High-throughput screening

High - throughput screening ( HTS ), also called high -throughput screening, is a mainly engaged in pharmaceutical research applied, automated method to millions of molecules biochemical, genetic or pharmacological tests are performed in a high throughput of tens of thousands. If more than 100,000 substances per day examined, one also speaks of ultra-high- throughput screening ( uHTS ). By means of the high-throughput screening is sought in particular for new, biologically active substances, from which conductive structures can be derived in order to develop new drugs.

Implementation

When high-throughput screening extensive molecule libraries are searched, the search makes high demands on the automation of the test procedure and evaluation.

Test method

When high-throughput screening for the discovery of novel pharmacologically active substances are target -based or phenotype -based tests ( assays) application.

Target - based

Wherein target -based screening, the interaction of test compounds with specific defined targets (targets) is examined. Targets may include, for example, be proteins associated with a disease or a physiological process. Target -based screening in the pharmaceutical industry represent the most common form of screening of small molecule drugs to determine their biological activity. They are usually in microtiter plates with purified or unpurified proteins or indirectly with cells that make up the target protein is performed. The interaction of a test substance to the target can be directly in binding assays (usually via the displacement of a labeled reference ligand from the target ) or indirectly through the influence of the activated from the target protein signaling pathways (eg, activation of second messengers, protein -protein interactions protein phosphorylation and gene activation ) and enzymatic reactions are determined. Biochemical methods to be used in particular, in which a signal is measured as a change in color intensity, fluorescence, or luminescence. From the signal to noise ratio follows: The greater the change in the evaluated test signal intensity is, the more suitable test method. In luminescence based methods cause a multiple signal change and are therefore often more suitable than photometric and fluorimetric methods. Their color or autofluorescence of test substances degrade the signal -to-noise ratio of the photometric and fluorimetric measurement. Also szintimetrische test methods such as radioligand binding studies are highly sensitive. However, the seizures of radioactive waste is a central problem in the implementation of szintimetrischen tests. Further properties of the test substances, such as solubility and stability, play a crucial role and must be considered in the experimental design.

Phenotype - based

In phenotype -based screening the effects of test compounds are examined for living cells or tissue, so the effect of the administration of the test substance on the phenotype of the cell or tissue. The effect of a test substance is the basis of a phenotypic change, for example, the change in the cell shape, the cell growth or cell function evaluated. Here, it is not necessary to know in advance the molecular target, the screening method is used, however often the identification of the molecular target. In order not to distort the result of the screening, usually a large number of parameters must be controlled. With phenotype - based screening in particular molecule libraries are screened to contain high molecular weight compounds such as proteins, DNA and siRNA. Addition cells and tissues and whole organisms, such as fish embryos used as model systems. A phenotype - based screening is often performed with the help of automated microscopy ( High Content Screening ). Often a high content screening against a target - based screening is limited by the lower throughput.

Assessment

Since the screening of a complete molecule library often takes several days to weeks in a high- throughput screening, a consistently reliable operation of the test method is a critical requirement. Particularly with the use of cells must be expected to change with increasing cultivation time.

To assess the robustness of the data of a high-throughput screening control substances to be investigated. In the simplest case, this one hand vehicle or a known inactive substance (negative control) and on the other hand, a substance that results in maximum activation or inhibition of the test include (positive control). With the help of the Z'- factor

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Where? p and σn the standard deviations of the positive or negative control, and uP and ìN the mean values ​​of the positive or negative control represent the measurement window of the high-throughput screening can be assessed. Assays with Z'- factors of at least 0.5 are considered optimal. Even high -throughput screening in a Z'- factor from 0 to 0.5 may still be capable of distinguishing between active and inactive compounds.

Automation

High - throughput screening are complex and are now performed only with fully or at least partially automated laboratory automation systems. It can be used for automatic or robotic liquid handling, the data receiver ( reader, cameras ) and optionally the cell culture. The test volume is reduced, and microtiter plates with 384, 1536 or 3456 wells used to test more samples at a time and save cost and time.

Evaluation

The as a result of high-throughput screening collected data are analyzed statistically. Substances that provide measured values ​​beyond a certain threshold are classified as hits ( "hits" ). However, the occurrence of false-positive and false-negative results must be considered. In order to reduce the amount of false positive hits, a second, much smaller screening is usually performed, which is restricted to the hits of the first screening.

The data obtained from the screening are analyzed with the help of chemo- informatic methods. For this purpose, the hits are filtered based on their molecular properties. In this way, substances that are considered for example due to reactive groups (eg, aldehydes, Michael acceptors and nitro groups), or a failure to comply with Lipinski Rule of Five to be unsuitable for further development, are removed from the candidate list of Leitstrukutren. Finally, one selects the most promising hits for the development of a lead.

Restrictions

Few identified in a high- throughput screening hits have the quality to be classified as a lead structure. A hit is therefore not automatically lead structure and certainly not a drug. As high-throughput screening are usually carried out at a single test concentration using a single assay, and quantitative information about the potency (potency ) and selectivity of the test substances are not possible. Many necessary for the efficacy and safety of a therapeutic substance pharmacological parameters such as the cell membrane and tissue permeability and the absorption, distribution, metabolism, excretion ( ADME ) and toxicology are not or only insufficiently taken into account in high-throughput assays. The development of a hit to a drug and its approval usually takes about 10 to 12 years.