Monitoring Drug-Target Interactions through Target Engagement-Mediated Amplification on Arrays and in situ

2021 ◽  
Author(s):  
Rasel Al-Amin ◽  
Lars Johansson ◽  
Eldar Abdurakhmanov ◽  
Nils Landegren ◽  
Liza Löf ◽  
...  
Keyword(s):  
Drug Target ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Clinical Samples ◽  
Target Engagement ◽  
Rolling Circle Replication ◽  
Target Proteins ◽  
Dna Conjugates

Abstract Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to a panel of fixed adherent cells agreed with expectations from expression profiles of the cells. These findings were corroborated by competition experiments using kinase inhibitors with overlapping and non-overlapping target specificities, and translated to pathology tissue sections. We also introduce a proximity ligation variant of TEMA in which these drug-DNA conjugates are combined with antibody-DNA conjugates to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and tissues.

scholarly journals Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0134157 ◽  
Author(s):  
Marta Słoniecka ◽  
Sandrine Le Roux ◽  
Peter Boman ◽  
Berit Byström ◽  
Qingjun Zhou ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Human Keratocytes

Monitoring Drug Target Engagement in Cells and Tissues Using the Cellular Thermal Shift Assay

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 84-87 ◽  
Author(s):  
Daniel Martinez Molina ◽  
Rozbeh Jafari ◽  
Marina Ignatushchenko ◽  
Takahiro Seki ◽  
E. Andreas Larsson ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Transport ◽  
Drug Binding ◽  
Target Engagement ◽  
Tissue Samples ◽  
Target Proteins ◽  
Thermal Shift Assay ◽  
Cellular Thermal Shift Assay ◽  
Thermal Shift ◽  
In Cells

The efficacy of therapeutics is dependent on a drug binding to its cognate target. Optimization of target engagement by drugs in cells is often challenging, because drug binding cannot be monitored inside cells. We have developed a method for evaluating drug binding to target proteins in cells and tissue samples. This cellular thermal shift assay (CETSA) is based on the biophysical principle of ligand-induced thermal stabilization of target proteins. Using this assay, we validated drug binding for a set of important clinical targets and monitored processes of drug transport and activation, off-target effects and drug resistance in cancer cell lines, as well as drug distribution in tissues. CETSA is likely to become a valuable tool for the validation and optimization of drug target engagement.

The Role of PAX2 in Breast Cancer: A Study Based on Bioinformatics Analysis and in Vitro Validation

2021 ◽  
Author(s):  
Shan Yang ◽  
Wei Gao ◽  
Haoqi Wang ◽  
Xi Zhang ◽  
Yunzhe Mi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Growth ◽  
Expression Profiles ◽  
Enrichment Analysis ◽  
Mapk Signaling ◽  
Gene Set Enrichment Analysis ◽  
Functional Enrichment ◽  
Migration And Invasion ◽  
Clinical Samples

Abstract Background: Breast cancer (BC) is the most frequently diagnosed cancer in women and is the second most common cancer among newly diagnosed cancers worldwide. Studies have shown that paired box 2 (PAX2) participates in the tumorigenesis of some cancer cells. However, the functions of PAX2 in the BC context are still unclear.Methods: Transcriptome expression profiles and clinicopathological information of BC were download from the TCGA database. Then the expression level and prognostic value in TCGA database were explored. Gene Set Enrichment Analysis (GSEA) and functional enrichment analysis were performed to investigate the functions and pathways of PAX2. Moreover, RT-qPCR was used to determine the expression of PAX2 in BC tissues, and the predictive value of PAX2 in clinical samples was assessed. CCK-8 assay was used to evaluate cell growth. The migration and invasion capacities of cells were assessed by wound healing assay and Transwell assay.Results: PAX2 was up-regulated in the TCGA-BC datasets. GSEA analysis suggested that PAX2 might be involved in the regulation of MAPK signaling pathways and so on. Moreover, PAX2 was overexpressed in BC tissues, and PAX2 expression was associated with menopause. PAX2 deficiency could inhibit the growth, migration, and invasion of BC cells.Conclusion: This study suggested that PAX2 was up-regulated in BC, which inhibited BC cell growth, migration, and invasion. Thus, PAX2 could be a potential therapeutic target for BC.

Rapid discovery of drug target engagement by isothermal shift assay

2019 ◽  
Author(s):  
Kristofor J. Webb ◽  
Kerri A. Ball ◽  
Stephen J. Coleman ◽  
Jeremy Jacobsen ◽  
Michael H.B. Stowell ◽  
...  
Keyword(s):  
Drug Target ◽  
Drug Targets ◽  
Kinase Inhibitors ◽  
Rapid Identification ◽  
Living Cells ◽  
Experimental Designs ◽  
Living Systems ◽  
Target Engagement ◽  
Protein Targets ◽  
Drug Molecules

Identifying protein targets directly bound by drug molecules within living systems remains challenging. Here we present the isothermal shift assay, iTSA, for rapid identification of drug targets. Compared with thermal proteome profiling, a prevailing method for target engagement, iTSA offers a simplified workflow, 4-fold higher throughput, and multiplexed experimental designs with higher replication. We demonstrate application of iTSA to identify targets for several kinase inhibitors in lysates and living cells.

scholarly journals Colorectal Cancer Growth Retardation through Induction of Apoptosis, Using an Optimized Synergistic Cocktail of Axitinib, Erlotinib, and Dasatinib

Cancers ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1878 ◽  
Author(s):  
Robert H. Berndsen ◽  
Nathalie Swier ◽  
Judy R. van Beijnum ◽  
Patrycja Nowak-Sliwinska
Keyword(s):  
Colorectal Cancer ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Chorioallantoic Membrane ◽  
Treatment Strategies ◽  
Tumor Growth Inhibition ◽  
Clinical Implementation

Patients with advanced colorectal cancer (CRC) still depend on chemotherapy regimens that are associated with significant limitations, including resistance and toxicity. The contribution of tyrosine kinase inhibitors (TKIs) to the prolongation of survival in these patients is limited, hampering clinical implementation. It is suggested that an optimal combination of appropriate TKIs can outperform treatment strategies that contain chemotherapy. We have previously identified a strongly synergistic drug combination (SDC), consisting of axitinib, erlotinib, and dasatinib that is active in renal cell carcinoma cells. In this study, we investigated the activity of this SDC in different CRC cell lines (SW620, HT29, and DLD-1) in more detail. SDC treatment significantly and synergistically decreased cell metabolic activity and induced apoptosis. The translation of the in-vitro-based results to in vivo conditions revealed significant CRC tumor growth inhibition, as evaluated in the chicken chorioallantoic membrane (CAM) model. Phosphoproteomics analysis of the tested cell lines revealed expression profiles that explained the observed activity. In conclusion, we demonstrate promising activity of an optimized mixture of axitinib, erlotinib, and dasatinib in CRC cells, and suggest further translational development of this drug mixture.

scholarly journals AL355338 acts as an oncogenic lncRNA by interacting with protein ENO1 to regulate EGFR/AKT pathway in NSCLC

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Qian Hua ◽  
Dongliang Wang ◽  
Lin Zhao ◽  
Zhihui Hong ◽  
Kairu Ni ◽  
...  
Keyword(s):  
Aerobic Glycolysis ◽  
Transcript Abundance ◽  
Metabolic Reprogramming ◽  
Clinical Samples ◽  
Abnormal Metabolism ◽  
Considerable Morbidity

Abstract Background Non-small cell lung cancer (NSCLC) is a malignancy with considerable morbidity and mortality. Abnormal metabolism is a hallmark of cancer; however, the mechanism of glycolysis regulation in NSCLC progression is not completely understood. Recent studies suggest that some dysregulated long non-coding RNAs (lncRNAs) play important roles in tumor metabolic reprogramming. Methods To identify glycolysis-associated-lncRNAs in NSCLC, we compared RNA-sequencing results between high 18F-fluorodeoxyglucose (FDG)-uptake NSCLC tissues and paired paratumor tissues. The transcript abundance of AL355338 in 80 pairs of clinical samples was evaluated by quantitative real-time PCR assay and fluorescence in situ hybridization. The biological role of AL355338 on NSCLC cells were evaluated by functional experiments in vitro and in vivo. Moreover, RNA pull-down, mass spectrometry and RNA immunoprecipitation (RIP) assays were used to identify the protein interacted with AL355338. Co-immunoprecipitation, in situ proximity ligation assays and western blotting were applied to define the potential downstream pathways of AL355338. Results AL355338 was an upregulated glycolysis-associated lncRNA in NSCLC. Functional assays revealed that AL355338 was critical for promoting aerobic glycolysis and NSCLC progression. Mechanistic investigations showed that AL355338 directly bound with alpha-enolase (ENO1) and enhanced the protein’s stability by modulating its degradation and ubiquitination. A positive correlation was observed between AL355338 and ENO1 in NSCLC, and ENO1 was subsequently confirmed to be responsible for the oncogenic role of AL355338. Furthermore, AL355338 was capable of modulating ENO1/EGFR complex interaction and further activating EGFR-AKT signaling. Conclusions This study indicates that AL355338 confers an aggressive phenotype to NSCLC, and targeting it might be an effective therapeutic strategy.

Identifying resistance biomarkers against five clinically approved tyrosine kinase inhibitors in 45 cell lines.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e21005-e21005
Author(s):  
Zsófia Pénzváltó ◽  
Bálint Tegze ◽  
Attila Marcell Szasz ◽  
Reinhold Schäfer ◽  
Balazs Gyorffy
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Resistance Index ◽  
Cancer Therapeutics ◽  
Immunohistochemical Analysis ◽  
Clinical Samples

e21005 Background: Clinical studies show low overall response rates of 10-47% to targeted cancer therapeutics. Our aim was to identify new biomarkers for predicting sensitivity against five already approved tyrosine kinase inhibitors. Methods: Sensitivity to sunitinib, erlotinib, lapatinib, sorafenib and gefitinib were tested in 45 cancer cell lines, and a resistance index was calculated for each cell line. The gene expression profiles (data were obtained by interrogating the raw microarray data of the caArray database) in the subset of resistant vs. sensitive cell lines were compared for each drug. Feature selection was carried out using significance analysis of microarrays and rank products. The results were validated by qPCR in the cell lines and - in case of four sunitinib resistance associated genes - in clinical samples by immunohistochemistry. Results: A set of 63 genes was identified as associated with resistance against the examined drugs. Overall classification accuracy of the prediction was 92.8% in a leave-one-out cross validation using prediction analysis of microarrays. The expression of the genes was validated by qPCR in the cell lines. The results confirmed 45/63 of the microarray-based resistance associated genes and 7/32 of literature based genes. All together 48 sunitinib-treated metastatic renal cell carcinomas were collected. The immunohistochemical analysis in these confirmed the correlation of the expression of RAB17, LGALS8, and EPCAM with overall survival. Conclusions: We identified new predictive biomarker candidates for five tyrosine kinase inhibitors, and validated a set of sunitinib resistance associated genes in an independent patient cohort.

Differential expression of microRNAs induced by trastuzumab emtansine (T-DM1) during megakaryocytopoiesis.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 645-645 ◽  
Author(s):  
Hirdesh Uppal ◽  
Kaushiki Mahapatra ◽  
Sock-Cheng Lewin-Koh ◽  
Steven Olsen ◽  
Mark X. Sliwkowski ◽  
...  
Keyword(s):  
Mirna Expression ◽  
Expression Profiles ◽  
Clinical Samples ◽  
Human Stem Cells ◽  
Antibody Drug Conjugate ◽  
Stem Loop ◽  
Real Time Analysis ◽  
Previously Treated ◽  
Grade 3

645 Background: Treatment with the HER2-targeted antibody–drug conjugate T-DM1 resulted in significantly longer PFS and OS vs lapatinib + capecitabine in patients previously treated with trastuzumab and a taxane in the phase 3 study EMILIA. Thrombocytopenia (TCP) was the dose-limiting toxicity for patients treated with T-DM1, although platelets do not express HER2. In EMILIA, grade 3/4 TCP was observed in 12.9% of T-DM1-treated patients. We have previously shown that T-DM1 inhibits megakaryocyte (Mk) production and differentiation. Here, we investigated the effect of T-DM1 on microRNAs (miRNAs) associated with megakaryocytopoiesis. Methods: Human stem cells (HSCs; CD133+/CD34+) from 8 donors were differentiated into Mks in the presence of T-DM1, trastuzumab, or vehicle. Total RNA was extracted using the miRNeasy MiniKit. cDNA was prepared using the Taqman miRNA RT Kit, FAM-MGB probes, and stem-loop RT primer pool set. miRNA expression was measured using the 96.96 Dynamic Array Chip on the Biomark HD Reader. Data were analyzed using Fluidigm real-time analysis software Spotfire 5, and SAS 9.2. hsa−let−7g and hsa−miR−671−3p were chosen as reference miRNAs due to their low variation between treatments and time points. Median normalization was also applied. Results: A total of 526 miRNA RT-qPCR assays were used to map miRNA expression during differentiation of HSCs from 8 separate donors to Mks in vitro over 30 days. Several miRNAs demonstrated temporal changes in their expression profiles during maturation, suggesting these miRNAs are potential drivers of Mk differentiation. T-DM1 treatment inhibited Mk production and differentiation. Concomitant modifications in the expression of specific miRNAs were observed. These modifications were not present in trastuzumab- or vehicle-treated cells, suggesting these miRNAs may be involved in the development of T-DM1–induced TCP. Conclusions: These results suggest that the miRNAs have the potential to be used as biomarkers for TCP in patients treated with T-DM1 and possibly other DM1 conjugates. Specific miRNA alterations related to T-DM1 treatment will be discussed following investigations using clinical samples to validate these preliminary data.

scholarly journals Systematic analyses reveal long non-coding RNA (PTAF)-mediated promotion of EMT and invasion-metastasis in serous ovarian cancer

2018 ◽  
Vol 17 (1) ◽  
Author(s):  
Haihai Liang ◽  
Xiaoguang Zhao ◽  
Chengyu Wang ◽  
Jian Sun ◽  
Yingzhun Chen ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Expression Profiles ◽  
In Vivo Studies ◽  
Clinical Samples ◽  
Orthotopic Mouse Model ◽  
Protein Coding ◽  
Mesenchymal Transition ◽  
Protein Coding Genes

Abstract Background A deeper mechanistic understanding of epithelial-to-mesenchymal transition (EMT) regulation is needed to improve current anti-metastasis strategies in ovarian cancer (OvCa). This study was designed to investigate the role of lncRNAs in EMT regulation during process of invasion-metastasis in serous OvCa to improve current anti-metastasis strategies for OvCa. Methods We systematically analyzes high-throughput gene expression profiles of both lncRNAs and protein-coding genes in OvCa samples with integrated epithelial (iE) subtype and integrated mesenchymal (iM) subtype labels. Mouse models, cytobiology, molecular biology assays and clinical samples were performed to elucidate the function and underlying mechanisms of lncRNA PTAF-mediated promotion of EMT and invasion-metastasis in serous OvCa. Results We constructed a lncRNA-mediated competing endogenous RNA (ceRNA) regulatory network that affects the expression of many EMT-related protein-coding genes in mesenchymal OvCa. Using a combination of in vitro and in vivo studies, we provided evidence that the lncRNA PTAF-miR-25-SNAI2 axis controlled EMT in OvCa. Our results revealed that up-regulated PTAF induced elevated SNAI2 expression by competitively binding to miR-25, which in turn promoted OvCa cell EMT and invasion. Moreover, we found that silencing of PTAF inhibited tumor progression and metastasis in an orthotopic mouse model of OvCa. We then observed a significant correlation between PTAF expression and EMT markers in OvCa patients. Conclusions The lncRNA PTAF, a mediator of TGF-β signaling, can predispose OvCa patients to metastases and may serve as a potential target for anti-metastatic therapies for mesenchymal OvCa patients.

Targeting PSG1 to enhance chemotherapeutic efficacy: new application for anti-coagulant the dicumarol

2016 ◽  
Vol 130 (24) ◽  
pp. 2267-2276 ◽  
Author(s):  
Dong-xu He ◽  
Feng Gu ◽  
Jian Wu ◽  
Xiao-Ting Gu ◽  
Chun-Xiao Lu ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Patients ◽  
Transforming Growth Factor ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Transforming Growth Factor Β ◽  
Clinical Samples ◽  
Breast Cancer Patients

Chemotherapeutic response is critical for the successful treatment and good prognosis in cancer patients. In this study, we analysed the gene expression profiles of preoperative samples from oestrogen receptor (ER)-negative breast cancer patients with different responses to taxane-anthracycline-based (TA-based) chemotherapy, and identified a group of genes that was predictive. Pregnancy specific beta-1-glycoprotein 1 (PSG1) played a central role within signalling pathways of these genes. Inhibiting PSG1 can effectively reduce chemoresistance via a transforming growth factor-β (TGF-β)-related pathway in ER-negative breast cancer cells. Drug screening then identified dicumarol (DCM) to target the PSG1 and inhibit chemoresistance to TA-based chemotherapy in vitro, in vivo, and in clinical samples. Taken together, this study highlights PSG1 as an important mediator of chemoresistance, whose effect could be diminished by DCM.

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