CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) genes among 120 patients taking warfarin. A new coding variant was identified by sequencing CYP2C9. The novel A > G mutation at nucleotide position 14,277 led to an amino acid substitution of isoleucine with valine at position 213 (I213V). The functional consequence of the variant was subsequently evaluated in vitro. cDNA of the novel variant was constructed by site-directed mutagenesis and the recombinant protein was expressed in vitro using a baculovirus–insect cell expression system. The recombinant protein expression was quantified at apoprotein and holoprotein levels. Its enzymatic activities toward tolbutamide, warfarin and losartan were then assessed. It exhibited changed apparent Km values and increases of 148%, 84% and 67% in the intrinsic clearance of tolbutamide, warfarin and losartan, respectively, compared to wild-type CYP2C9*1, indicating dramatically enhanced in vitro enzymatic activity. Our study suggests that the amino acid at position 213 in wild-type CYP2C9*1 may be important for the enzymatic activity of CYP2C9 toward tolbutamide, warfarin and losartan. In summary, a patient taking high-dose warfarin (6.0 mg/day) in order to achieve the target international normalized ratio was found to have a mutation in the CYP2C9 gene.Warfarin is a widely prescribed anticoagulant but the doses required to attain the optimum therapeutic effect exhibit dramatic inter-individual variability. Pharmacogenomics-guided warfarin dosing has been recommended to improve safety and effectiveness. We analyzed the cytochrome P450 2C9 ( CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) single-nucleotide polymorphisms. Subsequently, statements from both the US FDA and the Clinical Pharmacogenetics Implementation Consortium (CPIC) further confirmed the importance of genetic factors when determining optimum warfarin dosing  of 2–3 for most indications . This van dosing . Althougenotypes , especiaenotypes . ConsequVKORC1. It thereby interferes with the vitamin K-dependent carboxylation of glutamic acid residues in coagulation factors II, VII, IX, and X. In clinical practice, warfarin is used as a racemic mixture of both R and S-enantiomers (Warfarin plays a role in anticoagulation by inhibiting the oxidoreductase (OR) encoded by ntiomers . The S-entiomers . CYP2C9,ntiomers .VKORC1 and CYP2C9 genotypes of 120 patients taking warfarin. A novel A > G mutation at nucleotide position 14,277 was identified in exon four of CYP2C9 in a single patient, resulting in an amino acid substitution from an isoleucine to a valine at position 213 of the CYP2C9 protein (I213V). In vitro enzymatic activity assays showed that this mutation significantly increased the intrinsic clearance of the CYP2C9 probe drugs tolbutamide, warfarin and losartan.In this retrospective study, we analyzed the Spodoptera frugiperda (Sf) 21 cells, fetal bovine serum, Sf-900™ III SFM insect culture medium, Cellfectin II Reagent, and the Bac-to-Bac™ Baculovirus Expression System were purchased from Invitrogen . Tolbutamide, 4-hydroxy tolbutamide, losartan, losartan carboxylic acid, warfarin, 7-hydroxy warfarin, diazepam and midazolam  were purchased from Toronto Research Chemicals . Rabbit polyclonal anti-CYP2C9 antibody and mouse monoclonal anti-OR antibody were obtained from Abcam  and Santa Cruz Biotechnology , respectively. A SuperSignal West Pico Trial Kit was obtained from Thermo Scientific . A regeneration system for the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) was obtained from Promega . High-performance liquid chromatography-grade organic solvents and liquid chromatography–mass spectrometry-grade acetonitrile were purchased from Merck . All other chemicals and reagents used were of the highest purity available.TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit, PrimeSTAR HS DNA polymerase, restriction enzymes, and T4 DNA ligase were purchased from Takara Bio Inc. . CYP2C9 were amplified and sequenced according to previously described methods  using Lasergene software .Genomic DNA samples from peripheral leukocytes were acquired from 120 subjects at the Department of Cardiology of Beijing Hospital (2016BJYYEC-049-02). All subjects provided written informed consent. The study was approved by the ethics committee of Beijing Hospital. After extraction of genomic DNA from the whole blood cells using the TaKaRa MiniBEST Whole Blood Genomic DNA Extraction Kit  according to the manufacturer’s instructions, the whole exons of  methods . BrieflyTTCC-3’ . All acqSf21 insect cells using the Bac-to-Bac™ Baculovirus Expression System that included the pFastBac™ Dual Expression Vector. The dual expression vector pFastBac-OR-CYP2C9*1 was obtained from our previous study (CYP2C9 variant open reading frame (ORF) used in this study, overlap extension polymerase chain reaction amplification was employed, with wild-type CYP2C9*1 as the template. CYP2C9*3 (I359L) has been reported to show reduced enzymatic activities toward substrates such as tolbutamide, losartan, and warfarin , 1 mM phenylmethanesulfonyl fluoride, and 0.25 M sucrose, and sonicated for 40 s on ice using a Vibra-Cell™ sonicator  at 25% of its full power. The homogenate was centrifuged at 13,000 × g for 20 min at 4°C, and the supernatant was then collected and ultracentrifuged at 100,000 × g for 1 h at 4°C. The pellet was resuspended in 100 mM KPO4 (pH 7.4) containing 20% glycerol and then stored at −80°C.Recombinant baculovirus expressing CYP2C9 and OR proteins were prepared using the Bac-to-Bac™ Baculovirus Expression System. After infecting The expression of the CYP2C9 apoenzymes and OR protein was detected by western blotting following standard procedures . As descSf21 cells was evaluated using the classical CYP2C9 probe drugs tolbutamide, warfarin and losartan. Optimized reaction conditions  were performed based on previous studies  287 → 74.02 for 4-hydroxy tolbutamide, a m/z of 437.2 → 235.2 for losartan carboxylic acid, a m/z of 325 → 178.98 for 7-hydroxy warfarin, a m/z of 284.91 → 153.9 for diazepam, and a m/z of 326.1 → 291.1 for midazolam (IS). The cone voltage and collision energy were set at 30 V and 15 eV for 4-hydroxy tolbutamide, 35 V and 15 eV for losartan carboxylic acid, 20 V and 15 eV for 7-hydroxy warfarin, 10 V and 30 eV for diazepam, and 60 V and 30 eV for midazolam, respectively. The gas temperature was set to 500°C at a flow rate of 1000 L/h when detecting 4-hydroxy tolbutamide or losartan carboxylic acid, but was set to 600°C at a flow rate of 800 L/h when testing 7-hydroxy warfarin and diazepam.The levels of 4-hydroxy tolbutamide, losartan carboxylic acid, 7-hydroxy warfarin, diazepam and midazolam were measured by ultra-high-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS). The detection of 4-hydroxy tolbutamide, losartan carboxylic acid and midazolam (IS) was performed using an ACQUITY I-Class UPLC system coupled to a XEVO TQD triple quadrupole mass spectrometer , and fitted with an Acquity UPLC BEH C18 column . An ACQUITY I-Class UPLC system coupled to XEVO TQS triple quadrupole mass spectrometer, fitted with an Acquity UPLC BEH C18 column , was used to quantify the level of 7-hydroxy warfarin and diazepam (IS). The mobile phases involved water with 0.1% formic acid (A) and acetonitrile (B), with a flow rate of 0.4 ml/min and an injection volume of 5 µl. A gradient elution program was used to separate the 4-hydroxy tolbutamide and midazolam, as follows: 0–1.4 min (60–10% A) and 1.4–2.6 min (10–60% A). The total run time was 4 min. To separate losartan and midazolam, we used an isocratic elution of 25% A, with a flow rate of 0.5 ml/min; the total run time was 2.5 min. We used another gradient elution program for 7-hydroxy warfarin and diazepam: 0–0.5 min (10–10% B), 0.5–1.0 min (10–90% B), 1.0–2.0 min (90–90% B), 2.0–2.1 min (90–10% B), and 2.1–3.0 min (10–10% B); the total run time was 3 min. The target compounds were detected using positive electrospray ionization based on multiple reaction monitoring transitions of a mass/charge ratio (apparent Km), and maximum reaction velocity (Vmax), were estimated using Prism 5  based on non-linear regression analyses. The intrinsic clearance (CLint) was calculated as Vmax/apparent Km. The enzymatic activity of the wild-type and variant CYP2C9 was compared by one-way analysis of variance with Dunnett’s post-hoc test using SPSS software , with p < 0.05 representing statistical significance.The enzymatic kinetic parameters, including the Michaelis–Menten constant  warfarin to achieve an INR of 2.0–3.0 in order to prevent thrombogenesis. The novel mutation was located in CYP2C9 exon 4, resulting in an amino acid substitution at position 213 of the protein (I213V) (https://www.ebi.ac.ul/ena/browser/view/PRJEB40955). The novel CYP2C9 mutation has also been submitted to the PharmVar Consortium (https://www.pharmvar.org/gene/CYP2C9). Regarding the patient’s VKORC1 c.-1639G > A status, the A/A genotype was detected.During an investigation of pharmacogenomics-guided warfarin dosing among 120 randomly selected Chinese patients taking warfarin at the Department of Cardiology of Beijing Hospital, the subjects underwent  (I213V) . The datSf21 cells. The enzymatic activity of the recombinant novel CYP2C9 variant was then assessed. To conduct the enzymatic assays, recombinant CYP2C9 holoenzymes  along with the OR protein were successfully coexpressed in the Sf21 cells. Western blotting indicated that the expression levels of the three apoenzymes were similar , warfarin (183.6%) and losartan (166.5%) compared to wild-type CYP2C9*1. All enzymatic kinetic parameters are shown in The enzymatic activities of the recombinant CYP2C9 holoenzymes were evaluated using the three CYP2C9 probe drugs: tolbutamide, warfarin and losartan. The novel CYP2C9 variant (I213V) exhibited significantly changed CYP2C9 gene in a patient taking warfarin. The A > G transition at nucleotide position 14,277 results in an amino acid mutation at position 213, i.e., the substitution of isoleucine with valine. The patient was heterozygous regarding CYP2C9, with wild-type CYP2C9*1 and the novel CYP2C9 variant (I213V). As far as we know, this variant might be the first gain-of-function allele found in a patient and has not previously been submitted for inclusion in the nomenclature database for CYP450 alleles managed by the PharmVar Consortium. CYP2C9 is one of the most polymorphic enzymes, with 62 allelic variants identified and named so far by the PharmVar Consortium. In humans, it metabolizes approximately 20% of clinical drugs in the liver  to estimate the initial therapeutic dose of warfarin in a hypothetical patient possessing CYP2C9*1/*1 and VKORC1 A/A genotype. The clinical factors of the hypothetical patient required in the model were consistent with those of the patient detected in the study carrying the I213V mutation, including age, sex, ethnicity, weight, height, smoking status, indications, INR, and drug combination. The estimated initial therapeutic dose of warfarin was 3.0 mg/day for the hypothetical patient, according to the algorithm based on clinical factors and the CYP2C9 and VKORC1 genotypes (in vitro enzymatic activity of the variant (I213V) toward warfarin led us to speculate that the novel CYP2C9 mutation (I213V) found in the patient was likely to represent a gain-of-function mutation. The high dosage of warfarin required to achieve the target INR for the patient may be explained by taking into account both clinical factors and the novel CYP2C9 variant (I213V) which dramatically enhanced the in vitro enzymatic activity of CYP2C9.Genetic variation in genotype . Notablygenotype . In thisgenetics . The meagenetics . In addienotypes . The meaenotypes  were simCYP2C9 was identified, giving rise to a substitution of isoleucine with valine at position 213 in the protein. The recombinant variant exhibited dramatically increased intrinsic tolbutamide, warfarin and losartan clearance in vitro. The hypothesis that the novel variant affects the warfarin dosage required to achieve an optimum therapeutic effect remains to be validated in future studies.In summary, a novel A > G mutation at nucleotide position 14,277 in


MED13 is able to bind to CCND1 regulatory elements thus influencing the expression. The resistance of MED13 KO cells is directly dependent on the cyclin D1 overexpression, and its down-regulation is sufficient to re-sensitize the cells to alkylating agents. We further demonstrate the therapeutic potential of MED13-mediated response, by applying combinatory treatment with CDK8/19 inhibitor Senexin A. Importantly, the treatment with Senexin A stabilizes MED13, and in combination with alkylating agents significantly reduces viability of cancer cells. In summary, our findings identify novel alkylation stress response mechanism dependent on MED13 and cyclin D1 that can serve as basis for development of innovative therapeutic strategies.Alkylating drugs are among the most often used chemotherapeutics. While cancer cells frequently develop resistance to alkylation treatments, detailed understanding of mechanisms that lead to the resistance is limited. Here, by using genome-wide CRISPR–Cas9 based screen, we identify transcriptional Mediator complex subunit 13 (MED13) as a novel modulator of alkylation response. The alkylation exposure causes significant MED13 downregulation, while complete loss of MED13 results in reduced apoptosis and resistance to alkylating agents. Transcriptome analysis identified   Exposure to exogenous and endogenous alkylating agents results in damage of fundamental biomolecules including DNA . ApproxiGenomic phenotyping and interaction mapping in yeast identified transcription as one of the several novel pathways for alkylation resistance ,8. We veCCND1 and consequent cyclin D1 overexpression, were associated with both resistance to DNA damaging agents, and genome instability  promotes resistance to alkylating agents: methyl methanesulfonate (MMS), TMZ and 1,3-bis[2-chloroethyl]-1-nitrosourea . The resistance to alkylation is accompanied by reduced apoptosis of MED13 KO cells. Accordingly, upon the exposure wild-type (WT) cells downregulate MED13 to survive. Comparison of MED13 WT and KO transcriptomes identified 2 and 37°C. HAP1 cells were cultured in Iscove's modified Dulbecco's medium , supplemented with 10% fetal bovine serum (FBS) , 1% Penicillin–Streptomycin  and 1% l-glutamine . T98G cells were cultured in minimum essential Eagle's medium  supplemented with 10% FBS, 1% Penicillin–Streptomycin, 1% l-glutamine, 1% sodium pyruvate , 1% MEM nonessential amino acids . HEK293T, HeLa and U2OS cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% FBS and 1% Penicillin–Streptomycin, with addition of 1% l-glutamine to HeLa and U2OS cultures. G144 cells were grown on PDL and Laminin coated standard six-well plates. The plates were coated with 5 μg/ml PDL  overnight and 5 μg/ml Laminin  for 2 h. G144 cells were maintained in GBM medium [50% Dulbecco′s modified Eagle′s medium nutrient mixture F-12  and 50% neurobasal medium ] that was supplemented with 1% Penicillin–Streptomycin, 1% MEM nonessential amino acids , 1× Glutamax supplement , 1× N2 supplement (17502001 ThermoFisher), 1X B-27™ supplement without vitamin A , 50 μM 2-mercaptoethanol , 50 μg/ml bovine serum albumin (BSA) , 4 μg/ml heparin , 20 ng/ml epidermal growth factor (EGF) (236-EG-200 R&D Systems), 20 ng/ml fibroblast growth factor (FGF) .Near-haploid HAP1 cell line, derived from the KBM-7 cell line, was obtained from Horizon Genomics, Cambridge, UK. Human embryonic kidney 293T (HEK293T), glioblastoma T98G, cervical adenocarcinoma HeLa (CCL-2) and osteosarcoma U2OS cells were obtained from ATCC (USA). Glioblastoma-derived neural stem cells G144 . All ceHEK293T cells were seeded at 40% confluency the day before transfection. Transfection was performed using VSV.G  and psPAX2  plasmids, pooled human CRISPR Knockout (GeCKO v2) libraries A and B  , and lentivirus carrying GeCKOv2 library A and B added, to achieve multiplicity of infection (MOI) 0.4 and incubated at 37°C overnight. The total number of transduced cells was calculated to achieve at least 300× coverage per each single guide (sg) RNA construct. Next day, cells from each well were transferred to T175 flasks and cultured in IMDM medium supplemented with 0.75 μg/ml puromycin (InvivoGen). Puromycin selection was performed continuously for 7 days. Upon selection 5 × 107 cells were collected as the pre-treatment control, while the rest was transferred to new T175 flasks in three technical replicates and incubated overnight to reach 50% confluency on the day of treatment. Cells were treated for 3 or 7 days with 125 μM MMS , or IMDM medium alone in the case of the control. Next, 5 × 107 cells were collected from each condition, represented in triplicate and genomic DNA isolated using DNA Isolation Kit for Cells and Tissues . Illumina libraries were prepared using two step PCR method with Herculase II fusion DNA polymerase . The primer sequences are listed in 2 × 10To define genes as hits based on shRNA depletion data an RNAi gene enrichment ranking (RIGER) score tool was used . Java imMED13 were designed using the Optimized CRISPR Design tool (http://tools.genome-engineering.org). The oligo pairs encoding the sgRNAs  or media alone for 72 h. Control wells in TMZ and BCNU viability assay were treated with 0.5% DMSO. At the end of the treatment PrestoBlue™ Cell Viability Reagent was added and after 2 h incubation the absorbance measured at 570 nm with reference wavelength 600 nm. The cell viability was calculated using Eq. (1):Cell viability was evaluated using the PrestoBlue™ Cell Viability Assay . 2 × 10300 cells/well were plated in six-well plates. Cells were treated for 72 h with increasing concentrations MMS, TMZ or BCNU . Control wells in TMZ and BCNU experiments included 0.5% DMSO. TMZ and BCNU were diluted in DMSO. Upon treatment cells were allowed to form colonies for 5 days. Cell culture plates were then gently washed with PBS and colonies stained with 0.5% crystal violet solution in 6% glutaraldehyde for 30 min. Excess stain was removed by washing repeatedly with PBS. The colony area percentage was quantified using ColonyArea plugin for ImageJ , accordiThe graphs are presented as normalized colony area percentage calculated using Eq. (3):® . Total RNA was diluted to a concentration of 10 ng/μl and reverse transcribed to complementary DNA (cDNA) using high capacity cDNA reverse transcription kit : 10 μl of total RNA were mixed with 4 mM dNTPs and 50 units of multiverse reverse transcriptase in a final reaction volume of 20 μl in RT buffer supplemented with random primers. The reaction was incubated for 2h at 37°C and 5 min at 85°C. The resulting cDNA was then diluted 1:10 and 2 μl were used for real time-quantitative polymerase chain reaction (RT-qPCR) using Power SYBR Green master mix  in a total volume of 10 μl with 0.2 μM primers. qPCR experiments were performed in technical triplicates using StepOnePlus v2.3 real-time PCR system (Applied biosystems) following thermocycling parameters: initial step at 95°C for 10 min, followed by denaturation 95°C for 15 s; annealing and extension for 60°C for 1 min, for a total of 40 cycles. Primers targeting MED13, CCND1, RELN and GAPDH transcripts are depicted in GAPDH.HAP1 cells were treated with 125 μM MMS or media alone for 72 h, washed twice with PBS, pelleted, snap frozen and stored at −80°C. For mRNA levels analysis total RNA was isolated using RNeasy mini kit , with inclusion of on-column DNase I digestion , according to the manufactured protocol. Total RNA integrity was evaluated via gel electrophoresis analysis of the rRNA 28–18 s ratio and quantified using NanoDrop6 cells in 15 μl hypotonic lysis buffer ) and incubated on ice for 5 min, followed by three freeze-thaw cycles. Cell suspension was supplemented with 140 mM NaCl and 0.5% NP-40 and incubated 20 min on ice. Next, cell pellet was diluted with 50 μl lysis buffer containing 140 mM NaCl and sonicated using Bioruptor (Diagenode) . Samples were centrifuged 10 min, 12 000g at 4°C and supernatant representing WCE collected. For the immunoblot analysis, proteins were separated on NuPage 4–12% Bis–Tris polyacrylamide gel , and transferred to 0.45 μm PVDF membrane in transfer buffer . The proteins of interest were detected using specific primary antibodies , anti-Tubulin , anti-β-actin , anti-cyclin D1 antibody , anti-Phospho Ser15 (P-S15) p53 , anti-P-Ser727 STAT1 ), and corresponding secondary antibodies: polyclonal swine anti-rabbit immunoglobulins/HRP  and infrared (IR) Dye-conjugated secondary antibodies . The signal was visualized in case of HRP antibodies by SuperSignal West Femto Maximum Sensitivity Substrate  and ChemiDoc imager system (Bio-Rad), and the IR signal by the Odyssey Scanner, LI-COR Biosciences. Protein levels were quantified using ImageJ software.Cells were treated with 125 μM MMS, or media alone in case of negative control, for 72 h. Next, the whole cell extracts (WCE) were prepared by resuspending pellet from 0.5 × 106 HAP1 WT or HAP1 MED KO clones (cl.) 10 and 17 were seeded per six-well plate. Next day cells were treated with 125 μM MMS, or media alone in case of negative control, for 72 h. Total RNA was purified with RNeasy mini kit , in presence of DNase I , according to the manufacturer's protocol. RNA library preparation, and sequencing of HAP1 samples, were carried out by the BGI Genomics, China. The libraries were screened on the Bioanalyzer (Agilent), pooled in equimolar concentrations, and sequenced using a paired-end (PE), 100 bp stranded mRNA library protocol (B02) on a BGIseq platform. PE FASTQ files were processed to remove adapter sequences and low-quality reads using Trimmomatic v0.33  and incubated 15 min on ice, and 15 min at 30°C. Next, nuclei were pelleted and washed with buffer A , followed by buffer B . Nuclei were lysed in lysis buffer  and chromatin sheared to 200–250 bp DNA fragments by sonication with Bioruptor (Diagenode) for 30 min; 30 s ON per minute. 25 μg of chromatin was next precleared for 2 h at 4°C, and incubated with 2 μg antibody  or rabbit IgG  in ChIP buffer  overnight at 4°C. The DNA–protein–antibody complexes were isolated using A dynabeads  and washed using sequentially: low salt wash buffer , high salt wash buffer  and LiCl wash buffer . Proteinase K treatment was performed for 1 h at 50°C with 10 mM EDTA, 40 mM Tris–HCl pH 6.5 and 20 μg proteinase K . The DNA was purified with phenol-chloroform, ethanol precipitated and analyzed by qPCR. The qPCR experiments were performed in technical triplicates using following thermocycling parameters: 95°C for 10 min, followed by 40 cycles 95°C for 15 s; 60°C for 1 min, in StepOnePlus v2.3 Real-Time PCR System (Applied Biosystems). Levels of immunoprecipitated DNA is expressed as relative occupancy = (% input of tested condition)/(% input of control condition). The ‘% input’ value represents the enrichment of MED13 on specific region of the genome. Primer sequences are listed in the 6 HAP1 WT and MED13 KO cl.10 cells were seeded in T25 flasks and next day treated with 125 μM MMS or IMDM medium alone for 72 h. After treatment, cells were collected, washed once with 1 ml cold PBS, fixed in 1 ml of ice-cold 100% methanol and centrifuged at 200g at 4°C. Cell pellets were washed once with PBS and treated with 0.1 mg/ml RNase  at 37°C for 30 min in 200 μl. Next, 200 μl of 50 μg/ml propidium iodide (PI) solution  was added and samples incubated for 30 min at 37°C, followed by FACS analysis. Cells were analyzed using FlowJO 10.6.1. Forward scatter FSC-A and side scatter SSC-A were used to identify cell population; PI fluorescence pulse area (PI-A) and PI fluorescence pulse width (PI-W) were used to identify single cells. Cell cycle phases were analyzed in PI histogram plot. Data is available in FlowRepository under accession FR-FCM-Z32R.1 × 10d-lysine  and 0.35 × 106 HAP1 WT or MED13 KO cl.10, cl.17 cells seeded per well. Next day, cells were treated with 125 μM MMS for 72 h. After the treatment, cells were washed two times with PBS and fixed with 4% paraformaldehyde  for 15 min. Cells were washed three times with PBS and permeabilized with 0.1% Triton X-100 in PBS for 40 min at RT. The blocking was performed with 0.1% Triton X-100, 5% BSA and 5% goat serum  in PBS for 40 min at RT. Cells were incubated 2 h at RT with primary antibody Anti – pSer139 H2A.X clone JBW301  diluted in 0.1% Tween-20, 0.5% BSA and 0.5% goat serum. Cells were washed with 0.1% Triton X-100 in PBS and incubated 1 h at RT with secondary antibody Alexa Fluor 488 goat anti-mouse IgG1  diluted in 0.1% Triton X-100 in PBS. Cell were washed three times with 0.1% Triton X-100 in PBS, stained and mounted with Prolong Gold Antifade reagent with DAPI . The samples were imaged using a Zeiss LSM880 confocal microscope  at a 40x magnification (1.4 NA). Quadratic images with a side length of 354.25 μm were taken in 24+/–2 z-planes with a z-plane-interval of 0.5 μm . Images were taken at three random places per slide  by an experimenter not acquainted with the study design. After three-dimensional rendering using the software Imaris 8.2 , nuclei were identified by modelling spherical objects (‘spots’) with a diameter of 10 μm around all DAPI-positive areas. DAPI areas >50% outside the imaging area were not considered for counting. The spots were used to define the intranuclear area by ‘masking’ the channel containing the γH2AX/Alexa488 fluorescent signal, setting the voxels outside the spots to a fluorescent signal equaling zero. Within the masked channel, high-intensity spots were identified by modelling spherical objects with a 1 μm diameter. Two random images with a relevant number of double-strand break spots were used as a reference measurement, in which spots were first identified using the automatic spot detection method provided by Imaris , with an automatic thresholding algorithm  (Sigma) according to the manufacturer's protocol, incubated for 15 min at room temperature and analyzed on a BD FACS Aria II (BD Biosciences). The fraction of apoptotic cells was determined by using the FlowJo, LLC software (USA). Unstained and single stained cells were used as controls. PI negative and Annexin V positive cells we considered apoptotic, and cells that are positive to both PI and Annexin V considered necrotic. Data is available in FlowRepository under accession FR-FCM-Z32T.Twenty-four hours after seeding HAP1 cells were treated with 125 μM MMS for 72 h. Treated and untreated cells were the harvested, washed with cold PBS and diluted to 1 × 10CCND1 (cyclin D1) targeting siRNA are listed in HAP1 WT and MED13 KO cells were seeded to reach 80% of confluency on the day of transfection. Cells were transfected using 7.5 pmol of siRNA and 7.5 μl Lipofectamine 3000 transfection reagent . The sequences of scrambled and HAP1cells were transfected with pcDNA3-Myc-cyclinD1(WT)  or pEGFP-C1 (Clontech) as mock and transfection control using Lipofectamine 3000 transfection reagent. Twenty-four hours post-transfection, cells were seeded into 96-well plate for viability assay, and a T75 flask for RT-qPCR and immunoblot analysis to confirm overexpression. Next, cells were treated with MMS (concentrations indicated in the figures) or media alone for 72 h for viability assay.HAP1 WT and MED13 KO cl. 10, HeLa, T98G or U2OS cells were seeded in 96-well plates to reach 15% confluency the next day. Cells were first pretreated with Senexin A  or 0.125% DMSO for 2 h, followed by addition of modified media without, or with MMS or TMZ and 70 h incubation; all respective drug amounts are indicated in the Figures. Viability and immunoblot analysis were performed as described above.et al.  enriched gene targets at 3- and 7-day time points, respectively  (2O2), but not to hydroxyurea in HAP1 cells  . As in Hd cl.12) . InteresP1 cells . In concP1 cells  and F anP1 cells  treatmenP1 cells . To deteP1 cells  and I. AP1 cells . ImmunobP1 cells  and K. AP1 cells , while pP1 cells . In summSince MED13 is a subunit of large transcriptional Mediator complex, to investigate a mechanism of MED13 KO resistance to alkylation, we performed RNA sequencing analysis. Transcriptomes of MED13 deficient and proficient cells were compared in the absence and presence of MMS. After bioinformatic processing, at ≥2-fold change and FDR≤0.1, 446 differentially expressed genes (DEGs) were identified in untreated MED13 KO, when compared to WT HAP1 cells Figure  and B. UCCND1 expression, and consequently cyclin D1 mRNA levels, RT-qPCR analysis was performed in MED13 proficient and deficient cells. In accordance with the RNA sequencing results , LMNA (Lamin A) and MED13  expression (CCND1, thus having the ability to directly regulate its expression (Figure Alkylating agents belong to the oldest group of chemotherapeutics frequently used to treat cancer. Despite their use in clinics, cancer cells often develop resistance to alkylating drugs ,6. To im3 Figure . In contkylation ,49, MED12 Figure . By anal2 Figure –G. Work e stress . Since le stress . On the e stress . Cyclin e stress . Fruthere stress ,54. Simipression ,56. MED1n Figure . By analn Figure . Both shn Figure ,57. Impon Figure , while on Figure -H inducen Figure ,58,59. Bn Figure ,25. Furtn Figure . Recent n Figure , which cβ-catenin in human colon cancer HCT116 cells (in vivo tests with several currently available inhibitors reported severe adverse effects in animals (Since downregulation of MED13 is essential to promote survival upon alkylation exposure Figure , we hypo16 cells ,64. Simi16 cells . While C animals . In addi animals . Thus, tTaken together, our findings identified MED13 as a novel modulator of alkylation response, a downregulation of which promotes resistance to DNA damaging agents. Moreover, we provide mechanistic insights to the MED13 mediated alkylation response, by demonstrating link between MED13 status and cyclin D1 expression relevant for the cell survival. Finally, we propose an innovative strategy for potential combinatory treatment with CDK8/19 inhibitors that stabilize MED13, and significantly potentiate cancer cell killing by alkylating drug. The full potential of CDK8 inhibition in combination with alkylating chemotherapeutics remains to be explored.The RNA sequencing data reported in this paper are available in GEO under accession GSE147366. Data associated with FACS analysis is available in FlowRepository under accession FR-FCM-Z32R and FR-FCM-Z32T.gkaa1289_Supplemental_FileClick here for additional data file.


Mobile phase consisted of 0.1% acetic acid in water (10%) and methanol (90%) at a total flow rate of 0.25 mL/min. The mass spectrometer was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using precursor ion > product ion transitions of 324.10 > 168.30 m/z for MP1 and 411.95 > 224.15 m/z for PL-3. The MS/MS response was linear over the concentration range from 0.2–500 ng/mL for MP1, correlation coefficient (r2) of 0.988. Precision (% RSD) and accuracy (% bias) were within the acceptable limits as per FDA guidelines. MP1 was stable under storage and laboratory handling conditions. The validated method was successfully applied to assess the solubility, in-vitro metabolism, plasma protein binding, and bio-distribution studies of MP1.MP1 is a novel marinopyrrole analogue with activity in MYCN amplified neuroblastoma cell lines. A rapid, selective, and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method was developed and validated for quantitation of MP1 in mouse plasma. Analyte separation was achieved using a Waters Acquity UPLC Neuroblastoma (NB) is a rare embryological malignancy derived from neural crest cells. NB is mainly diagnosed before the age of 5 years, with a median age of diagnosis of 18 months. NB is the second most common extracranial malignant tumor in childhood and accounts for about 15% of pediatric cancer deaths ,3,4. AppSeveral genetic alterations have been observed in NB cells, including amplification of MYCN . MYCN amplification is associated with a poor survival rate of only 14–34%, even in patients with favorable outcome profiles ,6. AmpliMarinopyrroles are novel alkaloids isolated from a marine Streptomyces (actinomycete strain CNQ-418) with an uncommon 1,3′-bipyrrole pharmacophore . They weIn order to characterize in-vitro properties and assess the pharmacokinetics and biodistribution of MP1, we developed a rapid and sensitive bioanalytical method for the quantification of MP1 with a short run time (~6 min). The results of these studies provide the needed information to guide MP1 dosing in animal models of NB. Further, these findings in correlation with biological response will be useful for further evaluation of the therapeutic use of MP1.− of MP1 were seen in abundance, having a precursor ion > product ion combinations of 324.10 > 168.30 m/z and 411.95 > 224.15 m/z for PL3, respectively  detection, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) conditions were tested. Signal intensities of MP1 and the IS were found to be the highest using ESI in the negative ionization electrospray ionization mode (ESI−) (data not shown). MS/MS conditions were optimized using 1 µg/mL of MP1. De-protonated molecules [M–H]ectively .®BEH C18 column, 1.7 µm, 100 × 2.1 mm, Waters, Inc. Milford, MA, USA) column equipped with an Acquity UPLC C18 guard column . Final mobile phase consisted of 0.1% acetic acid in water , and MeOH , utilizing an isocratic elution at a total flow rate of 0.25 mL/min. The retention time of MP1 was 1.9 min and 4.5 min for PL3. The method incorporated an additional 2.1 min after the final analyte peak, to wash out any residue and assure complete evaluation of the compound, bringing the total run time to 6.0 min. No interference from endogenous components were found in the biological matrix at MP1 or the IS retention time , additives , gradient parameters, and analytical columns . Final separation conditions were achieved on an Acquity UPLCion time .To optimize sample preparation, protein precipitation, liquid–liquid extraction and solid-phase extraction (SPE) methods were evaluated. The final SPE method was selected to prepare plasma and tissue samples due to its high extraction recovery. By comparing the SPE rate with various organic washing and eluting solvents, MeOH was finally selected as a reliable eluting solvent for SPE because it had good recovery with no matrix effect ,12. The To analyze the specificity of the method, blank extracted plasma and tissue homogenate was analyzed for potential interferences at the retention times of 1.9 min (MP1) and 4.5 min (IS). There was no significant interference or co-eluting peaks that were >20% of the analytes area at the Lower limit of quantification (LLOQ) level, and no co-eluting peaks >5% of the area of IS were observed, meeting the acceptance criteria and indicating the specificity of the method.The LLOQ for MP1 was determined to be 0.2 ng/mL. The intra–run precision at the LLOQ plasma samples containing MP1 was 10.8%. The intra–run accuracy at the LLOQ plasma samples was 7.3%. The intra and inter-day variability results are tabulated in 2) was greater than 0.987.The MP1 calibration curve was linear over the range of 0.2 to 500 ng/mL . The corIn order to assess carry-over, blank samples (running buffer only) were analyzed immediately after running high quality control (HQC) samples. No significant peaks, ≥20% LLOQ, were observed. Thus, it was concluded that carry over was negligible in the current method (<5% of LLOQ).Intra- and inter-day accuracy and precision for MP1 was assessed at four different concentrations in mouse plasma LLQC (0.2 ng/mL), LQC (0.6 ng/mL), MQC (100 ng/mL) and HQC (375 ng/mL) levels. The %RSD of precision values ranged from 1.4 to 13.6%, indicating acceptable assay precision. The accuracy of the quantitative analysis of the compounds was varied from −13.4–7.3%, within the acceptance limits at all concentration levels. The accuracy and precision results are listed in Analyte recovery for MP1 and IS from spiked plasma samples was calculated using three control samples . The % extraction mean recovery for MP1 in LQC, MQC and HQC was 95.8 ± 2.6, 89.1 ±10.6 and 90.8 ± 3.4%, respectively. The mean recovery of all QC levels was >90%, whereas the mean recovery of IS was 87.3 ± 3.8%. The matrix effect was negligible and within an acceptable ±15%. Stability of MP1 was assessed by several means including 4 h bench-top at room temperature 21 °C, three freeze-thaw cycles, autosampler stability for 24 h at temperature of 4 °C, and long term stability for 12 months frozen −80 °C, MP1 showed no appreciable instability, as concentrations were within the accepted ±15% standard .The dilution integrity of the samples was ensured by diluting QC samples above the upper limit of quantification (ULOQ) with like matrix to bring to within quantitation range 0.2–500 ng/mL. The mean analyzed value of all diluted samples was within 15% of nominal concentration, with precision of the replicates ±15% coefficient of variation (data not shown).The solubility of MP1 in phosphate buffer (pH 7.4) was approximately 50 µg/mL (Mean ± SD 51.19 ± 2.11 µg/mL).The stability of MP1 was evaluated at pH 1.2, 6.8, and pH 7.4. MP1 was found to be stable with >99% parent remaining after 1 h in simulated gastric fluid (SGF), pH 1.2, and 2 h of incubation in simulated intestinal fluid, (SIF) pH 6.8, and in buffer at pH 7.4. The blood:plasma (Kb/p) ratio was determined for MP1 at a concentrations of 1 µM (325 ng/mL). The Kb/p ratio was 0.40, 0.68 and 0.63 after 0, 30- and 60-min incubations at 37 °C . MP1 was highly bound (>95%) to mouse plasma proteins at both 1 µg/mL and 10 µg/mL concentrations. MP1 was stable in mouse plasma following a 5 h incubation . There w½ of MP1 was 15.64 ± 0.46, 18.1 ± 3.73, 27.47 ± 2.68 min in mouse, rat, and human, respectively. The in-vitro half-life (t½), intrinsic clearance (CLint) and in-vitro hepatic clearance  of MP1 in mouse, rat, and human microsomes are summarized in In-vitro metabolic stability of MP1 was investigated using mouse, rat, and human liver microsomes. The time-dependent metabolic depletion of MP1 in the interspecies microsomes is shown in 1/2 of MP1 was 4.9 ± 0.2 min and CLint of 142.3 ± 5.6. CLint was calculated using the MP1 depletion over time from initial substrate concentration of 1 µM (In-vitro metabolic stability of MP1 was investigated using mouse S9 fractions. The result of the metabolic stability study was expressed as the % parent remaining at different time points relative to the parent at 0 min (100% parent) b. The in of 1 µM . Positiv− was recorded, and structural information was collected by fragmentation of all precursor ions. The mass of each predicted metabolite was used to generate post-acquisition extracted ion chromatograms (EICs).Mouse S9 fraction samples were run in a 35 min gradient method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in positive and negative ESI mode. Each peak, retention time, and precursor ion peak [M–H]− with a retention time of 25.39 min. Two metabolites were identified based on their LC/MS data and putative structure via possible biotransformation, precursor ion, and retention times  software simulating through non-compartmental analysis. The mean non-compartmental pharmacokinetic parameters are shown in max was 4714.7 ± 2343.5 ng/mL at a mean Tmax of 0.6 h. MP1 t1/2 was 9.2 ± 1.7 h and CL was 1.1 ± 0.3 L/h/kg.Pharmacokinetic data was processed with PhoenixConcentrations of MP1 was detected in all studied tissues, with the concentration the highest in the liver at all-time points evaluated . Concent2) greater than 0.987 for all calibration curves. The method was validated according to FDA guidelines [We successfully developed and validated a rapid, selective, and sensitive LC-ESI-MS/MS method for the quantitation of MP1 in mouse plasma and tissues. The response was linear over the concentration range of 0.2–500 ng/m with a correlation coefficient  in mouse, rat, and human liver microsomes respectively. In-vitro mouse S9 metabolic investigations exhibited rapid metabolic degradation with two metabolites being detected, glucuronidation being the major metabolic pathway ,22.max of 0.6 h with a t½ of 9.2 h. MP1 was found in the systemic circulation up to 48 h. In addition, MP1 showed good penetration in all the tested tissues, with the highest concentration in the liver and the lowest accumulation in the brain.The method was successfully applied to pharmacokinetic studies of the oral administration of MP1. We found that MP1 was rapidly absorbed with a TWe did not observe any phase I metabolites  in these initial studies. The disappearance rate of MP-1 in the S9 fraction studies was much faster compared to that observed in the MLM, RLM and HLM in the presence of UDPGA or NADPH, respectively . The glu50). One of the limitations of this study is that our drug concentrations represent total drug concentrations. Typically, free drug concentrations correlate with therapeutic effects better than total drug concentrations. Future studies will assess tumor drug penetration and cellular uptake of MP1 to bridge the gap between drug dose, free drug concentrations, and tumor response.Following the pre-clinical studies investigating the biodistribution of MP1, we found that MP1 is rapidly distributed throughout the body. These studies provide the framework for future studies to design dosing regimens that achieve MP1 concentrations associated with activity in cell lines  and PL3 (purity: ≥99.9%), used as an internal standard (IS), were generously provided by Dr. Rongshi Li (UNMC). Methanol (MeOH), acetonitrile (ACN) and sodium acetate were purchased from Fisher Scientific . All solvents were HPLC grade or higher. Water was purified using a Barnstead GenPure water purification system . Bond Elute C18 cartridges (50 mg per 1 mL) were purchased from Agilent . Mouse blood and plasma was purchased from Equitech-Bio, Inc. . All other materials and reagents were purchased from standard chemical suppliers and of analytical grade or higher.m/z for MP1 and 411.95 > 224.15 m/z for IS. The final optimum MS parameters are shown in An LC-MS/MS 8060 system  equipped with a dual ion source (DUIS) was used to perform mass spectrometric detection. LabSolutions LC-MS software Version 5.8  was used for data acquisition and quantitation. To achieve the desired sensitivity, the compound dependent mass spectrometer parameters, such as temperature, voltage, gas pressure, etc., were optimized by auto method optimization via product ion search for MP1 and IS using a 0.5 µg/mL solution in MeOH. All analytes were detected in ESI− with the mass spectrometer source settings as follows: nebulizer gas: 2.0 L/min; drying gas: 10 L/min; heating gas: 10 L/min; interface temperature: 300 °C; desolvation line (DL) temperature: 250 °C; heat block temperature: 400 °C. The MS/MS system was operated at unit resolution in the multiple reaction monitoring (MRM) mode, using precursor ion > product ion combinations of 324.10 > 168.30 ®BEH C18 column  equipped with an Acquity UPLC C18 guard column . The mobile phase consisted of 0.1% acetic acid in water (mobile phase A) and methanol (MeOH) (mobile phase B) operated in isocratic mode with 10% A and 90% B with a total flow rate of 0.25 mL/min. The total run time was 6 min. The injection volume of all samples was 2 μL.All chromatographic separations were performed with an Acquity UPLCStock solutions of MP1 and IS were prepared at 1 mg/mL concentrations in DMSO. The stock solutions were diluted with MeOH to make working standard solutions and used to prepare the calibration standards (CSs) and quality control samples (QCs). CSs and QCs were prepared by spiking 10 µL of working standard solution and 10 µL of IS working solution (0.5 µg/mL) into 100 µL of blank mouse plasma or tissue homogenate to obtain a concentration range of 0.2–500 ng/mL. The final CS concentrations were: 0.2, 0.5, 1, 5, 10, 50, 200 and 500 ng/mL in bio-matrices. The QC comprised four concentrations: 0.2 ng/Ml—LLOQ, 0.6 ng/mL—LQC, 100 ng/mL—MQC, and 375 ng/mL—HQC. QCs were prepared separately in five replicates, independent of the calibration standards. CSs, QCs and all the main stock solutions were freshly prepared prior to use. Intermediate stocks, spiking calibration, and QC stock solutions were kept at −20 °C.g for 10 min at 4 °C, and the was supernatant transferred to HPLC vial for injection and analysis.All analytes were extracted from CSs, QCs, mouse blood, and tissue samples by SPE using Agilent bond Elute C18 cartridges. Samples were prepared by spiking 10 µL of appropriate calibration stock in 100 μL blank bio-matrix (blank mouse plasma or tissue homogenate) and 10 µL of IS solution (0.5 µg/mL) and diluted with 600 µL of 1% acetic acid. A Cerex solid-phase extraction manifold from Varian  with nitrogen to modulate flow was used for sample processing. Samples were vortexed for 2 min and then loaded onto SPE cartridges pre-conditioned with MeOH (2 mL), followed by water (1 mL). Loaded cartridges were washed with 15% MeOH (1 mL) and eluted with MeOH (2 mL). The eluate was evaporated under vacuum at room temperature then reconstituted in 200 µL of mobile phase  then vortexed for 30 s and centrifuged at 13,400× Each tissue sample was accurately weighed and then homogenized with de-ionized water at a 5- fold dilution factor for liver, spleen, brain, lungs, and kidney using a TissueLyserII . Heart tissue was homogenized at a 6-fold dilution factor. The resultant tissue homogenate (100 µL) was spiked with 10 µL of IS solution and prepared for SPE as describe above for plasma samples.g for 10 min. A 100 µL aliquot of the clear supernatant was transferred to an HPLC vial and 2 µL injected into the LC-MS/MS system.S9 fraction and microsomal incubation buffer samples were extracted using simple protein precipitation. S9 fraction samples (100 µL) were obtained and mixed with 300 µL of ice-cold ACN spiked with 10 µL of IS (0.5 µg/mL) in a 1.5 mL-centrifuge tube. The samples were vortexed for 2 min, then centrifuged 17,940× The LC–MS/MS method was validated in accordance with the Food and Drug Administration (FDA) guidelines for Bioanalytical Method Validation  with resAssay selectivity and specificity was determined by comparing the chromatogram of six different blank mouse plasma or tissue homogenate samples with that MP1 and IS-spiked plasma or tissue homogenate samples. Peak interference at the retention time of analytes and internal standard was investigated by comparing analyte response of interference-free blank plasma, plasma spiked with MP1 and the IS, and neat stock solutions in reconstitution solvent as described in The sensitivity of the method was determined from the signal-to-noise ratio (S/N) of the response of MP1 to blank matrix. The S/N ratio was calculated by comparing the baseline noise at the vicinity of the retention time of analyte with that of the LLOQ peak response. The lower limit of detection (LLOD) and LLOQ were defined as a three- and ten-fold S/N ratio, respectively. y-axis versus concentration of MP1 on the x-axis. The calibration curve was generated using weighted linear regression (1/x2). Each calibration curve consisted of a blank sample, a zero sample (blank + IS), and 12 non-zero concentrations consisting of eight CSs and four QCs. The acceptance criteria for each CS concentration was ±15% standard deviation (SD) from the nominal value, except at LLOQ, which was set at ±20%. The calibration curve had to have a correlation coefficient (r2) of 0.988 or better for MP1. The assay was linear over the range of 0.2–500 ng/mL.The calibration curves were established by a linear plot of the relation between the peak area ratio (MP1/IS) on the Residual carry-over was assessed by injecting two consecutive “zero samples” after running the HQC (375 ng/mL). The response of the first zero sample was required to be <20% of the response of a processed LLOQ sample to conclude no carry-over.Intra and inter–assay accuracy and precision were evaluated by analyzing five replicates of QC samples at four different concentrations  in mouse plasma or tissue homogenate for three consecutive days. Precision was defined as the percent relative standard deviation (%RSD) with acceptance criteria of ±15% (except ±20% at LLOQ). Accuracy was defined as the percent bias (% Bias) with the same acceptance criteria as precision. %bias was calculated according to the following Equation (1): Extraction recovery was calculated at three different QC concentrations (LQC 0.6 ng/mL), (MQC 100 ng/mL) and (HQC 375 ng/mL) for both MP1 and IS (0.5 µg/mL) by comparing the mean peak area of an analyte spiked before extraction to the mean peak area of an analyte spiked post-extraction multiplied by 100. To assess the effect of the matrix on analyte response, blank mouse plasma and tissue homogenate were processed as described and the dry post-extract was spiked with analyte prepared equivalent to the QCs. The average peak area from analytes spiked in the extracted blank matrix was compared to the response for QCs prepared in reconstitution solvent. The absolute matrix effect (ME) and IS normalized ME were calculated as described in Equations (2) and (3).Storage stability of MP1 in mouse plasma samples was determined at three different QC concentrations. The following stabilities were evaluated: bench-top storage (4 h at room temperature 21 °C), three freeze–thaw cycles , and long-term storage (12 months at −80 °C). Additionally, 24 h auto-sampler stability of extracted samples (at 4 °C) was determined. The sample concentration for MP1 under each condition was tested and mean values for accuracy and precision calculated. The dilution integrity of samples was tested on six replicates of three levels of dilution: two- (2× HQC), five- (5× HQC), and ten-fold (10× HQC) dilutions of the high QC concentration. The calculated concentration measurements were compared to the nominal concentration at each dilution level. g for 15 min. Supernatant was aspirated and mixed with equal volume of acetonitrile. Calibration curve samples were prepared in buffer, acetonitrile, DMSO media for analysis. Solubility concentration was calculated via the calibration curve using analyte/IS peak area ratio and concentration at which the analyte precipitated out, which was considered to be the upper soluble concentration. The solubility of MP1 was assessed by spiking 1% DMSO stock solutions (5 µL) into 100 mM phosphate buffered saline  in triplicate to produce final assay concentrations of 1, 5, 10, 20, 40, 50, 60, 80 µM. Spiked samples were kept at room temperature for 2 h. At pre-determined times, each microcentrifuge tube was centrifuged at 10,000× Simulated gastrointestinal fluids were prepared according to USP specifications. Briefly, SGF was formulated consisting of sodium chloride (0.2 g), pepsin (3.2 g), hydrochloric acid (7 mL) in water . SIF was formulated with potassium dihydrogen phosphate (6.805 g), sodium hydroxide (0.896 g), pancreatin (10 g) in water . MP1 at 10 µM concentration was incubated with SGF, SIF and phosphate buffer  at 37 °C on a shaking water bath each in triplicate. Samples (200 µL) were collected at 0, 15, 30, 60 and 120 min. Immediately after sample collection, five volumes of MeOH containing IS were added and samples prepared for analysis. Stability was determined as a percentage parent compound detected at each time point relative to the concentration at 0 min in buffer (100%) .g for 10 min at 4 °C to separate 50 µL plasma for analysis. Blank plasma (50 µl) was added to the blood aliquot, and 50 µL of blank blood was added to the separated plasma for matrix match and calibration in 100 µL. The whole blood and plasma samples were further processed by SPE as described above. The analyte peak areas ratios were used to calculate the blood-to-plasma (B/P) ratio using Equation (4).The mouse blood to plasma partitioning of MP1 was determined at concentrations of 1 µM. Fresh mouse blood (800 µl) was incubated in a water bath maintained at 37 °C for 10 min. After incubation, blood was spiked with the drug from stock solutions of 1 µM (325 ng/mL) to achieve final concentrations and to maintain organic content <1%. At different time points , aliquots (50 µL) were collected for blood analysis and an additional sample (120 µL) was transferred into a micro-centrifuge tube and centrifuged at 4000× A Rapid Equilibrium Dialysis (RED) device system  was used to evaluate PPB by adding buffer to the buffer chamber and dosing solution to the sample chamber. The buffer chamber contained 350 µL of phosphate-buffered saline . The sample contained mouse plasma spiked with 1 µM or 10 µM MP1 and was added to the sample chamber. The RED kit top was then sealed and incubated at 37 °C on an orbital shaker at 100 rpm for 5 h.Plasma stability of MP1 was assessed along with the non-specific binding potential of MP1 to the dialysis membrane and cell chambers by spiking drug into buffer while incubating at 37 °C for 5 h and comparing it with 0 h concentration. At pre-determined times, aliquots (40 µL) were removed from the sample and buffer chambers and mixed with an equal volume of buffer or blank plasma, respectively. The samples were further processed by SPE as described above and analyzed by LC-MS/MS. The stability, equilibrium, device recovery and PPB were calculated using following equations .(5)% Ren = 3. Reactions were performed at a final incubation volume of 1000 µL and initiated with the addition of the S9 fraction, 50 µL of 20 mg/mL protein concentration. Immediately after mixing the MP1 into the incubation mixture, a sampling point was taken, representing t = 0. Subsequent sampling points were performed at the following intervals: 5, 15, 20, 30, 45, 60, 90 and 120 min. To stop the reaction, 100 µL of the sample were added to 1.5 mL-centrifuge tube containing 300 µL of ice-cold ACN spiked with 10 µL of IS (0.5 µg/mL). All the samples were vortexed and centrifuged at 13,000× g for 10 min. The resultant supernatant was transferred to an autosampler vial and 2 µL injected on the LC-MS/MS. Testosterone, 7-HC, and diclofenac were utilized as positive controls to ensuring proper incubation conditions were maintained.In-vitro phase I and II metabolism were assessed using mouse liver S9 fractions . Briefly, 1 µM MP1 was pre-mixed with a solution mixture of potassium phosphate buffer  at 37 °C containing the following: 1 mM NADPH, 4 mM saccharolactone, 1 mM uridine 4′-diphospho-glucuronic acid, and 0.1 mM 3′-phosphoadenosin-4′-phosphosulphate. Incubation was performed in triplicate, g for 15 min, supernatant collected and transferred to an autosampler vial and injected (2 µL) onto the LC-MS/MS system. Testosterone and diclofenac were used as positive controls to ensure that HLM and incubation conditions were appropriate to conduct metabolism studies.Metabolic stability was assessed using mouse, rat, and human liver microsomes  for phase I metabolism. Briefly, the buffer solution was prepared containing potassium phosphate buffer , 25 µL of microsomal protein (20 mg/mL), magnesium chloride (10 mM) and NADPH (2 mM) in a final volume of 0.5 mL was pre-incubated at 37 °C for 10 min in water bath maintaining at 60 rpm. The reaction was started by adding 2µL of MP1 (1 µM). Serial samples (50 µL) were collected at selected time intervals  and quenched with 200 µL of acetonitrile spiked with 10 µL of IS (0.5 µg/mL). All the samples were vortexed and centrifuged at 13,000× −1). The half-life (t1/2) was calculated using Equation (9). The in-vitro intrinsic clearance (CLint) was determined by using Equation (10). The intrinsic clearance was further extrapolate to in-vitro hepatic clearance  using a scaling factors and Equation (11) [Both the S9 Fraction and HLM metabolic stability was expressed as the percentage of drug remaining at each time point. The in-vitro metabolic elimination rate constant was calculated from the first-order plot of a natural logarithm of the area ratio versus time. The slope of the linear regression equation was used to determine elimination rate constant “k” (minion (11) ,25.(9)tTo identify MP1 metabolites, we performed a mass shift analyses using the same LC-MS/MS system. A 35 min gradient time profile was used for metabolite identification via Q1 scan mode, which ranged of 250 to 1000 Da. The gradient profile of the mobile phase was held at 10% mobile phase B (MeOH) for 1 min then increased up to 95% mobile phase B over 28 min, then brought back to 10% B over 1 min, followed by a 4 min equilibration. The parent drug MS/MS spectrum was used as a template model for metabolites structural identification and assuming the fragmentation pattern of the parent drug, MP1, from the product-ion spectrum, which was used to deduce the structures of the metabolites ,22. The v/v). MP1 (15 mg/kg) was administered by oral gavage, the dose was selected based on previous pharmacological reports in mice [g at 4 °C for 10 min. The collected plasma samples were stored at −80 °C until analysis.The University of Nebraska Medical Center Institutional Animal Ethics Committee approved all animal studies (protocol number 17-046-06-FC). Female BALB/c mice, with weights ranging from 24 to 30 g were used for biodistribution studies. Animals were housed in the University of Nebraska Medical Center animal facility, for at least 7 days prior to the experiments, in order to acclimatize the animals to the laboratory conditions, at a temperature of 23–24 °C, relative humidity of 40–70% and 12/12 h light/dark cycles. The dosing solution was made of DMSO-Polyethylene glycol 400 (PEG400)-Propylene glycol (PG)-EtOH-Cremophore-PBS  were collected at 2, 4, 8- and 48-h following dosing. Tissue samples were rinsed with phosphate buffered saline to remove blood and then blotted with filter paper. After weighing, each tissue sample was individually homogenized with de-ionized water, 5-dilution factor for liver, spleen, brain, lungs, and kidney while the heart was homogenized at a 6-dilution factor using a TissueLyser II  then all tissues were stored at −80 °C until analysis. Plasma concentrations (ng/mL) and tissue concentrations (ng/g) were determined for each time point collected. Drug accumulation in tissue was determined by calculating a tissue to plasma concentration ratio (Kp) for each tissue.® 8.2  to determine plasma pharmacokinetic parameter of MP1 in plasma. Peak plasma concentration (Cmax) and time for the peak plasma concentration (Tmax) was obtained from visual inspection of the concentration-time plot. The area under the curve (AUC0–∞) was estimated using the linear trapezoidal method from 0–tlast and extrapolation from last time point to infinity based on the observed concentration at the last time point divided by the terminal elimination rate constant (k).Non-compartmental pharmacokinetics analysis was performed on Phoenix1/2) was calculated using Equation (12). Clearance (CL) was calculated using Equation (13) and the apparent volume of distribution of the elimination phase (Vd) were calculated using Equation (14). The tissue to blood (Kp) ratio was calculated by using Equation (15).The elimination half-life (tIn conclusion, we successfully developed and validated a rapid, selective, and sensitive LC-ESI-MS/MS method for the quantitation of MP1 in mouse plasma and tissues. The assay was linear over the concentration range of 0.2–500 ng/mL. The LC–MS/MS bioanalytical method was validated according to FDA guidelines, showed acceptable selectivity, no significant matrix effects, and acceptable accuracy and precision. In addition, we applied the validated method to the assessment of MP1 in-vitro and in-vivo ADME properties in biological samples with nominal concentrations as low as 0.2 ng/mL.


Proteomics profiling disclosed the molecular mechanism underlying beef poor meat quality. This study aimed to identify protein markers indicating the quality of beef during postmortem storage at 4°C. Beef longissimus dorsi samples were stored at 4°C. The meat water holding capacity (WHC), pH value and moisture content were determined at different time points during the storage period. The iTRAQ MS/MS approach was used to determine the proteomics profiling at 0, 3.5 and 7 d during storage at 4°C. Bioinformatics analysis was performed to investigate the potential correlated proteins associated with meat quality. Storage at 4°C gradually decreased the pH value, WHC, and hence the moisture content. The iTRAQ proteomic analysis revealed that a cluster of glycolytic enzymes including malate dehydrogenase, cytoplasmic, L-lactate dehydrogenase, phosphoglycerate mutase and pyruvate kinase, and another cluster of proteins involved in oxygen transport and binding (myoglobin) and hemoglobin complex  were decreased during the postmortem storage. These results suggest that the decreased glycolysis, oxygen, and heme-binding activities might be associated with the beef muscle low quality and the decline of tenderness during postmortem storage at 4°C. The freshness of meat is a major factor influencing the purchasing decisions . Many exProteomics analysis of factors associated with meat quality has implied that storage methods or intrinsic factors influence the meat quality and color stability –10. ProtBeef meat accounts for a high proportion of meat sales worldwide. Among the attributes of beef quality, tenderness is the most important one affecting the acceptability the consumer , 15. WeiAs such, we performed this study to investigate the differential abundance of proteins related to the quality of beef muscle (longissimus dorsi). Muscle samples were stored at 4°C. Proteomics profiling was analyzed and the bioinformatics analysis was performed to identify the biomarkers related to the beef quality.Beef longissimus dorsi muscles were obtained from a local slaughterhouse and were brought to our laboratory within 4 h after slaughter. The samples were divided into three parts randomly and then stored at 4°C for 0, 12, 24, 36, and 48 h, respectively. They were stored at 100% humidity on sterilized disks without packaging .The changes in color, pH, and WHC during storage were determined according to our previous methods . In brieMinced beef was set on sterilized disks at 4°C for 0 d, 3.5 d, and 7 d (3 replications at each time point). After complete grounding, homogenates were then precipitated and incubated in chilled acetone (10% trichloroacetic acid) at -20°C for 2 h. After centrifugation at 4°C and 20,000 g for 20 min  the centrifugal precipitates were washed with chilled acetone, lysed in lysis buffer [Protein aliquots (100 μg) were digested into peptides using 3.3 μg Trypsin (Thermo Scientific.) at 37°C for 24 h, following with centrifugation . Peptide precipitation was vacuum-dried, diluted in 0.5 M tetraethylammonium bromide, and then prepared using an iTRAQ kit  according to the recommendation from the manufacturer. The samples were pooled and vacuum-dried.2PO4 in 25% acetonitrile) and trapped onto a strong cation exchange (SCX) column . The elution of peptides from the trapping column was performed in a gradient buffer B . The peptides were trapped onto C18- reversed-phase column  and eluted onto another reversed-phase C18 column using a gradient buffer B . The fractions of the peptides were then collected and subjected to a nanoelectrospray ionization tandem mass spectrometry . Data acquisition was performed at 70,000 resolution in full scan mode and 35,000 resolution in MS 2 mode.The dried peptide samples were dissolved to form a solution A (10 mM KHThe raw MS/MS data was downloaded and processed using Proteome Discoverer (Thermo Fisher Scientific). Peptides sequences were aligned and searched in the UniProt database  using the criteria of full tryptic specificity and tolerance of one missed cleavage (15 ppm and 0.02 Da). Proteins with fold discovery rate (FDR) <0.01 were retained and used for the identification of the differentially expressed proteins (DEPs).http://www.geneontology.org), Kyoto Encyclopedia of Genes and Genomes  databases and Interpro (IPR) protein domain database at p < 0.05. Heatmap analysis was carried out using heatmap (R package version 0.7.7). Enrichment analysis in GO and KEGG was performed to identify the biological functions and pathways associated with the DEPs, with at p < 0.05.The DEPs were identified at p-value ≤ 0.05 by the t-test and fold change (FC) ≥ 1.2 or < 0.83. The properties of the DEPs were characterized in Gene Ontology . There was also a significant decrease in WHC and moisture content of beef muscle during the first 24 h storage at 4°C .Beef muscle pH significantly decreased during the 48 h storage at 4°C , and the mass of most peptides was between 10 and 100 kDa . Annotation in GO  and KEGG  showed that these proteins were enriched in various biological processes and pathways involved in cellular processes, environmental information processing, and metabolism. The search for InterPro domains resulted in 465 proteins annotated with protein domains including SH3, WD40 repeat, and immunoglobulin .Three samples of 0, 3.5, and 7 d at 4°C storage, respectively, were extracted and used for proteomic analysis. Proteomic data generated 513,662 total spectra, corresponding to 13,345 single peptides and 1,667 proteins. The length of most peptides was between 6 and 32 aa (S1 Table). The bidirectional clustering heatmap showed the significantly differential expression profiles of these DEPs, and most of them gradually decreased during the 7-day storage at 4°C . Venn figure showed that protein Q32KU9 was downregulated at 3.5 d and 7 d compared with 0 d, and at 7 d compared with 3.5 d .Comparative analysis between groups showed that 102 proteins were differentially expressed between groups, including 11 DEPs between 0 and 3.5 d, 91 DEPs between 0 and 7 d, and 34 DEPs between 3.5 and 7 d, respectively (S2 Table).DEPs between the samples at 3.5 d and 0 d were associated with chondrocyte differentiation , chondrocyte proliferation , sarcoplasmic reticulum , and phosphopyruvate hydratase complex .DEPs between samples at 7 d and 0 d, including downregulated A6QLL8, Q3ZC87, F1N2F2, A0A3S5ZPB0, B3IVN4, Q3T145, P33097, and A0A3Q1M5R4, were involved in GO processes like glycolytic process (GO: 0006096), nucleoside metabolic process (GO: 0009116), organic acid metabolic process (GO: 0006082), and organic substance catabolic process (GO: 1901575). A0A3Q1MAU7 was associated with cellular aldehyde metabolic process (GO: 0006081) and small-molecule catabolic process .DEPs between samples at 7 d and 3.5 d were enriched into the carboxylic acid metabolic process (GO: 0019752), and organic substance catabolic process , UTP biosynthetic process , glycolytic process , oxygen transport , Glycolysis/Gluconeogenesis , and Complement and coagulation cascades . DEPs between samples at 7 d and 0 d, and 7 d and 3.5 d were associated with Malaria , Pyruvate metabolism , Glucagon signaling pathway (map04922) and Biosynthesis of amino acids . DEPs between 7 d and 0 d were also associated with the PPAR signaling pathway .KEGG pathways enrichment revealed that DEPs between the samples at 3.5 d and 0 d were associated with three pathways HIF-1 signaling pathway . We realized that the downregulated Q32KU9 and A6QR19, and upregulated Q4U0T9 were key proteins between samples stored for 3.5 d and 0 d. A6QLL8, Q3ZC87, A0A3Q1M5R4, A6QR19, A6QNN1, A0A1K0FUF3, Q3T145, Q3T0Q4, P01966, and P33097 were key proteins in the PPI consisting of the DEPs between 7 d and 0 d. A6QLL8, Q3ZC87, A0A3Q1M5R4, A6QR19, A0A1K0FUF3, Q3T145, Q3T0Q4, P01966, and P33097 were associated with the PPI consisting of DEPs between 7 d and 3.50 d.PPI network identified key DEPs between meat samples stored for 0 d, 3.5 d, and 7 d Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


It showed 65% inhibition of VEGF2 tyrosine kinase activity and demonstrated a potential antitumor activity in TAM-resistant, LCC2, and its parental MCF7 BC cells. KM6 retained the sensitivity of LCC2 through upregulation of key enzymes of apoptosis and proteins of cell death including caspases 3, 8, 9, P53, BAX/BCL-2 ratio and LDH in media. It downregulated mRNA expression of Ki-67, survivin, Akt, and reduced levels of ROS and glucose uptake. Moreover, KM6 reduced the levels of inflammation markers PGE2, COX2, IL-1β and IL6 and metastasis markers MMP-2 and MMP-9. In conclusion, KM6 is a promising compound for ER + and TAM-resistant BC with many potential antitumor and polypharmacological mechanisms.Breast cancer (BC) and endocrine resistance to chemotherapy are challenging problems where angiogenesis plays fundamental roles. Thus, targeting of VEGFR-2 signalling pathway has been an attractive approach. In this study, we synthesised a new sorafenib analogue, thieno[2,3-  The World Health Organisation considers breast cancer (BC) as the most common malignant tumour among women,,d]pyrimidine ring system constitutes an attractive chemical scaffold with several reported biological activities e.g. inhibition of inflammatory-related diseases, acting as potent ligands for 5-HT3 and/or 5-HT4 receptors and othersd]pyrimidines fused with lipophilic cycloalkyl ring  potentiate the anticancer activityKM6 with benzothienopyrmidine ring system replacing the pyridyl carboxamide moiety in sorafenib. The new derivative KM6 was tested for its cytotoxic activity against different BC cell lines namely MCF7, T47D, MDA-MB and LCC2 (TAM-resistant cells). Additional experiments were also performed to explore the different mechanisms where KM6 exerts its cytotoxic activity. Angiogenesis ensures sufficient supply of oxygen and nutrients to proliferating cells, resulting in tumour progression and metastasis1H-NMR spectra were recorded on a Bruker 400 MHz spectrometer in δ scale (ppm) using DMSO as a solvent and TMS as an internal standard. 13C NMR spectra was run at Joel 100 MHz spectrometer in δ scale J(Hz) using DMSO as a solvent and TMS as the internal standard. The LC–MS-MS system consisted of a Agilent 1200 HPLC system  with a quaternary gradient pump (Agilent 1260 infinity), an online vacuum degasser, a column oven and an autosampler (Agilent 1260 infinity), coupled to a ABSCIEX Q TRAP 3200 mass spectrometer  equipped with an electrospray ionisation (ESI) interface. Data acquisition was performed with analyst 4.0 software (ABSCIEX). Separation was performed using a Waters XBridge-C18-5 µm  reversed phase analytical column . The mobile phase was pumped at flow rate of 300 µl/min and it consisted of 0.1% formic acid/10 mM ammonium format (solvent A) and 100% acetonitrile (solvent B). Over all run time was 12 min. The mass spectrometer was operated in the positive ESI mode with spray voltage set at 4 kV, at a temperature of 350 °C and a sweep gas flow of 20 L/h. Calculations were completed by the Multiquant software programme. Serial dilutions of standards were prepared at concentrations ranging from 62.5 to 1000 ng/ml for sorafinib and KM6 to make calibration curves,All starting materials, reagents and solvents were purchased from  and used without further purification. Monitoring of chemical reactions was performed by analytical thin layer chromatography (TLC), on silica gel 60 F254 packed on Aluminium sheets, purchased from  and were visualised using U.V. lamp (254 nm). Column chromatography was conducted on silica 60 (35–70 microns) eluent gradient hexane: ethyl acetate 90:10 to 85:15). Melting points were determined by . TAM citrate was obtained from . It is a white powder stored at room temperature. TAM was dissolved in dimethylsulfoide (DMSO) and prepared as stock solution at concentration 10 mM and was kept at −20 °C. Immediately before use, it was serially diluted in RPMI1640 to yield a concentration range of 2–80 µM.KM6 was accomplished by a convergent synthesis through condensation of the diaryl urea derivative (1) with 4-chloro-5,6,7,8-tetrahydrobenzo[4–5]thienopyrimidine derivative (4). The diaryl urea (1) was obtained by reaction of p-aminophenol with 4-chloro-3(trifluoromethyl) phenyl isocyanate in dry 1,4-dioxane(4) was synthesised via three reaction steps starting by reaction of cyclohexanone, ethyl cyanoacetate and elemental sulfur in the presence of piperidine  to give the 2-amino tetrahydrobenzothiophene derivative (2). Compound (2) upon cyclisation with formamide gave the tetrahydrobenzothienopyrimidine derivative (3) which was chlorinated to afford compound (4)1H-NMR of KM6 revealed the appearance of two exchangeable protons corresponding to NHs at 9.8 and 10.0 ppm, aromatic protons at 7.08–8.34 ppm in addition to cyclohexyl protons at 1.78, 2.9 and 3.0 ppm. 13C NMR revealed the appearance of peaks at 168.03 (C4 pyrimidine), 163.51 (C2 pyrimidine), 153.10 (C = O), 22.27, 22.84, 25.45, 25.94 (cyclohexyl protons). Quantification was performed with multiple reaction monitoring (MRM) by using gas collision-induced dissociation and the following ion transitions: M/z 465.12/252.2 and 519.1/324.1 for sorafinib and KM6, respectively, with the cone voltages all set at 45 V and the collision energy at 35 eV. Typical chromatograms for detection of Sorafinib and KM6 are displayed in Supplementary Data SI. They were detected at retention times 0.98 and 11.1 min. Calibration curve was linear (r2 ≥ 0.99).As described in d]pyrimidine derivative (4)  was added slowly and the reaction mixture was heated for 6 h at 55–60 °C. The solvent was removed under reduced pressure and the residue was stirred with cold 4% NaOH solution  for 1 h. The solid product was filtered and purified with column chromatography (eluent hexane: ethyl acetate 90:10 to 85:15) to afford KM6 m.p. 250–253 °C. MS: (Mwt 518.94) (ES mass) (rel. int.) 519.08, , 1H-NMR  δ: 10.08 , 9.83 , 8.47 , 8:16 , 7.55–7.73 , 7.21 , 7.19 , 3.02–3.33 , 2.8–2.9 , 1.87–1.91 . 13 C-NMR  δ: 168.0, 163.5, 153.1, 152.5, 147.1, 140.2, 137.5, 136.0, 132.4, 127.4, 127.0 (2 C), 126.0 , 123.3, 122.6 , 120.0 (2 C),119.0 118.8, 117.1, 25.9, 25.4, 22.8, 22.2, Elemental Analysis: Calcd for C24H18ClF3N4O2S; C 55.55; H 3.50; N 10.80 Found; C 55.41; H 3.53; N 10.63 .A solution of the urea derivative (1)  and caesium carbonate  in dry acetonitrile (10 ml) was stirred for 1 h at room temperature. The 4-chloro thieno was prepared as stock solution at concentration 10 mM and was kept at −20 °C. Immediately before use, it was serially diluted in RPMI 1640 to yield a concentration range of 2–32 µM.2 in a humidified atmosphere at 37 °C.Human BC cell lines: MCF7, T47D and MDA-MB-231were obtained from the American Type Culture Collection  and were maintained at National Cancer Institute (NCI), Cairo, Egypt in RPMI1640 medium containing 10% foetal bovine serum and 1% penicillin-streptomycin. LCC2 was a generous gift from Dr Robert Clarke  and was cultured in EMEM medium containing 5% charcoal foetal bovine serum and 1% penicillin–streptomycin. All cell lines were routinely incubated in 5% COKM6 and TAM on BC cells were evaluated by sulphorhodamine-B (SRB) assay3 cells/well in 96-well microtiter plates. They were left to attach for 24 h before incubation with drugs. Next, cells were treated with different concentrations 2–32 µM of KM6, 2 − 14 µM of TAM for MCF7, T47D and MDA-MB-231 cells and by 5–80 µM of TAM for LCC2. For each concentration, three wells were used and incubation was continued for 48 h. DMSO was used as control vehicle (1% v/v). At the end of incubation, cells were fixed with 20% trichloroacetic acid, stained with 0.4% SRB dye. The optical density (O.D.) of each well was measured spectrophotometrically at 570 nm using ELISA microplate reader . The percentage of cell survival was calculated as follows: Survival fraction = O.D. (treated cells)/O.D. (control cells). The IC50 (concentration that produce 50% of cell growth inhibition) value of each drug was calculated using sigmoidal dose–response curve-fitting models .The antitumor activities of 50 concentrations of TAM and KM6 in both cell lines. Therefore, in MCF-7, 11 µM of TAM and 6.4 µM of KM6 were used, while in LCC2 the concentrations used were 67.6 µM of TAM and 3.6 µM of KM6.In all the mechanistic experiments in this study, we used the ICKM6 into the ATP binding site of VEGFR-2 kinase enzyme, molecular docking simulation study was performed using C-Docker protocol in Discovery Studio 2.5 Software to rationalise the VEGFR-2 inhibitory activity of KM6.To get insights of the binding mode, interaction and orientation of (4ASD), revealed that the substituted terminal phenyl ring lied in a deep extended hydrophobic pocket formed by the movement of Phe1047 residue of the “DFG” motif to induce the “DFG-out” conformation  in the VEGFR-2 kinase active site and aligning the lead compound's bioactive X-ray conformer with the best-fitted pose acquired from the docking run. The alignment displayed good coexistence between both posed with RMSD = 0.95A°, demonstrating the capability of the used docking protocol to fetch valid docking poses.(KM6) was prepared using ligand preparation protocol of Accelry’s Discovery Studio. Finally, docking process was done using C-Docker software in the interface of Accelry’s discovery studio 2.5. Ten docking poses of the docked compound (KM6) were generated and examined carefully for selecting the best binding mode similar to sorafenib in the VEGFR-2 binding pocket.To execute the docking protocol, the protein structure was prepared through protein preparation protocol that is integrated into the software. Water molecules were preserved due to their role in VEGFR-2/ligand interaction. The protein structure was also minimised via SMART minimiser algorithm. Afterwards, binding pocket and the surrounding amino acid residues were identified. Compound KM6 was shown to verify the essential key interactions, known for type II VEGFR-2 inhibitors and comparable to sorafenib. Thus, the thienopyrimidine moiety occupied the flat aromatic hinge region and a hydrogen bond between N1- nitrogen and the main chain NH group of Cys919 residue was observed. In addition, the urea tail was engaged in three key hydrogen bonds with Glu885 and Asp1046 residues. Finally, a Pi-cation interaction with Lys868 residue, as well as a Pi-sigma interaction with Leu 840 residue were established . The VEGFR tyrosine kinase activity at single dose concentration of 10 µM was performed, where VEGFR2 (KDR) (BPS#40301) served as the enzyme source and Poly  sodium salt,  (Sigma#P7244) served as the standardised substrate. Kinase-Glo Plus Luminescence kinase assay kit  was used according to the manufacturer’s instructions.The 4 cells/well. After treatment with drugs, the culture medium was aspirated and centrifuged at 10,000 rpm at 4 °C for 10 min and the resultant supernatant was used for determination of VEGF by an ELISA Human VEGF ELISA Kit  in accordance with the manufacturer’s instructions. The amount of VEGF was expressed as pg/ml. Level of VEGF was determined from calibration curve and the percentage of VEGF was calculated as follows:MCF-7 and LCC2 cells were plated in 24 well plates with 2.5 × 10The level of VEGF was calculated using sigmoidal dose–response curve-fitting models .Lipid peroxidation products were quantified by measuring MDA level in cell culture lysate of control and treated cells using lipid peroxidation (MDA) Assay Kit  following the manufacturer’s instructions. The absorbance was determined at 532 nm using a spectrophotometer . The MDA concentration was calculated using sigmoidal dose–response curve-fitting models .The amount of MDA present in the samples was determined from the standard curve.Sa = Amount of MDA in unknown sample (nmole) from standard curveSv = Sample volume (µl) added into the wellsC = Concentration of MDA in sampleD = Sample dilution factor.The activity of the antioxidative enzyme SOD was determined by SOD determination kit  in cell culture lysate of control and treated cells by following the manufacturer’s instructions. SOD level was calculated relative to the corresponding protein content. The absorbance of the supernatant was determined at 450 nm using a spectrophotometer . The SOD activity (inhibition rate %) was determined using the equation:Reduced glutathione was determined adopting Ellman’s methodNitric oxide was determined in culture media of the control and treated cells according to Miranda et al.Activities of caspases 3, 8 and 9 were measured spectrophotometrically at 450 nm in cell lysate using Colorimetric Assay Kits   following the manufacturer’s instructions. Percentage change in caspase activity was determined by comparing the results with the level of the untreated control. The experiment was carried out three independent times.In the cell culture medium of the control and treated cells, the concentration of glucose was determined using Colorimetric Assay Kit  (Cat No.GL 346) following the manufacturer’s instructions. The absorbance was recorded at a wavelength of 500 nm using a spectrophotometer . Glucose conc. (mg/dl) = 4 cells/well. After treatment with drugs, the medium was aspirated, centrifuged at 10,000 rpm for 10 min at 4 °C to remove any dead cells and was used for necrosis experiments. Cell pellets were lysed using protein lysis buffer and used for the determination of LDH. UV assay kit was used following manufacturer instructions . The activity was calculated relative to the corresponding protein content. The absorbance was recorded at 340 nm using a spectrophotometer .In this study, LDH was measured in the media for the detection of cytotoxicity (necrotic cell death). It was also measured intracellularly after cell lysis as a marker for glycolytic pathway. Thus, MCF-7 and LCC2 cells were plated in 24 well plates with 2.5 × 102 was calculated in comparison with a plotted standard curve and was expressed as pg/ml.The concentration of PGE2 in cell culture media of the control and treated cells was determined using the PGE2 Enzyme Immunoassay Kit  following the manufacturer’s instructions. The concentration of PGETotal RNA was extracted from cells using RNeasy Mini Kit . cDNA was prepared from RNA (200 ng) in a 20 µl reaction using High capacity cDNA archive kit . Real-time PCR of GAPDH, COX-2, IL-1β, IL-6, MMP2, MMP-9, Survivin, AKT and Ki-67 were performed in triplicate on an ABI 7500 fast real-time PCR System using GoTaq PCR master mix . Fast amplification parameters were as follows: an initial denaturation step at 95 °C for 10 min, followed by 45 cycles of denaturing at 95 °C for 15 s, annealing at 60 °C for 1 min, then extension at 72 °C. All primers used in this study were purchased from Invitrogen (CA) (Supplementary Table (SV)). Quantitative analysis of data was performed by using the ΔΔCt methodThe cycle threshold (Ct) was determined automatically.Agarose gel electrophoresis was used for assessing BAX, BCL2 and P53. Total RNA was extracted from cells using RNeasy Mini Kit . cDNA was prepared from RNA (200 ng) in a 20 µl reaction using high capacity cDNA archive kit . mRNA expression was measured by gel electrophoresis and β-actin was used as internal control. Primer sequences are displayed in Supplementary Table (SVI).After incubation of control and treated cells for the specified times, media were collected and stored at −80 °C and cells were harvested by trypsinization then lysed with RIPA lysis buffer  containing protease inhibitors. Protein concentrations in media and cell lysate were determined by Bradford Assay kit . All experiments were run in triplicate.After treatment of cells, cell pellet was resuspended in cold PBS and subjected to centrifugation, the pellets were collected. The cell pellets were lysed by resuspending in 1 ml complete RIPA buffer containing protease/phosphatase inhibitor cocktail and placed on ice for 30 min till complete lysis. The lysate in RIPA buffer was transferred to an Eppendorf and centrifuged for 15 min at 13,000 rpm at 4 °C. Extracted proteins were seperated by SDS-PAGE (12% acrylamide) and blotted onto PVDF membranes. The membranes were probed with IL-6 and β-Actin antibodies for normalisation. Band intensities were quantified using Win Image Studio Lite_5.2.5 software using β-actin as loading control. Data are represented as mean ± SD.MMP-2 and MMP-9 enzymatic activities in media of the control and treated cells were determined by SDS-PAGE gelatine zymographyP values less than 0.05 were statistically significant. Graphs were performed using Prism software programme  and analysis of data was performed using GraphPad InStat, version 5.Data are represented as means ± SD. The data were analysed using one-way analysis of variance (ANOVA) test. To assess the significance of differences, the Tukey post hoc test was used. KM6 were tested for their cytotoxicity against TAM sensitive MCF7 cells and TAM-resistant LCC2 cells. The IC50 of TAM in LCC2 was around 6 folds higher than in MCF7 (67.6 ± 0.07 µM vs 11 ± 0.05 µM) thus confirming the resistant phenotype of LCC2 cells (KM6 displayed a more remarkable antitumor activity than TAM on both MCF7 (IC50 = 6.4 ± 0.05 µM) and LCC2 (IC50 = 3.6 ± 0.03 µM) (Both TAM and C2 cells . Interes0.03 µM) .KM6 also demonstrated remarkable cytotoxic activity against other BC cell lines T47D (IC50 = 6.9 ± 0.04 µM) and MDA-MB-231 (IC50 = 10 ± 0.04 µM) ). In the subsequent experiments, we aimed to study the different mechanisms whereby KM6 exerted its cytotoxic activity in MCF-7 and LCC2 cell lines.This was in agreement with the study of Pedersen et al.KM6 was evaluated for its ability to inhibit VEGFR-2 tyrosine kinase enzyme activity in vitro at a single dose concentration of 10 µM as a measure to suppress angiogenesis. At such concentration, KM6 exhibited 65% VEGFR-2 kinase inhibition. Moreover, KM6 decreased VEGF level in both MCF7 and LCC2 culture media by 30.4 and 33%, respectively, compared to their corresponding controls. This is similar to sorafenib and other analogues that is a well-known inhibitor of VEGF in BCKM6 by 42.3% but no significant effect was observed on VEGF in LCC2 cells  production and an altered redox environment compared to normal cells. Most chemotherapeutics agents raise intracellular levels of ROS, and can alter redox homeostasis of cancer cellsKM6 on the apoptotic caspases was investigated. This included the caspases involved in the extrinsic pathway (caspase 8), intrinsic pathway (caspase 9) and executioner pathway (caspase 3). The results indicated that in MCF7, KM6 induced significant increase in caspases 8, 9 and 3 by 53.8, 49 and 76.4%, respectively. A similar pattern was observed in LCC2 where KM6-treated cells displayed increase of all the studied caspases. It's observed that the induction of caspases by TAM is more pronounced on MCF7 than KM6. However, LCC2 exhibited resistance to the apoptotic effect of TAM. This resistance was not observed in KM6-treated cells  Colorimetric assay of caspases Caspase-8, Caspase-9 and Caspase-3, respectively. (D) mRNA expression of survivin determined by real-time RT-PCR (E) mRNA expression of P53 determined by agarose gel electrophoresis.  Agarose gel electrophoresis of BAX and BCL-2, respectively. (H) BAX/BCL-2 ratio. (I) Effect of TAM and KM6 on necrosis as determined by measuring LDH levels in culture media of MCF7 and LCC2. Band analysis was analysed by Win Image Studio Lite_5.2.5software. Statistical significance of results was analysed using one-way ANOVA followed by Tukey’s multiple comparison test. aSignificantly different from control, bfrom TAM and (p < 0.05). TAM was used at IC 50 (11 µM for MCF7 and 67.6 µM for LCC2). KM6 was used at IC 50 (6.4 µM MCF7 and 3.6 µM for LCC2).Effect of TAM and KM6 on survivin (member of the inhibitor of apoptosis IAP family) was investigated as displayed in KM6. TAM also reduced the expression of survivin in MCF7 by 98-fold but increased its expression in LCC2. Concerning the tumour suppressor gene P53 which plays an important role in apoptosis inductionKM6 by 19.5% in MCF7 and by 13.9% in LCC2 compared to control ). This upregulation of P53 was previously observed by sorafenib in hepatocellular carcinomaThe effect of  control ). This uKM6 favourably modulates the gene expressions of the apoptotic protein BAX as well as the antiapoptotic proteins BCL-2. As shown in KM6 resulted in increasing BAX/BCL-2 ratio by 4.56 folds in MCF7 and by 3 folds in LCC2. This was comparable to BAX/BCL-2 ratio produced by TAM in MCF7 (4.52). However, in LCC2, KM6 was superior to TAM that decreased BAX/BCL-2 ratio to 0.438%. Previous studies demonstrated that sorafenib reduces the antiapoptotic protein Mcl-1, which is a member of BCL-2 family leading to induction of apoptosis in lung cancer cell lineThe current study indicated that KM6 by 37.2 and 78%, respectively. TAM also induced necrosis in both cell lines, yet its effect on LCC2 was very pronounced where it increased by 177.1% compared to control group.A key sign for necrotic cells is the permeabilization of the plasma membrane that can be detected in tissue culture by quantifying the release of the intracellular enzyme LDHAmong the metabolic changes exhibited by tumour cells is an increase in glucose metabolism and glucose dependence. Tumour cells exhibit high levels of glycolysis even in the presence of sufficient oxygen, a phenomenon termed aerobic glycolysis or Warburg effect. This involves increased glucose uptake, glycolytic rates, ATP production, lactate as well as several intermediates and enzymes that benefit rapidly proliferating cells. LDH is a key enzyme in this process that converts most of glucose stores into lactate. As a result, much of glucose metabolites are shifted from simple energy production to the promotion of accelerated cell growth and replicationKM6 resulted in decline in both glucose uptake and LDH levels by 44 and 32.95%, respectively, relative to their corresponding controls  showed a significant dramatic decrease in the gene expression of AKT by 232.4 folds in MCF7 and 106.9 folds for LCC2 as compared to control. In addition, a profound decline in AKT expression was observed compared to TAM-treated cells in both cell lines. It’s known that activation of AKT is a mechanism of resistance to sorafenib in hepatocellular carcinomaKM6 displays a promising potential in blocking AKT pathway and further studies are required to test its ability to reverse acquired resistance to sorafenib especially in hepatocellular carcinoma.In this study, treatment of MCF7 with controls . In LCC2 results ) showed ,The inflammatory environment affects cancer cell behaviour, including cancer formation, invasion and metastasisKM6 on several inflammatory mediators  has been evaluated . The reKM6 exerts a promising anti-inflammatory effect in BC.The effect of sorafenib on inflammatory markers in BC has not been widely explored. Recently, it was reported that sorafenib improves therapy with alkylating agents by blocking inflammation, invasion and angiogenesis in BC cellsKM6 to reverse this inflammatory effect. Further studies are thus required to elucidate the relation between TAM and inflammation in BC as well as the potential use of KM6 in modulating this effect.An interesting observation is an obvious increase mediated by TAM of all the studied inflammatory mediators in both MCF7 and LCC2 and the ability of KM6 as indicated in KM6-treated cells, which suggests that this drug may have some antimetastatic activities. Given the multiple pathways of metastasis, further studies are needed to confirm this mechanism.The high expression of proteinases such as MMP-9 and MMP-2 in the tumour microenvironment has a role in supporting migration, invasion and metastasis of cancer cells through ECM degradationd] pyrimidine based urea derivative KM6 is a potential therapeutic agent for ER + TAM sensitive and resistant BC, providing a basis for development of the compound as a novel anticancer agent. The mechanism of this activity is believed to be through targeting several key enzymes and proteins involved in tumour growth and progression. Through its polypharmacological properties, KM6 can affect several pathways of angiogenesis, redox activity, apoptosis, inflammation, glucose metabolism and metastasis. Further preclinical, molecular and toxicologic studies are needed to elucidate deeply the main mechanism of action and to determine the optimal dose, route and formulation for administering KM6.From this study it can be concluded that the newly synthesised thieno[2,3-Click here for additional data file.