linear model with MEANPCG, FPG, or 2-h PPG, but was similar to the simple linear model with MEANFCG.Our study analyzed the correlation of MEANTo our knowledge, our study was the first to use a ridge regression model to establish a model using FCG and PCG together to predict HbA1c in patients with type 2 diabetes. This new HbA1c prediction model was appropriate for patients who were not suitable for HbA1c measurement after adjusting hypoglycemic treatment for less than 3 months, especially when patients only reached FCG or PCG target. Individualized predicted HbA1c with SMBG results was used to determine whether the hypoglycemic treatment should be adjusted to promote HbA1c to reach the goal quickly. When the patient’s actual HbA1c was inconsistent with the predicted HbA1c, the cause should be actively sought, such as frequent hypoglycemia, anemia, hemolysis, pregnancy, hemoglobinopathy, etc. In these patients, the new HbA1c prediction model might be applied to evaluate long-term glycemic control.2 of the model developed using both FPG and 2-h PPG was lower than that of the new HbA1c prediction model using both MEANFCG and MEANPCG, indicating that SMBG at home was better than single intravenous blood glucose measurement in hospital when predicting HbA1c. A possible explanation was that FPG and 2-h PPG had day-to-day variability (Our study showed that FCG had a stronger predictive performance for HbA1c than FPG alone, which was similar to the results of a previous study . When coiability –14, so tCompared with the new prediction model, simple linear model with FCG showed comparable ability to identify patients with HbA1c <7%, but the simple linear model with PCG showed worse ability. It indicated that FCG had a higher value than PCG in determining whether patients had achieved an HbA1c goal of <7%. This was consistent with previous reports that premeal blood glucose was more closely related to HbA1c than postmeal blood glucose . In patiThere was a discrepancy between the actual HbA1c and predicted HbA1c by the new HbA1c prediction model in this study. In patients who had only FCG within control target range, about 1/3 of patients with actual HbA1c <7.0% had predicted HbA1c ≥7%. Compared to patients with actual and predicted HbA1c <7.0%, we found that more patients had experience hypoglycemia in those with actual HbA1c <7.0% and predicted HbA1c ≥7%. The possible explanation was that these patients had a higher risk of hypoglycemia, which lead to the actual HbA1c reaching the goal. At this time, the actual HbA1c might be inaccurate, suggesting that clinicians should not reduce blood glucose excessively to further increase the risk of hypoglycemia in this case. For those who had only PCG within control target range, 1/4 of the patients with actual HbA1c <7% had predicted HbA1c ≥7%. Limited by the number of people with hypoglycemia occurrence, it was difficult to speculate whether these people had an increased risk of hypoglycemia.Studies have found that blood glucose testing number correlated with HbA1c attainment in insulin-treated patients , 17. In The strength of this study was that it was a multicenter study, and only required type 2 diabetes with insulin-treated and non-insulin-treated to have at least three FCG and three PCG measurements, indicating that this study had good extrapolation in type 2 diabetes. Several limitations exist in the current study. First, SMBG measurements were performed by the patient’s glucose-monitoring devices. Improper operation during blood glucose measurement, expiration of the test strip, and failure to calibrate might affect the accuracy and precision of the glucose readings. Second, the SMBG values were collected from four points including fasting and after three meals for several days over 3 months, some higher or lower values might be missed. But each point glucose value had at least 3 measurements for each patient with stable hypoglycemic treatment, which avoid a snapshot of glucose variability and reduce the impact of intra-individual differences. Third, validation of the new HbA1c prediction model was performed internally, and further validation of the model in external populations will be required in the future. Fourth, the efficacy and safety of this new HbA1c prediction model remained to be investigated, and we are conducting clinical trials to evaluate its efficacy and safety in patients with type 2 diabetes who have just been adjusted for hypoglycemic treatment.In conclusion, we have established the association between FCG, PCG and HbA1c and developed a new HbA1c prediction model based on both FCG and PCG. The new HbA1c prediction model provided an available and convenient way to convert real-time SMBG readings to HbA1c. Applying the new HbA1c prediction model might help to make the most of SMBG information and promote HbA1c to reach the goal quickly in patients with type 2 diabetes.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Peking University People’s Hospital. The patients/participants provided their written informed consent to participate in this study.XZ designed this study. YY drafted the initial manuscript. XZ and LJ revised the manuscript. XZ, WJ, JZ, FZ, JD were responsible for collecting and managing the data of their hosipital. All authors contributed to the article and approved the submitted version.


INa,Late) caused by a mutation in the cardiac sodium channel (Nav1.5) has emerged as a contributor to electrical remodeling that causes susceptibility to atrial fibrillation (AF). Although downregulation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with AF, the molecular mechanisms underlying the negative regulation of INa,Late in AF remain unclear, and potential therapeutic approaches are needed. In this work, we constructed a tachypacing-induced cellular model of AF by exposing HL-1 myocytes to rapid electrical stimulation  for 6 h. Then, we gathered data using confocal Ca2+ imaging, immunofluorescence, patch-clamp recordings, and immunoblots. The tachypacing cells displayed irregular Ca2+ release, delayed afterdepolarization, prolonged action potential duration, and reduced PI3K/Akt signaling compared with controls. Those detrimental effects were related to increased INa,Late and were significantly mediated by treatment with the INa,Late blocker ranolazine. Furthermore, decreased PI3K/Akt signaling via PI3K inhibition increased INa,Late and subsequent aberrant myocyte excitability, which were abolished by INa,Late inhibition, suggesting that PI3K/Akt signaling is responsible for regulating pathogenic INa,Late. These results indicate that PI3K/Akt signaling is critical for regulating INa,Late and electrical remodeling, supporting the use of PI3K/Akt-mediated INa,Late as a therapeutic target for AF.An aberrant late sodium current ( Atrial fibrillation (AF) is a common heart arrhythmia characterized by structural and electrical remodeling that leads to cardiac diseases such as heart failure , 7, 18. v1.5) plays a pivotal role in the initiation and propagation of action potential (AP) via membrane depolarization . The voltage dependence of steady-state inactivation curves was measured using standard two-pulse protocols from 120 mV of holding potential, and cells were held ranging from − 140 to + 20 mV for 500 ms (pre-pulse) and then subjected to a − 40-mV test pulse for 20 ms. Currents (I) were normalized to maximum I (Imax), and curves were fitted by the Boltzmann function, giving values for the V1/2 and slope k where I/Imax = 1/[1 + exp ((V − V1/2)/k)].To record peak and late 2+ signals, HL-1 myocytes were loaded with 3 μM fluo-4 AM for 30 min. The cells were continuously superfused with 1.8 mM Ca2+ containing NT solution composed of (in mM) 137 NaCl, 5.4 KCl, 10 HEPES, 1 MgCl2, and 10 glucose (pH of 7.4) at 36.5 °C. HL-1 cells grown to > 80% confluence normally show regular occurrences of Ca2+ transients in the absence of electrical stimulation [2+ fluorescence was imaged at 60 or 120 Hz in two dimensions using a laser scanning confocal imaging system  attached to an inverted microscope  fitted with a × 60 oil immersion objective lens  [2+ increases, the average resting fluorescence intensity (F0) was calculated from several frames immediately before the Ca2+ upstroke. Tracings of local Ca2+ signals are shown as the average fluorescence of each region-of-interest normalized to the F0 (F/F0). The average F0, measured in normal HL-1 cells, was used to calculate the F/F0 and obtain Ca2+ traces in rapidly paced cells.To measure intracellular Camulation . Ca2+ fl, Nikon) . Dyes weCells grown on gelatin/fibronectin-coated coverslips were washed with phosphate-buffered saline (PBS) 2 times and fixed with 4% paraformaldehyde (Biosesang) for 20 min. The fixed cells were then blocked with PBS containing 1% bovine serum albumin (Gendepot) for 1 h. For intracellular proteins, 0.1% Triton X-100 was added in blocking buffer, and coverslips were incubated with primary antibodies , and phosphorylated Akt  mixed in each blocking buffer using titers recommended in the antibody datasheets. The coverslips were then washed with 1 × PBS buffer 2 times and incubated with secondary antibodies labeled with Alexa Fluor 488 (Abcam). The coverslips were PBS-washed 2 times, counterstained with diamidino-2-phenylindole (Abcam), and mounted on slide glasses. Visualization of the slides was performed under a fluorescence microscope (Olympus). Total fluorescence intensity was measured and analyzed with ImageJ.v1.5 , anti-PI3K p110ɑ , phosphorylated Akt , Akt , and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody . We used horseradish peroxidase–conjugated secondary anti-rabbit goat antibody (Enzo) to detect blots with enhanced chemiluminescence solution (Thermo). The blots were detected and analyzed with Chemidoc (Bio-Rad) and Image Studio™ analysis software (LI-COR Biosciences).The cells were washed with 1 × PBS 2 times and lysed in a Nonidet P-40 buffer (GenDepot) containing protease inhibitors (GenDepot) and a phosphatase inhibitor cocktail (GenDepot). The lysate was sonicated and incubated on ice for 5–15 min with intermittent vortexing, and then, the lysate was centrifuged at 14,000 rpm at 4 °C for 25 min. Supernatants were collected in clean microtubes. Total proteins were quantified via Bradford protein assay, and then, the proteins were size separated with acrylamide gel electrophoresis and transferred onto nitrocellulose membranes (Atto). The membranes were blocked for 1 h with 5% skim milk (Biosesang) in TBS-T (Biosesang) and incubated with primary antibodies overnight at 4 °C. The primary antibodies were as follows: NaAdult (10 weeks old) male C57BL/6 mice were used for this study. The mice were maintained in a specific pathogen-free animal facility at Korea Medical University under standardized conditions with 12-h light and dark cycles (light on from 8:00 am–8:00 pm) at 18–25 °C and free access to chow and water. All experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Republic of Korea. Experimental procedures were also approved by the Committee on Animal Research at Korea University College of Medicine.For in vivo ECGs, the mice were anesthetized by an intraperitoneal injection of Avertin . The mice were kept on a heating pad (37 ± 0.5 °C) and baseline ECGs were recorded using four subcutaneous electrodes for lead II. For injections, PI-103 and ranolazine were dissolved in DMSO (Biosesang) and injected at a dose of 10 mg/kg and 20 mg/kg for 2 weeks, respectively. Control (CTRL) mice were injected with the same volume of DMSO. Surface ECGs were recorded at baseline, 1, and 2 weeks. ECG data recorded the RR interval, PR interval, QRS interval, and QT interval according to Bazett’s formula using the Powerlab acquisition system, and were analyzed using LabChart 8 Pro software .P-values < 0.05 were considered to indicate significant differences between groups.All experimental data are presented as the mean ± standard error of mean (SEM). For comparisons between groups, statistical differences were evaluated by one-way ANOVA with Tukey’s multiple comparison test using Prism 8 . Rapid electrical stimulation is an established method for constructing AF models that has been used in many in vitro and in vivo studies . In thisINa,Late inhibition, we applied ranolazine (50 μM), an INa,Late blocker, for 2 h before the termination of tachypacing. We found that the reduction in PI3K p110α and pAkt expression induced by tachypacing was significantly recovered by INa,Late inhibition  that can lead to severe arrhythmic disorders [INa,Late inhibition on atrial excitability, we made AP recordings using the patch-clamp technique. Tachypacing caused severe DADs, and INa,Late inhibition markedly decreased DAD-induced arrhythmogenesis  and calculated normalized INa,peak between 50 and 150 ms, after subtracting the difference between that measured in the presence and absence of 10 μM TTX  Ca2+ loading status. In HL-1 myocytes treated with 6 h of tachypacing, we observed irregular Ca2+ releases of varying magnitudes and higher basal Ca2+ levels , which suppresses INa,Late, we measured the APs in HL-1 myocytes treated with PI-103 (100 nM) as a PI3K inhibitor or with co-application of PI-103 and ranolazine (50 μM) for 2 h and then divided the cells into three groups: control (CTRL), PI-103 treatment (PI), and co-application of PI-103 and ranolazine (PI + Ran). After PI3K inhibition, as shown in Fig. INa,Late inhibition completely reversed those changes to the CTRL level, demonstrating that INa,Late inhibition upregulated PI3K/Akt signaling , the second messenger produced by PI3K. Increasing INa,Late in tachypacing-induced HL-1 myocytes was recovered by infusion of PIP3 to non-pacing levels  from mice, which were divided into 3 groups: control , PI (PI-103 injection), and PI + Ran (PI-103 with ranolazine injection). We intraperitoneally injected PI-103 (10 mg/kg) alone or PI-103 with ranolazine (20 mg/kg) every day for 2 weeks. We found that PI3K inhibition displayed prolongation of QT interval, which was significantly shortened by ion Fig. , b, leadion Fig. –e. Our rINa,Late regulation, which prevents electrophysiological dysfunction and arrhythmic effects under AF conditions.Taken together, our findings support the anti-arrhythmic effect of PI3K/Akt signaling on pathogenic INa,Late reversed abnormal Ca2+ handling and AP (prolonged APD and DADs) in tachypacing-induced HL-1 myocytes and that this recovery effect is related to the enhancement of PI3K/Akt signaling. Intriguingly, PI3K inhibition produced marked increases in INa,Late and subsequent contributions to aberrant AP morphology that were recovered by exposure to the INa,Late blocker ranolazine, suggesting that PI3K/Akt signaling plays a beneficial role in regulating pathogenic INa,Late and alterations in electrical remodeling. Together, these findings provide new insight into the potential role of the PI3K/Akt pathway in INa,Late and indicate that such regulation is critical to the treatment of AF (Fig. In this study, we demonstrated that inhibiting  AF Fig. .Fig. 8Scv1.5 mutations appear to play a pivotal role in the management of myocyte remodeling in AF. In particular, dysregulation of INa amplitude and duration contributes to incidence-triggered activity and prolonged AP and is related to severe AF [INa,Peak have been observed in arrhythmic disorders such as Brugada syndrome, long QT syndrome, and AF [INa,Late by altering biophysical properties [INa function contributes to arrhythmic activity [INa,Peak and enhanced INa,Late with biophysical defects in activation and inactivation in SCAN5A 1472del mutations of tsA201 cells [INa,Peak and increased INa,Late while causing no difference in the activation curves of atrial myocytes in patients with sleep-disordered breathing. Although previous findings in clinical and experimental studies reported the dysregulation of INa in arrhythmia, the underlying mechanism and potential therapeutic options remain unclear. To clarify the pathological role of INa regulation in AF and the effect of INa,Late inhibition on INa, we examined both INa,Peak and INa,Late amplitudes in tachypacing-induced HL-1 myocytes. In those cells, we observed decreased INa,Peak and increased INa,Late, along with decreased kinetics of activation and inactivation and a depolarized shift rightward compared with non-paced HL-1 myocytes. Importantly, the consequences of pathological remodeling were all significantly reversed by INa,Late inhibition. Moreover, INa,Late inhibition recovered irregular Ca2+ handling and AP morphology. These results identify INa,Late as an efficient modulator of INa function and changes in electrical remodeling that could be used to prevent arrhythmic effects [Naevere AF , 24, 30., and AF , 11, 28.operties . Thus, t01 cells . Moreove01 cells  demonstr effects .INa,Late is also a contributor to rising intracellular Na+, which induces intracellular Ca2+ accumulation and ultimately induces DADs and severe AF [INa,Late contributes to Ca2+ dysregulation and the incidence of DADs, and those detrimental effects were abolished by INa,Late inhibition, suggesting that INa,Late could also be crucial in the regulation of Ca2+ handling. Of note, excessive intracellular Na+ overload caused by increasing INa,Late could be a result of reduced PI3K signaling, which leads to further intracellular Ca2+ overload [2+ signaling had any effect on PI3K signaling, it is possible that PI3K-mediated INa,Late regulation is responsible, at least in part, for alternating irregular Ca2+ release with basal Ca2+ levels. Further studies will be needed to examine the role of PI3K signaling in Ca2+ signaling under AF conditions.Importantly, a sustained increase in evere AF , 32. We overload . AlthougINa,Late through alterations of the INa gating property and exacerbates the cardiac remodeling related to arrhythmias [INa,Late and treating AF. Our data support the hypothesis that PI3K/Akt signaling plays an important role in INa,Late regulation for the management of AF in tachypacing-induced HL-1 myocytes. That loss of PI3K signaling significantly enhanced INa,Late due to decreased kinetics of activation and inactivation, which further contributed to prolonged APD and DADs, which are related to AF. Of note, PI3K inhibition is implicated as a consequence of INa,Late dysfunction and subsequent arrhythmia [INa,Late in canine ventricular myocytes. In addition, Yang et al. [INa,Late and further contributed to prolonged APD and triggered activity. Our results demonstrate that PI3K inhibition leads to increased INa,Late, aberrant APD, and the occurrence of DADs, which are all abolished by INa,Late inhibition and the consequent upregulation of PI3K 110α and pAkt. Moreover, we discovered that a reduction in INa,Late occurred after application of PIP3 in tachypacing- or Akt-inhibited HL-1 myocytes. This suggests that a reduction in PI3K/Akt signaling contributes to the enhancement of INa,Late and the dysregulation of INa open probability, which can lead to further accumulation of intracellular Na+ under AF conditions.Reducing or inhibiting PI3K/Akt signaling induces an increase in hythmias . Thereforhythmia . In factg et al.  demonstrINa,Late regulation as well as an increase in APD.Our study has several limitations. First, the constructed cellular AF model induced myocyte remodeling and thus might not fully represent the environments and circumstances of AF in vivo. Because upregulation of the PI3K/Akt pathway is involved in oncogenesis, appropriate dosages and the potential side effects of using ranolazine to treat AF need to be further investigated . Second,INa,Late and suggest ways that it might be used against AF. This study supports the identification of PI3K/Akt as an effective factor for regulating pathogenic INa,Late and preventing AF caused by aberrant electrical remodeling.In conclusion, these findings highlight the role of PI3K/Akt signaling in pathogenic


Oxidative damage by reactive oxygenspecies generated during RET underpins ischemia reperfusion injury,but as RET relies on several converging metabolic pathways, littleis known about its mechanism or regulation. Here, we demonstrate Δp-linkedRET through complex I in a synthetic proteoliposome system for thefirst time, enabling complete kinetic characterization of RET catalysis.We further establish the capability of our system by showing how RETin the mammalian enzyme is regulated by the active-deactive transitionand by evaluating RET by complex I from several species in which directassessment has not been otherwise possible. We thus provide new insightsinto the reversibility of complex I catalysis, an important but littleunderstood mechanistic and physiological feature.Respiratory complexI is an essential metabolic enzyme that usesthe energy from NADH oxidation and ubiquinone reduction to translocateprotons across an energy transducing membrane and generate the protonmotive force for ATP synthesis. Under specific conditions, complexI can also catalyze the reverse reaction, Δp-linked oxidationof ubiquinol to reduce NAD The energy from the redox reaction is harnessed totranslocate four protons across the inner mitochondrial membrane,or prokaryotic cytoplasmic membrane, contributing to the proton motiveforce (Δp) required for ATP synthesis and transport processes.3Respiratorycomplex I (NADH:ubiquinone oxidoreductase) is a redox-coupledproton pump central to oxidative phosphorylation in mitochondria andaerobic bacteria.5 Furthermore,ROS production by complex I during “reverse electron transfer” hasbeen shown to underlie the tissue damage occurring in strokes andheart attacks, during ischemia reperfusion (IR) injury.6 The physiological role of RET is now a majordiscussion in complex I function, with recent developments implicatingthe ROS produced by RET in redox signaling during inflammation,7 uncoupling of mitochondria in brown adipose tissue,8 oxygen sensing,9 andaging (in flies).10As a major source of reactive oxygen species(ROS), complex I contributessignificantly to cellular oxidative stress. In combination with itscentral metabolic role, this makes complex I dysfunctions one of themost frequent causes of mitochondrial disease.11 Thereare two key prerequisites: a highly reduced Q-pool ([QH2] > [Q]) to provide the electrons and a high Δp to driveprotonsback through the complex in the opposite direction to forward catalysis.Together, the high QH2/Q ratio and high Δp providethe thermodynamic driving force required for RET: the proton transferfree energy (4Δp) overcomes the redox potential free energy(2ΔE) to drive electrons from QH2 (+80 mV) to NAD+ (−320 mV). When the two energiesare equal, there is no net reaction, and catalysis switches from forwardto reverse at this point.12Complex I catalysishas long been known to be reversible, but theRET redox reaction is energetically unfavorable, and so RET only occursunder specific metabolic conditions.13 However, studiesin these systems are all limited by complexity and inaccessibility:a multitude of proteins interact with the Q pool and Δp; mammalianmitochondria are difficult to manipulate genetically; and key kineticparameters such as the membrane Q concentration cannot be varied.These limitations have precluded detailed mechanistic investigationsof the RET reaction.So far, studiesof the kinetics and thermodynamics of RET havemainly focused on mammalian complex I, either in intact mitochondriaor submitochondrial particles (SMPs).10. Incorporationalso of Trypanosoma brucei brucei alternativeoxidase (AOX), a quinol-oxidizing enzyme, allowed us to study Q10-linked forward catalysis.15 Further incorporationof Escherichia coli ATP synthase (F1FO) then enabled us to use complex I to drive Δp-coupledATP synthesis.16 Here, we replace AOX withthe quinone-reducing enzyme glycerol 3-phosphate dehydrogenase (GlpD),which catalyzes glycerol-3-phosphate:ubiquinone oxidoreduction. Byrunning the ATP synthase in reverse to generate Δp, we thusdescribe the first reconstituted system that efficiently catalyzesRET, enabling full kinetic characterization of the RET reaction andestablishing a rationale for direction-dependent inhibition. We thendemonstrate the potential of our system. First, we explore the regulationof RET to confirm that the deactive state, a resting state that formsin the absence of turnover, is neither RET competent nor can be reactivatedby RET conditions. Second, we exploit the modular nature of our systemto exchange complex I from Bos taurus for other homologues; we provide the first direct demonstrationof RET (as NAD+ reduction) by complex I from Mus musculus and Pichia pastoris and thereby validate RET as a conserved mechanism across a rangeof species.Here, we have engineered a modular, minimalsystem to study both“forward” and “reverse” catalysis by complexI under precisely defined conditions. We used established protocolsto incorporate bovine complex I into proteoliposomes containing itshighly hydrophobic natural substrate, ubiquinone QE. coli (GlpD), which has previously been overexpressed, purified, and shownto bind liposomal membranes,18 to drive quinone reductionin our RET system .To first test the ability of GlpD to drive RET, we incorporated itinto bovine SMPs, native-membrane vesicles well-established for studyingboth forward and reverse complex I catalysis.20 GlpD bound spontaneously to the SMP membrane(producing GlpD-SMPs) and, upon addition of G3P, catalyzed the reductionof Q10, activating downstream catalysis and proton pumpingby complexes III and IV (We selectedthe monotopicglycerol 3-phosphate (G3P) dehydrogenase from –1 GlpD butthen declined rapidly. When RET was induced by G3P instead of succinate,the rate first increased with increasing GlpD but again declined rapidlyabove ∼5 μg mL–1. To avoid the decline,which we ascribed to the n-dodecyl-β-d-maltopyranoside -β-cyclodextrin(β-cyclodextrin or β-CD)21 wasadded to sequester the DDM. Succinate-linked RET then became insensitiveto GlpD concentration, and the rate of G3P-linked RET continued torise to a plateau at ∼80 g mL–1 GlpD  and to the Δp-linkedgeneration of ATP by the same reaction as NADH-linked ATP synthesis.By combiningco-reconstituted proteoliposomes containing complex I and ATP synthasewith ATP to generate Δp by ATP hydrolysis and GlpD to reduceQomplex I 3A, insetomplex I 3A. FurthB.taurus for NADH-linked ATP synthesis and RET:(i)Figure S2A),below the excessive concentrations at which both RET  and FET  are affected. While β-CD did not benefit the maximalrate of NADH-linked ATP synthesis in AOX titrations , it was beneficial at higher AOX concentrations.A total of 2.5 mM β-CD was used from hereon.As for SMPs, GlpDspontaneously associatedwith the proteoliposomal membrane, and β-CD was needed to sequesterits accompanying detergent. Maximal rates of RET were achieved at1–2.5 mM β-CD ((ii)1FO ATP synthase in the reconstitution (at set complexI concentration) gave maximal rates for both NADH-linked ATP synthesisand RET (1FO ATP synthase per complexI. A standard ratio of three was therefore used from hereon.Titrating the amount of Fand RET 3Bat abo(iii)–1 (10 μg AOXper μg of outward facing complex I (CIout)) thenbegins to decrease. A similar observation was previously ascribedto uncoupling by the DDM transferred along with the AOX.16 The inclusion of β-CD now argues againstthat interpretation, and a matching decrease was observed here forFET activity, both in the presence and absence of gramicidin A tointentionally abolish Δp . A total of 10 μg mL–1 AOX was thereforeused from hereon, to avoid the decrease, perhaps due to AOX aggregation.For GlpD, the rate of RET plateaus above ∼40 μg mL–1 (∼8 μg GlpD per μg of CIout). Similar mass ratios of AOX and GlpD are thus required, relativeto complex I, to drive their reactions at the maximum rate. Concentrationsof 80–100 μg mL–1 GlpD were used fromhereon to ensure maximal quinone reduction during RET experiments.The rates of NADH-linkedATP synthesisand RET were optimized by titrating the amounts of AOX and GlpD added,respectively, to the proteoliposomes in the presence of β-CD3C. Both The conditions of proteoliposome preparationand catalysis were optimized using complex I from 10 concentration. Previously,the Q10 concentration was varied in CI-AOX proteoliposomes,relying on AOX to keep the quinone pool oxidized, to determine theMichaelis–Menten parameters for FET.15 Here, we extend the approach by using our modular system to determinethe kinetics for FET and RET together using common proteoliposomepreparations. 2) and NAD+ reduction. The concentrationsof complex I, phospholipids, and Q10 were individuallydetermined for each preparation, to define its individual membrane-Q10 concentration. It was assumed that 1 mg of phospholipidoccupies ∼1 μL, so that 1 nmol of Q10 permg phospholipid is equivalent to 1 mM Q10 in the membrane.22 The KM (Q10) of 1.65 ± 0.20 mM thereby determined for quinone reductionduring NADH oxidation is consistent with values of 0.45–3.9mM reported for complex I from B. taurus determined for NADH-linked ATPsynthesis by the bovine enzyme.16 Therewas no substantial change in KM (Q10) when gramicidin A was included to collapse Δp , but Vmax was higher when it was present (24.4 ±0.85 vs 18.3 ± 0.65 μmol min–1 (mg CIout)−1). For RET, KM (Q10H2) for quinol oxidation by complexI was determined to be 9.13 ± 1.62 mM, a striking 5.5 times higherthan KM (Q10), with a Vmax of 0.50 ± 0.04 μmol min–1 (mg CIout)−1.Before this study, SMPs and intact mitochondria werethe only systems in which RET could be measured routinely, but theydo not allow manipulation of the QB. taurus,15,23 anKM (Q10H2) forRET relies on GlpD holding the Q-pool reduced, an assumption supportedby our observation that increasing the GlpD concentration affectsneither the KM (Q10H2) nor Vmax , so GlpD catalysis is not rate limiting. However, therate of RET is also sensitive to the reduction potential of the Q-pool,13 a thermodynamic not kinetic effect . Therefore, the Q10H2/Q10 ratio was quantified using mass spectrometry.25 Following complex I preactivation  and a 30 s or 3 min period of RET , ∼90% of the Q-pool was found to be reduced.In comparison, only ∼50% of the quinone was reduced when NADHwas added (in the absence of AOX), and quinone reduction was abolishedby piericidin A  = 0.14 ± 0.02 mM) confirms thatNAD+ concentration is not rate limiting in our standardconditions. (ii) The pH dependence of G3P-linked RET in proteoliposomes revealed a maximum at pH ∼8,and matching pH-dependencies were observed for both G3P- and succinate-linkedRET in GlpD-SMPs. (iii) Using Q10-free liposomes, we demonstratedG3P-driven NAD+ reduction with either 100 μM decylubiquinone(DQ) or 100 μM Q1 but at lower rates  than for ∼10 mM membrane-bound Q10 .We completed our basickinetic characterization of RET as follows:(i) The dependence of the rate on NAD23 and studies using bovine heart SMPs26 have suggested it is a more potent inhibitorof RET than FET. Here, we directly assessed direction-dependent inhibitionby piericidin A, with the Q-pool held either oxidized or reduced. 50 value is lower during RET  than during FET . However, the difference may result simplyfrom competitive inhibition against a weaker binding substrate (the KM for Q10H2 during RETis lower than for Q10 during FET): it does not necessarilymean that the inhibitor binding affinity is direction/state-dependent.Finally, we usedour modular system to investigate the efficacyof a canonical complex I Q-site inhibitor in both directions of catalysis.The competitive binding mode of piericidin A has been establishedby combined structural, computational and kinetic approaches27 The active/deactive (A/D) transitionof complex I is a biochemically30 and structurally31 defined phenomenon where a catalytically“ready-to-go” resting state, the A state, relaxes spontaneouslybut slowly into a pronounced resting D state in the absence of turnover;D requires reactivating by both NADH and ubiquinone to return to catalysis.19 Typically, investigations of the A/D transitionare conducted in native preparations, owing to the much better stabilityof the membrane-bound enzyme compared to in detergent; this is particularlyrelevant when the enzyme is deactivated in a substrate-free incubationat 30 or 37 °C.31 Proteoliposomesnow offer a stable environment in which A/D transitions can be studiedin a minimal, well-defined system. N-ethylmaleimide (NEM) assay used routinely to evaluate the relativeamounts of A and D present.29 NEM derivatizesCys39 of ND3 in the D state, rendering the enzyme inactive and resistantto reactivation.29 Here, the NEM assayshowed that in a sample of “as-prepared” proteoliposomes,∼36% of the FET activity was retained after NEM treatment andso ∼64% of the complex I was in D. This value is consistentwith data from cryoEM analyses of bovine complex I,32 and it was consistent across several batches of proteoliposomes,both with and without ATP synthase, and unaffected by gramicidin A. Incubation of the proteoliposomesat 37 °C for 15 min increased the proportion of D to 97%. Importantly,the D enzyme regained full activity upon addition of NADH, confirmingthat complex I in proteoliposomes survives deactivation without lossof activity or integrity. In contrast, detergent-solubilized bovinecomplex I displayed a similar A/D ratio but underwent substantialunrecoverable loss of activity when incubated similarly at 37 °Cto deactivate it  and two yeast species (Yarrowia lipolytica and Pichia pastoris) were incorporated into proteoliposomes, in place of the bovineenzyme ((i)35 shows closestructural similarityto the bovine enzyme, and proteoliposomes created using mouse complexI and our standard protocol showed matching physical properties totheir bovine counterparts (Table S1). TheirFET activity was lower (–1 mg–1 for NADH:DQ oxidoreduction23 vs ∼20–25 μmol min–1 mg–1 for bovine complex I31), and their rates of ATP synthesis were thus also lower  and Δp formation by the mouse proteoliposomes were also lower, suggesting lower retentionof ATP synthase; further optimization was not pursued due to limitationsin the availability of mouse heart tissue. Approximately 75% of theas-isolated mouse complex I was found to be in the A state (usingthe NEM assay), substantially higher than for bovine complex I, consistentwith observations from cryoEM studies Y.lipolytica and P. pastoris are similar in size and subunit composition to bovine complex I38 and have been incorporated into coupled proteoliposomes previously.39 Initial (standard) preparations of P. pastoris complex I proteoliposomes exhibited comparable rates of NADH oxidationand ATP synthesis , and matching physical properties(Table S1). Near identical rates were alsoobserved for RET in P. pastoris complexI  was found to provide maximal rates of RET and ATP synthesisfor P. pastoris complex I. The sametreatment also stimulated bovine complex I, but it was less effective(∼15%) than for P. pastoris,where both rates increased by ∼50%. The close similarity tobovine complex I was further confirmed by the Michaelis–Mentenparameters  for bothFET and RET. The KM values for both Q10 and Q10H2 were both lower for P. pastoris, but the KM for Q10H2 was again significantly higher thanfor Q10 (ratio of 3.5 for P. pastoris).Complex I from ynthesis 6A, B to ynthesis S5, and m(iii)Y.lipolytica consistently showed remarkably high ratesof NADH oxidation in proteoliposomes (Table S1), considerably higher than for any other complex I tested. However,we have been unable to detect RET by Y. lipolytica complex I . Although rates of NADH-linkedATP synthesis from Y. lipolytica complexI are comparatively low, they are greater than from mouse complexI , and the results for both ATP hydrolysisand Δp formation were consistent . Therefore, we considered whether the A/D ratio of complexI may preclude RET in Y. lipolytica complex I. Complex I from Y. lipolytica is known to have a lower energy barrier to deactivation than bovinecomplex I,40 and its deactive state is lessdeveloped structurally.42 According to the NEMassay, Y. lipolytica complex I waspresent entirely in D Comp24 inhibition,44 ROS production,45 protonpumping,48 and ion transfer.49 The modular proteoliposome systems describedhere contain minimal respiratory chains designed to study complexI catalysis with the native, hydrophobic Q10 substrateincorporated in the membrane. They enable the study of both forwardand (for the first time in proteoliposomes) reverse catalysis by complexI and of the kinetics, mechanism, and regulation of the RET reaction.Complex I proteoliposomes combine the advantagesof a purifiedenzyme system  with catalysis in a native-like membrane that sustainsa proton-motive force (Δp). As such, they have already beenused to investigate multiple aspects of complex I function includingthe kinetics of turnover and ubiquinone reduction,10 to Q10H2, to rapidly recyclethe Q10 formed by complex I. We demonstrated that the Q-poolwas held highly reduced during RET, focusing our kinetic data on thefunction of complex I, not GlpD. In contrast, succinate:ubiquinoneoxidoreduction by complex II during RET in intact mitochondria couldonly hold the Q-pool ∼60% reduced.27 By accurately quantifying the concentrationsof complex I, phospholipids and Q10, we determined thekinetic parameters for ubiquinol oxidation during RET, and by exploitingour modular system to switch GlpD for AOX, we simultaneously determinedcorresponding parameters for FET. Our observation that the KM value for Q10 during FET is ∼5times higher than for Q10H2 during RET indicatesa clear kinetic bias for forward catalysis. Although this appearsconsistent with the substantially lower rates of catalysis observedfor RET than FET, the rates have not been normalized for their thermodynamicdriving forces or for the relative amounts of FET/RET-active complexI, precluding a fair comparison of turnover numbers.  The difference in KM values providesa simple rationale for the relative efficacy of competitive complexI (Q-site) inhibitors, such as piericidin A, that inhibit RET morestrongly than FET because they compete better against a less effectivesubstrate. Notably, such inhibitors are emerging as a promising routeto protect against the deleterious production of RET-ROS, for example,in IR injury.51 Our modular proteoliposome systemnow enables quantitative assessment of candidate drugs and inhibitorsfor both FET and RET, as well as mechanistic investigation of direction-dependentinhibitors. However, we note that our system is currently unsuitedto study RET-driven ROS production, which is implicated in redox signalingand oxygen sensing,9 cardiac IR injury,6 and aging in flies.10 GlpD  also produces ROS,52 and its high concentration in our assays causes substantialbackground ROS production.To drive RET in proteoliposomes, we incorporated GlpD to reducethe Q13 In our modularsystem, Δp can be created by ATP hydrolysis, and the directionalityof complex I catalysis is determined by the choice of partner enzyme(AOX or GlpD) that, through essentially irreversible catalysis, setsthe Q-pool (near) fully oxidized or reduced. As a result, our “binary”system can only be switched from one direction to the other, not titratedbetween the two extremes. The effects of the Q-pool potential andthe Δp can therefore be probed independently of one another.Due to its energetic demands, the rate of RET is exquisitely dependenton both the Q-pool potential and Δp, as demonstrated by measurementsof the thermodynamic driving force for RET-associated ROS productionin whole mitochondria.+ reduction.19 Although SMPs fromyeast species have been described,53 theyhave not been used to demonstrate RET, and measurements on D. melanogaster have focused only on RET-ROS production.The only non-mammalian species for which RET has been observed directlyis the bacterium P. denitrificans.34 Here, we provide the first direct evidence of RET by complex I fromtwo new species, M. musculus (mouse)and the yeast P. pastoris. Althoughour results further support the conservation of reversibility in complexI catalysis, complex I from Y. lipolytica was not able to catalyze RET under the conditions tested, highlightingdifferences in complex I regulation between species. Previously, weobserved that the P25L-ND6 complex I mouse variant is protected againstIR injury because P25L-ND6 complex I, despite being capable of catalyzingFET normally, is unable to catalyze RET-ROS formation.27 The protective effect was assigned to the increasedtendency of the enzyme to drop into D-like states, as a result ofthe mutation perturbing the stability around the π-bulge inND6, a hallmark of D. Notably, this protective mechanism relies onthe conditions for RET not being capable of reactivating the D enzymeto initiate RET, which we have demonstrated here to be the case. Parallelsbetween P25L-ND6 mouse and Y. lipolytica complex I thus lead us to ask if RET is prevented by the same mechanism.A lack of diversity in experimental systems able to support RETmeans it has so far only been studied widely in mammalian complexI. Direct observations of RET have typically been investigated usingSMPs, based on the accessibility of the outward-facing substrate bindingsites, their ability to sustain high Δp, and ease of monitoringNADY. lipolytica complex I is less stable than its mammalian counterpart,40 but the reasons are not well understood. Structuraldata revealed that, although the extent of the deactive transitionis less in Y. lipolytica, importanthallmarks of the deactive enzyme are present in the as-prepared enzyme,41 including the characteristic π-bulge inTMH3 of the ND6 subunit, and this was also conserved in structuralanalyses of a proposed “turnover” state.42 Therefore, rapid deactivation may explain theinability of Y. lipolytica complexI to catalyze RET. We note that, in mammalian complex I, conformationalrearrangements reposition the NDUFA10 and NDUFA5 supernumerary subunitsrelative to each other during the A/D transition,54 and the absence of NDUFA10 in Y. lipolytica may contribute to its low energy barrier for deactivation. However, P. pastoris lacks NDUFA1037 and yet displays a substantial proportion of complex I in the Astate, and P. denitrificans lacks bothNDUFA10 and NDUFA5 and appears fully in the A state.24 Therefore, there is a clear cross-species correlation betweenthe ability to catalyze RET and the existence of the A state but nocorrelation with the presence of NDUFA10 and NDUFA5. Finally, massspectrometry data have revealed that an arginine residue on a loopin subunit NDUFS7 (Arg77 in B. taurus), which forms part of the ubiquinone binding site and changes conformationbetween the A and D states, is post-translationally hydroxylated in B. taurus, M. musculus, P. pastoris, and P. denitrificans conformation. Extending the lists of specieswith RET-active (or inactive) complexes I is thus required to betterunderstand these correlations and to elucidate the subtle structuralvariations that occur and that may underpin mechanisms of complexI catalysis and regulation.It has long been known that the A state in trificans,56 but nIn summary, we havedemonstrated Δp-linked reverse electrontransfer by complex I in a proteoliposome system for the first timeand exploited the well-defined and quantifiable nature of our systemto determine previously inaccessible kinetic parameters. The modularnature of our proteoliposome system allows different forms of complexI to be incorporated and analyzed during FET, RET, and the A/D transition.Our system thereby enables investigations of complex I from less experimentallydeveloped species, and opens the door to further detailed characterizationof the effects of mechanistic and clinically relevant mutations inboth directions of catalysis.E. coli gene encoding GlpD (glpD) was synthesized and insertedinto a pET-28a expression vector  using the NdeIand BamHI restriction sites by a commercial service(GenScript). Two stop codons (TAG and TGA) were added at the C-terminus.The GlpD-pET28a plasmid was transformed into E. coli strain BL21(DE3) pLysS, and then GlpD was overexpressed and purifiedusing a method adapted from Yeh et al.17 Starter cultures were grown overnight at 37 °C in 200 mL LBbroth supplemented with 50 μg mL–1 kanamycinand 25 μg mL–1 chloramphenicol. The cellswere diluted 100-fold into terrific broth (containing 50 μgmL–1 kanamycin and 25 μg mL–1 chloramphenicol) and grown to OD600 ≈ 0.7 at 37°C. GlpD overexpression was induced by the addition of 1 mM isopropylβ-d-1-thiogalactopyranoside (IPTG), and the culturewas grown for a further 4 h. Cells were harvested at 6000 g for 10 min at 4 °C, washed in 50 mM HEPES (pH 7.4),100 mM NaCl then pelleted and stored at −80 °C.The 4,0.5 mM PMSF, and a few crystals of DNase1 were added. The cells werelysed with a French press .Cell debris and unbroken cells were removed by centrifugation , and the supernatant was ultracentrifugedat 100,000 g . The membrane pelletswere resuspended in 120 mL resuspension buffer and homogenized, and40 mL aliquots were frozen in liquid N2. Individual aliquotswere thawed, and the membranes were solubilized on ice for 60 minby the addition of 1.3% octyl glucoside. The suspension was clarifiedby ultracentrifugation ,filtered (0.22 μm), and loaded onto a 5 mL HisTrap HP columnequilibrated with GlpD Buffer A (20 mM HEPES (pH 7.4), 20 mM KCl,200 mM NaCl, 0.05% DDM). The column was washed with 37.5% Buffer B(Buffer A + 400 mM imidazole), and then GlpD was eluted with 100%Buffer B. GlpD-containing fractions were collected, pooled, concentratedin an Amicon Ultra-15 (10 MWCO), and dialyzed overnight at 4 °Cagainst 2 L of Buffer A (3.5 kDa MWCO). 20% glycerol was added forstorage at −80 °C.Cells from 12 L of culture were thawed and resuspended in 120 mL50 mM HEPES (pH 7.4), 100 mM NaCl, and then 1 mM MgSOB. taurus,15M. musculus,35 and Y. lipolytica(P. pastoris complex I was purified using a method adapted from published procedures.59 See Supporting Information for further details.Complex I was purified from ipolytica usingesE. coli membranes bysolubilization with octyl-glucoside and purified by Twin-Strep tagaffinity chromatography as described previously.15Recombinantly expressed AOX wasprepared from E. coli strain DK8 and the pBWU13-βHis plasmidwas prepared as describedpreviously.60ATP synthase from c were prepared as described previously62 and suspended in 10 mM MOPS (pH 7.5), 50 mM KCl, and 250 mM sucrose.SMPs supplementedwith cytochrome 16 A total of 10 mg of lipids , 1,2-dioleoyl-sn-glycero-3-phospho-ethanolamine(DOPE), and 18:1 cardiolipin (CDL)) in chloroform were combined withQ10 from a chloroform stock (10 nmol (mg lipid)−1), and the solvent was evaporated off with N2 before dryingunder vacuum for >1 h. The lipids were hydrated in 1 mL of proteoliposomebuffer (10 mM MOPS (pH 7.5), 50 mM KCl) for 30 min with frequent vortexingand then sonicated on ice using a Q700 probe sonicator (QSonica) equippedwith a 1.6 mm microtip ).The resulting liposomes (10 mg mL–1) were then reconstituted by partially solubilizing the lipidswith 0.5%  sodium cholate for 10 min on ice.Complex I (50:1 (w/w) lipid to protein) was then added, alongsideF1FO ATP synthase  total lipid toprotein ratio), and incubated for a further 10 min. The detergentwas removed using a PD10 desalting column (Cytiva). The sample wascentrifuged  followedby resuspension in proteoliposome buffer. Proteoliposomes were kepton ice or at 4 °C before use; no loss in activity was observedduring overnight storage.Proteoliposomes wereprepared using a protocol adapted from Biner et al.14 Total Q10 content was determinedusing an HPLC system equipped with a Thermo Scientific Dionex Ultimate3000RS Electrochemical Detector.16 Q10 concentrations are expressed in mM relative to the volumeof the membrane, where 1 mM Q10 (membrane) = 1 nmol Q10 per mg of phospholipid.22 Theredox state of the Q pool was determined using a mass spectrometryassay following Q10 extraction.25 Complex I content and orientation in proteoliposomes were estimatedby comparing the rate of NADH:APAD+ oxidoreduction to therate from a standard detergent-solubilized complex I sample, assayedin proteoliposome buffer with 100 μM NADH, 500 μM APAD+, 500 nM piericidin A and 0.2% (w/v) DDM.24 Proteoliposome APAD+ reductionrates were measured without DDM under otherwise identical conditions,with 15 μg mL–1 alamethicin added to determinethe (inside/outside) orientation.Total phospholipidcontents were determined using the Ames phosphate assay.2 oxidoreductionactivity was measured spectrophotometrically (ε340–380 = 4.81 mM–1 cm–1) using a SpectraMaxplus 348 96-well plate reader (Molecular Devices). Standard measurementswere carried out in 10 mM MOPS (pH 7.5), 50 mM KCl at 32 °C,with complex I and AOX (10 μg mL–1). Turnover was initiatedby addition of 200 μM NADH. Uncoupled rates were determinedwith 0.5 μg mL–1 gramicidin A. Inhibitionmeasurements were conducted with 2 μg mL–1 outward facing complex I and 2.5 mM β-CD for both FET andRET systems. We note that β-CD has the capacity to interactwith hydrophobic inhibitors and artificially increase the absoluteIC50 values; here, we observed the ratios of IC50 values in FET and RET to be unaffected.NADH:O–1 in 10 mM MOPS (pH 7.5), 50 mM KCl. They were activated by 3 minincubation with 10 μM NADH at 32 °C prior to the additionof RET substrates , and thereaction followed spectrophotometrically at 340 and 380 nm. For standardproteoliposome experiments, CI-F1FO proteoliposomes(5 μg mL–1 outward facing complex I) weretreated with 2.5 mM β-CD and GlpD (80–100 μg mL–1), preactivated by 3 min incubation with 10 μMNADH at 32 °C, and then RET was initiated as in the SMP experiments.In experiments with P. pastoris complexI, liposomes were incubated at 37 °C for 15 min before activationwith NADH.For SMP experiments,(GlpD)-SMPs were used at 20 μg protein mL–1 outward facing complex I) orDDM solubilized complex I (17.1 mg mL–1) at 37 °Cfor 15 min. N-Ethylmaleimide (NEM) was dissolvedat 400 mM in DMSO before dilution to 100 mM in proteoliposome buffer.Soluble complex I was diluted to 200 μg mL–1, and samples were treated with 1 mM NEM  and incubated on ice for 20 min before determining theNADH:O2 or NADH:DQ activity.31 NADH:O2 activity was performed as described above. NADH:DQactivity was performed with 0.5 μg mL–1 complexI, 200 μM NADH, 200 μM DQ, and 0.15% (w/w) of each ofasolectin  and CHAPS in 20 mM Tris–HCl(pH 7.5 at 32 °C).Deactivation of complex I was achieved by incubationof proteoliposomes(200 μg mL–1) was incubated in 10 mM MOPS(pH 7.5), 50 mM KCl at 32 °C with 200 μM Q1.Where indicated, 0.15% (w/w) of each of asolectin and CHAPS was addedto the reaction mixture. Catalysis was initiated by the addition of120 mM rac-G3P, and the reduction of Q1 was monitored at 275 nm (ε = 13.7 mM–1 cm–1)63 using a quartz plate.GlpD(0.1 μg mL16 with slight modifications.The assay comprised 20 mM Tris–PO4 (pH 7.4), 5 mMMgCl2, 2.5 mM β-CD, 50 μM ADP, 20 μLmL–1 luciferase reagent , 10 μg mL–1 AOX, and 1μg mL–1 of complex I (outward-facing). ATPsynthesis was stopped by the addition of 1 μg mL–1 gramicidin A.ATP synthesis was performed aspreviously described,64 Equivalent volumes of proteoliposomes were dilutedinto 10 mM MOPS (pH 7.5), 50 mM KCl containing 1 mM ATP, 1 mM MgSO4, 2 mM K2SO4, 200 μM phosphoenolpyruvate(PEP), 50 μg mL–1 lactate dehydrogenase (LDH)from bovine heart, 40 μg mL–1 pyruvate kinase(PK) from rabbit muscle, and 2 μM piericidin A to prevent complexI mediated NADH oxidation.ATP hydrolysiswas performedat 32 °C using a coupled assay system that oxidizes NADH (measuredat 340 and 380 nm) in response to the production of ADP.2 ACMA assays, AOX was added at 5 μg mL–1 andproton pumping initiated by addition of 500 μM NADH. Δpwas dissipated by the addition of 10 μg mL–1 alamethicin. For ATP hydrolysis, 1 mM MgSO4 was addedto the buffer and proton pumping was initiated by the addition of1 mM ATP.ACMA assays to assessproton pumping qualitatively were performed using a RF-5301PC spectrofluorometer(Shimadzu) at 32 °C. Proteoliposomes were transferred to buffer(10 mM MOPS (pH 7.5), 50 mM KCl) containing 0.5 μM ACMA and0.1 μM valinomycin with constant stirring. For NADH:O


In the published article, there was an error in the author list, and authors Abudureyimujinag Aili, Yao Xue Yan were erroneously excluded from co-first authorship. Jie Wen, Abudureyimujiang Aili, Yao Xue Yan should be co-first authors. The corrected author list appears below.1*†, Abudureyimujiang Aili2†, Yao Xue Yan3†, YuLin Lai4, Shaoqing Niu4, Shasha He4, Xiaokai Zhang5, Guixiong Zhang5 and Jiaping Li5*Jie Wen†These authors have contributed equally to this work and share first authorship.In the published article, there was an error in the Funding statement. There was a grammatical error and two funders were missed. The correct Funding statement appears below.This work was Funded by China Postdoctoral Science Foundation to Jie Wen (2020M683115), the National Natural Science Foundation of China , the Guangdong Medical Research Foundation (No. A2022121 to Shaoqing Niu), the Major Science and Technology Program of Guangdong Province (No. 2020B0101130016 to Jiaping Li) and the Guangzhou Key research and development Projects (No. 202103000021 to Jiaping Li).Methods,Malondialdehyde (MDA) measurement, line 239-245 in Paragraph 4 of Page 6. This sentence previously stated:In the published article, there was an error in the description of Malondialdehyde (MDA) measurement. A correction has been made to “The calculation method was as follows:Δod was as follows calculation mΔod was as follows calculation mΔod waA600 (sample)-A600 (blank)MDA (nmol/mgprot) =5 × 12.9ot) =55) =5t) =5A600 .”The corrected sentence appears below:“ΔA450=A450 (sample)-A450 (blank)ΔA532=A532 (sample)-A532 (blank)ΔA600=A600 (sample)-A600 (blank)MDA (nmol/mgprot) =5×(12.9×(ΔA532-ΔA600)-2.58×ΔA450)÷Spr .”The authors apologize for these errors and state that they do not change the scientific conclusions of the article in any way. The original article has been updated.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Although various treatments such as surgery and chemotherapy exist for advanced or unresectable HCC, most patients suffer from intractable diseases, having a poor prognosis. While immunotherapy using immune checkpoint inhibitors was recently proposed for HCC, only a small percentage of patients respond. Thus, there remains an unmet need for the development of therapeutic agents for the treatment of liver cancer. Here, we presented multi-RTKi MBP-11901, an innovative targeted anticancer agent for HCC, suggesting it as a new therapeutic strategy for the treatment of liver cancer.Hepatocellular carcinomas (HCCs) are aggressive tumors with a poor prognosis. Approved first-line treatments include sorafenib, lenvatinib, and a combination of atezolizumab and bevacizumab; however, they do not cure HCC. We investigated MBP-11901 as a drug candidate for HCC. Cell proliferation and cytotoxicity were evaluated using normal and cancer human liver cell lines, while Western blotting and flow cytometry evaluated apoptosis. The anticancer effect of MBP-11901 was verified in vitro through migration, invasion, colony formation, and JC-1 MMP assays. In mouse models, the tumor volume, tumor weight, and bodyweight were measured, and cancer cell proliferation and apoptosis were analyzed. The toxicity of MBP-11901 was investigated through GOT/GPT and histological analyses in the liver and kidney. The signaling mechanism of MBP-11901 was investigated through kinase assays, phosphorylation analysis, and in silico docking simulations. Results. MBP-11901 was effective against various human HCC cell lines, leading to the disappearance of most tumors when administered orally in animal models. This effect was dose-dependent, with no differences in efficacy according to administration intervals. MBP-11901 induced anticancer effects by targeting the signaling mechanisms of FLT3, VEGFR2, c-KIT, and PDGFRβ. MBP-11901 is suggested as a novel therapeutic agent for the treatment of advanced or unresectable liver cancer. Hepatocellular carcinoma (HCC) is a dominant health problem. Detection of HCC at an early stage is difficult and most tumors are detected at an advanced stage due to their rapid progression and high metastasis, resulting in a poor prognosis for affected individuals. The main options for the clinical treatment of HCC include surgery, radiation therapy, transarterial chemoembolization, and molecular targeted therapy. First-line drugs include sorafenib (approved in 2007) and lenvatinib (approved in 2018), as well as the most recently approved combination therapy of atezolizumab and bevacizumab (approved in 2020). In addition, a number of second-line or tertiary drugs, which are targeted small molecular drugs, such as cabozantinib (2019), regorafenib (2017), the monoclonal antibody ramucirumab (2019), and the immune checkpoint inhibitors nivolumab (2017) and pembrolizumab (2018), have been approved by the U.S. Food and Drug Administration (FDA) as a follow-up treatment for patients pretreated with sorafenib ,4. Thus,®), was reported to prolong the survival period of patients with advanced HCC [In general, the pathogenesis of HCC includes the induction of tumor growth, metastasis, and angiogenesis through the binding of growth factors to cell surface receptors, which activate the proliferative signaling system. The development of therapeutics targeting such signal transduction pathways has been actively studied over the last years. For instance, a multiple kinase inhibitor (TKI), sorafenib  was obtained from ATCC . RPMI-1640 medium was obtained from Welgene . Fetal bovine serum (FBS) was obtained from Hyclone . Antibiotic–antimycotic solution was obtained from Hyclone (Cat. SV30079.01). Antibodies against β-actin (Cat. sc-47778) were purchased from Santa Cruz Biotechnology . Antibodies against phospho-FLT3 (Cat. #3461), Ras (Cat. #3965), phospho-c-Raf (Cat. #9421), phospho-MEK1/2 (Cat. #9121), phospho-ERK1/2 (Cat. #9101), phospho-Akt (Cat. #9271), phospho-mTOR (Cat. #5536), phospho-VEGFR2 (Cat. #3770), caspase-3 (Cat. #9662), cleaved caspase-3 (Cat. #9661), and PARP (Cat. #9542) were purchased from Cell Signaling Technology . Phospho-PDGFRβ (Cat. LF-PA0035) was obtained from BioVendor Laboratory Medicine Inc. .® HB-8065™) and Hep3B (HBV(+), p53 null, ATCC® HB-8064™) were obtained from ATCC. Huh-7 (HBV(−), p53 mut., KCLB No. 60104) and PLC/PRF.5 (HBV(+), p53 mut., KCLB No. 28024) cells were obtained from the Korean Cell Line Bank .All cancer cell lines and corresponding normal cell lines were purchased from the American Type Culture Collection (ATCC). Hepatocellular carcinoma cell lines, HepG2 (HBV(−), p53 wt, ATCC® HTB-38™), adenocarcinoma human alveolar basal epithelial cells , and prostate cancer cells  were cultured in RPMI1640 medium. Human breast adenocarcinoma cells , rattus brain glioma cells , and human renal carcinoma cells  were cultured in Dulbecco’s modified Eagle’s medium . Mouse brain neural cells  and human cervix adenocarcinoma cells  were cultured in EMEM (ATCC® 30-2003™), while human embryonic kidney cells  were cultured in minimum essential medium . All media contained 10% (v/v) FBS and antibiotic–antimycotic solution (1 mL per 100 mL of cell culture medium). Mouse liver normal cells  were cultured in Dulbecco’s modified Eagle medium/nutrient mixture F-12  containing 10% FBS (Hyclone), 1× ITS , 40 ng/mL dexamethasone , and 1% antibiotic–antimycotic solution (Hyclone). Human breast epithelial cells  were cultured in DMEM/F-12 (WelGENE) supplemented with 5% horse serum , 20 ng/mL epidermal growth factor , 500 μg/mL hydrocortisone (Sigma-Aldrich), 100 ng/mL cholera toxin (Sigma-Aldrich), 10 μg/mL insulin , and 1% antibiotic–antimycotic solution (Hyclone). Human colon normal epithelial cells  were cultured in DMEM/F-12 (WelGENE) supplemented with 10% FBS (Hyclone), 10 ng/mL cholera toxin (Sigma-Aldrich), 20 ng/mL epidermal growth factor , 100 μg/mL hydrocortisone (Sigma-Aldrich), 1× ITS  and 1% antibiotic–antimycotic solution (Hyclone). HepG2 and Hep3B were cultured in EMEM medium, whereas Huh-7 and PLC.PRF.5 cells were cultured in RPMI-1640 medium. All media contained 10% (v/v) FBS and antibiotic–antimycotic solution (1 mL per 100 mL of cell culture medium). Cells were maintained at 37 °C in a humidified incubator under an atmosphere of 5% CO2.Human colorectal adenocarcinoma cells . After attachment and stabilization of cells for 24 h, the medium was switched with fresh containing various concentrations  of MBP-11901, and cells were incubated for 22 h. Next, CCK-8 solution was added to each well, and cells were incubated for another 2 h. Absorbance was then measured at 450 nm using a microplate reader . The IC50 and log IC50 values were calculated using GraphPad Prism . All experiments were independently performed 3 times. The graph of log IC50 values represents average values.Cell proliferation was detected using the Cell Counting Kit-8  according to the manufacturer’s instruction. To evaluate cytotoxicity, cells were seeded in a 96-well plate  mixture was placed on the filter membrane in the upper chamber and incubated  to seed cells. Cells were exposed to MBP-11901 and sorafenib for 2 h. Then, the medium in the upper chamber was changed to serum-free medium, whereas the medium in the lower chamber was changed to a medium containing 10% FBS. The medium was replaced once every 2–3 d. Following 6 d incubation, the upper surfaces of the filters were scraped 5 times with cotton swabs to remove nonmigrated cells, whereas cells crossing the membrane into the lower chamber were stained with 1% (w/v) crystal violet for 30 min. Crystal violet was removed and cells were washed with tap water. For the migration assay, after drying at 25 °C, 10% acetic acid solution was added to cells, and left to react in a shaker for 30 min. Absorbance was measured at 590 nm using a microplate reader . For the invasion assay, the number of migrated cells was counted.For the migration assay, HepG2 cells were seeded into the upper Transwell chamber at a density of 2 × 106 HepG2 cells were suspended in 100 mL PBS, and then 200 mL 95% ethanol was added while vortexing. Cells were incubated at 4 °C for 1 h, washed with PBS, and resuspended in 250 mL 1.12% sodium citrate buffer  supplemented with 12.5 μg RNase. Incubation was continued at 37 °C for 30 min. Cellular DNA was then stained by applying 250 mL propidium iodide (50 μg/mL) for 30 min at 25 °C. Stained cells were analyzed on a FACScan flow cytometer for relative DNA content based on increased red fluorescence.Approximately 1.5 × 104 cells per well and AML12 cells at 2.0 × 104 cells per well in 96-well plates. After 24 h of incubation, MBP-11901 at indicated concentrations was added to each well in a final volume of 100 μL. After 24 h, floating cells in the supernatants were removed by centrifugation at 600× g for 5 min. Control cells were lysed by adding 10 µL lysis buffer before centrifugation. Each supernatant (10 µL) was transferred to a new well in a 96-well plate. Finally, 100 µL LDH reaction mixture (1:50 ratio of WST substrate to LDH assay buffer) was added and samples were incubated at 25 °C for 30 min. Absorbance at 450 nm was measured using a microplate reader. Three independent experiments were performed in duplicate at different time points.The LDH assay was performed using the D-Plus™ LDH cell cytotoxicity assay kit . Briefly, HepG2 cells were seeded at 1.2 × 104 cells/well and incubated for 24 h. Cells were treated with MBP-11901 and sorafenib for 2 h and media was changed with fresh one. Cells were incubated for 8 d and the medium was replaced once every 2–3 d. The medium was carefully removed, cells were washed with PBS, and fixed with 100% methanol for 30 min. After removing methanol, cells were stained with 0.5% (w/v) crystal violet for 30 min, and washed with tap water. The plate was dried at 25 °C and images were captured under a microscope . The stained area was automatically measured using ImageJ. This experiment was independently performed 3 times. The area of the colony was calculated as a percentage of the total area of the well.HepG2 cells were seeded in a 6-well plate at a density of 2 × 102 dish at a density of 7.5 × 105 cells/dish and incubated for 24 h. Cells were treated with MBP-11901 for 2 h and media was changed with fresh one. Briefly, after 2 d incubation, cells were washed with 1× dilution buffer, and incubated with 1 μM JC-1 in 1× dilution buffer for 30 min at 37 °C. Cells were then washed 3 times with 1× dilution buffer. Fluorescence was measured under a fluorescence microscope  using an excitation filter of 530 nm ± 15 nm and an emission filter of 590 nm ± 17.5 nm. The fluorescence intensity of immunostaining was quantified using ImageJ.Alterations in MMP were investigated utilizing the cationic dye tetraethylbenzimidazolylcarbocyanine iodide (JC-1) according to the manufacturer’s instructions . HepG2 cells were seeded in a 35 mmSectioned tumor tissues (5 μm) were blocked with TBS supplemented with 5% normal goat serum (NGS) and 5% BSA for 2 h, and incubated overnight with primary antibodies  in blocking solution (5% NGS and BSA) at 4 °C. Sections were then washed 3 times in TBS, and incubated for 1 h in the presence of Alexa Fluor-conjugated IgG-labeled secondary antibodies . Sections were washed and mounted with Vectashield mounting medium containing DAPI . Images were captured using a fluorescence microscope  and fluorescence intensity of immunostaining was quantified using ImageJ.Sectioned tumor tissues (5 μm) were deparaffinized in xylene  and rehydrated through an ethanol series. Endogenous peroxidase activity was inactivated by incubation in 0.3% H2O2 (Sigma-Aldrich) in methanol for 10 min. Sections were then rinsed in 0.1 M TBS (pH 7.4) and boiled in citrate buffer (pH 6.0) containing 0.03% Tween-20 for 4 min. Finally, sections were incubated with blocking solution (5% NGS and BSA in TBS) at 25 °C for 1 h, and subjected to indirect immunohistochemistry using an antibody against anti-Ki-67(diluted 1:100) for 1 h. For the negative control, the primary antibody was omitted and slides were incubated with a blocking solution. Sections were then incubated with HRP-conjugated anti-rabbit IgG for 1 h at 25 °C, stained with VECTOR1 NovaRED , and counterstained with hematoxylin . Sections were dehydrated, cleared, and mounted with Permount . Images were captured under a fluorescence microscope  and the fluorescence intensity of immunostaining was quantified using ImageJ.Male Balb/c-nude mouse weighing 20–22 g  were used for all experiments. Animals were housed under controlled environmental conditions in an ambient temperature of 23 ± 1 °C and relative humidity of 50 ± 10% under a 12 h light/dark cycle and were given ad libitum access to food and water. All animal experiments were approved by and performed in accordance with the guidelines issued by the Institutional Animal Care and Use Committee (IACUC) of Kyungpook National University .6) were suspended in 100 μL serum free EMEM:Matrigel (1:1) and then subcutaneously injected into the upper-left flank region of nude mice. When the tumor reached a mean size of about 100–150 mm3, mice were randomized into each experimental group according to tumor size. Mice were orally administered Nexavar® , MBP-11901 , or saline as control (once every 2 d). Tumor size was measured with a caliper rule twice a week and body weight was measured simultaneously. The tumor size was calculated as follows: tumor size (mm3) = (L × W2)/2, where L is the longest and W is the shortest radius of the tumor in millimeters. At the end of the experiment, mice were euthanized, blood samples were collected via the abdominal vena cava, and tumors were removed and weighed.For the generation of a tumor xenograft, HepG2 cells  for histologic examination and immunohistochemical staining or immediately frozen at −80 °C in a deep-freezer and stored until protein extraction.x/V1, where Vx is the volume in mm3 at a given time and V1 at the start of treatment. Mean TGI (%) and standard deviation were calculated for each group.Drug efficacy was expressed as the percentage of tumor growth inhibition (% TGI), calculated using the equation: 100 − (T/C × 100), where T is the mean relative tumor volume (RTV) of the treated tumor and C is the mean RTV in the vehicle control group at the time of sacrifice. RTV = VThe levels of GOT and GPT were measured using enzymatic assay kits . The collected blood was allowed to clot and the serum, which was separated by centrifugation at 4000 rpm for 15 min after 2 h at 25 °C, was used for analysis.7. The first group served as the saline control. The second and third groups were orally administered MBP-11901 (82 mg/kg) and Nexavar® (164 mg/kg) once every other day for 4 weeks. Tumor progression/regression was recorded once weekly by quantifying the bioluminescence signals. Experimental animals were sacrificed by CO2 inhalation.The orthotopic HCC mouse model was established by placing a small piece of tumor-derived HepG2-Luc cells. Mice were randomly distributed into 3 groups upon bioluminescence signal reaching 10Tumors were fixed in 4% PFA for 72 h, and treated with an alcohol concentration gradient , xylene , and paraffin for 30 min, respectively. Sectioned tumor tissues (5 μm) were treated with xylene and an alcohol concentration gradient  for 1 h and 10 min, respectively, in an oven at 65 °C. Hematoxylin and eosin (H&E) staining  was performed according to the manufacturer’s instructions and stained tissues were observed under a microscope . For Sirius Red staining, tissues were immersed in Picro Sirius Red Stain  for 30 min, washed for 1 min in 0.1 N HCl, followed by dehydration and mounting of slides.https://www.rcsb.org) (accessed on 21 July 2021) under the PDB IDs of 4RT7, 3VHE, 3SLS, and 4U0I, respectively. Due to the lack of an experimentally revealed structure, the three-dimensional structure of PDGFRβ was obtained from the AlphaFold Protein structure database  (accessed on 21 July 2021). These structures were optimized for in silico simulation using the Prime protein modeling application [Crystal structures of FLT3, VEGFR2, MEK5, and KIT were obtained from the RCSB Protein Data Bank website . Binding constants (Kd) were calculated by DiscoverX using KdELECT technology, a widely used assay for comparing selectivity across the kinome. The DiscoverX platform reports the thermodynamic Kd to facilitate direct comparison of inhibitor affinity across the kinome, independent of ATP concentration.p < 0.05 was considered statistically significant.Data were evaluated using one-way or two-way ANOVA followed by Tukey’s or Dunnett’s test. Analyses were performed using GraphPad Prism . Data are expressed as the mean ± SD (standard deviation) or standard error of the mean (SEM), and 50) values of MBP-11901 in HepG2, Hep3B, Huh-7, and PLC/PRF5 were 5.16 ± 1.37, 16.82 ± 0.88, 29.41 ±1.13, and 18.55 ±0.81 μM, respectively. As HepG2 cells exhibited the lowest IC50 values and were the most sensitive to MBP-11901 among the four cancer cell lines tested, we used HepG2 cells in our subsequent experiments.We conducted a cell-based screening, examining the effects of MBP-11901 on the viability of various tumor and normal cells . We usedTo test whether treatment with MBP-11901 induces the apoptosis of hepatocellular carcinoma cells, we evaluated the levels of protein expression of apoptosis markers in HepG2 cells, such as cleaved PARP1 and active caspase-3. Our results showed that treatment with MBP-11901 led to high levels of cleaved PARP and active caspase-3 in a time- and dose-dependent manner A,B, sugg50 = 13.44 ± 2.77 μM), whereas the cytotoxicity of MBP-11901 was significantly reduced (IC50 = 65.70 ± 1.62 μM), as indicated by the LDH assay  and a normal liver cell line, AML12. We observed that MBP-11901 showed a similar level of anticancer effect in liver cancer cell lines as that shown by sorafenib . HoweverDH assay B,C. In aDH assay D–F. The DH assay F.We also measured the mitochondrial membrane potential of cells exposed to MBP-11901 using JC-1 staining. As shown in ® (164 mg/kg) group showed a tumor growth inhibition of 38.77 ± 0.37% (® group (164 mg/kg) was 0.144 ± 0.073 g  was steadily increased without significant differences between the two groups . Presumably, for this reason, it has the advantage of exhibiting sufficient efficacy even at low concentrations and with no associated toxicity. We are currently conducting additional research for conducting phase 1 clinical trials.In conclusion, the treatment trend for HCC has been changing. In addition to sorafenib, administration of lenvatinib has increased, as well as treatment options, including the addition of atezolizumab and bevacizumab combination therapy. However, compared with other carcinomas, HCC is difficult to cure, has limited treatment options, a poor prognosis, and a high recurrence rate, making it a highly unmet medical problem. Animal experiments confirmed the high reactivity and perfect therapeutic effect of MBP-11901, suggesting that it could be used as a new therapeutic strategy for the treatment of advanced or unresectable HCC.