Our biosciences division focuses on efficacy screening using cell based assays. We specialize in 2D and 3D cell-based approaches and have expertise in a wide range of techniques to cover phenotypic-based screening, target-based screening, and exploratory biology and mechanistic studies.
Phenotypic based drug discovery occurs prior to target identification. Typically, cell-based assays are used to measure biological effects in cells and this often involves monitoring multiple relevant targets and pathways simultaneously. Translation to human disease can be more straightforward than target based screens.
Target-based drug discovery occurs once the target has been identified, and involves target validation, assay development and high throughput screening against the target. The assays typically use recombinant proteins or cells over expressing the target of interest.
Cell-based assays enable a good understanding of the complex biological processes within the body and how molecules interact with these processes. Using our expertise we can design, develop and validate cell-based approaches to:
|Step 1: Initial Discussions|
|Step 2: Design Assay and Establish Validation Criteria|
|Step 3: Screening|
|Step 4: Reporting|
Cyprotex regularly design and develops new assays for our clients. We typically employ a 4 stage process:
Step 1: Our experienced scientists evaluate your needs and discuss the scientific rationale of the project and how your goals can be achieved.
Step 2: We plan and design an assay to match your requirements. We jointly establish the development process and validation criteria.
Step 3: We screen your test articles in the assay using robust QC processes within short turnaround times. We can assist in training, transfer and cross validation of the assay to a client’s site with ongoing support if required.
Step 4: Our reporting options are flexible – ranging from full written reports to customised data formats which can feed directly into your databases. We can assist you in interpreting the data and highlighting potential next steps.
Cyprotex has extensive experience in a wide range of platforms to support routine and customised bioscience services which include:
High content screening uses fluorescent dyes, fluorescently labelled antibodies or reporter based fluorescent protein systems to track processes or components within the cell. Cyprotex can offer both conventional and confocal high content screening techniques, the latter being beneficial for imaging 3D cell models. We have previous experience developing many phenotypic screening assays as well as novel protein-protein interactions target screening assays:
Microelectrode array instruments monitor whole cell electrophysiology allowing multiple ion channels to be monitored simultaneously. Cyprotex have developed cardiac and neuronal models using iPSC-derived cardiomyocytes and cortical neurons respectively.
A shift between respiration and glycolysis is observed in several pathological states including cancer, obesity, diabetes, and mitochondrial, cardiovascular, and neurodegenerative diseases. Cellular bioenergetics also play a role in aging, immune response, hypoxia and drug toxicity. The Seahorse Flux Analyzer measures the metabolic phenotype of cells by simultaneously quantifying respiration and glycolysis in real time. Cyprotex is able to develop methods using the Seahorse XFe Flux Analyzer to investigate specific disease states and the mechanisms behind the cellular bioenergetics effects.
Cyprotex offer a wide variety of cell types including stem cell derived cells, primary cells and cell lines. We can source additional cell lines on customer request. We specialize in both 2D and 3D cell-based approaches, including both mono-culture and co-culture models.
Cyprotex use the Applied Biosystems 7900HT system to monitor and quantify either up-regulation or down-regulation of genes. These effects may be a consequence of disease, drug- or chemical-induced effects or physiological effects.
A client required a cell-based assay to screen compounds for breaking up the protein: protein interaction of p53 and hDM2. Following discussion with the client, an appropriate study design was proposed, and the assay was developed and validated based on this agreed design. For this assay design, p53 is anchored in the nucleus using a NLS (nuclear localization sequence) and tagged with GFP. hDM2 shuttles between the nucleus and the cytoplasm using a NLS (nuclear localization sequence)/NES (nuclear export sequence) shuttling component and is tagged with RFP. If the hDM2 binds to the p53 then it is unable to move back to the cytoplasm and is retained in the nucleus.
Nutlins are only found to be active in cells with active wt p53, and were found to induce p53 dependent pathways such as cell cycle arrest and apoptosis. Nutlin-3 was discovered as a small molecule which interfered with the p53:hDM2 interaction. This is illustrated in the video below:
In this time lapsed video, initially the hDM2 is retained in the nucleus by binding to the p53 protein (the yellow color is the overlapping color of the GFP and RFP). On the addition of nutlin-3 to the incubation, the hDM2 is displaced and is free to move out of the nucleus into the cytoplasm (shown by the red color). The p53 remains in the nucleus (shown by the green color).
After completing the validation, the assay was adapted into a high throughput screen by creating the components in adenovirus. This allowed a high throughput campaign to be undertaken with the University of Pittsburgh on a 220,000 compound library5. Other protein:protein interaction biosensors have since been developed in a similar format2.
Microelectrode array technology is becoming a popular approach for measuring neuronal activity. Measurements such as spiking, bursting, plasticity and organization can be used to assess a wide range of neurotransmitter along with pharmacological agonists and antagonists. Cyprotex has developed a model of seizurogenic response using this system.
The first set of traces shown below represent Raster plots of spike activity in five individual electrodes before drug treatment (A), and after 1hr treatment with the GABAA antagonist picrotoxin, 10µM (B).
B. Treatment with GABAA antagonist, picrotoxin (10 µM)
The results show an increase in bursting organization as well as synchrony which is characteristic of a seizurogenic response. This assay can be applied to the development of testing for anti-seizurogenic potental. This platform also creates an opportunity to create phenotypic assays for pain using Dorsal Root Ganglia (DRG) neurons as well as assays for neurodegenerative disease or neuronal signaling models using tool compounds to induce responses in various rodent neurons or stem cell derived neurons.
iPSC-derived cardiomyocytes have transformed in vitro measurements of cardiac electrical activity due to the fact that they beat, exhibit an ECG-like response and respond to exogenous agents.
Using iPSC derived cardiomyocytes in combination with microelectrode array technology, hERG channel blockers such as cisapride and E-4031 are able to induce arrhythmias, through prolongation of the QT interval (or field potential duration (FPD)) and subsequent early after-depolarizations (EAD) events as illustrated in Table 1.
|Test Article||Concentration (µM)||Beat Period||Fast Na* Slope||Fast Na* Amplitude||FPD||EAD|
This arrythmia model has potential uses in the development of antiarrythmic drug therapy and mechanistic studies to understand specific cellular cardiac responses.
For their oncology project, a client was interested in evaluating the potential inhibitory effects of their compounds on cell motility in U87-MG cells (a human glioma cell line). Cyprotex designed and developed a high content screening assay to assess the client’s objectives.
Cells were seeded onto commercially available plates where a stopper prevented cell growth in the center of the well. Once the stopper was removed, migration of the cells into the cell free area was assessed in the presence of various concentrations of test compounds to determine the inhibitory effects.
The positive control compounds, cytochalasin D and alendronate, showed a concentration dependent decrease in cell motility of U87-MG cells. The data for cytochalasin D are illustrated below.
Communication between cells is important in maintaining homeostasis by controlling cell differentiation and proliferation. Inhibition of GJIC (gap junction intercellular communication) is known to be involved in tumor promotion.
A client requested a specific protocol within a publication to assess the impact of their chemicals on GJIC. Cyprotex cross validated the assay described in the publication and then worked closely with the client in transferring the assay over to their site.
The assay evaluated inhibition of GJIC in NHBE donor cells stained with a calcium sensitive, fluorescent dye. The transfer of the dye to NHBE acceptor cells was monitored.
High content images are illustrated in Figure 6 below. In the figure the bright green dye represents the donor cells with the lighter green cells in the background representing the acceptor cells. In the cells treated with the GJIC inhibitor, TPA (12-O-tetradecanoylphorbol-13-acetate), a decreased transfer of dye to the acceptor cells is observed.
Assessment of combined cell cycle analysis: cell proliferation is a direct indicator of cell growth and division. Cyprotex have worked with 2 different clients to design and develop cell proliferation assays for their oncology projects using high content imaging technology.
The first client was working in the area of colorectal cancer, and Cyprotex designed an assay to study the effects of their test articles on cell proliferation of two colorectal carcinoma cell lines, HT29 and HTC116 using cell proliferation markers p-H3, alkynyl nucleoside EdU and Ki-67 antigen.
The second client was working in the area of breast cancer, and Cyprotex designed an assay to investigate the effects of their test articles on cell proliferation of MCF7, a human breast adenocarcinoma cell line.
The data below illustrate the effect of etoposide and vinblastine on phosphohistone 3 (p-H3) levels in MCF7 cells over 24 and 72 hr. Etoposide causes G2 arrest with a decrease in p-H3 levels in the nucleus and a decrease in cell proliferation. Vinblastine arrests cells in the M-phase and therefore a corresponding increase in p-H3 is observed in the nucleus. This is accompanied by a cytotoxic response to the cells.
1 Terstappen GC et al., (2007) Target deconvolution strategies in drug discovery. Nat Rev Drug Disc 6; 891-903
2 Hua Y et al., (2014) High-content positional biosensor screening assay for compounds to prevent or disrupt androgen receptor and transcriptional intermediary factor 2 protein-protein interactions. Assay Drug Dev Technol 12(7); 395-418
3 Hua Y et al, (2015). High content screening biosensor assay to identify disruptors of p53-hDM2 protein-protein interactions. Methods Mol Biol 1278; 555-565.
4 Dudgeon DD et al., (2010) Characterization and optimization of a novel protein-protein interaction biosensor high-content screening assay to identify disruptors of the interactions between p53 and hDM2. Assay Drug Dev Technol 8(4); 437-458.
5 Dudgeon DD et al. (2010) Implementation of a 220,000-compound HCS campaign to identify disruptors of the interaction between p53 and hDM2 and characterization of the confirmed hits. J Biomol Screen 15(7); 766-82.