Cell-based Reporter Assays

Cell-based assays can offer an efficient and valuable means to be used to assess a variety of biochemical and functional effects of a substance in vitro, such as cell division and proliferation, signal transduction cascade, activity of different enzymes and reporter genes, antibody- dependent (ADCC) and complement dependent (CDC) cytoxicity, pathways of programmed cell death, the most concerned cellular phenomena among others.

The cellular reporter assay unit monitors the cellular events associated with signal transduction, consists of a reporter or marker gene fused with the gene of interest under influence of an appropriate promoter which binds to a transcription factor that has been resulted in response to a binding event or signaling cascade activation. Binding of the transcription factor with the promoter region triggers the basal level reporter gene expression.

The reporter gene should be small enough to deliver through a vector, and, the desired product should be nontoxic, nonimmunogenic to the cell and, have dynamic, reliable and, unique characteristic and/ or enzymatic activity that can be easily distinguished from the pool of whole other extra- and intracellular proteins. The activity and stimulation of the reporter gene depends upon the optimum combination of that with a suitable promoter.

Nowadays a popular choice of promoter is that does not bind any native transcription factor to the cell of interest. A common example is the yeast Gal4 system, which needs the transfection of the Gal4 transcription factor DNA binding domain coupled to a regulatory or binding domain with a reporter gene whose expression is controlled by Gal4 response elements (cis- regulatory sequences).

Recently commercially available reporter genes are classified as intracellular and extracellular reporter genes. Intracellular reporter gene products such as Luciferase, Chloramphenicol Acetyltransferase(CAT), β-Galactosidase, Aequorin, Green fluorescent protein, are expressed and retained in the cellular systems. Both beta-Galactosidase and Luciferase reporters are now in use for the purpose of highly sensitive colorimetric and luminometric detection assays.

The extracellular reporter genes include Secreted Placental Alkaline Phosphatase (SPAP), β-Lactamase, which is secreted into the cell culture medium. The advantage of extracellular reporter gene assays makes this distinctive by the facility to identify the reporter protein activity in culture medium without disturbing the cells. This allows repeated experimentation and sampling of the culture medium using the same cell population.

Luciferase is nowadays a commonly used reporter system because of its convenient mode of action and, cost effectiveness that is broadly applicable in the field of cell based assay and molecular biology as well. Luciferases express a class of oxidative enzymes found in several species of bacteria and firefly that enable the organisms to expose “bioluminescence,” or emit light. The most important enzyme among these is the firefly luciferase. Fireflies are capable to emit light due to a chemical reaction in which luciferin is converted to oxyluciferin by the luciferase enzyme. Some of the energy releases in the form of light by this reaction.

With the help of Luciferase Assay Reagent which is specifically formulated to use with Luciferase Reporter Cell Line products and, designed to produce highly sensitive, prolonged and intensive signal. As one-step glow assay reagent, the Luciferase Assay Reagent can be directly added to cell culture plates compatible with the luminometer being used without diluting or transferring culture supernatants or cell lysates to other plates. 

This cell based technique can be used readily to assess particular gene transfer and expression or separately the included steps such as promoter analysis, gene delivery and imaging of gene expression. The system is very convenient to directly estimate the relative permeability, stability, potency and, screening of mechanism of action of different biological and chemical compounds and small molecules in the selected target cells for the early stages of drug discovery and bio-similar therapeutics. These systems can mimic physiological conditions, are robust and advantageous to provide important concepts about different receptor- ligand interactions, allosteric modulators, signaling pathways of cell division and proliferation as well as cell death, and acute cytotoxicity of the substance which cannot be availed from the conventional methods like secondary messenger and receptor binding assays.

Thus these assays represent a well compromise between a whole organism and biochemical assays and, produce physiologically relevant functional result of compound activity, or that may have reliable biological importance because live cells are used. Due to these characteristics the reporter system is most commonly used to screen a vast library of chemicals/ small molecule to find a suitable target in a very short span. This high through put screening method not only successfully reduces the lead detection time but also helps in finding an ideal drug target in the duration of early drug discovery.

The journey of drug discovery from its original idea to FDA approval and commercialization is complicated, time consuming and costly. It usually takes several years and well over globally several million dollars for the research and establishment of a new life saving drug. On average, approximately 250 lead compounds screened from a million compound library, enter pre-clinical testing, if succeed then 10 of them proceed to clinical trials, and only one will be certified by Food and Drug Administration (FDA)

Many drugs do not succeed during the clinical trial because either they do not elicit any significant effect upon binding or not being safe. For such reasons, proper action and functional target of the drug compound such as DNA, RNA, protein of the particular cell and/or tissue, need to be identified and validated for the reproducibility of basic data. Recently G- Protein Couples Receptors (GPCRs), anti- sense RNA are the most concerned target for discovered drug molecules.

After the identification and validation of the target, the compound screening assays are required during hit detection and lead discovery phase. A ‘hit’ molecule in the drug discovery field can be defined as being a compound which has the desired activity in library screening with reproducible data confirmation. Various screening paradigms exist to identify hit molecules. High throughput screening (HTS) involves cell based assay for the screening of the entire compound library directly against the drug target that in turn reduces the duration of the lead discovery that cannot be availed by using the conventional biochemical assays.

With the advancement of molecular biology techniques the reporter gene constructs are made and transfected in different mammalian cell lines. These stable cell lines expressing the reporter and the targeted gene products upon signal activation in response to bind desired compound, are now the basic instrumental to pharmacological companies to carry on high -throughput screening(HTS) to identify new high quality hits or leads which can act in fairly little concentration, out of a large number of compound library in an automated manner to study alteration in the regulation of functional synthesis and activity of both the extracellular and intracellular receptors upon interactions with these agonist and antagonist ligands.

The major task of HTS is not really to detect the drug rather the identification and optimization of the lead according to the information of its potency and efficacy that are also reliant upon the preliminary structure-activity relationship of the compound. Thus, finally medicinal chemistry and pharmacological study are required to convert a compound that elucidates from HTS into a useful drug.

High throughput screening assays are mainly categorized into two types:

• Target-based biochemical assays, such as receptor-ligand binding assays and enzyme inhibition assays, have been the basis of HTS campaigns in the pharmaceutical industry. In this assay, the binding tendency or affinity of the desired compounds, specific to the interested target is performed in homogeneous reactions which permit the efficiency with fewer variations. However, the biochemical assays are applicable to some because all targets cannot be purified or prepared in a method that fits for biochemical measurement.
• Cell-based assays for HTS include mainly three types of assays: second messenger assays, reporter gene assays, and cell proliferation assays, which are the described in following table.

The components of the cell based reporter gene assays for high throughput screening are thus cells, cell-culturing system and, detection device for the quantification of the cellular activities. The reproducibility of the overall throughput data can be achieved by carrying out several assays at a time, optimizing the throughput of the final detection step [3, 5].

Drug responses tested by biochemical methods cannot briefly characterize tissue-specific responses since the activity of a small molecule in a biochemical assay is different from the activity in a cellular context. Therefore, the toxicity testing market is slowly proceeding towards in vitro cell-based assays, as they can give a brief and early hint of the toxicity profile of the drug candidates.

To facilitate the research in drug discovery, Abeomics provides extensively validated and  functionally characterized several Reporter Cell Lines which are most advanced system available for screening STATs, Treg, Th17, TLR, DC-SIGN, Tim-1 and inflammatory pathway mediators. Abeomics also undertake Drug Discovery Screening Services/ Compound screening services for different pharma companies using the above cell lines.