scholarly journals A statistical framework for assessing pharmacological response and biomarkers using uncertainty estimates

2020 ◽  
Author(s):  
Dennis Wang ◽  
James Hensman ◽  
Ginte Kutkaite ◽  
Tzen S. Toh ◽  
Jonathan R Dry ◽  
...  
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Drug Response ◽  
Biomarker Discovery ◽  
Cancer Cell Line ◽  
Cancer Therapies ◽  
Statistical Framework ◽  
Curve Fit ◽  
Uncertainty Estimates

AbstractDrug high-throughput screenings across large molecular-characterised cancer cell line panels enable the discovery of biomarkers, and thereby, cancer precision medicine. The ability to experimentally generate drug response data has accelerated. However, this data is typically quantified by a summary statistic from a best-fit dose response curve, whilst neglecting the uncertainty of the curve fit and the potential variability in the raw readouts. Here, we model the experimental variance using Gaussian Processes, and subsequently, leverage this uncertainty for identifying associated biomarkers with a new statistical framework based on Bayesian testing. Applied to the Genomics of Drug Sensitivity in Cancer, in vitro screening data on 265 compounds across 1,074 cell lines, our uncertainty models identified 24 clinically established drug response biomarkers, and in addition provided evidence for 6 novel biomarkers. We validated our uncertainty estimates with an additional drug screen of 26 drugs, 10 cell lines with 8 to 9 replicates. Our method is applicable to drug high-throughput screens without replicates, and enables robust biomarker discovery for new cancer therapies.

scholarly journals A statistical framework for assessing pharmacological responses and biomarkers using uncertainty estimates

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Dennis Wang ◽  
James Hensman ◽  
Ginte Kutkaite ◽  
Tzen S Toh ◽  
Ana Galhoz ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dose Response ◽  
Biomarker Discovery ◽  
Statistical Framework ◽  
Curve Fit ◽  
Uncertainty Estimates ◽  
Drug Response Biomarkers ◽  
Novel Biomarkers ◽  
Response Biomarkers

High-throughput testing of drugs across molecular-characterised cell lines can identify candidate treatments and discover biomarkers. However, the cells’ response to a drug is typically quantified by a summary statistic from a best-fit dose-response curve, whilst neglecting the uncertainty of the curve fit and the potential variability in the raw readouts. Here, we model the experimental variance using Gaussian Processes, and subsequently, leverage uncertainty estimates to identify associated biomarkers with a new Bayesian framework. Applied to in vitro screening data on 265 compounds across 1074 cancer cell lines, our models identified 24 clinically established drug-response biomarkers, and provided evidence for six novel biomarkers by accounting for association with low uncertainty. We validated our uncertainty estimates with an additional drug screen of 26 drugs, 10 cell lines with 8 to 9 replicates. Our method is applicable to any dose-response data without replicates, and improves biomarker discovery for precision medicine.

Design, Synthesis, Molecular Docking, and Anticancer Evaluation of Pyrazole Linked Pyrazoline Derivatives with Carbothioamide Tail as EGFR Kinase Inhibitors

2020 ◽  
Vol 21 (1) ◽  
pp. 42-60
Author(s):  
Farah Nawaz ◽  
Ozair Alam ◽  
Ahmad Perwez ◽  
Moshahid A. Rizvi ◽  
Mohd. Javed Naim ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Kinase Assay ◽  
Cervix Uteri ◽  
Egfr Kinase

Background: The Epidermal Growth Factor Receptor (known as EGFR) induces cell differentiation and proliferation upon activation through the binding of its ligands. Since EGFR is thought to be involved in the development of cancer, the identification of new target inhibitors is the most viable approach, which recently gained momentum as a potential anticancer therapy. Objective: To assess various pyrazole linked pyrazoline derivatives with carbothioamide for EGFR kinase inhibitory as well as anti-proliferative activity against human cancer cell lines viz. A549 (non-small cell lung tumor), MCF-7 (breast cancer cell line), SiHa (cancerous tissues of the cervix uteri), and HCT-116 (colon cancer cell line). Methods: In vitro EGFR kinase assay, in vitro MTT assay, Lactate dehydrogenase release, nuclear staining (DAPI), and flow cytometry cell analysis. Results: Compounds 6h and 6j inhibited EGFR kinase at concentrations of 1.66μM and 1.9μM, respectively. Furthermore, compounds 6h and 6j showed the most potent anti-proliferative results against the A549 KRAS mutation cell line (IC50 = 9.3 & 10.2μM). Through DAPI staining and phase contrast microscopy, it was established that compounds 6h and 6j also induced apoptotic activity in A549 cells. This activity was further confirmed by FACS using Annexin-V-FITC and Propidium Iodide (PI) labeling. Molecular docking studies performed on 6h and 6j suggested that the compounds can bind to the hinge region of ATP binding site of EGFR tyrosine kinase in a similar pose as that of the standard drug gefitinib. Conclusion: The potential anticancer activity of compounds 6h and 6j was confirmed and need further exploration in cancer cell lines of different tissue origin and signaling pathways, as well as in animal models of cancer development.

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals Synthesis and Experimental Validation of New Designed Heterocyclic Compounds with Antiproliferative Activity versus Breast Cancer Cell Lines MCF-7 and MDA-MB-231

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Vincenza Barresi ◽  
Carmela Bonaccorso ◽  
Domenico A. Cristaldi ◽  
Maria N. Modica ◽  
Nicolò Musso ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Anticancer Agents ◽  
Cancer Cell Line ◽  
Breast Cancer Cell Lines ◽  
Design And Synthesis ◽  
Human Breast Cell ◽  
Mcf 7

Recent drug discovery efforts are highly focused towards identification, design, and synthesis of small molecules as anticancer agents. With this aim, we recently designed and synthesized novel compounds with high efficacy and specificity for the treatment of breast tumors. Based on the obtained results, we constructed a Volsurf+ (VS+) model using a dataset of 59 compounds able to predict the in vitro antitumor activity against MCF-7 cancer cell line for new derivatives. In the present paper, in order to further verify the robustness of this model, we report the results of the projection of more than 150 known molecules and 9 newly synthesized compounds. We predict their activity versus MCF-7 cell line and experimentally verify the in silico results for some promising chosen molecules in two human breast cell lines, MCF-7 and MDA-MB-231.

In vitro chemotherapeutic and antiangiogenic properties of cardenolides from Acokanthera oblongifolia (Hochst.) Codd

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Maha M. Soltan ◽  
Howaida I. Abd-Alla ◽  
Amal Z. Hassan ◽  
Atef G. Hanna
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Cancer Cell Lines ◽  
Enzyme Inactivation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Anticancer Properties ◽  
Mechanistic Investigation ◽  
G2m Phase

Abstract Acovenoside A and acobioside A were isolated from Acokanthera oblongifolia. Their anticancer properties were explored regarding, antiproliferative and antiangiogenic activities. The study included screening phase against six cancer cell lines followed by mechanistic investigation against HepG2 cancer cell line. The sulforhodamine-B (SRB) was used to determine their growth inhibitory power. In the other hand, flow cytometry techniques were recorded the cell death type and cell cycle analysis. The clonogenic (colony formation) and wound healing assays, enzyme-linked immunosorbent assay (ELISA) and molecular docking, were performed to evaluate the antiangiogenesis capability. Both compounds were strongly, inhibited four cancer cell lines at GI50 less than 100 nM. The in vitro mechanistic investigation against HepG2 resulted in cell accumulations at G2M phase and induction of apoptosis upon treating cells separately, with 400 nM Acov-A and 200 nM Acob-A. Interestingly, the same concentrations were able to activate caspase-3 by 7.2 and 4.8-fold, respectively. Suppressing the clonogenic capacity of HepG2 cells (20 and 40 nM) and inhibiting the migration of the colon Caco-2 cancer cells were provoke the results of vascular endothelial growth factor receptor2 (VEGFR2) kinase enzyme inactivation. The docked study was highly supportive, to the antiangiogenic approach of both cardenolides. The isolated cardenolides could orchestrate pivotal events in fighting cancer.

scholarly journals EVALUATION OF [11C]MPC-6827 AS A MICROTUBULE TARGETING PET RADIOTRACER IN CANCER CELL LINES

2019 ◽  
pp. 43-47
Author(s):  
J. S. DILEEP KUMAR ◽  
JAYA PRABHAKARAN ◽  
NARESH DAMUKA ◽  
JUSTIN W. HINES ◽  
STEVEN J. KRIDEL ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Prostate Cancer ◽  
Cell Lines ◽  
Cancer Cell ◽  
Specific Binding ◽  
Cancer Cell Line ◽  
Prostate Cancer Cell Line ◽  
Microtubule Targeting Agents ◽  
Pc3 Cells

Objective: The objective of this study was to evaluate the uptake and specificity of [11C]MPC-6827, a MT targeted PET ligand in prostate, glioblastoma and breast cancer cells. Methods: [11C]MPC-6827 was synthesized by reacting corresponding desmethyl precursors with [11C]CH3I in a GE-FX2MeI/FX2M radiochemistry module. In vitro binding of [11C]MPC-6827 was performed in breast cancer MDA-MB-231, glioblastoma (GBM) patient-derived tumor (GBM-PDX), GBM U251 and prostate cancer 3 (PC3) cell lines at 37 °C in quadruplicate at 5, 15, 30, 60, and 90 minute incubation time. The nonspecific bindings were determined by incubation with unlabeled microtubule targeting agents MPC-6827, HD-800, colchicine, paclitaxel and docetaxel (5.0 mM). Results: [11C]MPC-6827 provided the highest binding in the breast cancer cell, MDA-MB-231, among all the cells studied, with 90% specific binding. [11C]MPC-6827 binds to glioblastoma PDX and U251 cells with ~50% and 40% specific binding, whereas, prostate cancer cell line, PC3 cells showed 40% specific binding. [11C]MPC-6827 also exhibits binding to the taxane and colchicine binding sites of MTs, in MDA-MB-231 cells. Conclusion: These data indicate that [11C]MPC-6827 can be a promising PET radiotracer for preclinical imaging of the brain and peripheral cancers.

scholarly journals Somatostatin and dopamine receptor interaction in prostate and lung cancer cell lines

2010 ◽  
Vol 207 (3) ◽  
pp. 309-317 ◽  
Author(s):  
M Arvigo ◽  
F Gatto ◽  
M Ruscica ◽  
P Ameri ◽  
E Dozio ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Line ◽  
Membrane Receptors ◽  
Receptor Interaction ◽  
Inhibition Of Proliferation ◽  
Receptor Interactions

Somatostatin analogues inhibit in vitro cell proliferation via specific membrane receptors (SSTRs). Recent studies on transfected cell lines have shown a ligand-induced formation of receptor dimers. The aim of this study is 1) to evaluate the role of specific ligands in modulating receptor interactions in the androgen-dependent prostate cancer cell line, LNCaP, and in the non-small cell lung cancer line, Calu-6, by co-immunoprecipitation and immunoblot; and 2) to correlate the antiproliferative effect of these compounds with their ability in modulating receptor interactions. In LNCaP, we have demonstrated the constitutive presence of sstr1/sstr2, sstr2/sstr5, sstr5/dopamine (DA) type 2 receptor (D2R), and sstr2/D2R dimers. BIM-23704 (sstr1- and sstr2-preferential compound) increased the co-immunoprecipitation of sstr1/sstr2 and significantly inhibited proliferation (−30.98%). BIM-23244 (sstr2–sstr5 selective agonist) significantly increased the co-immunoprecipitation of sstr2/sstr5, and induced a −41.36% inhibition of proliferation. BIM-23A760, a new somatostatin/DA chimeric agonist with a high affinity for sstr2 and D2R and a moderate affinity for sstr5, significantly increased the sstr5/D2R and sstr2/D2R complexes and was the most powerful in inhibiting proliferation (−42.30%). The chimeric compound was also the most efficient in modulating receptor interaction in Calu-6, increasing the co-immunoprecipitation of D2R/sstr5 and inhibiting cell proliferation (−30.54%). However, behind BIM-23A760, BIM-53097 (D2R-preferential compound) also significantly inhibited Calu-6 proliferation (−17.71%), suggesting a key role for D2R in receptor cross talk and in controlling cell growth. Indeed, activation of monomeric receptors did not affect receptor co-immunoprecipitation, whereas cell proliferation was significantly inhibited when the receptors were synergistically activated. In conclusion, our data show a dynamic ligand-induced somatostatin and DA receptor interaction, which may be crucial for the antiproliferative effects of the new analogues.

Characterization Of FLT3/ITD Resistant Cells and Identification Of Novel Therapeutic Targets Utilizing High Throughput Drug Screening

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2522-2522
Author(s):  
Katherine Tarlock ◽  
C. Anthony Blau ◽  
Timothy Martins ◽  
Soheil Meshinchi
Keyword(s):  
Cell Lines ◽  
High Throughput ◽  
Drug Screening ◽  
Therapeutic Options ◽  
High Throughput Drug Screening ◽  
Mutational Profiling ◽  
Flt3 Inhibitors ◽  
Resistant Cells

Abstract The overall survival (OS) of pediatric acute myeloid leukemia (AML) exceeds 60%, however for high risk (HR) patients, including high allelic ratio FLT3/ITD+, survival remains poor. FLT3/ITD is one of the first genomic alterations in AML to be exploited for therapeutic benefit as it has greater sensitivity to the pro-apoptotic effects of FLT3-inhibitors. Children’s Oncology Group (COG) phase III AML trial AAML1031 is investigating the role of sorafenib in combination with chemotherapy in HR FLT3/ITD+ patients. In vitro and in vivo studies indicate that resistance to FLT3-inhibitors can develop through varying mechanisms including up-regulation of FLT3 receptor, acquisition of secondary mutations, or activation of alternate survival mechanisms leading to apoptotic escape. For FLT3/ITD+ patients who relapse despite treatment with FLT3-inhibitors, there are often no therapeutic options and survival is very poor. In evaluation of therapeutic options for those who relapse on sorafenib, we developed an in vitro resistance model using the FLT3/ITD+ cell line MV4-11. Resistance was induced thru long-term exposure to incrementally increasing doses of sorafenib. Two distinct cell lines with resistance at 10 and 100 fold above the IC50 of naïve MV4-11 were generated for experimental evaluation. Genotypic and phenotypic characterization of the resistant cells was conducted by multidimensional flow cytometry (MDF), conventional karyotyping, and mutational profiling. MDF revealed an overall similar immunophenotype, however the resistant cells were significantly more homogeneous for expression of HLA-DR and had significantly higher CD11b expression compared to their naïve counterparts. CD135 expression was minimally increased in the resistant cells. In comparison of the karyotypes, the resistant cells were a more homogenous population with emergence of one dominant clone and disappearance of a number of pre-existing sub-clones. Mutational profiling by Sanger sequencing revealed a novel N841Y mutation in activation loop, an area implicated in TKI-resistance. Using a high throughput drug screening assay, we explored sensitivity profiles of the naïve and resistant MV4-11 cells to 163 oncology agents, including 45 FDA approved and 118 investigational agents that target a number of key pathways regulating cell growth, differentiation, and survival. The naïve MV4-11s had a sorafenib IC50 of 1.3 nM (published 1-5nM) and resistant cells had IC50 of approximately 2-log folds above the naïve, which was consistent to what we had seen in our lab-based validations. We initially assessed whether resistance to sorafenib induces cross-resistance to other TKIs. Agents in the panel with previously demonstrated efficacy for FLT3/ITD included quizartinib (AC-220), tandutinib, ponatinib, sunitinib, and midostaurin, and in all cases sorafenib-resistant cells were also more resistant to these agents. We then examined whether we could identify agents with efficacy in the resistant cells. We identified 5 novel agents to which the resistant cells retained sensitivity. Two bcl-2 inhibitors tested maintained sensitivity in the resistant cells with IC50s in the 20-100nM range. In addition, YM-155, a survivin inhibitor, also maintained sensitivity in the resistant cells with IC50s of approximately 25-50nM across the cell lines. Survivin over-expression is associated with AML stem progenitor cells and decreased OS in adults, and transcription regulation has been linked to the FLT3/STAT5 pathway. Two CRM inhibitors, a novel class of agents which inhibit nuclear export to restore tumor suppressor function, also maintained sensitivity in the resistant cell lines with an approximate 3-fold increase in IC50 from 12nM in the naïve to 32-40nM in the resistant cells. Experience with the use of directed therapy to target specific somatic events has provided evidence that leukemic evolution can continue under this selection pressure and therapeutic options for patients with emergent disease is often insufficient. Using the high throughput drug assay in a FLT3/ITD+ cell line as an in vitro model for sorafenib-resistant FLT3/ITD patients, we identified classes of targeted agents that maintain sensitivity in resistant cells. Further validation of the targets in specimens from those with resistance to such TKIs can inform on the class of agents that can be used to treat or prevent refractory disease FLT3/ITD+ patients. Disclosures: No relevant conflicts of interest to declare.

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