Assay, development

Quality assay development is crucial for successful drug development. An assay is a method by which the activity of a compound is measured. As compounds are tested, or screened, in assays, compounds that show activity help steer the medicinal chemistry 

In the initial stages of searching for active compounds, the number of compounds to be screened is often greater than 100,000 and can exceed 500,000. Even 1 million compounds or more may be screened in a single assay.14 For this reason, biochemical screens must be inexpensive and quick. Ideally, the screen is automated and performed by a robot. Solutions of all necessary reagents and compounds to be tested are loaded into the instrument. The robot, called a liquid handler, dispenses reagents and solutions into microtiter plates. 

Microtiter plates have a standard size of approximately 3.4 X 5.0 . Within the area of the plate are typically 96 wells (8 X 12) for reagents. As technology has improved, 384-well (16 X 24) and 1,536-well (32 X 48) plates have become more common. More wells per plate translates to smaller volumes of reagents and reduced costs. However, very small volumes are difficult to dispense accurately, and the screens can lose reliability. Continued improvements in robotic technology are making the 1,536-well plate the standard format.15 

Assays are often performed in an aqueous medium with water as the solvent. Most drugs are organic molecules with poor water solubility. To improve solubility or at least evenly disperse the compound throughout the assay well, compounds are commonly dissolved in dimethyl sulfoxide (DMSO). DMSO has several attractive properties. DMSO dissolves organic compounds well, has a low vapor pressure to limit evaporation, and is fairly inert. Furthermore, DMSO freezes around 18 °C. A solution of a compound in DMSO can be easily frozen for long-term storage. Because high concentrations of DMSO may affect the reliability of an assay, compound solutions must be prepared such that, when mixed in the assay, the final DMSO concentration is 1% or less. 

While biochemical assays can provide receptor binding or enzyme inhibition information, they cannot determine how a compound behaves in a more complex cellular environment. Assays involving whole cells are required for this information. Compounds that show activity in a biochemical screen but fail in a cellular assay likely suffer from interference from the cell 

Amino terminus this region in most instances is involved in activating or stimulating transcription. 

DNA binding domain amino acids in this region are responsible for binding of the receptor to specific sequences of DNA. 

Carboxy terminus, or ligand-binding domain this is the region that binds ligands. 

To study drug-receptor/enzyme interaction, it is not always convenient or appropriate to use a living system of the target receptor. Instead, biochemical assays can be devised to mimic the target. Very often, the assays use multicolor luminescence or fluorescence-based reagents. In this way, the reaction path can be followed in space and time to enable quantitative evaluation of the reaction. 

Many parameters can be monitored, for example, free-ion concentrations, membrane potentials, activities of specific enzymes, rate of proton generation, transport of signaling molecules, and gene expression. 

Design a starting protocol based on prior literature or experimental information on substrate specificity test the protocol for enzyme-based or ligand-stimulated activity and use reference inhibitors when possible. 

Gain knowledge of kinetic and mechanistic parameters as guidelines for assay optimization. 

Set-up matrix experiments by varying pH, ionic strength buffers, and various parameters mentioned below to optimize signal-to-background ratios. 

Choose volumes appropriate for HTS workstations or automated systems. 

Test a screening protocol in a pilot study and determine assay quality based on the coefficient of variations across the assay wells, the Z prime measurements (Zhang et al.,1999) and dynamic range toward various control or reference compounds. 

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The basic elements and considerations for assay development, validation, and specification assignment are reviewed briefly. Assay development produces a method that requires validation for the analysis and release of materials (bulk or formulated finished product) for use in clinical development. The cumulative analysis of materials and stability considerations is then used to established specifications for internal and regulatory submission. 

Additional information on biomarker approaches and new technologies, such as microarray assays, developed to address complex pollution problems... 

Keywords Assay development, Automation, Data management, High throughput screening, Statistics... 

In most cases, in consultation with the HTS group, the research area laboratory will develop a benchtop assay that is at least compatible with the HTS format of choice for their target. This tends to facilitate project transitions and provides a tool that the research area laboratory will use later to follow up hits and develop SAR. In other cases, the HTS assay development group will assume all responsibility for assay development. The formality of the transfer of the project from the research area to the HTS group varies between organizations, but the outcomes are quite similar. All of the details of the prototype assay are reviewed by both teams, and, where applicable, reagents, protocols, and even plates or pipette tips are exchanged. 

Kinases are enzymes that place a phosphate group on a serine/threonine or a tyrosine residue of a protein or peptide. All kinase reactions use ATP as the phosphate source. Therefore there have been assays developed that monitor the loss or gain of the peptide/protein substrate (LANCE, ULight) [23], the loss of ATP (easylite luminescence kinaseGlo, Perkin Elmer) [20], or the gain of ADP (Tran-screener TR-FRET) [24]. Many of these formats are applicable to cell based assays. 

Fig. 1. Competition binding screens Bead/ligand compatibility test in early assay development. The beads PVT-WGA, Type A and Type B are based on polyvinyltoluene (PVT) not polyethyleneimine (PEI). PVT, polyvinyltoluene WGA, wheat germ agglutinin Ysi, yttrium silicate.

The concept that different structural domains on the heparin chains are principally involved for optimal activity in the foregoing interactions could not be perceived in early work on structure-activity correlations, because the activity of heparin has been most frequently evaluated only with whole-blood-clotting tests (such as the U.S.P. assay). Development of assays for specific clotting-factors (especially Factor Xa and thrombin) has permitted a better insight into the mechanism of action of heparin at different levels of the coagulation cascade. 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

Assays developed for the study of long distance charge transfer through DNA have to include the following experimental steps ... 

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