Assay Development

Did you know it could take around 12-15 years and anywhere near $1 billion for a drug to reach the market?

In that period, it takes 3 to 6 years to decide the efficacy of a drug candidate in an animal model after the identification, validation, optimization, and efficiency testing of the drug compound. However, even after such extensive research, one-third of the developed drugs fail at the first clinical stage, and half of them fail as they may show toxicity in humans. This is mainly because of the assumption that the chosen drug candidates are non-toxic compounds for humans after testing their efficacy in animal models.

The other half of the drugs fail due to their inefficacy in humans during late-stage clinical trials. Because of all such reasons, testing the chosen drug candidate's effectiveness in the early stages of drug development using in vitro assays and human cells and tissues poses a best practice to accelerate the drug discovery and development process and save money.

Assays are procedures that assess the effectiveness of the chosen drug candidate on the desired target, which can be molecular or biochemical targets. It can also be defined as the process of “hit” discovery (Figure 1). After target identification and validation, assays are developed for the efficient optimization of the chosen compound. The assay should be efficient for high-throughput screening, biologically relevant, and economical.

In this article, we review the types of assays developed during drug development, their process, and what challenges drug manufacturers face while designing an efficient assay to assess drugs.

Types of Assays in Drug Discovery

Assay development, or creating a test system to assess the effects of chosen drug candidates on desired biological processes, including cellular-based, and biochemical processes (Figure 2), is one of the first steps of drug development.

High throughput screening of compound libraries enables researchers to perform pharmacological or genetic tests using automated software robotic machines, and sensitive detectors. The process narrows down to pinpoint compounds with therapeutic properties or functions that contribute to human health or are associated with various diseases. These tested compounds (also known as probes) are further optimized for their application in the drug development pipeline as therapeutic candidates.

Cell-based assays

Cell-based assays help evaluate the efficacy of drug molecules in a more effective way than biochemical assays. The in vitro cell cultures are more reliable and provide deeper insight into the effect of the small molecule on humans. To obtain substantive information and keep track of cellular activities in terms of temporal (time of occurrence) and spatial (location) resolution, researchers often combine advanced microscopic techniques with cell-based assays.

Some cell-based assays used in the drug development process include:

• On-chip, cell-based microarray immunofluorescence assay: Used for high-throughput target protein analysis.
• Beta-lactamase protein fragment complementation assays: To study protein-protein interaction
• The ToxTracker assay: Used to study the toxicity of the chosen drug candidates
• Fluorescence-based IHC assays: To study morphologic features and molecular target activities.
• Reporter gene assay: To detect primary signal pathway modulators.
• Mammalian two-hybrid assay: To study mammalian protein interactions in the cellular environment.

These assays can be applied in a 2-D cell culture environment and in 3-D cell culture systems as well.

Featured Product

Biochemical assays

Biochemical assays are used to test the binding affinity or inhibitory activity of the tested drug candidate with the target enzyme or receptor molecule.

Some assays extensively used in drug discovery and development processes are:

• Quenched fluorescence resonance energy transfer (FRET) technology-based assay: Used to screen inhibitors and monitor proteolytic activity of
• High-performance liquid chromatography (HPLC) technique: Used to assess proteolytic action (mainly for chromogenic compounds), and it can also be used to screen inhibitors to a certain extent.
• Enzyme-linked immunosorbent assay (ELISA): Used to analyze the inhibitory activity of the tested or chosen drug compound.
• Surface plasmon resonance (SPR) techniques: Used to study the interaction of the lead compound with the target protein.

Featured Product

In silico assays

In silico methods are computation-based approaches. They are effective techniques used to screen a large library of compounds and evaluate their affinity and efficacy even before they enter the development process, based on their structure. They have massive applications in assessing the pharmacodynamic (PD) and pharmacokinetic (PK) properties of molecules. The two types of virtual screening methods used in drug development are ligand-based methods (utilizing topological fingerprints and pharmacophore similarity) and target-based methods. Docking and consensus scoring are some target-based approaches used to estimate the binding affinity and inhibitors of the selected compound. Quantitative Structure-Activity Relationship (QSAR) is another technique that predicts the quantitative relationship between a chemical structure and its biological activity.

What are the factors responsible for variation of Assays?

Factors influencing cell-based assays are as follows:

• Culturing Media

Culturing conditions influencing cell-based assay methods are as follows:-

• Culturing media
• Culturing conditions
• Serum
• Cell Cycle
• Passage number

Factors influencing biochemical assay are as follows:-

• Temperature
• PH
• Concentration of Ions
• Solubility of Reagents
• Stability of Reagents
• Aggregation of Reagents

Assay Development Process

The first step in the drug development process is the identification and validation of targets, such as DNA, enzymes, receptors, and ion channels for diseases. This is followed by designing assays to evaluate the pharmacokinetics, molecular, and biological activity of the hit molecule.

Different biochemical and cell-based assays are designed to assess compounds’ effect on the desired target. After designing the assay, several validation steps are implemented to improve the chances of success for a drug candidate. The assay conditions and procedures are optimized to reduce or minimize the influence of potential factors that could introduce errors in measuring a specific substance (analyte) or a biological endpoint. The rate of false-positive and false-negative determines the selectivity and sensitivity of an assay. Furthermore, various other factors, including assay automation, reagent stability, pipetting, and data analysis models, play roles in determining the validity of an experiment.

To facilitate compound screening, a range of assay formats are available that are chosen by researchers based on the goal, available facility and equipment, and screening scale.

When choosing an assay for drug development, it is important to consider the following factors :

• Pharmacological Relevance: Conduct studies with a known ligand and the target to assess if the assay pharmacology predicts the disease state and identifies compounds with desired potency and mechanism of action.
• Assay Reproducibility: The assay must demonstrate reproducibility across plates, screen days, and the entire duration of the drug discovery program, which may span several years.
• Assay Quality: It’s assessed using the Z' factor, with values above 0.4 considered robust for screening. Monitoring pharmacological controls is crucial, and high-quality assays result from simple protocols, stable reagents, and optimal instrumentation.
• Assay Costs: Assays typically involve microtiter plates, with reagents and volumes selected to minimize costs. The format (e.g., 96-well or 384-well) depends on the context (academia, industry, or high-throughput screening).
• Effects of Compounds: Assays need to be configured to be insensitive to solvent concentrations. Cell-based assays tolerate up to 1% DMSO, while biochemical assays can handle up to 10%. Based on the false negative and false positive hit rate, the assays are reconfigured or optimized for their further applications in the drug development process.

Ensuring the reproducibility and transferability of measurements relies heavily on the integration of standard operating procedures (SOPs) and comprehensive method documentation. In this context, bioinformatic support is unquestionably pivotal, playing a critical role in both configuring assays and analyzing data.

Challenges in Assay Development

Assay development is the process of designing assays and optimizing their environment and procedure for the specific hit molecule or drug compound. These assays designed for drug development purposes are of three types: biochemical assays that provide insight into the chemistry of the drug molecule, a cell-based assay that offers insight into the efficacy and toxicity of drug molecules in humans, and computation-based approaches that predict the chemistry of the lead molecule with the target in advance.

In recent times, High-Throughput Screening (HTS) campaigns have leaned towards employing target-directed and specialized libraries, along with assay formats designed to minimize artifacts and yield more comprehensive information. Further, primary cells and 3D cultures have become more common in high-content screening of lead compounds.

Besides improving current technologies, researchers are actively seeking out new detection methods in High-Throughput (HT) formats. This ongoing exploration covers possibilities such as localized surface plasmon resonance (SPR), time-resolved anisotropy analysis (TRA), intrinsic protein fluorescence, multi-photon excitation techniques, and electrochemical screens. The refinement of analysis software is also seen as a way to elevate the effectiveness of various High-Throughput Screening methods.

Conclusion and Future Directions in Assay Development

The assay development stage is a crucial drug development stage, which determines the success of a drug candidate in further drug development processes. Thus, one needs to ask the right questions and carefully design the assay protocol based on their goal and requirements. However, this is not easy and poses several challenges.

There is not one assay without limitations; thus, to ensure the reliability and accuracy of data one needs to design several assays to evaluate the drug candidate. For example, biochemical screening assays face limitations in capturing the intricate dynamics of living systems, which necessitates the development of more sophisticated biological assays for accurate evaluation of the biological activity of the hit molecule or lead compound and its potential toxicity in humans.

Further, the application of advanced approaches needs years of study to assess the effectiveness of their procedure in high throughput screening. For example, the use of 3D cell culture in HCS and HTS campaigns is still challenging as there are no high-throughput methods for analyzing cells within a 3D environment.

Understanding that all assays have limitations, it's crucial to create counter-assays. These counter-assays are essential for filtering out compounds that work in undesirable ways. Additionally, to learn in-depth about the complexity of biological responses, the development of secondary assays is required.