By :Waqar Ahmad BamkhelviSession: 2011-2016University Of Swabi

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High-throughput screening Technology

•Commonly used terms in drug discovery

• High throughput screen: an optimised, miniaturised assay format that enables the testing of > 100,000

chemically diverse compounds per day.

• Assay: a test system in which biological activity can be detected

• Hit: a molecule with confirmed concentration-dependent activity in a screen, and known chemical

structure. The output of most screens

• Progressible hit: a representative of a compound series with activity via acceptable mechanism of action

and some limited structure-activity relationship information

• Lead: a compound with potential (as measured by potency, selectivity, physico-chemical properties,

absence of toxicity or novelty) to progress to a full drug development programme

Drug Discovery Process:

Drug Discovery Process:•The key steps of drug discovery are:

•research - average 2 to 3 years •pre-clinical testing - average 1 year •clinical trial testing (involving human patients) - average 10 years

•regulatory approval - average 2 years

What is High throughput Screening ?• High throughput screening (HTS) is a tool for early-stage drug

discovery.

•Definition : HTS is process by which large number of

compounds are rapidly tested for their ability to modify the properties of a selected biological target.

History

• HTS was invented by Dr Gyula Takatsky in 1951, who machined 6 rows of 12 wells in Lucite to make the first microtiter plate.

• The microtiter plate has further grown to include standardized 96, 384, 1536 well formats, with additional 3072 well nanoplate formats.

• “Twenty 384-well plates are currently run daily on the Accuri C6 HyperCytometer combination. We could run up to 40 plates in a standard 8 hour workday, over 12,000 compounds.

•High throughput screening for drug discovery

• Why High throughput screening need arises ?

• FACT 1: recent understanding of disease mechanisms has dramatically increased no. of protein targets for new drug treatment

• FACT 2: new technologies have increased the no. of drugs that can be tested for activity at these targets.

Goal of HTS

Goal is to identify ‘hits’ or ‘leads’

• - affect target in desired manner

• active at fairly low concenteration ( more likely to show specificity)

• - new structure

• The greater the number and diversity of compounds screened, the more successful screen is likely to be.

• HTS = 50,000-100,000 compounds screened per day!!!

The majority of drug targets are :

• a) G-protein coupled receptors

• b) nuclear receptors

• c) ion channels

• d) enzymes

Explanation• High-throughput screening is a method for scientific experimentation especially

used in drug discovery and is relevant to biology and chemistry. This process in combination with robotics, data processing and control software, liquid handling devices and sensitive detectors allows a researcher to quickly conduct millions of chemical, genetic or pharmacological tests.

• High-throughput screening can rapidly identify active compounds, antibodies or genes which modulate a particular bimolecular pathway. It can be considered - a process in which batches of compounds are tested for binding activity or biological activity against target molecules.

• High-throughput screening is a process of screening more compounds against more targets per unit time, which should generate more hits, which in turn will generate more leads, subsequently generating more products.

• Various technologies like high-throughput screening defined by the number of compounds tested to be in the range of 10,000-100,000 per day, ultra high-throughput screening is defined by screening more than 100,000 data point generated per day. These two technologies play a vital role in drug discovery to find new chemical compounds.

Which strategy is best for hit identification?

When a target is identified, a decision has to be made about which chemicals to screen, in order to identify potential lead compounds.

Random screening :All possible drug molecules screened against target.This is simply not possible.Focussed screening :A limited number of compounds are pre-selected for screening.Has proved successful as a hit generation strategy.Useful when 3D structure of target is known (e.g. crystal structure of a receptor)

Procedure• High-throughput screening in drug discovery is used to screen :• Novel biological active compounds• Natural products• Combinatorial libraries (Ex: peptides; chemicals)• Biological libraries• DNA chips• RNA chips• Protein chips• High-throughput screening’s main lab ware is the microtiter plate.

Modern microplates for high-throughput screening assays are performed in automation-friendly microtiter plates with a 96, 384, 1536 or 3456 well format. These wells contain experimentally useful matter, often an aqueous solution of dimethyl sulfoxide (DMSO).

• For most drug discovery labs, the library collection has grown from 400,000 to 1 million or more compounds. The standard paradigms used to screen these libraries have evolved to automated 384 wells or higher density single compound test formats.

• Primary screen is designed to rapidly identify hits from compound libraries. The goals are to minimize the number of false positives and maximize the number of confirmed hits.

• Depending on the assay, hit rates typically range between 0.1 – 5 per cent. This number also depends on the cutoff parameters set by the researchers, as well as the dynamic range of a given assay.

• Primary screens are run in multiplets of single compound concentrations. Hits are then retested, usually independently from the first assay.

• If a compound exhibits the same activity, it is coined as confirmed hit, which proceeds to secondary screens or lead optimization. The results from lead optimization are used to decide which substances will make it on to clinical trials.

• In combination with bioinformatics, it allows potential drugs to be quickly and efficiently screened to find candidates that should be explored in more detail. Initial screening of these compounds for their binding ability is the job for high-throughput screening.

• The key to high-throughput screening is to develop a test, or assay, in which binding between a compound and a protein causes some visible change that can be automatically read by a sensor. Typically the change is emission of light by a fluorophore in the reaction mixture.

• One way to make this occur is to attach the fluorophore to the target protein in such a way that its ability to fluoresce is diminished (quenched) when the protein binds to another molecule. A different system measures the difference in a particular property of light (polarization) emitted by bound versus unbound fluorophores. Bound fluorophores are more highly polarized and this can be detected by sensors.

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Procedure

The sets of compounds produced by combinatorial chemistry are generally referred to as libraries, which depending on how the solid-phase is handled, may be either mixtures or individual compounds. There are a range of options for testing the libraries in a biological assay.

Test mixture in solutionTest individual compounds in solutionTest compounds on the beads

Test mixture in solution :

• All the compounds are cleaved from the beads and tested in solution.

• If activity in a pharmacological screen is observed, it is difficult to find out which compounds are active. To identify the most active component, it is necessary to resynthesize the compounds individually and thereby find the most potent. This iterative process of resynthesis and screening is one of the most simple and successful methods for identifying active compounds from libraries.

Test individual compounds in solution• A second method is to separate the beads manually into individual

wells and cleave the compounds from the solid-phase. These compounds can now be tested as individual entities.

•Test compounds on the beads :

• A third method for screening is testing on the beads, using a colorimetric or fluorescent assay technique. If there are active compounds, the appropriate beads can be selected by color or fluorescence.

Applications :• High-throughput technology can also be put to use in

other areas besides drug development.• Genomics Applications • DNA Sequencing• Protein Analysis

Applications

Conclusion :• HTS is became an effective technique and competitive with

the latest, upcoming related technologies in the market. The growing importance of this process is cost effectiveness of drug-discovery and development, operating processes for development of homogeneous, fluorescence-based assays in reduced formats.

• The usage of 384, 1536 and 3456 wells density plates and robotics made the HTS process through which compounds can be screened more than 100,000 data points per day.

• The number of higher density plates used in the drug-discovery process is inversely proportional to the samples required for the process; thereby it reducing the initial setup costs.

• The combination with robotics, data processing and control software, liquid handling devices, TR-FRET, FRET, Fluorescence polarization techniques has added a significant valued to each data point generated by high throughput screens.

•Thanks For All Of You.