CDH1 and EPCAM in A2780cis compared to the cisplatin-sensitive parental cells. These changes were concomitant with gene expression down-regulation. DNA hypomethylation associated with transcription up-regulation of the mesenchymal marker TWIST2 was observed in the resistant cells. Azacytidine treatment confirmed DNA methylation role in regulating gene expression of CDH1, EPCAM and TWIST2 genes. A2780cis cell line undergoes EMT like changes, and EMT genes are regulated by DNA methylation. To that end, a better understanding of the molecular alterations that correlate with chemoresistance may lead to therapeutic benefits such as chemosensitivity restoration.Ovarian cancer is one of the lethal gynecologic cancers. Chemoresistance is an essential reason for treatment failure and high mortality. Emerging evidence connects epithelial-mesenchymal transition (EMT) like changes and acquisition of chemoresistance in cancers. Including EMT, DNA methylation influences cellular processes. Here, EMT-like changes were investigated in cisplatin-resistant A2780 ovarian cancer cells (A2780cis), wherein role of DNA methylation in some EMT genes regulations was studied. Cell viability assay was carried out to test the sensitivity of A2780, and A2780cis human cancer cell lines to cisplatin. Differential mRNA expression of EMT markers using qPCR was conducted to investigate EMT like changes. CpG methylation role in gene expression regulation was investigated by 5-azacytidine (5-aza) treatment. DNA methylation changes in EMT genes were identified using Methylscreen assay between A2780 and A2780cis cells. In order to evaluate if DNA methylation changes are causally underlying EMT, treatment with 5-aza followed by Cisplatin was done on A2780cis cells. Accordingly, morphological changes were studied under the microscope, whereas EMT marker’s gene expression changes were investigated using qPCR. In this respect, A2780cis cell line has maintained its cisplatin tolerance ability and exhibits phenotypic changes congruent with EMT. Methylscreen assay and qPCR study have revealed DNA hypermethylation in promoters of epithelial adhesion molecules  In 2021, 2% of all new cancer cases was estimated to be ovarian cancers2. Patients with ovarian cancer have a poor prognosis and high mortality rate due to its deceptive onset and lack of early diagnosis3. The low survival rate of ovarian cancer is due to metastasis and acquisition of chemoresistance4. Cisplatin (cis-diamminedichloroplatinum [II]) is a platinum compound widely used in solid tumors treatment many (including ovarian cancer)6. Cisplatin induces DNA lesions and activates several signaling pathways such as DNA repair mechanisms7. When DNA damage fails to be repaired, cells undergo apoptosis8. Cisplatin resistance is the main obstacle that limits the treatment effectiveness9. The mechanisms that underlie the platinum drug resistance are multifactorial. Several cellular processes are responsible for the resistant phenotype, such as drug influx or efflux alterations, DNA repair, cell cycle, and apoptosis11.Ovarian cancer is one of the deadliest gynecologic cancers17. EMT is a cellular procedure where epithelial cells lose their cell–cell adhesion and cell polarity and gain a metastatic capability18. The molecular signatures of EMT are the loss of epithelial cell markers CDH1 and the up-regulation of mesenchymal markers, which comprise EMT transcription factors SNAIL and TWIST20. Increased findings implicate EMT in promoting aggressiveness in ovarian cancer. Reports observed activation of EMT during the progression of ovarian cancer23. Expression signatures of enriched EMT genes have been observed and used to specify poor prognosis groups of ovarian cancer24. EMT induced an invasive phenotype in ovarian cancer cells through downregulation of CDH1 and up-regulation of CDH2 in Hey and OVCA433 cells25. EMT’s transcription factor SNAI1 induced EMT in ovarian cancer cells (SKOV3 cells) and enhanced the invasiveness26. Furthermore, EMT like changes have been reported in various chemoresistant cancers such as gastric cancer27, non-small cell lung cancer29, nasopharyngeal carcinoma cells30, breast cancer cells32, and ovarian cancer cells17. Many reports showed that ovarian cancer cells undergoing chronic exposure to cisplatin or paclitaxel generate cells that exhibit an EMT phenotype and molecular feature33. Additionally, multiple epigenetic events, such as DNA methylation, histone modifications, and non-coding RNA could contribute to the acquisition of chemoresistance36.Recent evidence suggests that epithelial-mesenchymal transition (EMT) processes might play a role in ovarian cancer progression and chemoresistance development37. Commonly, gene promoter hypermethylation is associated with reduced expression, while hypomethylation results in increased gene expression39. DNA methylation changes at CpG islands associated with transcriptional silencing have been described in cisplatin-resistant cancer cell lines42. For example, DNA methylation of several genes, including  was linked to ovarian cancer initiation and chemotherapy resistance44. A study in ovarian and breast cancer cell lines with doxorubicin tolerance described methylation changes in genes contributing to chemoresistance and identified hyper-methylation of CDH1, BRCA1, SULF2 and DNAJC15 genes, in addition to hypo-methylation of APC, ABCB1 and HIC1 genes35. Based on these considerations, the need to study the linkage between EMT and DNA methylation regulation has become a key to understanding the chemoresistant phenotype.DNA methylation is the most well-known epigenetic mechanism that occurs at CpG islands influencing cellular processes by regulating gene expressionThis study aims to determine if there are any significant differences in the expression of EMT gene regulators between two commercially-available ovarian cancer cell lines (A2780 and A2780cis), which differ in the degree of chemoresistance. This work also focuses on CpG methylation changes in these EMT genes.2, a circularity of 0.81 and an aspect ratio of 1.19. However, A2780cis cells were larger and more elongated with a cell area of 11,600 μm2 with a circularity of 0.43 and an aspect ratio of 4.26 , MRP1 (MDR-related protein 2) and MRP2 (MDR-related protein 2) using qRT-PCR might help understanding if there is a correlation between cisplatin tolerance and expression of ABC transporter genes in A2780cis cell line.ABC transporter genes are responsible for chemotherapy cellular response. Therefore, assessing the mRNA expression level of MDR1 transcript level (5.8 fold) in A2780cis cell line compared to the parental cell line. On the other hand, MRP1 and MRP2 expression did not significantly change in A2780 and A2780cis cell lines.Figure CDH1, and EPCAM, was significantly reduced by 0.02 and 0.013 fold, respectively in the A2780cis cells compared with A2780 cells. The expression of mesenchymal markers, SNAIL, and TWIST2, were higher by 4.9 and 20.3 fold, respectively in the A2780cis cells compared with the parental cell line. Still, no significant expression changes in CDH2 nor VIM genes could be observed . However, no significant changes were observed in CDH2 nor in VIM expression in MDA-MB-231 in comparison with MCF7 cells  in T98G, the more resistant cell line, whereas SNAIL and TWIST2  were up-regulated  determines assay sensitivity and it was represented by ΔCt between the Rsd and R0 reactions and it ranged from 3.2 to 13.6.CDH1 assay revealed a 13.47% hypermethylation in the densely methylated portion after the acquisition of cisplatin resistance. The methylation of the region from (− 306 to − 82 bp), which contains  restriction sites, increased from 24.76 to 39.11% in A2780 and A2780cis, respectively  region is unmethylated in A2780 cell line and no significant differences in methylation between A2780cis and its parental cell line  that contains  restriction sites was 3.37% hypermethylated, 88.98% intermediate methylated and 7.65% unmethylated. In A2780cis, In A2780cis, the fraction of unmethylated DNA was 21.76%  was implicated as a core mechanism mediating drug resistance53. EMT is a biological mechanism characterized by loss of cell adhesion, as well as loss of cell polarity and gain motility18. EMT results in changes in cell morphology associated with alterations in epithelial and mesenchymal markers expression54. EMT like phenotype has been reported in chemoresistant variant cell lines generated upon multiple rounds of chemotherapy treatment such as; cisplatin resistant ovarian cancer cells 15, A2780/PTX, NOS-PR, TAOV-PR, and SKOV-PR paclitaxel resistant ovarian cancer cells17. Similarly, chemotherapeutic resistance promotes EMT like changes in other cancer cell lines, including non-small cell lung cancer29, gastric cancer27 nasopharyngeal carcinoma cells30, and breast cancer cells32. Partial EMT has been proposed as an important mechanism in cancer metastasis and chemoresistance57. Cells that have partial EMT phenotype display properties of both epithelial and mesenchymal cells, indicating that EMT is being induced but not completed58. Galle et al. discussed the role of partial EMT in resistance acquisition, they showed that 3 ovarian cell lines  underwent partial EMT when they acquired chemoresistance42. In this present study, it was proven that the resistant variant A2780cis cell line underwent partial EMT. This was confirmed by observing a morphological change from small circular shaped to elongated shaped cells and changes in molecular markers of EMT; significant reduction in CDH1 and EPCAM and upregulation of the transcription factors, SNAIL and TWIST2. Additionally, no significant expression changes were observed in the studied mesenchymal markers such CDH2, VIM, ZEB1, ZEB2, SLUG, TWIST1 that should be upregulated in completed EMT.In this study, it was demonstrated that the A2780cis cell line still has the tolerance capacity to cisplatin and its resistance is accompanied by an increase in the expression of 64. From this observation, The EMT like changes detected in A2780cis were compared with changes in breast and glioma cell line models known as resistant cancer cells47. MDA-MB-231 and T98G cancer cells were used as reference cell lines models to figure if the acquired changes observed in A2780cis are common in other cancer cells, resistant to therapy. The same molecular changes consistent with morphological alteration in cisplatin resistant MDA-MB-231 cells were detected compared with the sensitive MCF7 cells. The same transcripts changes in CDH1, EPCAM, SNAIL and TWIST2 genes were also detected in chemoresistant T98G cell line in comparison with the less cisplatin tolerance cells U-87-MG. This indicates the importance of CDH1, EPCAM, SNAIL and TWIST2 genes in EMT mechanism associated with cisplatin resistance. Similarly, no significant expression changes were observed in the studied mesenchymal markers in the resistant cancer cells such CDH2, VIM, ZEB1, ZEB2, SLUG, TWIST1 that which are upregulated in completed EMT. These data indicate that these cell lines have a partial EMT state.Multiple studies have shown that naïve cancer cell lines and clinical tumor samples can be divided according to their mesenchymal/epithelial phenotype, this sorting could determine the sensitivity to chemotherapy in various cancers, including ovarian, breast, and lung cancersCDH1 is a Ca2+ dependent adhesion molecule that binds by its extracellular domain to CDH1 on the adjacent cell creating a bridge between the cell’s cytoskeletons65. Many studies revealed that CDH1 down-regulation may be associated with cancer cells resistant to chemotherapy that can be attributed to the EMT mechanism activation66. Acquisition of paclitaxel chemoresistant induces EMT phenotypic changes and CDH1 down-regulation in NOS-PR and A2780/PTX ovarian cancer cell line17. EPCAM is an epithelial cell surface transmembrane glycoprotein that mediates homophilic cell–cell adhesion68. In ovarian cancer cells, EPCAM upregulation is connected to a more favorable prognosis and more effective platinum-based therapy69. Galle et al., found EPCAM expression is down-regulated in addition to CDH1 in resistant variant cancer cells due to EMT process activation42. EMT transcription factors such as SNAIL are considered as direct repressors of CDH1 as they bind to E-boxes existing on the CDH1 promoter72. Hojo et al., observed that ovarian cancer cell lines OVCAR8 and COV318 with high Snail/CDH1 showed more motile and cisplatin resistant phenotypes than OVSAHO and Kuramochi cell lines that have low SNAIL/CDH159. TWIST2 is considered as a direct repressor of CDH1, it can bind directly on E-boxes existing on the CDH1 promoter to suppress its expression and it can repress CDH1 expression indirectly through activation of other signaling pathways73. Studies show that TWIST2 expression is a prognostic indicator for overall survival and disease-free survival and its overexpression correlates with poor prognosis and is associated with CDH1 down-regulation giving mesenchymal cell phenotype on ovarian cancer tumors75. Wang et al., demonstrate that TWIST2 plays a critical role in the cisplatin resistance of ovarian cancer. They found that TWIST2 expression was up-regulated in resistant variant C13K ovarian cancer cell line compared to the cisplatin sensitive ovarian cancer cell line OV200876.77. Upon initiation of EMT, DNA methylation of the genome selectively undergoes CpG site methylation changes, which regulate transcription of EMT-related genes79. In the current study, the investigation of methylated CpG islands role in the modulation of gene expression of EMT regulated genes was done in A2780 cancer cell lines. DNA methyltransferase inhibitor 5-aza induces gene expression of CDH1, EPCAM, SNAIL, and TWIST2, which indicates that DNA methylation may regulate these genes. Chang et al., used gene expression profiling after cancer cells treatment with 5-azadeoxycytidine, they identified genes that were dysregulated in cisplatin resistant cancer cells and reactivated by the DNA methyltransferase inhibitor80. Here, it found that epithelial gene promoters CDH1 and EPCAM became significantly more methylated in A2780cis compared to the parental cell line. These promoter methylation changes correlate with significant gene expression down-regulation. Boettcher et al., profiled DNA methylation of 800 selected CpG islands and identified hypermethylation in CDH1 CpG islands in breast and ovarian doxorubicin resistance cancer cells35. EPCAM overexpression has been linked to promoter hypomethylation EPCAM-negative cells treated with a DNA methyltransferase inhibitor prompted EPCAM expression in various cancers types including ovarian cancer83. A recent study reported consistent methylation changes across multiple cancer cell lines that differed in chemoresistace. Specifically, hypermethylation of epithelial marker genes such as CDH1 and EPCAM promoters and hypomethylation of mesenchymal marker genes such as SNAIL in resistant versus parental cell lines42. Analysis of SNAIL promoter region predicted a CpG island surrounding the TSS, the DNA methylation status in the genomic region (− 688 to − 165 bp) was examined, no methylation was observed in CpGs located in this region of SNAIL promoter in A2780 and its resistant variant cells, although there were changes in SNIAL expression between the two cell lines and after azacytidine treatment. Literature has described changes in the histone modifications regulating SNAIL gene expression84. A single study described changes in the methylation of CpG island located in the first intron after 1000 pb from TSS in EMT models of cancer cells85. The differential methylation in SNAIL gene could be possibly identified in the intron region in A2780 cell line. CpGs island of mesenchymal transcription factor TWIST2 promoter showed DNA hypomethylation in A2780cis compared to the parental cells, this hypomethylation coincides with gene expression up regulation due to EMT activation. TWIST2 methylation changes were observed in various cancers such as colorectal cancers, prostate cancer, and chronic lymphocytic leukemia, this epigenetic event might be the underlying mechanism for TWIST2 transcriptional regulating87.DNA methylation is one of the best described epigenetic mechanisms that regulate gene expression. Aberrant DNA methylation is observed in CpG dinucleotides clustered around the TSS of genes, called CpG islands, leading to gene expression dysregulationCDH1, SNAIL) which indicates that the treatment of demethylating agent under the pressure of cisplatin cause a partial reversing of EMT with cell shape changes. This causality of methylation changes, EMT and cell shape changes caused by acquired chemoresistance to cancer therapy were noted in Galle et al. study by exposing treatment-resistant cell lines during cell divisions to the demethylating agent 5-aza-2′-deoxycytidine, and observing the reversing of EMT42.To test whether DNA methylation changes are causally underlying EMT induced by acquired chemoresistance, A2780cis cells were exposed to noncytotoxic doses of the demethylating agent 5-aza under the pressure of cisplatin. The changes in cell shape of A2780cis cells towards epithelial shape was observed. This treatment caused a reversal in the expression pattern of two EMT markers . Morphological phenotypes of cell lines were assessed when the cell density was up to 70% confluence using Eclipse TS100 inverted light microscope . The Shape descriptors of the cells were assessed using Fiji image analysis software88, three parameters were investigated: cell area for cell size, circularity which is defined as 4*(area/perimeter2) and aspect ratio which is defined as the ratio of the major to the minor axis of a fitted ellipse as a measure of cell shape. Particles that have perfect circular shapes have circularity and aspect ratios equal to 1. Values of aspect ratios that are higher than 1 are associated with elongation and lower circularity values are associated with cellular bumps.Human ovarian cancer cell lines A2780  and A2780cis (the resistant variant), human breast cancer cell lines MCF7 and MDA-MB-231, and human glioblastoma cancer cell lines U-87 MG and T98G were purchased from European Collection of Authenticated Cell Cultures (England). A2780 and A2780cis cell lines were grown in complete RPMI-1640 medium containing 10% FBS, 2 mM glutamine, 0.1 mg/ml each of penicillin and streptomycin. To maintain A2780cis resistance to cisplatin, 1 μM of cisplatin  was added to the media every 3 passages. MCF7 and MDA-MB-231 cell lines were grown in EMEM containing 10% FBS, 2 mM glutamine, 1% non-essential amino acids (NEAA), 0.1 mg/ml each of penicillin and streptomycin. U-87 MG and T98G cell lines were cultured in EMEM, 2 mM Glutamine, 10% FBS and 1% Sodium Pyruvate (NaP). All cell cultures were kept in a 5% (v/v) CO4 cells/well) for MTT assay and allowed to attach O/N. Different concentrations of cisplatin were used to treat cells for 24 h. MTT solution  was added to each well and incubated for 4 h. at 37 °C. Then absorbance values were measured at 550 nm using Multiskan Ascent absorbance plate reader . Cell viability was determined as follows:Cells were plated into 96-well-plates , and ABC transporters (ATP-binding cassette transporters) genes; , quantitative real-time PCR was performed using Maxima™ SYBR™ Green/ROX 2 × qPCR Master Mix  for 40 amplification cycles using StepOne™ Real-Time PCR System . Relative transcript fold changes were calculated using the ΔΔCt method with GAPDH as a reference gene. All reactions were run in triplicate. Primers sequences are detailed in Tables Total RNA was extracted from A2780, A2780cis, MCF7, MDA-MB-231, U-87 MG, T98G cell lines, from A2780 treated with 5-azacytidine (5-aza), and from A2780cis treated with cisplatin and 5-aza using the RNeasy Mini kit . First-strand cDNA synthesis was carried out from 3 μg total RNAs were reverse transcribed to cDNAs using M-MLV reverse transcriptase  for 2 h. at 37 °C. To calculate the relative expression of EMT regulating genes; . The culture medium was removed every 24 h. and replaced with a fresh medium containing 0.1 μM 5-aza. Treated and mock treated cells were collected after 5 treatment days and total RNAs were extracted as described above.Genomic DNAs from A2780 and A2780cis cells were extracted using the QIAamp DNA Mini kit  according to the manufacturer’s instructions. Isolated DNAs were quantified using NanoVue Plus .89. Genomic DNA of A2780 and A2780cis cells were divided into four parts and treated with different digestions: (1) Rs: two methylation-sensitive enzymes MSRE (HhaI + AciI) or (HpaII + AciI) depending on the frequency of their restriction sites within the studied fragments, which are cutting only unmethylated DNA, (2) Rd: one methylation-dependent restriction enzyme McrBC (MDRE), which is cutting only methylated DNA or (3) Rsd: both MSRE and MDRE enzymes , and (4) R0: neither MSRE nor MDRE (mock control). illustrations of the studied section of CDH1, EPCAM, SNAIL, and TWIST2 genes as shown in Supplementary  from European Bioinformatics Institute (EMBL-EBI) to identify the CpG sites associated with the proximal promoter and transcription start site (TSS) for four genes. Sets of locus-specific PCR primers were designed to amplify gDNA at proximal CpG located within 1000 bp (±) of the transcription start site for each gene. Primers sequences, genomic loci, numbers of CpGs nucleotides and number of restriction sites contained in amplified amplicons are listed in Table Quantitative PCR-based methylation analysis (Methylscreen assay) was performed to analyze DNA methylation of genes that have differential expression between A2780 and the resistant variant cells and that expression increased after 5-aza treatment. Methylscreen assay is based on combined restriction digestion of DNA with methylation sensitive and methylation dependent restriction enzymes, MSRE and MDRE respectivelyementary . Each 50The Ct values from R0, Rs, Rd and Rsd, reactions were used to calculate the initial amount of DNA in each digest before PCR as follows:The DNA methylation (%) was calculated as follows:90.Hypermethylated DNA fraction (HM) = Rs/(R0 − Rsd) × 100; unmethylated DNA fraction (UM) = Rd/(R0 − Rsd) × 100; intermediately methylated DNA fraction (IM) = 1-HM-UM. If or ΔCt(Rd − R0) or ΔCt(Rs − R0) < 1.0, The DNA methylation (%) was calculate as following: HM = 1-UM, UM = 1-HMCDH1, EPCAM, SNAIL1, TWIST2 were studied by qPCR.In order to find out if DNA methylation changes are causally underlain EMT, A2780cis cells were cultured and treated with 0.2 μM 5-Azacytidine . The culture medium was removed every 24 h and replaced with a fresh medium containing 0.2 μM of 5-aza for 5 days, subsequently, cells were treated with 10 μg/ml cisplatin. Treated and mock treated cells were photographed and collected for RNA extraction and cDNA synthesis as described above. The relative expression changes of EMT regulating genes P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001.GraphPad Prism Version 7.0  was used to generate graphical figures and to perform statistical analysis. In MTT assay, non liner regression was used in the statistical study. For MethylScreen assay and qPCR study one-tailed student t test was used in the statistical study. Data are expressed as the mean ± SD. Statistical significance was defined as * = Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


For the above reasons, the obtained compounds can be considered a new group of promising anticancer agents, useful in the fight against p53-dependent drug resistance in cancers. They can also be treated as convenient, leading structures suitable for further optimization and searching for more active and selective molecules.Benzodiazepines that consist of one α- and one β-amino acid residues linked together in a seven-membered heterocyclic ring could be treated as small, rigid, cyclic dipeptides capable of exhibiting a wide range of biological activities. During our research on novel analogues of anthramycin, a tricyclic antibiotic benzodiazepine, we developed the synthesis of two benzodiazepine dimers, obtained through the cyclization of appropriate linear tripeptides. The synthesized compounds were tested on a panel of seven cancer and normal cell lines. The developed molecules exhibited promising cytotoxic activity against the lung cancer cell lines A549 and NCI-H1299 and the epidermoid carcinoma cell line A-431. Moreover, they showed significant selectivity compared to the reference cell lines . When tested on two isogenic cell lines, HCT116 and HCT116p53 The ratio of stereoisomers in the NMR spectrum was 54:46; 1H NMR  δ 11.06 , 8.86–8.76 , 8.55–8.47 , 8.40–8.29 , 7.30 , 4.09–3.98 , 3.75 , 3.09–2.99 , and 2.83–2.72 . 13C NMR  δ 170.65, 170.64, 165.1, 143.0, 142.1, 127.1, 127.0, 126.6, 126.2, 122.2, 51.93, 51.86, 35.1; 35.0, 28.4, and 28.3. HRMS (ESI): m/z [M + H]+ calculated for C21H19N6O8S2: 547.07003, found: 547.07069.3,3′-((methylenebis(sulfanediyl))bis(methylene))bis (11). The ratio of stereoisomers in the NMR spectrum was 58:42; 1H NMR  δ 10.81 , 8.89–8.76 , 8.05–7.90 , 4.05–3.95 , 3.73 , 3.08–2.98 , and 2.81–2.71 . 13C NMR  δ 170.85, 170.83, 166.2, 149.34, 149.33, 137.6, 132.62, 132.59, 131.35, 131.34, 118.1, 116.0, 115.9, 51.8, 35.2, 35.1, 28.5, and 28.4. HRMS (ESI): m/z [M + H]+ calculated for C21H19N6O8S2: 547.07003, found: 547.07034.3,3′-((methylenebis(sulfanediyl))bis(methylene))bis, lung carcinomas A549 (ATCC® CCL-185) and NCI-H1299 (ATCC® CRL-5803), colorectal carcinomas HCT116 (ATCC® CCL-247) and HCT116p53−/−, normal skin fibroblasts BJ (ATCC® CRL-2522), and normal lung fibroblasts MRC-5 (ATCC® CCL-171). The HCT116 p53−/− cell line was a kind gift from Professor Bert Vogelstein . The remaining cell lines were purchased from ATCC . The cells were cultured in minimal, Dulbecco’s modified Eagle’s, or Ham’s F-12K medium supplemented with 10% fetal bovine serum  and grown under standard culture conditions  in a humidified incubator with an oxygen concentration of 21%. All of the experiments were performed with mycoplasma-free cells.The following cell lines were used in this study: epidermoid carcinoma A-431  were performed for 72 h. The MTT stock solution  was added at a final concentration of 0.5 mg/mL. After 4 h of incubation at 37 °C, the water-insoluble formazan was dissolved in a lysis buffer containing 20% SDS, 50% DMF, 2.5% hydrochloric acid, and 2.5% acetic acid. The optical densities were measured at 570 nm using a scanning multi-well spectrophotometer . The ratio of the absorbance value of each tested group to the absorbance value of the untreated control was calculated, and each tested group’s cell viability was expressed as a percentage of the control. The cell viability assays were performed at least in triplicate using independent cell cultures.The viability of the cells was assayed by measuring the conversion of MTT -2,5-diphenyltetrazolium bromide) to formazan . The cells were seeded in 96-well culture plates at a density of 3500–5000 cells per well 24 h before the treatment. Six wells were considered for each experimental point . Treatments with increasing concentrations of https://www.sartorius.com/en/products/live-cell-imaging-analysis/live-cell-analysis-resources/live-cell-imaging-and-analsis-handbook (accessed on 21 October 2022)). The HCT116 and HCT116 p53−/− cells were seeded in 96-well culture plates at a density of 5000 cells per well 24 h before the treatment. Four wells were considered for each experimental point. Treatments with 10, 11, cisplatin (40 μM), or dimethyl sulfoxide  were carried out for up to 72 h, and images were recorded every 2 h during this time. The experiment was performed twice.The cell proliferation and death were estimated in the IncuCyte S3 live-cell imaging system using the SYTOX™ Green Dead Cell Stain  and analyzed using the IncuCyte 2019B Rev2 software , according to the manufacturer’s instructions , followed by a post-hoc Tukey’s test. The differences were considered significant if the p-value was ≤ 0.05. All of the statistical analyses were performed using the Statistica software package .The data are presented as means ± SDs. The differences among the variables were analyzed using a Student’s 10 and 11 utilizes a commercially available, naturally occurring, and non-proteogenic amino acid, known as djenkolic acid (12), which is isolated from legume seeds. In the first step, the djenkolic acid (12) was reacted with 5-nitroisatoic anhydride (13) or 4-nitroisatoic anhydride (14) in the presence of potassium carbonate, which led to the formation of the linear tripeptides 15 and 16 , and the following two non-cancer derived cell lines: normal skin fibroblasts BJ (a reference line to A-431) and lung fibroblasts MRC-5 (a reference line to A549 and NCI-H1299). A549 cells are used as models for studying lung cancer and developing drug therapies against it. HCT116 is also a cell line used in therapeutic research and drug screenings. The viability results and obtained half-maximal viability inhibitory concentrations (IC50) are shown in 10 in the lung cancer cell lines. The IC50 values varied from 1.07 ± 0.25 μM (NCI-H1299) to 1.6 ± 0.21 μM (A549) and were much lower than in the case of cisplatin . Thus, compound 10 was 5.33 times more cytotoxic for the NCI-H1299 cell line than cisplatin and 3.72 times more cytotoxic for the A549 cell line than cisplatin. The least cytotoxic effect was observed for the normal cell lines, BJ and MRC-5, where the IC50 values were, respectively, 16.72 ± 2.77 μM and 16.85 ± 2.96 μM for 10 and 23.1 ± 2.34 and 28.7 ± 7.6 μM for 11. Interestingly, the IC50 values of the BJ and MRC-5 cells exposed to cisplatin were significantly higher compared to the treatments with 10 or 11 .Compounds 10 showed very high selectivity indices, which are as follows: 10.54 ± 1.54, p < 0.001 for A549 and 16.08 ± 3.43, p < 0.001 for NCI-H1299, respectively . Contrary to cisplatin, the cytotoxic effects of 10 and 11 were not dependent on p53 and were comparable for both the HCT116 and HCT116p53−/− cell lines , was more sensitive to 10 than to cisplatin , which was used as a negative control, the HCT116 and HCT116p53−/− cells grew to near full confluence, reaching an average of 90% or 84% and 88% or 83% confluence, respectively, on the third day. When cultured in the presence of 40 µM of drugs, a drastic reduction in the confluence occurred in both cell lines, reaching only up to 16% confluence  than cisplatin; (ii) the calculated selectivity indices for compounds 10 and 11, obtained by comparing the values for the tumor and normal lines, were, in most cases, higher than those for cisplatin; (iii) contrary to cisplatin, the observed cytotoxic effect was not dependent on the p53 protein, which is one of the factors responsible for the drug resistance of tumors. We would emphasize that the synthesized products are low-molecular-weight compounds with lead-like properties. The applied molecular framework could serve as a convenient privileged structure for designing and developing novel bioactive molecules suitable for drug design, development, and optimization. The results suggest that this class of compounds could be applied to the treatment of lung cancers.During our studies, we identified compounds based on a unique, dimeric benzodiazepine scaffold that exhibited exceptional anticancer effects against various cell lines and were superior to the reference cancer drug cisplatin in the following three ways: (i) compound


Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties–minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects. Ganglioside GD2 is a validated clinical target overexpressed in multiple cancers such as neuroblastoma, glioma, breast cancer, sarcomas, small cell lung cancer, and melanoma. Its postnatal expression on healthy body cells is generally limited to the central nervous system, peripheral neurons, and skin melanocytes. Ganglioside GD2 performs complex biological functions in normal cells, including participation in cellular communication and adhesion, formation of lipid rafts, and modulation of signaling pathways. Being a glycosphingolipid, GD2 is directly synthesized from its precursor ganglioside GD3 by the enzyme GM2/GD2 synthase; however, its content in the cell is determined by the combined action of the various enzymes involved in ganglioside biosynthesis . The mosThe monoclonal antibodies dinutuximab and naxitamab, chimeric and humanized antibodies from the 14.18/14G2a and 3F8 families, respectively, are the only GD2-directed therapeutics that have been granted regulatory approval. Extensive research is underway to develop alternative GD2-specific strategies, including bispecific antibodies, immunocytokines, CAR T and NK cell therapies, and radioimmunotherapy .I1 drug, which was conjugated to the modified lysines of the antibody, and the conjugate was shown to significantly suppress liver metastases in a disseminated mouse model of neuroblastoma. In our recent work [Only two works regarding GD2-targeting conjugates with small cytotoxic molecules are present in the open literature. Lode et al.  developeent work , ADCs weChanging the antibody format within ADCs from full-length IgG molecules to smaller antibody fragments that results in antibody fragment-drug conjugates (FDCs) represents an important strategy for improving the efficacy of ADC therapy. All major antibody fragment formats such as Fab fragments, minibodies, diabodies, scFvs, and nanobodies, as well as smaller antigen-binding peptides, are being actively explored in preclinical studies as targeted molecules within drug conjugates; however, it is yet to be evaluated whether they represent a viable alternative to full-length mAbs in drug conjugates ,9. The pNo data exists on FDCs directed to GD2-expressing tumors. In this work, such FDCs were developed for the first time based on two antibody fragment formats: the scFv fragment from the clinically approved antibody ch14.18, and the corresponding minibody, which is the bivalent dimer of the same scFv fragment fused to the hinge region and the CH3 domain of the heavy chain of IgG1 antibody. MMAE and MMAF were site-directly conjugated to cysteines of the minibody hinge region and the terminal cysteine introduced into the scFv structure via a cleavable maleimide linker. We generated the FDCs with specific drug-to-antibody ratios (DAR) and evaluated their stability and binding to GD2 by the enzyme-linked immunosorbent assay (ELISA) and to cells with or without GD2 expression by flow cytometry and confocal microscopy. Furthermore, the cytotoxic effects of the conjugates were analyzed in GD2-positive and GD2-negative tumor cell lines by the MTT and propidium iodide (PI) assays.3S)4 linker for construct flexibility and protease resistance, the FLAG octapeptide tag (the sequence DYKDDDDK) for detection, and a C-terminal unpaired cysteine for site-directed thiol-maleimide conjugation were introduced into the sequence (3S)4 linker identical to and in the same orientation as those in the scFv fragment 14.18, with the addition of the IgG1 hinge region, a second GS linker (GGGSSGGGSG), and the IgG1 CH3 constant domain (Both the scFv antibody fragment and the minibody were designed based on the sequence of the light (VL) and heavy (VH) chain variable domains of the GD2-specific antibody 14.18 reported by Bolesta et al. . The scFsequence A. The mit domain A.Following transient gene expression in Expi293F cells and purification by protein L chromatography, both the minibody B and theWe also analyzed the scFv fragment and the minibody in 10% polyacrylamide gel electrophoresis (SDS-PAGE) C,E. The Several methods were used to confirm the specific binding of the obtained antibody fragments to the antigen. First, conjugates of the scFv 14.18 and minibody 14.18 with fluorescein 6-maleimide (FAM) were generated, and with these conjugates we directly demonstrated in flow cytometry and in confocal microscopy that both the scFv fragment and the minibody did not bind the GD2-negative B16 melanoma cell line, but manifested high binding to the B78-D14 melanoma cell line overexpressing GD2 A,B. The Additionally, the specificity of binding of the obtained antibody fragments was studied in direct ELISA using individual purified gangliosides GD2, GD3, GD1b, and GM2 C. When gWe generated the FDCs by conjugating MMAE or MMAF microtubule inhibitors to the C-terminal cysteine of the scFv fragment 14.18 or the reduced interchain cysteines of the minibody 14.18 by a maleimide linker that contains the cathepsin-cleavable valine-citrulline site. The reaction scheme and the FDC structures are presented in n = 4), while the average DAR for minibody-MMAE and minibody-MMAF in optimized conditions constituted 2.0 ± 0.1 and 2.1 ± 0.1, respectively. The absorption spectra of the representative scFv-based and minibody-based FDCs normalized at 280 nm together with the corresponding naked antibody fragment are shown in Based on the ultraviolet-visible (UV-VIS) spectroscopy analysis, the average drug-to-antibody ratios for both the scFv-MMAE and scFv-MMAF generated in optimized reaction conditions constituted 0.9 and were highly consistent between batches  and GD2-negative (B16 and NGP-127) cell lines were incubated for 48 h with a constant concentration of scFv fragment-based FDCs (scFv-MMAE and scFv -MMAF) and minibody-based FDCs (Mb-MMAE and Mb-MMAF) A.In the PI assay, all FDCs induced cell death in GD2-positive B78-D14 and IMR-32 cells, while none of them increased the level of cells with fragmented DNA in GD2-negative B16 and NGP-127 cell lines under the selected FDC concentration (100 nM) A. MiniboThe MMAE and MMAF drugs used to generate the FDCs belong to the family of dolastins, the mechanism of activity of which comprises inhibition of microtubule polymerization that leads to arrest in the G2 phase of the cell cycle and subsequent cell death ,21. In oChanging the format of the vector molecule in the ADCs from full-length antibodies to antibody fragments is an important strategy used to improve their efficacy ,22. VariResearchers are primarily guided by several criteria when choosing a small vector molecule format for developing FDCs. These include a balance between the circulation time of the vector molecule in the blood and its ability to penetrate into solid tumors. In addition, in the case of antibody fragments, it is necessary that they retain high affinity for the antigen and a significant rate of internalization, criteria which are also crucial for full-length antibodies . An addiIt is often difficult to predict in advance which antibody fragment format will be optimal in a particular case, even for tumor markers of protein nature. In the context of the ganglioside GD2, a relatively small glycosphingolipid molecule located only on the outer leaflet of the plasma membrane, this aspect demands even more consideration. In addition, the number of research papers and experimental data regarding anti-GD2 ADCs is minimal. Therefore, we formulated two main goals in the present work: 1) to analyze the fundamental possibility of GD2-directed FDCs to induce cytotoxic antigen-specific effects in tumor cells, and 2) given that the aforementioned effects are present, to compare the efficacy of FDCs created on the basis of different variants of GD2-binding antibody fragments. Results from our previous studies served as prerequisites for the following work.In our earlier paper, an scFv antibody fragment was generated based on the sequence of the ganglioside GD2-targeting murine antibody 14.18 which retained specific binding of the original antibody to GD2 . We haveFDCs based on both GD2-binding moieties, minibodies and scFv fragments, were successfully generated by site-direct conjugation to MMAE or MMAF via a cathepsin-B cleavable maleimide linker and with specific drug-to-antibody ratios . We confirmed specific binding of the FDCs to ganglioside GD2 in direct ELISA and to GD2-positive cells in flow cytometry and confocal microscopy analyses. Both formats induced selective cytotoxic and cytostatic effects in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. The IC50 values for both formats were in the nanomolar range in GD2-positive tumor cell lines.When analyzed in the MTT assay, the FDCs induced significant inhibitory effects in the B78-D14 mouse melanoma cell line that overexpresses GD2, in contrast to the GD2-negative B16 mouse melanoma cell line. Moreover, the FDCs based on both scFv fragments and minibodies conjugated to MMAF manifested stronger cytotoxic effects in this cell line than those conjugated to MMAE. The inhibitory effects of the FDCs were also significant in the GD2-positive human neuroblastoma cell line IMR-32. However, the FDCs based both on scFv fragments and minibodies were more effective in eliminating IMR-32 cells when they contained MMAE, in contrast to the FDCs with MMAF, which demonstrated a more prominent activity in B78-D14 cells. As in the case of GD2-negative mouse melanoma B16, cells of the GD2-negative human neuroblastoma NGP-127 were insensitive to our fragment-drug conjugates even at high concentrations.In order to differentiate the mechanisms of action of the FDCs, we used the PI assay which enabled us to study the contribution of cytotoxic and cytostatic effects to the overall activity of the anti-GD2 FDCs on the tumor cells. All FDCs induced cell death in GD2-positive B78-D14 and IMR-32 cells, while none of them increased the level of cells with fragmented DNA in GD2-negative B16 and NGP-127 cell lines. Minibody and scFv conjugates with MMAF showed the strongest effects in B78-D14 cells; FDCs containing MMAE manifested a less strong yet a pronounced effect. The FDCs also demonstrated a marked increase in cell death in a different GD2-positive cell line, IMR-32, characterized by a lower level of GD2 expression; however, the effects were expectedly weaker than in the B78-D14 cell line overexpressing ganglioside GD2. The results from the PI assay indicated a greater efficiency of the conjugates with MMAF for the B78-D14 cell line, and of those with MMAE for the IMR-32 cell line, which is in agreement with the results obtained in the MTT assay. The results also confirmed the absence of activity towards the GD2-negative B16 and NGP-127 lines at selected concentrations.We next set out to evaluate the effects of our FDCs on the cell cycle, for which the GD2-positive IMR-32 cells treated with the conjugates were analyzed in the PI assay. The distribution of cells with unfragmented DNA in the control samples and in the samples incubated with the FDCs containing MMAE clearly showed that most control intact cells were at the G0/G1 stage of the cell cycle, while the FDCs induced the transition of cells with unfragmented DNA that had not yet entered the late stages of apoptosis and had unfragmented DNA to the G2/M stage of the cell cycle. Moreover, the minibody-MMAE which had the strongest effect on the viability of these cells caused the highest accumulation in the G2/M stage. The data from the PI assay indicate that the mechanism of activity of the FDCs is directly related to the arrest of cells in the G2 phase of the cell cycle and that the total inhibitory effect of the conjugates observed in the MTT assay consists of both the cytotoxic and the cytostatic effects of MMAE or MMAF.The minibody-based FDCs with both MMAE and MMAF demonstrated more pronounced cytotoxic effects compared to scFv-based FDCs in all GD2-positive tumor cell lines within the analysis. This can be explained both by the higher DAR in minibody-based FDCs and by their stronger antigen-binding properties, resulting from the bivalent interaction of the minibodies with the antigen in contrast to monovalent interaction for scFv fragments. The valency of the interaction is an important criterion for the high binding affinity of an antibody or antibody fragment when interacting with ganglioside GD2, a carbohydrate antigen . In thisScFv fragments of GD2-specific antibodies 14.18 were produced by transient gene expression in modified HEK293 cells , as described previously [Minibodies were produced in the same expression system. To this end, the minibody monomer sequence was cloned between the XbaI and AgeI restriction sites of the eukaryotic expression vector pcDNA3.4, followed by verification of the construct identity by analytical restriction digests. The vector was then purified by QIAGEN Plasmid Maxi Kit , and transfection was performed in 50 mL cell culture volume following the protocol for the expression system. The cell culture was routinely checked for optical density and the percentage of viable cells, while the target protein content in the medium was assessed by polyacrylamide gel electrophoresis. The Antibody-Expressing Positive Control Vector from the expression system kit was employed for additional monitoring of protein expression level under recommended conditions.ScFv fragments and minibodies were isolated from the cell culture supernatants by protein L chromatography  as described previously , followeConjugation of the scFv fragments and the minibodies to MC-VC-PABC-MMAE or MC-VC-PABC-MMAF , further referred to as MMAE or MMAF, was carried out by thiol-maleimide chemistry. First, reduction of the C-terminal cysteine introduced intro the scFv fragment and the cysteines forming disulfide bonds between minibody chains was performed by 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP) in PBS buffer, for 1.5 h at room temperature (RT) and with agitation. The reducing agent was then removed by a Zeba Spin Desalting Column, 7K MWCO , followed by an immediate reaction of 1–2 mg/mL proteins with 4:1 molar excess of the corresponding auristatin drug over each antibody fragment (for FDCs with optimized DAR) in PBS buffer at pH 6.0 with addition of 0.2 g/l EDTA, for 3 h at RT and with agitation. The percent of DMSO from the drug stock solutions was kept below 3% in the reaction mix. The FDCs were then transferred to PBS in Zeba columns. Both scFv fragments and minibodies were additionally conjugated to fluorescein 6-maleimide , further referred to as FAM, in reaction conditions identical to those described above.Product purity and stability were analyzed by size-exclusion chromatography, as described above, and by gel electrophoresis. Visualization of the naked antibody fragments and the FDCs by SDS-PAGE was performed either in reducing or in non-reducing conditions, with modifications to . For redAverage drug-to-antibody ratio (DAR) for antibody fragments conjugated with MMAE or MMAF, as well as degree of labeling (DOL) for conjugates with FAM-maleimide, were calculated by UV-VIS spectroscopy  on a Bio450 in GM2-, GD1b-, or GD3-coated wells to OD450 in GD2-coated wells.Nunc MaxiSorp high protein-binding capacity 96-well ELISA plates  were coated with gangliosides GD2, GM2, GD1b, and GD3 at a concentration of 0.25 μg in 100 μL of 96% ethanol per well. GD2, GD1b, and GD3 were purchased from Sigma-Aldrich, and GM2 was obtained according to our previous work . FollowiL-glutamine, 100 μg/mL penicillin, and 100 U/mL of streptomycin .B16 and B78-D14 mouse melanoma cell lines were cultured in RPMI-1640, IMR-32 human neuroblastoma in EMEM, and NGP-127 human neuroblastoma in DMEM medium. All culture media were supplemented with 10% heat-inactivated fetal bovine serum, 2 mM IMR-32 and B16 cell lines were obtained from ATCC, and the NGP-127 cell line was kindly provided by Anton Buzdin . The GD2-positive B78-D14 mouse melanoma cell line generated by transfection of the GD2-negative B16 line with genes coding for GD3 and GD2 synthases  was a kiStaining of B16, B78-D14, IMR-32, and NGP-127 cells with FAM-labeled scFv fragments and minibodies was performed as described previously . In brieStaining of GD2-positive and GD2-negative cell lines for immunofluorescence analysis was performed by a modified protocol from our previous work . In brie4 cells/well, Greiner, Kremsmünster, Austria) with serial dilutions of FDCs for 72 h under standard culture conditions. Following incubation, the MTT solution  was added to each sample for 3 h. Reaction OD was assessed by Multiscan FC microplate reader at a wavelength of 540 nm. Cell viability was calculated using the formula (ODtreated cells − ODblank)/(ODcontrol cells − ODblank) × 100%, where ODblank represents OD in control wells containing no cells. SigmaPlot  was used to generate dose–response curves. All MTT experiments were reproduced at least three times.FDC-induced decrease in cell viability was analyzed by colorimetric MTT  assay previously described by Denizot and Lang , with mo6 cells per sample) were incubated with FDCs at concentration 100 nm for 48 h under standard culture conditions. Cells were subsequently fixed and permeabilized with ice-cold ethanol for 60 min at 4°C, and washed twice with PBS by centrifugation for 10 min at 300× g. Cell pellets were resuspended in DNA staining buffer , 20 μg/mL RNase A ) and further incubated for 30 min at 4 °C. For all samples, cell death and cell cycle were performed in triplicate. In each sample, at least 10,000 events were collected. The quantitation of DNA content was evaluated with BD FACSCalibur flow cytometer .Analysis of cell death or cell cycle was performed using propidium iodide (PI) staining in accordance with modifications  to the pp < 0.05 were considered statistically reliable.Graphs were created using SigmaPlot, GraphPad Prism, and MS Excel software. The data are represented as mean ± SEM of at least three independent experiments, or as one representative experiment from three. Statistical analysis was performed by unpaired Student’s t-test. Significance levels of


CRTC1-MAML2 chimeric gene was determined. Furthermore, the susceptibility of head and neck mucoepidermoid carcinoma to standard treatment drugs such as cisplatin, 5-fluorouracil, and cetuximab was investigated. We successfully established unique MEC cell lines, AMU-MEC1, from an initial surgical specimen and AMU-MEC1-R1 and AMU-MEC1-R2 from the recurrent biopsy specimen in the same patient. These cell lines exhibited epithelial morphology and developed in vitro-like cobblestones. They shared eight chromosomal abnormalities, including der(19)ins, which resulted in a chimeric CRTC1-MAML2 gene, indicating the same origin of the cell lines. The susceptibility of all cell lines to cisplatin and 5-fluorouracil was low. Interestingly, EGFR dependency for cell growth decreased in AMU-MEC-R1 and AMU-MEC-R2 but was retained in AMU-MEC1. These cytogenetic and biochemical findings suggest that the established cell lines can be used to investigate the disease progression mechanisms and develop novel therapeutics for MEC.Patients with advanced/recurrent mucoepidermoid carcinoma (MEC) have a poor prognosis. This study aimed to establish and characterize human mucoepidermoid carcinoma cell lines from the initial surgical specimen and biopsy specimen upon recurrence from the same patient to provide a resource for MEC research. MEC specimens from the initial surgical procedure and biopsy upon recurrence were used to establish cell lines. The established cell lines were cytogenetically characterized using multi-color fluorescence in situ hybridization and detection, and the sequence of the  CRTC1  gene at 19p13 or CRTC3 gene at 15q26 in-frame to exons 2–5 of the Notch coactivator mastermind like gene 2 (MAML2) gene at 11q21 resulting in the expression of a novel CRTC1/3-MAML2 fusion gene [CRTC1/3-MAML2 fusion genes are more prevalent in low- and intermediate-grade tumors than in high-grade tumors [Mucoepidermoid carcinoma (MEC) accounts for 5–15% of all salivary gland malignancies but less than 1% of all head and neck cancers . MEC is ion gene ,9,10,11.e tumors ,13. The e tumors ,14. CRTCe tumors .Only surgery is the standard treatment for MEC, though radiation is sometimes used as postoperative adjuvant therapy for advanced cases. Chemotherapy and radiation therapy can be combined, although the overall survival is not improved compared to radiation alone ,18,19,20Phase contrast microscopy images of the MEC cell lines are depicted . All thrCRTC1-MAML2 chimeric gene (detailed in the next section). Each of the three cell lines, AMU-MEC1, AMU-MEC-R1, and AMU-MEC-R2, shared seven distinct chromosomal abnormalities, including der(19)ins (The karyotypes of eight randomly selected cells from each cell line are listed in ) , suggest) . The uni) . CRTC1-MAML1 chimeric gene [CRTC1-MAML1 chimeric gene corresponded to the previously reported sequence (GenBank: AY040324.1) [CRTC1-MAML2 chimeric gene. All MEC cell lines displayed RT-PCR amplification of a 194bp gene product, which was consistent with the predicted length of the forward and reverse primers A,B. The ric gene , but not40324.1)  . Thus, MEC cell lines exhibited extremely limited growth . OSCC cell lines showed a drug concentration-dependent reduction in viability. IC50 of CDDP in HSC-2, HSC-3, and HSC-4 was approximately 6.0 µM, 2.0 µM, and 4.8 µM, respectively. The IC50 of 5-FU in HSC-2, HSC-3, and HSC-4 was approximately 2.1 µM, <0.3 µM, and <0.3 µM, respectively. However, MEC cell lines maintained 60–100% viability even at high CDDP and 5-FU concentrations . Thus, tCetuximab blocked EGF-EGFR signaling but not proliferation in AMU-MEC1-R1 and AMU-MEC1-R2 cells in all MEC cell lines treated with exogenous EGF; cetuximab inhibited EGFR phosphorylation. Cetuximab also suppressed the EGFR downstream protein, STAT1 phosphorylation A. These PD-L1 expression was not shown in all MEC cell lines. IFN-γ induced PD-L1 expression in AMU-MEC1 and AMU-MEC1-R1 but not in AMU-MEC1-R2 .CRTC1-MAML2 chimera is typically caused by the t translocation [CRTC1-MAML2, their chromosomal structural abnormality was der(19)ins instead of t. This is the first report of MEC with der(19)ins-generated CRTC1-MAML1. However, this investigation did not compare the biological and clinical aspects of translocation and insertion cases. These investigations need to be approached in the future. Low- and intermediate-grade patients have more CRTC1-MAML2 chimeric gene-positive cases than high-grade cases [CRTC1-MAML2 positive. Comparisons of these different histological-grade cell lines could be beneficial in the future. We established the AMU-MEC1 cell line from the surgical specimen of a MEC patient and the AMU-MEC1-R1 and AMU-MEC1-R2 cell lines from biopsy specimens at the time of recurrence of the same patient. The MEC-specific location ,14. Alththe t11;1 has been extensively used for various tumors. However, few clinical trials and basic research have focused on MEC immunotherapy, and its effects are unknown . RecentlCRTC1-MAML2 chimeric gene as a result of der(19)ins instead of t, and 3) the response to EGF or IFN-γ was heterogeneous among these three cell lines. MEC is a rare cancer with a favorable prognosis. However, there are few treatment options for advanced/recurrent cases, necessitating the development of novel targeted therapies. These cells could be used to study the mechanisms of MEC tumorigenesis and progression and to develop novel therapeutics. The three MEC cell lines established in this study are distinct in three ways, as described below. 1) The cell lines were derived from both surgical and biopsy specimens upon recurrence in the same MEC patient, 2) all cell lines generate the The patient is a 69-year-old male with a history of hypertension, hyperlipidemia, pneumonia, and herpes zoster. His father, sister, and aunt had a history of malignant lymphoma. Two months ago, he presented to the otorhinolaryngology clinic with a chief complaint of pain on swallowing and bilateral cervical lymphadenopathy. A laryngeal fiberoptic scan revealed a neoplastic lesion in close proximity to the epiglottis . After bMEC cell line, AMU-MEC1 was derived from surgically-resected salivary MEC tissue on 21 May 2019, while AMU-MEC1-R1 and AMU-MEC1-R2 were derived from recurrent carcinoma biopsy specimens on 15 October 2019 . The fol5 and 2 × 104 cells per well, respectively, serial concentrations of cisplatin (CDDP)  or 5-fluorouracil (5-FU)  were added in triplicate and cultured for five days. The supernatant in each well was completely aspirated, and the cells were incubated with 100 μL of WST-1 solution  diluted with DMEM (10% FBS) at 37 °C for 2 h. Absorbance was measured at 450 and 620 nm using a microplate reader . Cell viability was calculated using the following formula: % viability = 100 × (E − S)/(M − S), where E, M, and S denote the absorbances of the experimental well, well without drugs , and well with medium alone (without cells). After MEC and OSCC cells suspended in DMEM (10% FBS) were seeded in 96-well plates at 105 MEC and OSCC cells were seeded in 24-well plates and cultured for four days in DMEM (10% FBS). After seeding (days 1–4), the cells were treated with 10 μM BrdU (Fuji Film) and incubated at 37 °C for 1 h. After fixing cells with 70% ethanol at −20 °C for 30 min, double-stranded DNA was denatured with 2N HCl at room temperature for 30 min. After washing three times with buffer (PBS containing 0.2% HSA and 2mM EDTA), the cells were incubated with FITC-conjugated anti-BrdU monoclonal antibody  at 4 °C for 30 min. The cells were washed and resuspended in 1 μg/mL propidium iodide-containing washing buffer. For detection of PD-L1, 2 × 105 MEC and OSCC cells were seeded to 24-well plates, cultured for two days in DMEM (10% FBS), and treated with 25 ng/mL of interferon (IFN)-γ for two days. The cells were collected and incubated with PE-conjugated anti-PD-L1 monoclonal antibody  at 4 °C for 20 min. Then, flow cytometry was performed using BD LSRFortessa flow cytometer (BD Biosciences), and the data were analyzed using the FlowJo software v10.8.1 . Two × 105 cells/lane) and transferred to polyvinylidene fluoride membranes using an iBlot 2 Dry Blotting System . After blocking in tris-buffered saline (TBS) with 1% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies: EGFR , p-EGFR , p-STAT1 , and β-actin . β-actin was used as the internal control. After four TBS washes, the membranes were incubated with peroxidase polymer anti-mouse IgG or anti-rabbit IgG  at a 1:100 dilution for 30 min at room temperature. The chemiluminescence signal was detected using ECL Prime Western blotting Detection Reagent  and captured using an Amersham Imager 600 . The cell lysates in protein extraction buffer , and 2 mM 2-mercaptoethanol (pH 7.4)) were resolved on SDS-PAGE gels  was approached to conduct the staining and analysis. Cells were sequentially treated with 0.2 µg/mL colcemid for 3 h and 75 mM KCl for 10 min, then fixed using Carnoy’s solution. The fixed cells were denatured with 2 × SSC at 72 °C for 30 min and hybridized with the 24XCyte Human multicolor FISH probe  at 37 °C for 42 h. Images were captured using a Zeiss Axio Imager. Z2 microscope . 3 dishes, and 5 × 106 cells were collected per 1.5 mL tube. RNA was extracted using the Nucleo Spin RNA Kit , and Reverse transcription (RT)-PCR was performed using the Prime Script One-Step RT-PCR Kit ver.2  according to the manufacturer’s instructions. A 194-base-pair amplicon was generated to detect the CRCT1-MAML2 chimera using the primer sequences 5′-ATG GCG ACT TCG AAC AAT CCG CGG AA-3′(forward) and 5′-CCA TTG GGT CGC TTG CTG TTG GCA GGA G-3′(reverse). The amplified RT-PCR products of the CRCT1-MAML2 chimera were detected using 2% agarose gel electrophoresis. PCR products were sequenced using an ABI Prism 377 Genetic Analyzer .MEC cells were plated in 100 mmt-test. p < 0.05 was considered statistically significant.Differences between the two groups were examined with the Student


We screened a focused array of compounds based on the 4-anilinoquin(az)oline scaffold against both U-CH1 and the epidermal growth factor receptor (EGFR) inhibitor resistant U-CH2. To prioritize the hit compounds for further development, we screened the compound set in a multiparameter cell health toxicity assay. The de-risked compounds were then screened against a wider panel of patient derived cell lines and demonstrated low micromolar efficacy in cells. We also investigated the properties that gave rise to the toxophore markers, including the structural and electronic features, while optimizing for EGFR in-cell target engagement. These de-risked leads present a potential new therapeutic avenue for treatment of chordomas and new chemical tools and probe compound  Protein kinases have presented promising drug targets, with more than 70 inhibitors targeting the ATP binding site of kinases approved for use in the clinic3. This promising treatment avenue is currently being explored to treat chordomas using afatinib4.Cancer is the leading cause of death worldwide, responsible for nearly 10 million deaths in the last year5. Treatment of chordoma is challenging, with initial treatment focused on radical resection combined with proton beam therapy. Surgical and chemotherapeutic options are limited, meaning recurrent or metastatic disease is often fatal6.Chordoma is an osseous cancer, contained within the spine and central nervous system, with invasive and metastatic potential which is driven by the notochord transcription factor brachyury8. Several quinazoline-based clinical kinase inhibitors whose main target is EGFR have been shown to inhibit chordoma cell lines, including gefitinib, erlotinib, afatinib and lapatinib  is highly expressed in 40% of chordomas, and targeting EGFR has been shown to be both effective in chordoma cell lines and murine xenograft modelsnib Fig. 4,8–12.Fi17. They are also not effective at targeting some of the more resistant patient derived cell lines12. While exploring cyclin G associated kinase (GAK) as a potential collateral target to treat chordoma, we identified a series of potent EGFR inhibitor starting points based on the 4-anilinoquinazoline scaffold, exemplified by 112.While these drugs were designed to target EGFR, they show similar or higher affinity for several other kinases, making them limited tools for direct interrogation of EGFR and other kinase biology23. We initially synthesized and tested a panel of compounds based around compound 1. We synthesized compounds 1–45 through nucleophilic aromatic displacement of 4-chloroquin(az)olines (Scheme 10 (23%) and 16 (21%) both containing the trifluoromethyl quinazoline, and the 6,7-difluoroquinazoline analogue 6 (20%), all of which were consistent with previous reports22.Our objectives are to simultaneously improve the chordoma cellular potency and compound preclinical safety profile. Our strategy was to interrogate the structure activity relationships (SAR) of these literature starting points, using our in-house GAK inhibitor knowledge and library, along with a safety risk estimation screen, to optimize the 4-anilinoquin(az)oline scaffold1 with a series of  modifications, on both U-CH1 and U-CH2 chordoma cell lines. N-(3-ethynylphenyl)-6,7-dimethoxyquinazolin-4-amine (1) was previously identified as a narrow spectrum inhibitor of EGFR with only two other off-targets (GAK and Receptor-interacting serine/threonine-protein kinase 2 (RIPK2)) in a kinome wide screen18. Compound 1 was shown to be a low micromolar inhibitor of the U-CH1 cell line, consistent with our previous screen , significantly reduced activity against U-CH1 compared with 11, with no inhibition activity at all (IC50 = > 100 μM) on U-CH2. The 7-trifluoromethyl 16 showed some improvement against U-CH1 with an IC50 = 12 μM and some inhibition of U-CH2 (IC50 = 15 μM). The 6-cyano 17 and 7-cyano 18 substitutions demonstrated a sixfold reduction in inhibition of U-CH1 compared with 16. The 6-methylsulfone 19 showed little improvement. However, reverting to an electron donating substituent cyclizing the catechol first with a methyl spacer 20 and then with an ethyl spacer 21 moved potency back toward single digit micromolar IC50 values against U-CH1. Compound 21 had a similar inhibition profile to erlotinib on U-CH1, but while erlotinib showed activity against U-CH2 (IC50 = 18 μM), 20 and 21 did not (IC50 = > 100 μM).Switching the 7-position back to the methoxy 10. The initial compound 4-((3-ethynylphenyl)amino)-6,7-dimethoxyquinoline-3-carbonitrile (22) with the 6,7-dimethoxy substitution showed good inhibition against U-CH1 with an IC50 = 4.1 μM, and a weaker inhibition of U-CH2 , so did the potency of the compound in both U-CH1 and U-CH2 cell lines. The 6-iodo substitution 28 was the most potent with U-CH1 IC50 = 8.7 μM and U-CH2 IC50 = 14 μM. The 6-methylsulfone 29 had only limited potency against both cell lines, while the 7-position halogens (30–32) showed no affect at all on either cell line. However, switching back to the 7-methoxy 33 recovered activity to levels observed in 22.We then switched to the 3-cyanoquinoline scaffold, to evaluate if it was possible to increase the potency on U-CH1 and U-CH2, as had been demonstrated previously24. In addition, we ran an EGFR in-cell target engagement assay, measuring in-cell EGFR phosphorylation, using the human epidermoid carcinoma cell line A431. This cell line endogenously expresses a high level of EGFR, and stimulation of these cells with human epidermal growth factor (EGF) results in receptor tyrosine autophosphorylation25.We then selected a subset of the most potent compounds on U-CH1 and U-CH2, along with some structurally diverse compounds Table , to screN-(3-ethynylphenyl)-6,7-difluoroquinazolin-4-amine (6) displayed weaker activity against EGFR in A431 cells (IC50 = 12 μM), and no potency in the four additional patient-derived chordoma cell lines or WS1 control (IC50 = > 100 μM). The 6-iodo 9 was more potent against EGFR (IC50 = 0.83 μM), but was still weak in the four additional chordoma cell lines (IC50 = > 100 μM) and WS1 (IC50 = 98 μM). Similar results were observed for 7-methoxy 11 and 7-iodo 15. The 6-trifluoromethyl 16, while showing limited EGFR activity (IC50 = > 20 μM), displayed moderate activity against all four patient-derived chordoma cell lines, but this appeared to be driven by non-specific toxicity . The 7-cyano 17 was selected for structural diversity but was inactive at the top concentration in all chordoma cell line assays (IC50 = > 100 μM). The closing of the 6,7-dimethoxy to form a 6-member ring 21 showed some activity against EGFR (IC50 = 1.9 μM), but only weak activity on UM-Chor1 (IC50 = 64 μM). Erlotinib performed consistently as previously described12, with good potency against EGFR (IC50 = 0.030 μM), weak activity in four of the patient-derived chordoma cell lines, but also with similar non-specific toxicity observed with compound 16 (IC50 = 19 μM). Switching to the 3-cyanoquinoline scaffold, 4-((3-ethynylphenyl)amino)-6-iodoquinoline-3-carbonitrile (28) showed an improvement in potency in three of the four chordoma cell lines: CH22 (IC50 = 8.4 μM), UM-Chor1 (IC50 = 6.9 μM), and U-CH12 (IC50 = 7.7 μM), with only moderate non-specific toxicity in the WS1 control cells (IC50 = 34 μM) despite an eightfold reduction in EGFR activity compared to erlotinib. The observed potency could be related to a target other than EGFR, as the corresponding 7-iodo 32 has similar EGFR activity to 28, but was not potent in the patient-derived chordoma lines or the WS1 control (IC50 = > 100 μM).26. The screen works by estimating the risk using a supervised machine learning (ML) classifier to assess a multiparametric cellular stress phenotype, produced by the test compound, relative to a training set of known compounds  screen was developed to assess human safety risk for candidate pharmaceutical small molecules at the early hit-to-lead stage of developmentds Table . This sc26. This external curation informed an outcome of “high-risk” or “low-risk”. All 300 compounds were also processed through the automated FC screen, populating each of the two classes with frequency distributions of empirical cell-based phenotypes.The ML classifier was trained with 300 historically known compounds , which were divided into two outcome classes based upon literature, clinical trials, and market histories where applicableTo estimate human safety risk for an unknown compound, the ML classifier assesses the similarity of the test compound’s cell-based phenotype to the distributions of phenotypes in the two outcome classes. The final quantitative score, or Cell Health Index (CHI), is the probability (0–1) that the test compound’s phenotype belongs in the distribution observed for the high-risk outcome class in the training set Table . PhenotyTo produce the Cell Health Index, the ML classifier uses all twelve FC parameters simultaneously in a multidimensional logistic regression model, so the CHI contains the most predictive power. In addition to the Cell Health Index, Table N-(3-ethynylphenyl)-6,7-dimethoxyquinazolin-4-amine (1) produced an intermediate risk estimate, consistent with the WS1 toxicity counter screening12. The toxicity is potentially driven by cell morphology changes, but this is currently unclear. Compounds 6, 9, 11, 17 and erlotinib were consistent and found to be non-toxic in both assays; in contrast liabilities were consistently found in 16 and 21. The 7-iodo derivatives 15, 28 and 32 all performed poorly in the cell health secondary screening but showed no toxicity in the initial WS1 (IC50 = > 100 μM) counter screen. Interestingly, the 6-iodo derivative 9, appears to be more tolerated in this assay (for the complete data set see Tables S2–S3).The starting point 9 meant the first set of analogues focused on small changes of the aniline on 9. Shielding the anilino N–H group with a fluoro at the distal ortho-position 34, maintained EGFR activity, but decreased U-CH1 inhibition by fourfold compared with 9. Moving the fluoro to the proximal ortho-position 35 between the acetylene and quinazoline recovered the activity on U-CH1. The para-fluoro 36 was a threefold improvement in U-CH1 inhibition with EGFR activity maintained. Interestingly, switching to a para-chloro 37 reduced activity on U-CH1 by 11-fold, however, demonstrated relatively high activity on the difficult to treat U-CH2 cell line12, with an IC50 = 5.0 μM compared with no activity from the rest of the mini-series .In order to optimize the scaffold further, the 6- and 7-positions on the quinazoline ring were fixed, and the aniline portion was modulated with different halogens Table . The enc15. A fluoro at the distal ortho-position 38 showed no overall improvement, whereas in the proximal ortho-position 39 the fluoro improved potency on U-CH1 by sixfold (IC50 = 2.4 μM). Compound 39 also showed activity on the difficult to treat U-CH2 cell line (IC50 = 15 μM)12. The para-fluoro analog 40 showed no activity on U-CH1 or U-CH2, whereas the para-chloro derivative 41 demonstrated moderate inhibition on both U-CH1 (IC50 = 9.1 μM) and U-CH2 (IC50 = 16 μM) with low single digit micromolar EGFR inhibition (IC50 = 3.7 μM).The encouraging results with the 6-iodoquinazoline led to the exploration of the 7-iodo based around 1 has demonstrated potent inhibition on U-CH1 below 1 µM, suggesting that this substitution pattern may yield a more potent compound across multiple cell lines. The distal ortho-fluoro 42 reduced activity on the U-CH1 by tenfold compared to 1, whereas there was a threefold increase on U-CH2. This was despite 42 showing a tenfold decrease in in-cell EGFR activity. The proximal ortho-fluoro between the meta-acetylene and quinazoline ring system 43 demonstrated a threefold increase across U-CH1 and U-CH2 with EGFR activity increased by fivefold. The para-substituted analogues 44 and 45 both lost activity on U-CH1, but maintained some activity on U-CH2, and EGFR inhibition was maintained. The loss of inhibition on U-CH1 for 41, 44 and 45 is not easily explained and highlights the degree to which target-ligand interactions in cells may be governed by energetic phenomena that classical models inadequately address27.The 6,7-dimethoxy substitution present in 34–45, we screened them against the four additional patient-derived chordoma cell lines  and WS1 as a nonspecific cytotoxicity control . However, the 6-iodoquinazoline, para-chloro aniline analogue 41 showed some of the most potent inhibition to date on CH22 (IC50 = 0.48 μM) and U-CH12 (IC50 = 0.96 μM), along with good inhibition on the other two cell lines UM-Chor1 (IC50 = 25 μM) and U-CH7 (IC50 = 8.0 μM). There was some observed toxicity on WS1 (IC50 = 36 μM), but the ratios of inhibition to toxicity are 75-fold in the best case (CH-22/WS1). The 6,7-dimethoxy substitution analogues 42–45 showed good potential against the four cell lines but also showed some nonspecific toxicity in some cases. Compound 42 demonstrated good inhibition of CH22 (IC50 = 4.2 μM) but showed evidence of some non-specific toxicity . The same was observed for 43, with good inhibition of CH22 (IC50 = 5.8 μM) but some toxicity . The para-fluoro analogue 44 demonstrated excellent potency, but this was unfortunately likely driven by nonspecific toxicity . However, this nonspecific toxicity liability was able to be modulated with the para-chloro analogue 45 that showed potent inhibition on CH22 (IC50 = 1.2 μM) and U-CH12 (IC50 = 3.0 μM) with no observed toxicity .In order to better characterize these optimized compounds ol Table . The 6-i34–45 on the toxicity profiling platform and compared the results to some existing clinical compounds that have activity against chordoma (Table N-(3-ethynylphenyl)-6-iodoquinazolin-4-amine (9) performed well in the toxicity profile platform, while a fluoro substitution in the distal ortho-position 34 and a para-position chloro 37 were not well tolerated. However, other substitutions, such as an ortho-position fluoro 35 and a para-position fluoro on the same core, were tolerated. Interestingly, moving the iodo from the 6-position 9 to the 7-postion 15 led to an increase in toxicity risk estimation in the assay. However, the modifications to the pendant aniline 38–41 were well tolerated, demonstrating that trends cannot always be easily defined. The original starting point 1 was also tolerated but near the middle of the Cell Health Index at 0.41. The modifications 42–45 were consistent with the profile of 1 showing an acceptable toxicity window consistent with the WS1 data. Erlotinib demonstrates low-to-intermediate risk for toxicity, but other relevant literature inhibitors show high toxicity risk, including gefitinib and tesevatinib30.To further understand the toxicity liabilities in this scaffold we screened the optimized analogues ma Table . N- to measure the kinase activity of the 320 protein kinases at two concentrations (10 μM and 1 μM) (Table S6). Compound 41 only inhibited EGFR at 1 μM (> 50%), while at 10 μM (> 50%), several other kinases in addition to EGFR were moderately inhibited including Aurora-B, ERBB4, MAP4K4, PRKG2 and SLK. Compound 45 had a similar profile with only EGFR potently inhibited at 1 μM (> 50%), with some moderate inhibition of ERBB2 and ERBB4. The higher concentration of 10 μM (> 50%) affected several additional kinases in addition to EGFR including Aurora-C, DDR2, EPHA6, EPHB2, LCK, MAP4K4, MAP4K5, MINK1, MKNK1, MKNK2, NIK, PIM1, RIPK2, SLK, and VEGFR2. Beyond the kinases that can be screened in this assay format, GAK20, NLK20, STK10 also known as LOK22 and ADCK3 also known as COQ8A32 could also be potentially inhibited by this scaffold.To understand the selectivity profile across the kinome we screened 12.Chordoma tumors are challenging to effectively treat. The integrated nature of the tumor, combined with slow growth, creates a uniquely complex situation where standard treatment options are not well suited. These factors, combined with highly variable presentation and susceptibility to treatment, prevent a readily available and generalizable therapeutic approach. There have been a number of clinical trials that have evaluated EGFR inhibitors to treat chordomas, but treatment outcomes are highly variable, and molecular mechanisms underlying this observed variability are not well understood12. This was supported by the observation that 6, 11, 21, 28 and erlotinib all had single digit micromolar inhibition on U-CH1 and good EGFR activity . However, in later optimized derivatives (34–45), more correspondence between EGFR and cell line inhibition was observed  but no activity against U-CH1 (IC50 = > 100 μM), again highlighting the complexity of chordoma biology. This was despite 45 showing exciting results across all other cell lines with limited toxicity on WS1 and potent in-cell activity against EGFR, with IC50 = 0.018 μM. U-CH2 proved difficult to inhibit with the most potent compound 11 already known10, and even the lead compounds presented here, 41 and 45, only moderately inhibited U-CH2 . Compounds 41 and 45 both have good inhibition across the remaining chordoma cell lines screened. This activity, combined with limited toxicity in WS1 , support from the flow cytometry toxicity assay and a very narrow kinome spectrum at 1 μM, are all desirable characteristics for a lead compound. Compounds 41 and 45 not only have potent inhibition of EGFR, and good in-cell target engagement , they also appear to have stronger retention in the EGFR binding site than erlotinib via MD simulation  is a probe quality compound for EGFR, fulfilling several of the following criteria: (1) in vitro biochemical IC50 = < 50 nM, (2) ≥ 30-fold selectivity relative to other kinases in a large assay panel, and (3) cellular activity or target engagement IC50 = < 1 μM17.The two lead compounds 41 and 45 (UNC-CA359) both show very narrow kinome profiles and excellent activities across an array of patient derived chordoma cell lines. This is a potentially exciting step forward for a disease with a poor clinical prognosis.This screening and optimization effort has produced a series of useful tools for further interrogation of EGFR biology, and more specifically, chordoma inhibitor development. The lead compounds 34. Briefly, U-CH1 and U-CH2 cell lines were cultured in 4:1 IMDM:RPMI medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in gel-coated flasks. The WS1 cell line (ATCC:CRL-1502) was cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. CH22, UM-Chor1, U-CH12, and U-CH7 cell lines were cultured in RPMI medium supplemented with 10% FBS and 1% penicillin/streptomycin. The cell lines all undergo regular mycoplasma screening9.Chordoma cell lines were cultured as described previously50 values were determined by four-parameter nonlinear regression analyses using GraphPad Prism™ version 8 software .Cells were seeded in 384 well plates and were treated with test compound in quadruplicate 24 h after plating. Cell viability was assessed at 72 h using alamarBlue . Fluorescence was measured using a Tecan Infinite 200 PRO plate reader with excitation at 535 nm and emission at 590 nm. ICComputational modelling was performed using Schrödinger Maestro software.31 was downloaded from RCSB, pre-processed and minimized using the protein preparation wizard tool of Schrödinger Suite 2020-4 . Structures of small molecule ligands were parametrized and minimized using LigPrep module  using OPLS3e force field35.Prior to docking studies, the selected x-ray structure of EGFR complexed with 4-anilinoquinazoline inhibitor erlotinib (PDB:1M17)Docking was computed using Induced Fit Docking workflow of Schrödinger employing SP-setting for Glide docking  and side chains 5 Å from initiative ligand poses were consider for conformational refinement with Prime module. At both stages of docking hydrogen bond was required to hinge amine of Met769. Graphical illustrations are made using PyMOL Molecular Graphics System, Version 2.4.1. Schrödinger, LLC.37. WaterMap links molecular dynamics (MD) simulations with statistical thermodynamic analysis of water molecules within a protein structure. Water molecules were analyzed within 6 Å from the docked ligand, and the 2 ns simulation was conducted with OPLS3e force field39.Schrödinger suite 2020-4 WaterMap  was used to evaluate the hydrations sites replaced upon ligand bindingsimulations were carried out using Schrödinger Desmond . Selected favorable Induced fit docking poses of corresponding EGFR inhibitors were selected as template structures. Prior to simulation set up each kinase-inhibitor complex structure was first minimized using Protein preparation wizard of Schrodinger with default heavy atom rmsd constraint of 0.3 Å and OPLS3e force field. In Desmond system builder the orthorhombic periodic the system was created, and it was solvated using TIP3 waters and 0.1 M NaCl– salt buffer. At the beginning of the simulation, the system was subjected to default relaxation protocol of Desmond and heated up to simulation temperature of 300 K. Unconstrained 1 s simulations were run using NPT protocol at temperature of 300 K, pressure of 1.01325 bar, Noe–Hoover thermostat and timestep of 2 fs. Trajectories were visually examined to see whether docking poses are stable, and interactions remain along the 1 s. The numerical analyzes were calculated in assistance of simulation quality analysis and simulation event analysis tools.Physical execution summaryAsedaSciences SYSTEMETRIC Cell Health Screen.2. Each dilution series was screened in duplicate, occupying a total of 20 wells, allowing 16 test compounds to be assayed per plate. Each row contained one positive and one negative control well, for a total of 16 replicate positive/negative control pairs on each assay plate. Compound formatting, cell deposition, and dye application were performed robotically so that final assay conditions comprised 100,000 cells in a 40 µL volume. After the 4 h compound exposure, cells were immediately stained with a panel of fluorescent dyes that report physiological signatures of both mitochondrial dysfunction and gross cell stress. Fluorescence and forward/side-scatter data were collected using automated FC with no gating. FC data are processed by an automated algorithm for producing quality control measures and ML classification of compound phenotypes.2.HL60 cell culture productionIn a 384-well platform, HL60 cells were exposed to a 10-step, 3 × dilution series of each test compound (5 nM–100 µM) for 4 h at 37 °C with 5% CO2 roller bottles with vented caps, at 1 RPM, 5% CO2, and 37 °C. Culture medium was RPMI 1640 without glucose, supplemented with 10 mM galactose and 10% dialyzed heat-inactivated FBS . Culture density was maintained at or below 1 × 106 cells/mL. Standard protocol for the Cell Health Screen is that a new production lineage of HL60 cells is started each month, and a crossover screen is performed in which the old and new production lineages are compared by using a set of 16 reference compounds to produce a known set of stress phenotypes (see supporting information). In this way, variation of screen performance is minimized by producing all screening cell populations within a narrow range of passage numbers, each checked for consistency of phenotypic performance with reference compounds. This process was performed prior to using cells to produce data for this study.3.Test compound format, cell exposure, and stainingHL60 cells were produced as suspension cultures in glass 850 cmP (Beckman Coulter) was used to deposit cells in all wells, at a density of 2.5 × 106 cells/mL, in a final assay volume of 40 µL per well . After cell deposition, each assay plate was sealed with breathable plate sealer, shaken at 2200 RPM for 10 s (Illumina® High-speed microplate shaker), and incubated for 4 h at 37 °C with 5% CO2.First fluorescent dye mix staining conditionsAll compound formatting, cell exposure, and staining with reporter dyes was performed in this study according to a standard protocol for the Cell Health Screen, which is described as follows. Compounds were formatted in groups of 16, with DMSO blanks loaded in unused screen positions for any smaller compound groups required to finish the complete study set. Each set of 16 test compounds was formatted in two replicate 384-well plates  for assays with two subsets of fluorescent dyes . Compounds in these replicate plates were identical except for the positive controls, which were chosen to produce an optimal response within each subset of fluorescent reporter dyes. Test compound dilution series and controls were formatted on a Biomek® 4000. Each compound was formatted as a 10-step, 3 × dilution series, in duplicate, on each of the two plates. Negative control wells contained the diluent used for both the test compound dilution series and positive controls. The diluent was RPMI 1640 (supplemented as above) with final working concentration of DMSO normalized to 1% in all wells. The positive and negative controls were distributed to plate wells from a single initial reservoir of each control mixture. Final assay concentration range for test compounds was 5 nM–100 µM. Prior to cell deposition, assay plates containing formatted compounds were sealed and stored at room temperature, protected from light, for 2 h, to allow binding equilibrium between serum components and test compounds. A Biomek NXP (Beckman Coulter) was then used to aspirate 20 µL of each well volume, after which 20 µL of dye mix was deposited in all wells. After dye deposition, the plate was re-sealed with its breathable plate sealer, shaken 2 × at 2200 RPM for 5 s each time , and incubated for 10 min at 37 °C with 5% CO2. The plate was then rapidly cooled to room temperature for 1 min in a shallow water bath, after which acquisition of flow cytometry data was started immediately.3b.Second fluorescent dye mix staining conditionsThe following protocol was applied to the first plate in each assay plate pair, inclusive of all compounds in this study. Dye mix buffer was 1 × PBS with 4% FBS, filter sterilized. The dye set consisted of Calcein AM, SYTOX™ Red, MitoSOX™ Red, and Monobromobimane . Dye concentrations were previously optimized, during the screen prototyping phase, to produce maximum dynamic range between positive and negative control wells. Prior to deposition of dye mix, the assay plate was removed from its 4-h incubation, and cells were gently pelleted at 300xg for 2 min. A Biomek NX2. The plate was then allowed to sit at room temperature for 15 min, protected from light. Acquisition of flow cytometry data was started immediately after this 15-min period.4.Acquisition of flow cytometry dataThe following protocol was applied to the second plate in each assay plate pair, inclusive of all compounds in this study. Dye mix buffer was 1 × PBS with 4% FBS, filter sterilized. The dye set consisted of JC-9, propidium iodide, and Vybrant® DyeCycle™ Violet . Dye concentrations were previously optimized, during the screen prototyping phase, to produce maximum dynamic range between positive and negative control wells. Cell pelleting and dye deposition were performed as above, in 3a. After dye deposition, the plate was re-sealed with its breathable plate sealer, shaken 2 × at 2200 RPM for 5 s each time , and incubated for 30 min at 37 °C with 5% CO26.5.Data processing and analysisFC data were acquired with a CyAn™ ADP flow cytometer (Beckman Coulter) with automated sampling performed by a HyperCyt® autosampler (Intellicyt). Autosampler settings were optimized to aspirate ≥ 10,000 cells per well. As described in section “Feature reductionAll well-specific FC data and matching plate map files were transferred to an EC2 server instance on Amazon Web Services (AWS). An automated algorithm converts the raw data to risk scores for each compound in two stages:26. This effectively quantitates the amount of change, in test cells relative to the negative control cells, that each concentration of the test compound caused in one of the 12 FC detection channels. For each FC detection parameter, the amount of change between each test well and the negative control was thus converted to a dose–response curve of QF distance values. The same process was executed for all 12 FC detection parameters, after which each of the 12 QF distance value curves was further reduced to two values: the point of the maximum rate of change and the range within which change occurs26. These two feature values for each FC parameter were then assembled into a vector representing all 12 FC parameters. This vector serves as the quantitative digital phenotype for the test compound, to be used in subsequent ML classification26.5b.Machine learning classificationFor each test each compound, all 12 ungated FC detection parameters were converted to a feature vector as follows. For each of the 10 concentration steps in a test compound dilution series, quadratic form (QF) distance was calculated between the empirical distribution of an FC detection parameter and that same parameter in the negative controlRisk scores were produced for test compounds with an ML classifier employing supervised learning, with a multidimensional logistic model. The classifier was trained on a set of 300 known compounds drawn from on-market pharmaceuticals, withdrawn drugs, research compounds, and a few industrial/agricultural compounds (representative set shown in Table 1–45) were prepared as previously reported and were consistent with previous reports22.4-Chloroquin(az)oline derivative (1.0 eq.), aniline (1.1 eq.) were suspended in ethanol (10 mL) and refluxed for 18 h. The crude mixture was purified by flash chromatography using EtOAc:hexane followed by 1–5% methanol in EtOAc. After solvent removal under reduced pressure, the product was obtained as a free following solid or recrystallized from ethanol/water. Compounds (Supplementary Information.