Glycol chitosan nanoparticles (CNPs) have emerged as an effective drug delivery system for cancer diagnosis and treatment. Although they have great biocompatibility owing to biodegradable chemical structure and low immunogenicity, sufficient information on in vivo toxicity to understand the potential risks depending on the repeated high-dose have not been adequately studied. Herein, we report the results of in vivo toxicity evaluation for CNPs focused on the number and dose of administration in healthy mice to provide a toxicological guideline for a better clinical application of CNPs.The CNPs were prepared by conjugating hydrophilic glycol chitosan with hydrophobic 5β-cholanic acid and the amphiphilic glycol chitosan-5β-cholanic acid formed self-assembled nanoparticles with its concentration-dependent homogeneous size distributions (265.36–288.3 nm) in aqueous condition. In cell cultured system, they showed significantly high cellular uptake in breast cancer cells (4T1) and cardiomyocytes (H9C2) than in fibroblasts (L929) and macrophages (Raw264.7) in a dose- and time-dependent manners, resulting in severe necrotic cell death in H9C2 at a clinically relevant highly concentrated condition. In particular, when the high-dose (90 mg/kg) of CNPs were intravenously injected into the healthy mice, considerable amount was non-specifically accumulated in major organs  after 6 h of injection and sustainably retained for 72 h. Finally, repeated high-dose of CNPs  induced severe cardiotoxicity accompanying inflammatory responses, tissue damages, fibrotic changes and organ dysfunction.This study demonstrates that repeated high-dose CNPs induce severe cardiotoxicity in vivo. Through the series of toxicological assessments in the healthy mice, this study provides a toxicological guideline that may expedite the application of CNPs in the clinical settings.The online version contains supplementary material available at 10.1186/s12951-023-01824-3. In the field of medicine, nanotechnology has shown great potential as a means for targeted drug delivery in cancer treatment –3. A var2 based on doxorubicin contents without any mention of the equivalent dose of materials for liposome formulation [Such negative consequence of low tumor-targeting efficiency of nanoparticles is directly related to manufacturing, drug price, therapeutic efficacy and especially toxicity . Althougmulation . HoweverRecently, chitosan polymers or its derivatives have been widely utilized in formulating nanoparticles over the last few decades owing to its high biodegradability, biocompatibility and low immunogenicity . In partGlycol chitosan nanoparticles (CNPs) were successfully prepared by conjugating hydrophobic 5β-cholanic acid (CA) to hydrophilic glycol chitosan polymer (GC) in the in the presence of chemical catalysts, wherein 150 ± 4.5 molecules of CA are conjugated into each GC polymer , and three types of normal cells, including rat cardiomyocytes H9C2), mouse fibroblasts (L929) and macrophages (Raw264.7). For subcellular tracking, the CNPs were further modified with near-infrared fluorescent dye, Cy5.5, with an average of 4.8 ± 0.7 molecules per CA-GC conjugates. In previous study, it was confirmed that modification with Cy5.5 is not influence on physicochemical and biocompatible characteristics of CNPs , 39. Whe, mouse f, and thrt1/2) of 13.8 ± 0.5 h in mice treated with 10 mg/kg of Cy5.5-CNPs, whereas a detectable amount of Cy5.5-CNPs remained for 72 h in the body with t1/2 of 21.4 ± 0.2 h when the administered dose was increased to 22.5 mg/kg  was also increased 12.4–12.64-fold and 5.2–5.27-fold compared to 10 or 22.5 mg/kg of Cy5.5-CNPs, respectively. These results indicate that considerable amount of CNPs are remained in the body for a long time along to the dose increase, which can potentially induce in vivo toxicity. Next, PD profiles after treatment with low- or high-dose of Cy5.5-CNP were evaluated by analyzing accumulation in major organs via ex vivo fluorescence imaging. As expected, only a small quantity of Cy5.5-CNPs was observed in the major organs  after 6 h of injection with 10 mg/kg, but considerable amount was accumulated in those organs in mice treated with 22.5 or 90 mg/kg Cy5.5-CNPs  profiles of CNPs in the low- and high-dose were assessed in healthy BALB/c mice. To evaluate the PK profiles, three different doses of Cy5.5-CNPs  were intravenously injected into the mice, and blood samples were collected at pre-determined time points. Interestingly, Cy5.5-CNPs in bloods were detected up to 48 h of injection with a half-life  saline; (ii) 10 mg/kg single-dose; (iii) 10 mg/kg multi-dose 10 mg/kg*3); (iv) 22.5 mg/kg single-dose; (v) 22.5 mg/kg multi-dose (22.5 mg/kg*3); (vi) 90 mg/kg single-dose; (vii) 90 mg/kg multi-dose (90 mg/kg*3). In the case of the multi-dose groups, mice were treated with Cy5.5-CNPs three times with 2 days-intervals. After 7 days of treatment, the histopathological changes in the organs stained with H&E were observed under microscopy Fig. A. The re; (iv) 22Encouraged by organ toxicity study, we performed additional histology with analysis in molecular levels to assess the cardiotoxicity after repeated high-dose of CNPs treatment at a whole region of heart tissues. Likewise, the mice were treated with 10, 22.5 or 90 mg/kg Cy5.5-CNPs three times with 2 days-intervals, and heart tissues were collected on day 7 after treatment. As expected, detectable fluorescence signals of Cy5.5-CNPs were not observed in whole heart tissues from mice treated with 10 mg/kg multi-dose due to its in vivo clearance, whereas Cy5.5-CNPs were clearly observed after treatment with 22.5 mg/kg multi-dose; notably, those accumulated in whole heart tissues were considerably increased in 90 mg/kg multi-dose group  focused on the number and dose of administration in healthy mice to provide reliable toxicological data that is crucial for clinical applications. First, we found that CNPs highly accumulated in cardiomyocytes compared to fibroblasts and macrophages, resulting in severe necrotic cell death. This in vitro cytotoxicity study can provide a minimum guideline for safe use of CNPs, but nanoparticles may show unpredictable adverse effects in vivo that include organ toxicity, genotoxicity, and immunotoxicity. Therefore, PK/PD profiles of CNPs in the low- and high-dose were carefully evaluated in healthy mice. The low-dose (10 mg/kg) of CNPs showed relatively fast in vivo clearance within 48 h, whereas high-dose (22.5 and 90 mg/kg) administration resulted in nanoparticle residue for a long-time in body. Hence, mice treated with high-dose of CNPs showed high non-specific accumulation of nanoparticles in major organs, which was sustainably retained with no excretion. As a result, high-dose of CNPs induced severe cardiotoxicity accompanying inflammatory responses, tissue damages, fibrotic changes and organ dysfunction; furthermore, these symptoms were more worsen at the increase of dose number. Through these series of toxicological assessments, this study provides a toxicological guideline that may expedite the application of CNP in the clinical settings.Glycol chitosan -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were purchased from Sigma Aldrich . Flamma 648 NHS ester was purchased from BioActs . Cell counting kit-8 (CCK-8) was purchased from Vitascientific . Tem grid (Carbon Film 200 Mesh copper) was purchased from Electron Microscopy Sciences . H9C2 (Rat cardiomyocyte), L929 (mouse fibroblast) and Raw264.7 (macrophage) cell lines were purchased from American Type Culture Collection . Fetal bovine serum (FBS), streptomycin, penicillin and RPMI 1640 medium were purchased from WELGENE Inc. . Antibodies against mouse TNF-α , mouse IL1b, mouse IL6 and mouse β-actin were purchased from BioLegend .Glycol chitosan nanoparticles (CNPs) were prepared by conjugation of 5β-cholanic acid to the primary amine groups of the glycol chitosan (GC). Briefly, 5β-cholanic acid , NHS , EDC  and GC  were dissolved in distilled water/methanol mixture . The resulting solution was stirred for 12 h at 37 °C, followed by dialysis for 3 days in distilled water/methanol mixture (1:3 v/v) using a cellulose membrane . Then, the solution was further dialyzed for a day in distilled water and lyophilized to yield glycol chitosan-5β-cholanic acid conjugates. The near-infrared (NIRF) fluorescence dye, Cy5.5, was additionally introduced in CNPs. Briefly, Cy5.5-NHS  was dissolved in 1 ml of DMSO and added dropwise to the CNPs (220 mg) in DMSO solution. The reaction buffer was stirred for 48 h at 37 °C, dialyzed in the distilled water condition using a cellulose membrane  for a day, and lyophilized for 3 days to obtain Cy5.5-CNPs. Then, it was dispersed in distilled water or mouse serum, followed by analysis of size distribution using a Zetasizer Nano ZS . The morphology of CNPs were observed by using transmission electron microscopy (TEM) .4 4T1, H9C2, L929 and Raw264.7 cells were seeded into 96-well cell culture plates. Then, each cell was treated with different concentration of Cy5.5-CNPs ranging from 0 to 900 μg/ml. After 24 h of incubation, the cells were further incubated with cell culture medium containing 10% of CCK-8 solution for 20 min. Finally, the cell viability was analyzed using a microplate reader  with a wavelength of 450 nm.The cellular uptake of Cy5.5-CNPs was investigated in three types of normal cells, such as breast cancer cells (4T1), rat cardiomyocytes (H9C2), mouse fibroblasts (L929) and macrophages (Raw264.7). Each cell was incubated with Cy5.5-CNPs  for 4 or 24 h. After treatment, cells were fixed with 4% paraformaldehyde for 15 min, and stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. The cellular uptake was observed using a Leica TCS SP8 confocal laser-scanning microscope . Quantitative analyses of the fluorescence images were performed using ImageJ software . The cytotoxicity was assessed by the Cell Counting Kit-8 (CCK-8) assay. Briefly, 5 × 10t1/2) were calculated using a WinNonlin software. The pharmacodynamic (PD) profiles were also assessed in mice by ex vivo fluorescence imaging of major organ  after 6 or 72 h of injection with Cy5.5-CNPs . The fluorescence intensities in the major organs were quantified using a Living Image software .The 5-week male BALB/c mice were purchased from NaraBio . Mice were bred under pathogen-free conditions in the Korea Institute of Science and Technology (KIST). All experiments with animals were performed in compliance with the relevant laws and institutional guidelines of Institutional Animal Care and Use Committee  in Korea Institute of Science and Technology (KIST). First, pharmacokinetic (PK) profiles were assessed after intravenous injection with Cy5.5-CNPs . After treatment, blood samples were collected from mice by cardiac puncture after deep anesthesia at pre-determined times and centrifuged at 2200 rpm to obtain plasma, followed by analysis of fluorescence intensity using an IVIS Lumina Series III system . The area under the curves (AUC) and half-life  were intravenously injected into BALB/c mice with single- or multi-dosage (three times). On day 7 after treatments, major organs  were collected from mice, and structural abnormalities in organ tissues were assessed by staining with H&E. In the case of hematological analyses, blood samples were collected from the mice on day 7 and centrifuged at 2200 rpm to obtain plasma. The following factors in blood samples were measured; alanine aminotransferase (ALT), blood urea nitrogen (BUN), alkaline phosphatase (ALP), aspartate Aminotransferase (AST), creatine kinase (CK) and troponin I. The cardiotoxicity by Cy5.5-CNPs was further analyzed after multiple-dosage. The heart tissues were collected from mice after treatment with 10, 22.5 or 90 mg/kg of Cy5.5-CNPs three times. The accumulation of Cy5.5-CNPs in heart tissues was observed using a Leica TCS SP8 confocal laser-scanning microscope. Collagen fiber in heart tissues were stained with Masson's trichrome. Briefly, heart tissues were incubated in Bouin's fixative for 30 min at 56 °C, and the nuclei were co-stained with Weigert's iron hematoxylin. Then, cytoplasm was stained with Biebrich scarlet-acid fuchsin, and then differentiated in phosphomolybdic–phosphotungstic acid. The collagen matrix in heart tissues was stained with aniline blue solution. The collagen in heart tissues were quantitatively analyzed using an Image Pro software, and collagen contents were presented in proportion to the total area of heart tissues.The statistical significance between two groups was analyzed using Student’s t-test. One-way analysis of variance (ANOVA) was performed for comparisons of more than two groups, and multiple comparisons were analyzed using the Tukey–Kramer post hoc test. Survival data was plotted as Kaplan–Meier curves and analyzed using the log-rank test. The statistical significance was indicated with asterisks  in the figures.Additional file 1: Figure S1. Synthetic route to prepare the glycol chitosan and 5β-cholanic acid conjugates. Figure S2. Detail information of average size of different concentrations of CNPs in the mouse serum. Figure S3. Detail information of average size of different concentrations of CNPs in the mouse serum (n=5). Figure S4. Flow cytometric results showing H9C2 cells stained with Annexin V/PI after treatment with CNPs for 24 h. Figure S5. Fluorescence image of major organs from mice treated with 90 mg/kg of CNPs for 7 days. Fluorescence intensities were normalized with the results of Figure 3B and 3C. Figure S6. Excretion profile of Cy5.5-CNPs after 90 mg/kg treatment. The urines were collected from the mice at the indicated time points, followed by analysis of Cy5.5 fluorescence intensity using HPLC. Figure S7. Detail information of hematological parameters on day 7 after single- or multi-dose of 10, 22.5 or 90 mg/kg CNPs. Figure S8. Detail information of complete cell count results on day 7 after single- or multi-dose of 10, 22.5 or 90 mg/kg CNPs (n=5). Figure S9. Uncropped images of western blot results in Figure 5E.


Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa compared to the first- and second-generation compounds A22 and CBR-4830, respectively. Large inocula of these four pathogens are associated with a low frequency of resistance (FOR) to TXH11106. The enhanced bactericidal activity of TXH11106 relative to A22 and CBR-4830 correlates with a correspondingly enhanced capacity to inhibit E. coli MreB ATPase activity via a noncompetitive mechanism. Morphological changes induced by TXH11106 in E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa provide further evidence supporting MreB as the bactericidal target of the compound. Taken together, our results highlight the potential of TXH11106 as an MreB inhibitor with activity against a broad spectrum of Gram-negative bacterial pathogens of acute clinical importance.The emergence of multi-drug-resistant Gram-negative pathogens highlights an urgent clinical need to explore and develop new antibiotics with novel antibacterial targets. MreB is a promising antibacterial target that functions as an essential elongasome protein in most Gram-negative bacterial rods. Here, we describe a third-generation MreB inhibitor (TXH11106) with enhanced bactericidal activity versus the Gram-negative pathogens  Acinetobacter baumannii, Pseudomonas aeruginosa, and various Enterobacteriaceae (including Escherichia coli and Klebsiella pneumoniae) [Ever since their discovery in the early 20th century, antibiotics have been the principal tools for treating bacterial infections. However, over the past several decades, antibiotic resistance has risen alarmingly ,2. The Wumoniae) ,6.Addressing the threat posed by MDR bacterial pathogens requires the development of new antibiotics. The majority of antibiotics in current clinical use target bacterial processes underlying cell wall, protein, or DNA synthesis . This liMreB is a highly conserved essential protein directly involved in cell wall formation and maintenance of most rod-shaped bacteria ,15. MreBE. coli growth [Caulobacter crescentus MreB (CcMreB) [P. aeruginosa relative to A22 [Iwai et al. identified the benzylisothiourea derivative A22  as an ini growth . A22 hasi growth ,22,23. L(CcMreB) . CBR-483e to A22 .E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa. The enhanced antibacterial activity of TXH11106 correlates with a correspondingly enhanced inhibition of E. coli MreB ATPase activity, with this inhibition being non-competitive in nature. Significantly, TXH11106 is also associated with a very low frequency of resistance (FOR). In the aggregate, our results highlight TXH11106 as a lead MreB inhibitor worthy of further development.In this study, we describe TXH11106 as a third-generation MreB inhibitor that incorporates the dichloro-substituted ring functionality of A22 (ring A) and the cyclohexylamine substituent (ring C) of CBR-4830 . A22 was obtained from Cayman Chemical Company, and CBR-4830 was synthesized as described previously [eviously . Luria–BH-carbazol-1-one (3) , ammonium acetate , and sodium cyanoborohydride  were dissolved in ethanol (10 mL). The reaction mixture was stirred overnight at 60 °C. After removal of solvent, the reaction mixture was then diluted with ethyl acetate. The organic layer was washed with 10% sodium hydroxide and brine, and then dried over sodium sulfate. The organic layer was then concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0–10% methanol/dichloromethane + 0.1% NH4OH) to give the product 5,6-dichloro-2,3,4,9-tetrahydro-1H-carbazol-1-amine (TXH11106) (4) as a white solid . 1H NMR (300 MHz) (CD3OD) δ 7.18 , 7.09 , 4.08–4.03 , 3.04–2.98 , 2.15–1.99 , 1.83–1.75 , 1.72–1.65 ; LC/MS RT = 2.72 (M-H-: 253/255).As depicted in (i)H-carbazol-1-one (3):5,6-Dichloro-2,3,4,9-tetrahydro-1The requisite intermediates were prepared as follows:tert-butyl 5,6-dichloro-1-oxo-1,2,3,4-tetrahydro-9H-carbazole-9-carboxylate (1)  in dichloromethane (20 mL), trifluoroacetic acid (4 mL) was added. The reaction mixture was stirred for an hour at room temperature. After removal of solvent, the mixture was diluted with ethyl acetate and was washed with 10% sodium hydroxide and brine. The organic layer was then dried over sodium sulfate and concentrated under reduced pressure to give the product (3) as a white solid . 1H NMR (300 MHz) (CDCl3) δ 7.90 , 7.47 , 3.33 , 2.64 , 2.24 .To a solution of (ii)tert-Butyl 5,6-dichloro-1-oxo-1,2,3,4-tetrahydro-9H-carbazole-9-carboxylate (1):H-carbazol-1-one (1) and 6,7-dichloro-2,3,4,9-tetrahydro-1H-carbazol-1-one (2) as a white solid .At 60 °C, a solution of hydrazine hydrochloride  in methanol (20 mL) was added slowly to a mixture of 1,2-cyclohexanedione  in acetic acid (44 mL) and concentrated hydrochloric acid (16 mL). The reaction mixture was stirred for 3 h at 60 °C. The reaction mixture was then diluted with ethyl acetate and washed with water followed by brine. The organic layer was then dried over sodium sulfate and concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0–20% ethyl acetate/hexane) to give a mixture of two regio-isomers, 5,6-dichloro-2,3,4,9-tetrahydro-1tert-butyl 5,6-dichloro-1-oxo-1,2,3,4-tetrahydro-9H-carbazole-9-carboxylate (1) as a white solid  (1H NMR (300 MHz) (CDCl3) δ 7.90 , 7.47 , 3.33 , 2.64 , 2.29–2.00 , 1.56 ) as well as tert-butyl 6,7-dichloro-1-oxo-1,2,3,4-tetrahydro-9H-carbazole-9-carboxylate (2) as a white solid  (1H NMR (300 MHz) (CDCl3) δ 8.23 , 7.68 , 2.91 , 2.67 , 2.29–2.21 , 1.62 ).To a solution of a mixture of two regioisomers  in tetrahydrofuran (20 mL), boc anhydride  and DAMP  were added. The reaction mixture was stirred for 3 h. The reaction mixture was then diluted with ethyl acetate and washed with saturated ammonium chloride followed by brine. The organic layer was then dried over sodium sulfate and concentrated under reduced pressure. The residue was purified on an ISCO chromatograph (0–10% ethyl acetate/hexane) to give 5 cells/mL to 96-well microtiter plates containing twofold serial dilutions of A22, CBR-4830, or TXH11106 in CAMH broth. Cultures were grown with shaking at 37 °C for 18 h, and bacterial growth was measured by recording optical density at 600 nm (OD600) using a VersaMax plate reader . MICs values were defined as the lowest compound concentration where growth was inhibited by ≥90%.MIC assays were performed in duplicate according to Clinical and Laboratory Standards Institute (CLSI) guidelines for broth microdilution assay . Briefly5 cells/mL).MBC values for each compound were determined by plating (in duplicate) 50 µL of the wells corresponding to 1×, 2×, and 4× MIC on TSA plates. Plates were incubated at 37 °C for 24 h, and counts of CFU/mL were determined. MBC values were defined as the lowest compound concentration that yielded ≥3 logs of kill relative to the initial inoculum  or 8× MIC (for P. aeruginosa). The plates were then incubated at 37 °C for 48 h and counted. In the absence of any observed colonies on the TXH11106-containing plates, the FOR was defined as ≤−1.FOR assays were performed using a large inoculum approach described previously . Briefly600 in 5 mL of CAMH broth, and TXH11106 was then added at a concentration of 1× MIC. An equivalent volume of DMSO was added to a separate culture as a vehicle control. The cultures were incubated with shaking for 3 h at 37 °C. A total of 1 to 5 mL of culture was pelleted at 15,000× g and washed twice with 1 mL of PBS. The pellets were then resuspended in 200 µL of PBS, and 8 µL was overlaid on pads of 1% agarose (in PBS) affixed to microscope slides. The bacteria were visualized by DIC microscopy using an Olympus BX50 microscope equipped with a 100× Olympus UPLSAPO oil immersion objective with a 1.40 aperture. The resulting images were captured using a QImaging Retiga R3 camera and processed using the Ocular version 2.0 software package . Image analysis and quantification was performed using the Fiji ImageJ software package.Log-phase bacteria were diluted to 0.1 ODp-value < 0.0001.The DIC microscopy results were quantified by determining the percentage of cells exhibiting a particular phenotype or morphology. Each percentage reflected an average of 5 different fields of view, with the average number of cells in each field of view ranging from 70 to 255 in the vehicle-treated groups and 110 to 258 for TXH11106-treated groups. The statistical significance of differences in cell morphology were analyzed using a one-way ANOVA test, with **** reflecting a mreB gene encoded by E. coli ATCC 25922 was PCR amplified from genomic DNA. Primers were designed to introduce an 8× His tag to the N-terminus of E. coli MreB (EcMreB). The PCR product was cloned into a pET-22b(+) plasmid  using the NEBuilder Hifi DNA assembly cloning kit  and transformed into NEB 5a electrocompetent E. coli cells (New England Biolabs). Transformed cells were plated on LB agar containing 100 μg/mL ampicillin and incubated at 37 °C overnight. Individual colonies were isolated and grown overnight in 5 mL of LB broth containing 100 μg/mL ampicillin. Plasmids were then isolated using the Monarch plasmid miniprep kit (New England Biolabs), and their sequences were verified by Sanger sequencing. The plasmids were then transformed into calcium competent E. coli BL21(DE3) cells.The 600 of 0.6, induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM, and incubated at 15 °C for 18 h. The cells were then pelleted at 5000× g and 4 °C for 15 min and subsequently resuspended in 10 mL of ice-cold Buffer A . The cells were lysed by ultrasonication on ice for 20 min at 60 W with an on/off cycle of 10 s using a Qsonica Q500 sonicator equipped with a 0.5-inch probe. Cell lysates were centrifuged at 10,000× g and 4 °C for 30 min, added to 7 mL of TALON metal affinity resin , and then incubated with gentle shaking at 4 °C for 1 h. The resin was washed with 50 mL of Buffer A and then added to a gravity flow column. A total of 15 mL of elution buffer  was added, and 500 μL fractions were collected at 4 °C. MreB-containing fractions were determined by SDS-PAGE and then dialyzed versus 4 L of dialysis buffer  with gentle stirring at 4°C overnight. Dialysates were concentrated using an Amicon Ultra-4 Ultracel 10K centrifugal filter , and protein concentration was determined with a Pierce BCA protein assay kit .Transformed cells were grown on LB agar plates containing 100 μg/mL ampicillin, and individual colonies were used to inoculate 50 mL of ampicillin-containing LB broth. The culture was grown at 37 °C overnight, with 40 mL being subsequently added to 4 L of ampicillin-containing LB broth. The resulting culture was incubated until it reached an ODmreB plasmid described above. Mutagenesis was performed using the site-directed mutagenesis kit from New England Biolabs, and the mutation was verified by sequence analysis. The pET-22b(+)-mreB(E143G) plasmid was then transformed into calcium-competent BL21(DE3) cells, and the E143G mutant EcMreB protein was expressed and purified as described above.To generate E143G mutant EcMreB, primers were designed to introduce a GAA to GGT mutation at the relevant amino acid residue in the pET-22b(+)-620 readings were corrected for the no protein background, and the standard curve was used to determine the total amount of phosphate produced by each reaction. Reaction velocities were determined from the phosphate produced over the course of 30 min. Compound concentrations that inhibit MreB ATPase activity by 50%  were determined by fitting plots of reaction velocity (v) versus compound concentration © with the following relationship:The ATPase activity of purified WT and E143G mutant EcMreB was characterized using an ATPase assay kit (Sigma-Aldrich). Briefly, 40 μL reactions in 96-well microtiter plates were initiated by adding 30 μL of 1.33 mM ATP to 10 μL of sample containing 8 µM WT EcMreB and differing concentrations of test compound . The final concentrations in each reaction well were 0 to 1000 μM compound, 2 μM EcMreB, and 1 mM ATP. Final reaction buffer conditions were 20 mM Tris-HCl pH 7.5, 40 mM NaCl, 4 mM magnesium acetate, and 0.5 mM EDTA. Negative controls included DMSO vehicle instead of compound and MreB dialysis buffer instead of protein (no protein background). A 0 to 50 μM phosphate standard curve was prepared to calculate the phosphate production in each well. All samples, controls, and standards were prepared in duplicate, and the reactions were run at room temperature for 30 min. After 30 min, the reactions were stopped by the addition of 200 μL of malachite green reagent, and the colorimetric product was measured at 620 nm using a SpectraMax M2 plate reader (Molecular Devices). Final ODmaxv is the maximal (uninhibited) reaction velocity and m is the Hill slope. The same assay was used to determine the impact of TXH11106 on the ATPase activity of E143G mutant EcMreB.In this equation, To analyze the impact of A22, CBR-4830, and TXH11106 on the catalytic activity of EcMreB, the reactions were set up and run as described above, except that compound concentrations were maintained at 30 μM and the final ATP concentrations ranged from 0.1 to 1 mM. Separate backgrounds were run for each concentration of ATP. All sample and control reactions were run in triplicate. At each ATP concentration, velocities were determined as described above, and catalytic parameters were calculated from the resulting velocities. The impact of 30 μM TXH11106 on the catalytic activity of E143G mutant EcMreB was determined as described above.E. coli (ATCC 25922), K. pneumoniae (ATCC 13883), A. baumannii (ATCC 19606), and P. aeruginosa (ATCC 27853). The MIC values of TXH11106 against these pathogens ranged from 4 µg/mL (versus E. coli) to 8 µg/mL , with these values being two- to fourfold lower than the corresponding values for CBR-4830 and 4- to 64-fold lower than the corresponding values for A22  and minimal bactericidal concentration (MBC) of TXH11106 relative to CBR-4830 and A22 against the Gram-negative pathogens  for A22 . TXH1110ruginosa . The corE. coli, K. pneumoniae, A. baumannii, and P. aeruginosa induced by differing concentrations of TXH11106. At 1× MIC, 24 h of treatment induced approximately 5 logs of kill in E. coli and 3 logs of kill in K. pneumoniae  of E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa following 48 h of exposure to TXH11106 at concentrations yielding total time-dependent kill (4× or 8× MIC). In E. coli, K. pneumoniae, and A. baumannii, no detectable colonies were observed with TXH11106 exposure at 4× MIC, resulting in FOR values ranging from <6.2 × 10−10 to <1.3 × 10−9  exhibited their expected rod shapes (n ranging from 550 to 1291) being spherical in shape and much fewer (1.9–27.9%) being rod-shaped  microscopy to examine the impact of TXH11106 treatment (at 1× MIC) for 3 h on the morphology of d shapes , with mad-shaped . The rem50 values of 14 ± 2, 49 ± 8, and 447 ± 87 µM for TXH11106, CBR-4830, and A22, respectively  EcMreB relative to A22 and CBR-4830. Like A22 and CBR-4830, TXH11106 inhibited the ATPase activity of EcMreB, as demonstrated by the concentration-dependent velocity profiles shown in ectively . Thus, Tm  compared to treatment with DMSO vehicle (Km = 147 ± 4 µM). By contrast, inhibitor treatment decreased both Vmax (from (9.66 ± 0.10) × 10−2 µM/s for DMSO vehicle to (4.46 ± 0.28) × 10−2, (6.05 ± 0.07) × 10−2, and (8.15 ± 0.19) × 10−2 µM/s for TXH11106, CBR-4830, and A22, respectively) and kcat (from (4.83 ± 0.05) × 10−2 s−1 for DMSO vehicle to (2.23 ± 0.14) × 10−2, (3.03 ± 0.04) × 10−2, and (4.08 ± 0.10) × 10−2 s−1 for TXH11106, CBR-4830, and A22, respectively). These collective effects are consistent with a non-competitive mechanism of inhibition. In addition, the impact of inhibitor treatment on kcat and Km yielded a corresponding decrease in kcat/Km from (3.29 ± 0.11) × 10−4 µM−1s−1 for vehicle to (1.56 ± 0.33) × 10−4, (2.02 ± 0.08) × 10−4, and (2.70 ± 0.21) × 10−4 µM−1s−1 for TXH11106, CBR-4830, and A22, respectively. Thus, at an equivalent concentration of 30 µM, TXH11106 reduced the kcat/Km of WT EcMreB by 2.1-fold, compared to only 1.6-fold for CBR-4830 and 1.2-fold for A22.To determine the mechanism of inhibition, we also compared how TXH11106, CBR-4830, and A22 impact the kinetics of WT EcMreB ATPase activity by examining catalysis as a function of ATP concentration in the absence and presence of 30 µM inhibitor. The results of these characterizations are presented in the form of the Eadie–Hofstee plots shown in K. pneumoniae (E143 in KpMreB), A. baumannii (E142 in AbMreB), and P. aeruginosa (E141 in PaMreB). These conserved Glu residues are highlighted in yellow in The Glu residue E143 in EcMreB is highly conserved among the MreB proteins of most rod-shaped Gram-negative pathogens, including max ((0.84 ± 0.04) × 10−2 µM/s), kcat ((0.42 ± 0.02) × 10−2 s−1), and kcat/Km ((0.25 ± 0.04) × 10−4 µM−1s−1) values compared to vehicle-treated WT EcMreB , followed by K. pneumoniae (MIC = MBC = 8 µg/mL), A. baumannii , and P. aeruginosa  , suggesting that the observed grow back did not reflect mutational resistance. Instead, the grow back may reflect a subpopulation of persister P. aeruginosa cells that became tolerant of TXH11106 at 4× MIC, perhaps by transitioning to an L-form. However, TXH11106 at 8× MIC was sufficient to prevent the emergence of persister subpopulations.We describe the antibacterial activity of the third-generation MreB inhibitor TXH11106 against the Gram-negative pathogens 2 µg/mL) . The actin 6–9 h A–C. A TXthin 3 h D. While E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa relative to the corresponding activities exhibited by the first- and second-generation MreB inhibitors A22 and CBR-4830, respectively. Significantly, our MIC and MBC characterizations demonstrated that TXH11106 is two- to fourfold more potent than CBR-4830 and 4- to 64-fold more potent than A22 (9–1010 CFUs) in the range that can be present in clinically relevant infections [E. coli, K. pneumoniae, and A. baumannii) or 8× MIC (in P. aeruginosa).We compared the activity TXH11106 against than A22 . Given tfections . No evidE. coli, K. pneumoniae, A. baumannii, and P. aeruginosa cells. In all four strains, treatment with TXH11106 induced a profound morphological change from the normal rod shape to a predominantly spherical shape    correlatctively) . This grE. coli, K. pneumoniae, A. baumannii, and P. aeruginosa  that are also highly conserved among Gram-negative bacteria  may play a key role in the catalysis of ATP hydrolysis. To test this hypothesis, we cloned and expressed an E143G mutant form of EcMreB and evaluated its ability to catalyze ATP hydrolysis compared to WT EcMreB. Significantly, E143G mutant EcMreB hydrolyzed ATP with a 13-fold reduced catalytic efficiency compared to WT EcMreB , confirmE. coli, K. pneumoniae, A. baumannii, and P. aeruginosa relative to the first- and second-generation agents A22 and CBR-4830, respectively. Enzymatic studies with WT and E143G mutant EcMreB indicate that the enhanced antibacterial potency of TXH11106 is correlated with a correspondingly enhanced ability to inhibit the catalytic efficiency MreB, with this inhibition being non-competitive in nature. Morphological changes induced by TXH11106 in E. coli, K. pneumoniae, A. baumannii, and P. aeruginosa further validate MreB as the antibacterial target of the compound. Large inocula (≈109–1010 CFUs) of these pathogens do not produce mutational resistance to TXH11106 with 48 h of exposure. Viewed as a whole, our results highlight TXH11106 as a lead MreB inhibitor of interest.Here, we show that the third-generation MreB inhibitor TXH11106 is associated with enhanced bactericidal activity against the clinically significant Gram-negative pathogens


Staphylococcus aureus has necessitated the development of innovative anti-infective agents acting on novel targets. Alpha-hemolysin (Hla), a key virulence factor of S. aureus, is known to cause various cell damage and death. In this study, with bioassay-guided fractionation, a pair of unusual epimeric lignan trimers, ligustchuanes A and B (1 and 2), were isolated from the rhizomes of Ligusticum chuanxiong Hort, together with two known phthalides being identified by UPLC-QTOF-MS. To the best of our knowledge, trimers with rare C8-C9″-type neolignan and ferulic acid fragments have not been identified in any natural product. Both of them were isolated as racemic mixtures, and their absolute configurations were determined by comparing experimental and calculated ECD spectra after enantioseparation. Ligustchuane B exhibited an outstanding inhibitory effect on α-hemolysin expression in both MRSA USA300 LAC and MSSA Newman strains at concentrations of 3 and 6 μM, respectively. Notably, a mouse model of infection further demonstrated that ligustchuane B could attenuate MRSA virulence in vivo.The emergence of antibiotic resistance in  Staphylococcus aureus is an ubiquitous human pathogen that causes a broad array of diseases, ranging from superficial infections to severe, life-threatening invasive infections such as endocarditis, septic arthritis, and sepsis (S. aureus (MRSA), along with a dearth of new antimicrobial agent discovery in recent decades, have exacerbated S. aureus infections , a major virulence-associated protein synthesized by S. aureus, forms heptameric transmembrane pores in target cell membranes , leading to cell damage and death -soluble fraction of the 80% EtOH extract of the rhizomes of Ligusticum chuanxiong Hort (chuanxiong), named LCA, was found to be a promising hit, as it could inhibit the expression of Hla at a concentration of 200 μg/mL against both methicillin-sensitive S. aureus (MSSA) and MRSA  to coculture with S. aureus , are rich in bioactive components which present opportunities for the discovery of novel drug candidates . A libraand MRSA . Moreovese model . However. aureus . The minS. aureus. However, the anti-virulence effect of chuanxiong and its bioactive components have not been investigated  on a UV-2401 PC spectrophotometer. ECD spectra were recorded in MeCN (50 μg/mL) on a Chirascan-plus spectrometer . NMR spectra were measured in DMSO-d6 on Bruker AV-400 and Bruker AV-600 spectrometers and calibrated by the solvent peak used. Mass spectrometry was performed on a SYNAPT G2-Si HDMS  with an electrospray ion source  connected to a lock-mass apparatus, which performed real-time calibration correction. Column chromatography was performed with CHP20P MCI gel  and Sephadex LH-20 . A Waters 2535 Series machine equipped with a X-bridge C18 column  was used for HPLC analysis, and a preparative X-bridge Prep C18 OBD column  was used for sample preparation. Enantioseparations were performed by a Waters Acquity UPC2 system  with a sample manager, binary solvent manager, compensation solvent pump and column manager on a Daicel Chiralpak IG column .Optical rotations were measured using an Autopol VI polarimeter. IR spectra were obtained from Perkin-Elmer 577 spectrometers. Ultraviolet absorption spectra were recorded in MeCN (25 Ligusticum chuanxiong Hort. A voucher specimen (herbarium No. 20180501) was deposited at the School of Pharmacy, Shanghai University of Traditional Chinese Medicine.The crude plant of chuanxiong was purchased from a Chinese herbal medicine market in Sichuan Province in China and identified by associate professor Hong-Mei Zhang at Shanghai University of Traditional Chinese Medicine as the rhizome of Ligusticum chuanxiong Hort (500 g) were soaked in 80% EtOH for 30 min at room temperature and then extracted by heat reflux (3 × 5 L). The obtained solutions were combined and concentrated in a rotary evaporator at 45°C to obtain the EtOH-soluble portion (149 g). The residue was suspended in H2O (300 mL) and extracted with PE (5 × 300 mL) to obtain the dried PE-soluble fraction named LCA (36 g), which was later applied to an MCI chromatography column and successively eluted with 50, 80 and 95% EtOH. The fraction eluted with 50% EtOH was named LCAI (25.8 g), while the fraction eluted with 80% EtOH was named LCAII (7.1 g). The last fraction eluted with 95% EtOH was named LCAIII (2.9 g). LCAII was then subjected to Sephadex LH-20 eluted with MeOH to yield subfraction LCAIIB (231 mg), which was later purified with preparative HPLC using a gradient of 45% MeCN-H2O (0.1% formic acid) at a flow rate of 20 mL/min and afforded ligustchuanes A (10.2 mg) and B (19.8 mg). As both compounds 1 and 2 were obtained as racemic mixtures, enantiomers (+) 1 (0.56 mg), (−) 1 (0.63 mg), (+) 2 (0.72 mg) and (−) 2 (0.77 mg) were separated via chiral-phase UPC2 on a Daicel Chiralpak IG column . Compound (+) 1 was eluted at 24.9 min, and compound (−) 1 was eluted at 15.7 min, while compound (+) 2 was eluted at 11.8 min and compound (−) 2 was eluted at 14.2 min.Air-dried and fragmented rhizomes of 1: yellow oil; [α]20D +4.0 ; (−) Ligustchuane A (−) 1: yellow oil; [α]20D −6.0 ; UV (MeCN) λmax (log ε) 271 (3.98), 287 (3.96), 315 (3.92) nm; IR (KBr) υmax 2972, 1695, 1596, 1511, 1452, 1429, 1372, 1267, 1156, 1120, 1030, 970, 849, 818 cm−1; 1H NMR data, see 13C NMR data, see m/z 563.2270 [M−H] − .(+) Ligustchuane A (+) 2: yellow oil; [α]25D +18.0 ; (−) Ligustchuane B (−) 2: yellow oil; [α]25D −16.7 ; UV (MeCN) λmax (log ε) 270 (4.30), 287 (4.26), 315 (4.23) nm; IR (KBr) υmax 2972, 1694, 1596, 1512, 1451, 1429, 1374, 1267, 1156, 1122, 1030, 968, 849, 817 cm−1; 1H NMR data, see 13C NMR data, see m/z 563.2292 [M−H] − .(+) Ligustchuane B (+) Z-ligustilide and senkyunolide A were purchased from the Chengdu Push Bio-Technology Co., Ltd. .The MSSA Newman strain and CA-MRSA USA300 LAC strain applied in the present study were cryopreserved at −80°C. Both strains were grown at 37°C in tryptic soy broth  with gentle shaking in a shaker (TENSUC TS-100B Shaker Incubator) at 250 rpm and maintained on tryptic soy agar  plates at 4°C. Z-ligustilide and senkyunolide A, were dissolved in dimethyl sulfoxide (DMSO) to a concentration of 40 mg/mL, while compounds 1 and 2 were dissolved to a concentration of 9.6 mM to serve as stock solutions. An overnight culture of the S. aureus strain was transferred (1:100) into fresh TSB and incubated at 37°C when an OD600 of ∼0.3 was reached. The culture was then treated with the indicated concentrations of the fractions or compounds, incubated for another 3 h, and shaken at 250 rpm, while the blank culture was treated with 1% DMSO alone as control. A final DMSO concentration was kept at 1% (v/v). After centrifugation , the supernatants were boiled with 5 × SDS–PAGE loading buffer, separated on 12.5% SDS–PAGE and transferred to nitrocellulose membranes . Ten percent dried skim milk was used to block the membranes for 1.5 h at room temperature, which were then stained by subsequent incubation with anti-α-toxin antibodies  overnight at 4°C and secondary antibodies  at room temperature for 1.5 h. Protein expression was detected by chemiluminescent reaction and analyzed via a luminescent image analyzer (GE ImageQuant LAS 4000 min).LCA, LCAII and two compounds, μL of washed 1% rabbit erythrocytes for 20 min at 37°C. The culture supernatant from untreated cells served as a positive control (100% hemolysis). Following centrifugation , the OD543 of supernatant fluid was determined. The hemolysis percentage was then calculated after comparison with the control.Bacterial culture supernatant samples, prepared as described in the western blotting assay section, were applied for the hemolysis assay. One hundred microliters of the supernatant of each sample was incubated with 900 S. aureus strains was examined in triplicate using the standard microdilution method recommended by the Clinical and Laboratory Standards Institute.The minimal inhibitory concentration (MIC) of LCA against 2 on α-hemolysin heptamer were investigated with the AutoDock Vina1.1.2 program (α-hemolysin was accessed through the protein data bank website (http://www.rcsb.org/pdb) (PDB ID: 6U49). From the crystal structure, water and ion molecules were removed, and appropriate hydrogen atoms were added under physiological pH conditions (pH = 7) using AutodockTools-1.5.6 software. In docking, the protein was considered rigid, while the ligands were flexible. To perform suitable docking for each ligand, we set the search space box parameters to 47.3–47.3–47.3 Å  centered at  Å.The interaction mechanisms of compound (±)  program . The 3D Based on the binding energy (∆G) results, the final docked conformations were ranked. The most favorable binding conformations had the lowest free energies, which were selected as suitable poses of binding and were later analyzed visually. Hydrogen bonding and hydrophobic interactions between ligands and proteins were visualized using PyMOL2.3.0 and LIGPLOT V 2.2.4 software .S. aureus were diluted 20-fold in fresh TSB in a 50 mL flask and then cultured at 37°C for approximately 3 h to an OD600 of 0.6. The bacteria were later washed twice with phosphate-buffered saline (PBS) after being harvested. After weighing, 1 × 108 CFU of the MSSA Newman strain suspended in PBS was administered to each female BALB/c mouse (18–20 g)  via intraperitoneal injection, recorded as day 0. From day −1 to day 2, these mice received intraperitoneal injections of LCA  or saline containing 20% PEG400 and 1% Tween 80 (mock). The animals were euthanized in day 3. The kidneys, spleens, hearts and livers were aseptically removed, recorded by photos, homogenized in PBS with 0.1% Triton X-100 to obtain single-cell suspensions, and quantified by the plating method.The experiments were performed according to our previous study . The ove8 CFU and 6.1 × 108 CFU of bacteria were used for the infection model and intraperitoneal injections of LCAII and compound 2, rather that LCA, were given once daily at 100 mg/kg/d and 10 mg/kg/d, respectively.With regard to MRSA strain USA300 LAC, all the procedures were similar to those of Newman, except 7.4 × 10All mouse experiments were performed according to institutional guidelines approved by the Committee for Animal Experiments, Shanghai University of Traditional Chinese Medicine, which are in accordance with guidelines for the ethical review of laboratory animal welfare approved by the General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (2-6-2018). The animal study protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Public Health Clinical Center (2019A00201).t test and the nonparametric Mann–Whitney test (two-tailed). One-way ANOVA followed by Dunnett’s multiple comparison test was carried out to compare three or more groups using GraphPad Prism 6 software . p values of <0.05 were considered statistically significant .Data were presented as the mean ± standard error (SE). Differences between two datasets were measured using the two-tailed Student’s μg/mL against MSSA Newman and 25 μg/mL against the MRSA USA300 LAC strain without exhibiting bacteriostatic activity against bacteria  showed stronger inhibition of Hla expression against .5 μg/mL . Two rar.5 μg/mL . Their s1) was obtained as a yellow oil, and its molecular formula was designated C32H36O9 according to the pseudomolecular ion peak at m/z 563.2270 [M−H] −  in its HRESIMS. The 1H NMR spectroscopic data of 1 , 6.79 , 6.70 , 7.29 , 6.79 , 7.07 , 6.94 , 6.67 , and 6.74 ], four olefinic protons , three methoxyl groups  and one methyl singlet [δH 1.11 ]. Further analyses using 13C NMR, DEPT and HSQC  and an ethoxyl group . The remaining carbon signals included two C6-C3 units, indicating the presence of a lignan dimer. Thus, 1 was undoubtedly composed of the three moieties mentioned above.Ligustchuane A  curves with the TDDFT-predicted curves. However, the measured ECD spectrum had no obvious cotton effect coupled with its specific rotation, [α]20D -1.5 , hinting at a racemic mixture  1  and (−) 1  -1, -1, -1, and -1. The calculated ECD spectrum of -1 agreed well with the experimental ECD spectrum of (+) 1, while the other showed similar Cotton effects to -1 (1 and (−) 1 were defined as  and , respectively.Given its flexible structure, the relative configurations at the chiral centers C-7 and C-8 could not be determined on the basis of NOESY data. Additionally, due to its oily texture, a suitable crystal was hard to acquire. Therefore, the absolute configuration of  mixture . Semi-pr5.7 min) . The spe7R,8S)-1 . Therefo2) was also isolated as a yellow oil. The molecular formula was determined to be C32H36O9 on the basis of a pseudomolecular ion [M−H] − in the HRESIMS at m/z 563.2292, which indicated that 2 is one of the isomers of 1. In addition, 1H and 13C NMR spectra   and (−) 2  (2 and (−) 2 were established as  and , respectively  . Two ena4.2 min) . By compectively . To dateethoxyl substituent groups were not common in the natural products, which might originate from the extract solution and isolation procedures. To determine whether 1 and 2 are artifacts, another sample of chuanxiong was extracted with 80% MeOH and subsequent fractionation and analytical processes were carried out in the absence of EtOH , liver (∼1.01 log10 CFU/organ reduction), kidney (∼0.81 log10 CFU/organ reduction) and heart (∼0.54 log10 CFU/organ reduction) of the infected mice. Furthermore, fewer abscesses were observed in the visceral organs of infected mice treated with ligustchuane B  and -enantiomers are shown in α-hemolysin heptamer, suggesting that ligustchuane B may attenuate the virulence of S. aureus partly by direct interaction with heptamer protein. Moreover, a comparison of the binding affinity of these enantiomers indicated that the -enantiomer (−8.5 kJ/mol) had a stronger binding affinity than the -enantiomer (−7.6 kJ/mol). Even though the antihemolytic activities of both  and -enantiomers were not determined in this study due to their small quantity obtained after enantioseparation, we speculated that the -enantiomer may show a stronger inhibitory effect because of its higher binding affinity.In the biological evaluation, given the outstanding antihemolytic activity of ligustchuane B against MRSA, molecular docking studies were also conducted to speculate its possible interaction with α-hemolysin inhibitor isolated from the rhizomes of Ligusticum chuanxiong Hort, which could effectively attenuate the virulence of the S. aureus both in vitro and in vivo. Moreover, this study not only suggested innovative lead compounds for anti-virulence therapy against S. aureus but also further explored the chemical components in chuanxiong and expanded its traditional medicinal use.In conclusion, we have discovered a natural potential small-molecule


Halomonas bluephagenesis is a versatile chassis for the production of fine chemicals because of its open growth property without sterilization.Red-colored lycopene has received remarkable attention in medicine because of its antioxidant properties for reducing the risks of many human cancers. However, the extraction of lycopene from natural hosts is limited. Moreover, the chemically synthesized lycopene raises safety concerns due to residual chemical reagents. H. bluephagenesis strain TD1.0 to engineer a bacterial host for lycopene production. A pTer7 plasmid mediating the expression of six MVA pathway genes under the control of a phage Mmp1P and an trcEscherichia coli P promoters and a pTer3 plasmid providing lycopene biosynthesis downstream genes derived from Streptomyces avermitilis were constructed and transformed into TD1.0. The production of lycopene in the engineered H. bluephagenesis was evaluated. Optimization of engineered bacteria was performed to increase lycopene yield.A heterologous mevalonate (MVA) pathway was introduced into S. lividans elevated the lycopene production to 0.70 mg/g DCW in the TD1.0/pTer7-pTer5 strain. Optimizing the Mmp1P promoter in plasmid pTer7 with a relatively weak trcP even increased the lycopene production to 1.22 mg/g DCW. However, the change in the trcP promoter in pTer7 with Mmp1P did not improve the yield of lycopene.The engineered TD1.0/pTer7-pTer3 produced lycopene at a maximum yield of 0.20 mg/g dried cell weight (DCW). Replacing downstream genes with those from H. bluephagenesis for the lycopene production. The co-optimization of downstream genes and promoters governing MVA pathway gene expressions can synergistically enhance the microbial overproduction of lycopene.We first engineered an  IPP and DMAPP are substrates for final lycopene production via the downstream enzymes, including geranylgeranyl diphosphate synthase (CrtE), phytoene synthase (CrtB), and lycopene synthase , mvaK1, mvaK2, and mvaD genes originated from Streptococcus pneumoniae (Sp), and idi gene derived from E. coli (Ec) was constructed in the pTer7 plasmid that was subsequently transformed into Halomonas bluephagenesis strain TD1.0, a non-carotenogenic bacterium that can grow pollution-free in unsterilized seawater , the engineered TD1.0/pTer7-pTer3 could successfully produce lycopene by a yield of 0.20 mg/g dried cell weight (DCW). Replacing downstream genes with those from S. lividans (Sl) considerably increased the lycopene production to 0.70 mg/g DCW. Further optimization of promoters involved in the MVA pathway even elevated lycopene yield to 1.22 mg/g DCW, which was a 6.1-fold increase relative to that produced by TD1.0/pTer7-pTer3. Our data showed that an engineered H. bluephagenesis is successfully generated for lycopene production. Changing the source of metabolism-responsible genes and optimization module gene promoters can effectively promote the microbial production of lycopene.As for metabolic engineering, heterogeneous genes involved in both upstream and downstream, modules of lycopene biosynthesis can be selected and constructed into a single chassis to generate a microbial host of interest . Many opseawater . After tH. bluephagenesis TD1.0 (CGMCC 4353), a novel T7-like RNA polymerase-integrated derivative of the wild-type H. bluephagenesis strain TD01 that was isolated from Aydingol Lake of Xinjiang province in China (Escherichia coli S17-1 was used to manipulate plasmids and served as the conjugation donor (E. coli strain was cultured at 37°C in the Luria-Bertani (LB) medium  or plated on LB agar for 16 h. H. bluephagenesis TD1.0 and its derivatives were cultivated with 60-LB medium (LB medium supplemented with 60 g/L NaCl) or 60-LB agar at 37°C for 16 h. Whenever necessary, the bacterial media were supplemented with chloramphenicol , kanamycin , or spectinomycin  for maintaining plasmids.The bacterial strains used in this study are listed in in China , was useon donor . E. coliEfmvaE, EfmvaS, SpmvaK1, SpmvaK2, SpmvaD, and Ecidi under the control of two trcE. coli P promoters cloned in pMVA2  and subjected to PCR verification and DNA sequencing. The relevant plasmid was designated as pTer1  and incubated at 37°C to achieve an optical density (OD) at 600 nm (OD600) of 0.5–0.6. The bacterial cells in 1 mL culture were recovered by centrifugation at 2,500 ×g for 5 min and resuspended with 50 μL of 20-LB without antibiotics. H. bluephagenesis TD1.0 was cultivated with 60-LB and prepared as mentioned above. H. bluephagenesis TD1.0 sample was 1:1 (vol/vol) mixed with the plasmid-carried E. coli S17-1 donor cells and inoculated into 20-LB agar without antibiotics. After 6 h of incubation at 37°C, the bacteria were collected and streaked onto 60-LB agar plate with appropriate antibiotics corresponding to the plasmid and cultured at 37°C for 16 h. The single colony was selected and cultured in 60-LB medium. The plasmid in the conjugated H. bluephagenesis TD1.0 was extracted and verified with PCR amplification.erformed . BrieflyH. bluephagenesis TD1.0 or its derivatives were cultured in a small conical flask (100 mL) with 60-LB broth at 37°C overnight with shaking. On the next day, the culture was inoculated (1%) to 20 mL fresh 60-LB medium. A total of 50 μL culture was taken every hour and dropped in triplicate into the wells of a 96-well plate, and the OD600 value was detected by a FLUOstar Omega microplate reader (BMG Labtech). The growth curve was drawn by plotting OD600 values vs. culture time.H. bluephagenesis TD1.0 or its variants were taken from a 60-LB agar plate for each experiment, inoculated individually into 2 mL of 60-LB medium, and then cultured at 37°C for 12–16 h. Subsequently, the culture was transferred to 20 mL of 60-LB medium in a 100-mL conical flask  and cultured at 37°C to an OD600 value of 0.6–0.8. Then, the secondary culture was inoculated into 50 mL fresh 60-MMG medium . The pH of the solution was adjusted to 8.0–8.5 using 5 M NaOH. Trace element solution I comprises 0.5% Fe (III)-NH4-citrate (m/vol) and 0.2% CaCl2 (m/vol) dissolved in 1 M HCl; Trace element solution II contains 0.01% ZnSO4·7H2O (m/vol), 0.003% MnCl2·4H2O (m/vol), 0.03% H3BO3 (m/vol), 0.02% CoCl2·6H2O (m/vol), 0.003% NaMoO4·2H2O (m/vol), 0.002% NiCl2·6H2O (m/vol), and 0.001% CuSO4·5H2O (m/vol) dissolved in 1 M HCl. Glucose was added at a final concentration of 3% (m/vol) at the beginning of the experiment in a 500-mL conical flask  and cultured at 37°C for 48 h with shaking. Then, 30 mL of fermentation culture was harvested and pelleted by centrifugation at 10,000 ×g for 10 min at 4°C in a sterile centrifuge tube (50 mL) that had been prepared and weighed. The supernatant was removed, the tube was sealed with plastic wrap and placed in a −80°C refrigerator for 1 h, then bacterial cells were treated with a lyophilizer at least 8 h. The total weight was weighed, and the dried cell weight (DCW) was calculated.Three colonies of 18 column  was used. The following parameters were set for HPLC analysis: The flow rate was 1 mL/min, the column temperature was kept at 30°C, the detection wavelength was 470 nm, and the mobile phase ratios of the solvents methanol/acetonitrile/isopropanol were 42/42/16, respectively. The peak area of the sample was substituted into the standard curve, and the lycopene yield was computed.After flask fermentation, 1 mL of the culture was centrifuged at 5,000 ×g for 5 min, and bacterial cells were resuspended and lysed with acetone solution (using 1% butylated hydroxytoluene), vortexed for 5 min. Then, the cell lysate was centrifuged at 10,000 ×g for 5 min, and the supernatant was filtered through a 0.22 μm filter (Millipore) into the liquid sample bottle. Lycopene standard was prepared and diluted with acetone for five concentration gradients  to draw standard curves, as previously described . An highH. bluephagenesis TD1.0 or its derivatives were cultured with 60-LB broth at 37°C for 16 h. The overnight culture was inoculated into 2 mL of fresh 60-LB medium  and cultured at 37°C. Diverse concentrations of IPTG were added when the OD600 reached 0.5–0.6. The bacterial cells were harvested and the total RNA was extracted using a RNAprep Pure Cell/Bacteria Kit . For each sample, three duplicate samples were prepared. The first cDNA was synthesized with a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). A quantitative polymerase chain reaction (qPCR) was conducted, and the relative expression level of each gene of interest was calculated as a ratio of its Ct value relative to that of the reference 16S rRNA gene.t-test, and the difference between groups was calculated with analysis of variance (ANOVA). Each experiment was conducted at least three times, and the data were presented as mean ± standard deviations (SD). The statistically significant was considered as a p-value < 0.05.All results were analyzed with GraphPad Prism 8. Results derived from samples between two groups were treated with unpaired two-tailed Student’s E. coli and Pseudomonas putida  derived from phage MmP1  involved in lycopene production have been characterized in microorganisms, such as s putida . H. bluealdehyde . Howevernd Ecidi . The E. age MmP1 . The resage MmP1 . After tquencing . Bacterification .crtE, crtI, and crtB derived from S. avermitilis in the pSEV434 plasmid to introduce the downstream of the lycopene synthetic pathway to H. bluephagenesis strain TD1.0  and analyzed via HPLC. The results showed that the wild-type TD1.0 produced no detectable lycopene. A maximum yield of approximately 0.20 mg/g (DCW) was achieved in TD1.0/pTer7-pTer3 under the induction of 5.00 μM IPTG  were prepared, and a standard curve was drawn . H. blue μM IPTG . Compara μM IPTG . These dcrtE, crtI, and crtB, for lycopene biosynthesis in pTer3 were replaced with those from S. lividans. The resultant plasmid pTer5  constructed in the pTer7 plasmid may disturb the expression balance of genes involved in the MVA pathway of lycopene biosynthesis in H. bluephagenesis. Therefore, we set out to promoter optimization. On the basis of the pTer7 plasmid, the trcP promoter from E. coli was replaced with the phage MmpI promoter (Mmp1P) to generate pTer14  into E. coli resulted in 198 ± 3 mg/g DCW lycopene yield  and alkaline pH (8.5), thus, avoiding possible contamination by other microorganisms even under open unsterile conditions , we found that the lycopene yield in TD1.0/pTer7-pTer3 or TD1.0/pTer7-pTer5 without IPTG induction was comparable with that of the bacteria treated with different IPTG concentrations  carrying pTer1 plasmid in TD1.0/pTer1-pTer5 responded well to the IPTG induction .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


First, the in vitro cytotoxic activity of Imatinib Mesylate (IM) in K-562 and FEPS cell lines was evaluated. Subsequently, membrane integrity and mitochondrial membrane potential assays were performed to assess the cytotoxic effects of CNN1 in K-562 and FEPS cell lines, followed by cell cycle, alkaline comet assay and annexin V-Alexa Fluor® 488/propidium iodide assays (Annexin/PI) using flow cytometry. RT-qPCR was used to evaluate the H2AFX gene expression. The results demonstrate that CNN1 was able to induce apoptosis, cell membrane rupture and mitochondrial membrane depolarization in leukemia cell lines. In addition, CNN1 also induced genotoxic effects and caused DNA fragmentation, cell cycle arrest at the G2/M phase in leukemia cells. No genotoxicity was observed on peripheral blood mononuclear cells (PBMC). Additionally, CNN1 increased mRNA levels of H2AFX. Therefore, CNN1 presented anticancer properties against leukemia multidrug resistant cell line being a potential anticancer agent for the treatment of resistant CML.The multidrug resistance (MDR) phenotype is one of the major obstacles in the treatment of chronic myeloid leukemia (CML) in advantage stages such as blast crisis. In this scenario, more patients develop resistance mechanisms during the course of the disease, making tyrosine kinase inhibitors (TKIs) target therapies ineffective. Therefore, the aim of the study was to examine the pharmacological role of CNN1, a  Since the advent and introduction of tyrosine kinase inhibitors (TKIs), significant improvements have occurred in the treatment of chronic myeloid leukemia (CML), increasing patient’s survival rate ,2,3,4. AApproximately 20–40% of CML patients treated with TKIs fail due to primary refractoriness (primary resistance), where patients exhibit lack of efficacy to TKIs from initiation of therapy, while secondary resistance (acquired) is defined as the loss of response ,12,13,14Juglone  is a natural 1,4- naphthoquinone found in the Juglandaceae, a family of compounds known for their vast biological application ,21,22,23TOP1) expression in both cell lines. Therefore, this study aimed to examine whether CNN1 is able to circumscribe the multidrug resistant and induce cell death in resistant leukemia cells.Previously, our research group synthesized and evaluated in silico and in vitro antitumor activities of a series of synthetic naphthoquinones . Among t50 value of 0.90 µM (CI 95% 0.34–1.27) and presented a strong cytotoxic activity against FEPS with an IC50 of 0.60 µM (CI 95% 0.48–0.80) when compared to K-562 that presented an IC50 value of 1.12 µM (CI 95% 0.90–1.38). On the other hand, IM showed an IC50 of 4.97 µM (CI 95% 3.69–5.70) to K-562-Lucena-1 and 9.66 µM (CI 95% 8.45–11.1) to FEPS, a much higher concentration when compared to CNN1 in MDR cell lines. Only to sensitive cell line, K-562, IM showed a CI50 of 0.03 µM (CI 95% 0.01–0.05)  .p < 0.001) and also in FEPS cells (p < 0.001) after 24 h of treatment (p < 0.01).Flow cytometry analysis of membrane integrity A showed reatment B,C. Addip < 0.001) disruption of mitochondrial membrane potential in K-562 and FEPS cell lines (p < 0.001) and FEPS (p < 0.001) cell lines. In order to investigate whether CNN1 compound was able to cause disruption of the mitochondrial membrane potential, rhodamine 123 was used after 24 h of treatment D. Naphthll lines E,F, whenp < 0.001) and induced a significant decrease in cells in the S phase (18.27%) in K-562 cells (p < 0.05) (p < 0.001) and increased the number of cells at G0/G1 phase to 25.66% (p < 0.05) (p < 0.01) and FEPS (p < 0.05) cells. In addition, negative control presented 7.46% of K-562 cell at sub-G1 and 5.84% of FEPS at sub-G1, while CNN1 treatment increases the number of cells at sub-G1 to 10.85% and 9.35% (p < 0.01), respectively (p < 0.01) and FEPS (p < 0.001) cells.Cell cycle analysis was measured to evaluate whether the CNN1 compound induces effects in the cell cycle of K-562 and FEPS cell lines. The negative control presented 32.21% of cells in G0/G1 phase, 30.30% in S phase and 25.67 in G2/M phase. We found that CNN1 (0.1 µM), after 24 h of treatment, triggered a significant accumulation of 39.9% of cells in G2/M phase ( < 0.05) A. On FEP < 0.05) B. Treatmectively C,D. The p < 0.001). Statistical analysis showed that CNN1 was more genotoxic to leukemia cells than to non-malignant peripheral mononuclear blood cells (PBMC). Moreover, IM showed significant differences between leukemia cells and PBMC, where IM present significant genotoxic effects only in K-562  and IM (0.1 µM) in K-562 and FEPS leukemia cell lines and PBMC after 3 h of exposure. CNN1 induced DNA damage in leukemia cell lines. CNN1 (0.1 µM) significantly induced DNA damage of K-562 and FEPS cells in comparison to negative control  in early apoptotic cells (Annexin-V positive/PI negative) and an increase in late apoptosis (Annexin V-positive/PI-positive) in K-562 and FEPS cell lines (p < 0.001)  B,C, whenH2AFX gene modulation after 18h of treatment. H2AFX is an important biomarker to monitor genotoxic events [H2AFX gene expression (p < 0.001) in K-562 and FEPS cell lines when compared to control, as show in ABCB1 mRNA expression, and no significant difference was found in ABCB1 expression after 18 h of drug exposure (p > 0.06) in K-562 and FEPS cell lines. The results also showed no significant differences in BCR-ABL1 expression in both K-562 (p > 0.27) and FEPS (p > 0.28) cell lines. We also investigated whether CNN1 induces c events . SamplesMDR is still a significant obstacle to provide effective treatment to many patients with cancer, including CML. In this sense, one strategy to overcome MDR is the use of compounds able to selectively target MDR cells [The naphthoquinones present different pharmacological properties, among them anticancer action, antifungal, antibacterial and antiviral activities, as well as allelopathic activities ,31,32,33DR cells .Our previous study showed that CNN1 is more potent to resistance cell lines FEPS (IC50 0.60 µM) and K-562-Lucena-1 (IC50 0.90 µM) when compared to a parental sensitive cell line K-562 (IC50 1.12 µM) . Moreovep < 0.001). In fact, CNN1 caused pronounced rupture in membrane integrity on leukemia cells compared to IM standard therapy. Some naphthoquinones can induce disruption of membrane integrity [To demonstrate the efficacy of CNN1 on resistant (FEPS) and sensitive (K-562) leukemia cell lines, experiments were performed to evaluate if CNN1 was able to induce apoptosis as a cell death mechanism. Analyzing the cell membrane integrity after treatment with CNN1, a significant difference was found between the leukemic cell lines when compared to positive control . Previous studies have reported a decrease in mitochondrial membrane potential induced by 1,4-naphthoquinone in liver cancer cells (Hep3B), leukemia cells (HL-60), and human breast adenocarcinoma cells (MCF-7) [p < 0.001), mediated by the loss of mitochondrial membrane potential.The next question was to determine whether disruption of membrane integrity would be linked with cell death by depolarization of mitochondrial membrane potential, considered one of the hallmarks of apoptosis . In the  (MCF-7) ,43,44. T (MCF-7) . The mec (MCF-7)  and the  (MCF-7) . These f(p < 0.001) and FEPS (p < 0.001) cell lines. Tumor cells present an increased rate of cellular proliferation, and this feature of uncontrolled cell division can be targeted to treat cancer patients [It was also investigated whether the pronounced effect of CNN1 on leukemia multidrug resistant cell line could reflect a cell cycle arrest. Our results showed that CNN1 induced cell cycle arrest at G2/M phase for both K-562 patients . The pripatients . Therefopatients . In agrepatients  and breapatients . TOP1 expression levels in both leukemia cell lines [TOP1 expression can be correlated with a genomic damage induced by CNN1 [Furthermore, CNN1 showed a possible preferential genotoxic effect against K-562 and FEPS cells and did not cause significant DNA damage in peripheral blood mononuclear cells (PBMC). Data from previous work by our group showed that CNN1 reduced ll lines . Thus, t by CNN1 . TOP1, that can result in DNA damage [Some naphthoquinones have exhibited topoisomerase inhibition effects by catalytic activity or trapping of A damage . These dA damage .p < 0.001) and FEPS (p < 0.001) cell lines, similar findings were reported on in vitro models of breast cancer [In this research, the apoptosis induced by CNN1 was confirmed by phosphatidylserine externalization in K-562  and FEPS (p < 0.01) cells, suggesting that this upregulation of H2AFX gene has a major role in initiating cell death in leukemia MDR cells through the DNA damage signaling pathway. To the best of our knowledge, our study is first to describe H2AFX gene overexpression in mediated apoptosis of resistant leukemia cells (FEPS). In addition to the mechanism of cell death induced by CNN1 in K-562 and FEPS cell lines mediated by DNA damage, we investigated the H2AFX biomarker . Our resSeveral studies have shown that deregulation of the DNA damage response (DDR) pathway causes genomic instability in non-neoplastic cells and thus compromising tumor cell sensitivity to anticancer therapy ,61,62. HH2AFX low expression compromise the apoptotic response to IM and promote blast crisis of CML, as well as the H2AFX deficiency, promotes B-cell tumorigenesis, suggesting that deregulation could make cells more sensitive to leukemogenic factors [H2AFX as a tumor suppressor is involved to its regulation of apoptosis [H2AFX dysfunction is considered one important event in cancer patients that develop resistance during treatment, including IM therapy, and demonstrated the importance of H2AFX in the apoptosis regulation process of CML cells. Other authors also described that  factors . In partpoptosis . Zangh apoptosis  reinforcP-glycoprotein (P-gp), encoded by ABCB1 , and the expression of BCR-ABL1 transcripts. In our results, CNN1 did not cause decreased expression of BCR-ABL1 and ABCB1 in both K-562 and FEPS cell lines. This result corroborates a previous in silico study by our group, where CNN1 was not considered inhibitor or substrate of P-gp [BCR-ABL1 compared to K562, indicating that the BCR–ABL1-independent mechanism of resistance, that occurs in CML patients with overexpression of P-glycoprotein (P-gp) is also found in the FEPS cell line model [ABCB1 mRNA, and they also failed to respond to subsequent treatment with nilotinib and dasatinib. Finally, recent studies demonstrated some evidence that IM, nilotinib, and dasatinib are Pgp-substrates [H2AFX modulation. Moreover, CNN1 can lead to new drugs that might be capable of overcoming the interference of efflux transporters in MDR phenotype. At last, we evaluated whether CNN1 could decrease the mRNA expression of  of P-gp . In addi of P-gp  describene model . This fine model . Furtherne model  showed tbstrates ,71. In t6 lymphocytes were seeded into six-well plates in Roswell Park Memorial Institute medium 1640 , supplemented with 10% fetal bovine serum , 100 U/mL of penicillin, 100 μg/mL of streptomycin (Gibco®) and phytohemagglutinin A  for 20 h at 37 °C.Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors, non-smoking individuals , after a signed written consent form to participate in the study which was approved by the Ethics Research Committee of the Federal University of Ceará (registration number 52352121.0.0000.5054). Blood samples were collected according to Fenech (2012) . The lym®). The cell lines were supplemented with 10% fetal bovine serum , 100 U/mL of penicillin, and 100 μg/mL of streptomycin (Gibco®). All cells were cultured at 37 °C and 5% CO2.Chronic myeloid leukemia cell lines (K-562), vincristine-resistant derivative (K-562-Lucena1), daunorubicin-resistant derivative (FEPS). K-562, K-562-Lucena1 and FEPS was provided by Prof. Dr. Vivian Rumjanek from the Federal University of Rio de Janeiro . The cel®, and Imatinib Mesylate (IM) (1mM) were used as a positive controls. In other studies conducted by our group, CNN1 demonstrated cytotoxic activity against K-562, K-562-Lucena1 and FEPS cells after 72 h. The concentration of CNN1 (0.1 µM) used in tests was based in CI50 determined before [Stock solution of CNN1 (1mM) was dissolved in DMSO (0.02%) from Sigmad before .ABCB1 overexpression [ABCB1 and ABCC1 overexpression [K-562 sensitive cell line, derived from a patient with CML. The K-562-Lucena-1 cell line is derived from the K-562 cell line following selection by vincristine and presenting pression , and thepression . In the pression . curve against leukemia cell lines (K-562 and FEPS). Cells were plated (5 × 103 cells/well) in a 96-well-plate, after 24 h cells were treated with compounds (0.312–20 µM) for 72 h. DMSO (0.02%) was used as a negative control. Cell viability was evaluated by the Alamar blue assay [The cytotoxicity of the CNN1 and IM were evaluated in a concentration-response ue assay . BrieflyCells (K-562 and FEPS) were seeded into 6-well plates at a density of 50 × 10³ per well and maintained for 24 h at 37 °C in incubator. After this period, cells were submitted to treatment with CNN1 (0.1 µM) for 24 h. IM (0.1 µM) was used as a positive control and DMSO (0.02%) as negative control. Posteriorly, cells were suspended in PBS and incubated in propidium iodide (1 μg/mL) solution for 30 min at 37 °C, in the dark. Fluorescence intensity (from 10.000 cells) was immediately analyzed by flow cytometry (BD FACSVerse) with 488-nm laser excitation .3 cells per well) were plated into 6-well for 24 h at 37 °C. Cells were then treated with CNN1 at concentration 0.1 µM for 24 h. IM (0.1 µM or 10 µM) was used as a positive control and DMSO (0.02%) as a negative control. Next, cell pellets were resuspended in rhodamine 123 solution (1 μg/mL) for 20 min at 37 °C, in the dark. Then, mitochondrial transmembrane potential (ΔΨm) was determined using flow cytometry system (BD FACSVerse) [K-562 and FEPS cell lines (50 × 10CSVerse) . 3 cells per well into 6-well plates for 24 h at 37 ˚C. After 24 h, cells were treated with CNN1 at 0.1 µM for 24 h. DMSO (0.02%) as negative control. IM (0.1 µM) was used as a positive control. Cells were pelleted and fixed with 70% alcohol for 30 min at 4 °C. After, cells were submitted to Triton- X100 (0.1% v/v in PBS). Subsequently, cells are incubated for 40 min at 37 °C and propidium iodide was added (50 µg/mL). Events (10.000) were evaluated by flow cytometry (BD FACSverseTM). All data were analyzed using FlowJo Software v.10 [Cells were plated at a density of 50 × 10are v.10 .3) per well for 24 h at 37 °C in a CO2 incubator. Then, cells were exposed to CNN1 at concentration (0.1 µM) for 3 h. IM (0.1 µM) was used as positive control and DMSO (0.02%) as negative control. Following exposure, the cells were suspended in 150 μL in 0.8% of low melting agarose (LMPA) in phosphate buffered saline (PBS) without calcium and rapidly spread on three slides pre-coated with 1.5% normal melting agarose (NMA) and waited to solidify at 4 °C for 5 min. After, slides containing cells were placed in the chilled lysis solution containing 100 mM EDTA, 2.5 M NaCl, 10 mM Tris, 10% DMSO and 1% Triton X-100, pH 10) at 4 °C for 24 h. The slides were incubated is fresh electrophoresis buffer  for 20 min in other to unwinding the DNA. Then, horizontal electrophoresis at 300 mA and 34 V for 20 min was performed. Finally, slides were washed with distilled water for 5 min and fixed with absolute ethanol. Thereafter, slides were stained with propidium iodide at 20 µg/mL A total of 100 comets were counted for each sample using a fluorescence microscope  [K-562, FEPS and PBMC were plated into 6-well plates at  .K-562 and FEPS cell lines were seed into 12-well plates at a 50 × 10³ per well for 24 h. After, cells were exposed to CNN1 (0.1 µM) for 24 h. The positive control used IM (0.1 µM) and DMSO (0.02%) was used as negative control. Subsequently, cells were washed with PBS and resuspended in 200 µL Annexin-binding buffer consisting of 4 μL Annexin V-FITC and then incubated with propidium iodide for 20 min. After incubation, cells were resuspended in 1× binding buffer. The fluorescent emission was measured by flow cytometry system (BD FACSVerse) .3 cells per well) were plated into 6-well plates for 24 h at 37 °C. After 24 h, cells were treated with CNN1 (0.1 μM) for 18 h, a time that does not modify cellular viability (® (Invitrogen™) according to the manufacturer’s protocol. The RNA concentration and quality were determined by using Nanodrop 2000 . From 20 ng of RNA, the cDNA was synthesized using HighCapacity cDNA Reverse Transcriptase kit  to convert the extracted and purified RNA to cDNA. The conversion step was performed in a Veriti® thermal cycler  [K-562 and FEPS cell lines  .®, Foster City, CA, USA) for H2A.X Variant Histone (H2AFX) gene and Beta-Actin (ACTB) gene as an endogenous control. The other genes selected for expression evaluation were ABCB1 (Hs00184500_m1), BCR-ABL1 (Hs03024541_ft) and the ABL gene (Hs99999002_mH) as the endogenous control, and each sample used the concentrations following: 3 μL of cDNA, 1 μL of each primer/probe, 12.5 μL of TaqMan® Gene Expression Master Mix or Fast SyberGreen kit (Life Technologies’), and 8.5 μI of Ultrapure water. The RT-qPCR was performed using QuantStudio5 Real-Time PCR system . Each sample was performed in triplicate for the validation of the technique and the values of CT and gene expression levels were calculated using the 2−ΔΔCT (delta-delta threshold cycle) [All requirements proposed in the Minimum Information for Publication of Quantitative Real-Time PCR Experiments–MIQE Guidelines were followed . The expd cycle)  using DMp < 0.05), and was performed by GraphPad Prism 5.0 software. Each assay was performed in triplicate from three independent experiments. All data were expressed as mean ± standard deviation (SD), the distribution of normality was verified by the Kolmogorov–Smirnov test and statistically compared to untreated control (DMSO) by Analysis of Variance (ANOVA) followed by Bonferroni’s considering a (H2AFX gene signaling, that seems to play a major role in initiating of apoptosis by intrinsic pathway. Therefore, these results indicated that CNN1 has a promising anticancer activity and as a potential lead compound to the development of a new treatment for CML patients who fail their TKI therapy due to intolerance and/or developed resistance, mainly in refractory disease advantage stages.CNN1 naphthoquinone presented cytotoxicity activity based on its ability to induce apoptosis in K-562 and FEPS cell lines. Briefly, we showed that CNN1 blocks cell cycle progression, inducing DNA damage followed by the recruitment of