High throughput screening
High-Throughput-Screening (HTS), also known as high-throughput screening , is an automated method used primarily in pharmaceutical research in which biochemical , genetic or pharmacological tests are carried out on tens of thousands to millions of substances at high throughput . If more than 100,000 substances are examined per day, this is also called ultra- high-throughput screening (uHTS). High-throughput screening is used in particular to search for new, biologically active substances from which lead structures can be derived in order to develop new drugs .
execution
In high-throughput screening, extensive molecular libraries are searched, whereby the search places high demands on automation, test procedures and evaluation.
Test procedure
Target-based or phenotype-based test methods (assays) are used in high-throughput screening to discover new pharmacologically active substances.
Target-based
In target-based screenings, the interaction of the test substances with certain defined target structures (targets) is examined. For example, targets can be proteins that are related to a disease or a physiological process. Target-based screenings represent the most common form of screening of low molecular weight substances in the pharmaceutical industry in order to determine their biological activity. They are usually carried out in microtiter plates with purified or unpurified proteins or indirectly with cells that produce the target protein. The interaction of a test substance with the target can be carried out directly in binding assays (usually by displacing a labeled reference ligand from the target) or indirectly by influencing the signal pathways activated by the target protein (e.g. activation of second messengers , protein-protein interactions , Protein phosphorylation and gene activation ) and enzymatic reactions can be determined. For this purpose, biochemical methods in particular are used in which a signal is measured as a change in color intensity, fluorescence or luminescence . From the signal-to-noise ratio it follows that the greater the change in the signal intensity evaluated in the test, the more suitable the test methods are. Methods based on luminescence cause a multiple signal change and are therefore often better suited than photometric and fluorimetric methods. Self-color or self-fluorescence of test substances worsen the signal-to-noise ratio of the photometric and fluorimetric measurement. Also szintimetrische test methods, such as radioligand binding studies are highly sensitive. The generation of radioactive waste is, however, a central problem when performing scintimetric tests. Other properties of the test substances, such as solubility and stability , play a decisive role and must be taken into account when planning the experiment.
Phenotype based
In phenotype-based screening, the effects of test substances on living cells or tissue are examined, i.e. the effects of the application of the test substance on the phenotype of the cell or tissue. The effect of a test substance is based on a phenotypic change, e.g. B. the change in cell shape, cell growth or cell function assessed. It is not necessary to know the molecular target in advance, but the screening process is often used to identify the molecular target. In order not to falsify the result of the screening, a large number of parameters usually have to be checked. With phenotype-based screenings, molecular libraries in particular are screened that contain compounds of higher molecular weight, such as proteins, DNA and siRNA . In addition to cells and tissue , whole organisms such as fish embryos are also used as model systems. A phenotype-based screening is often carried out with the help of automated microscopy ( high content screening ). Often a high content screening is limited compared to a target-based screening due to the lower throughput.
evaluation
Since the screening of a complete molecule library in a high throughput screening often takes several days to weeks, a consistently reliable operation of the test procedure is a critical requirement. In particular when using cells, a change must be expected with increasing cultivation time.
Control substances are examined to assess the robustness of the data from a high-throughput screening. In the simplest case, these comprise, on the one hand, a vehicle or a known inactive substance ( negative control ) and, on the other hand, a substance which leads to maximum activation or inhibition of the test ( positive control ). With the help of the Z 'factor
,
where σ p and σ n represent the standard deviations of the positive and negative controls and µ p and µ n represent the mean values of the positive and negative controls, the measurement window of the high throughput screening can be assessed. Assays with Z 'factors of at least 0.5 are considered optimal. High throughput screenings with a Z 'factor of 0-0.5 can also be suitable for differentiating between active and inactive compounds.
automation
High-throughput screenings are complex and are now only carried out with fully or at least partially automated laboratory automation systems . Robots or automatic machines are used for liquid handling , data acquisition (readers, cameras) and, if necessary, cell culture . The test volume is reduced and microtiter plates with 384, 1536 or 3456 wells are used to test even more samples at the same time and save costs and time.
evaluation
The data obtained as a result of the high throughput screening are statistically analyzed. Substances that deliver measured values beyond a certain threshold value are classified as hits ("hits"). However, the occurrence of false positives and false negatives must be taken into account. In order to reduce the number of false positive hits, a second, much smaller screening is usually carried out, which is limited to the hits from the first screening.
The data obtained from the screenings are also analyzed with the help of chemoinformatical methods. To do this, the hits are filtered based on their molecular properties. In this way, substances that are considered unsuitable for further development due to reactive groups (e.g. aldehydes , Michael acceptors and nitro groups ) or failure to comply with Lipinski's Rule of Five can be removed from the candidate list of lead structures. Finally, one selects the most promising hits for the development of a lead structure.
Restrictions
Few hits identified in a high throughput screening have the quality of being classified as lead structures. A hit is therefore not automatically a lead structure and certainly not a drug . Since high-throughput screenings are usually carried out at a single test concentration with the help of a single assay, quantitative statements about the potency and selectivity of the test substances are not possible. Many pharmacological parameters necessary for the efficacy and therapeutic safety of a substance, such as cell membrane and tissue permeability as well as uptake, distribution, metabolism, excretion (ADME) and toxicology, are not or only insufficiently taken into account in high-throughput assays. The further development from a hit to a medicinal substance and its approval usually takes about 10 to 12 years.
See also
Individual evidence
- ↑ Wunder F, Kalthof B, Müller T, Hüser J: Functional cell-based assays in microliter volumes for ultra-high throughput screening . In: Comb Chem High Throughput Screen . . 11, No. 7, August 2008, pp. 495-504. PMID 18694386 .
- ↑ Hanspeter Gubler: Methods for statistical analysis, quality assurance and management of primary high-throughput screening data . In: Gerd Folkers; Jörg Hüser; Raimund Mannhold; Hugo Kubinyi (Ed.): High-Throughput Screening in Drug Discovery (Methods and Principles in Medicinal Chemistry) . Wiley-VCH, Weinheim 2006, ISBN 3-527-31283-8 , pp. 151-206.
literature
- Gerd Folkers; Jörg Hüser; Raimund Mannhold; Hugo Kubinyi: High-Throughput Screening in Drug Discovery (Methods and Principles in Medicinal Chemistry) . Wiley-VCH, Weinheim 2006, ISBN 3-527-31283-8 .