Diese Präsentation wurde erfolgreich gemeldet.
Die SlideShare-Präsentation wird heruntergeladen. ×
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Anzeige
Nächste SlideShare
High Throughput Screening
High Throughput Screening
Wird geladen in …3
×

Hier ansehen

1 von 17 Anzeige

High Throughput Screening

High-throughput screening (HTS) is the name given to rapid semi-automated simultaneous primary screening of large numbers of compounds, mixtures or extracts for active compounds.
The process is based on the use of bio-microassays that are rapid to carry out and require very small quantities of the reagents and test compound.
These assays are carried out on 96- and bigger-well plates using specialised handling equipment.

High-throughput screening (HTS) is the name given to rapid semi-automated simultaneous primary screening of large numbers of compounds, mixtures or extracts for active compounds.
The process is based on the use of bio-microassays that are rapid to carry out and require very small quantities of the reagents and test compound.
These assays are carried out on 96- and bigger-well plates using specialised handling equipment.

Anzeige
Anzeige

Weitere Verwandte Inhalte

Diashows für Sie (20)

Ähnlich wie High Throughput Screening (20)

Anzeige

Weitere von ISF COLLEGE OF PHARMACY MOGA (20)

Aktuellste (20)

Anzeige

High Throughput Screening

  1. 1. High Throughput Screening ROHIT BHATIA ASSISTANT PROFESSOR DEPT. OF PHARMACEUTICAL CHEMISTRY ISF COLLEGE OF PHARMACY WEBSITE: - www.isfcp.org EMAIL: bhatiarohit5678@gmail.com ISF College of Pharmacy, Moga Ghal Kalan,GT Road, Moga- 142001, Punjab, INDIA Internal Quality Assurance Cell - (IQAC)
  2. 2. Flow of Contents Introduction General principles of HTS Types of microassays in HTS Biochemical Assays Whole Cell Assays Hits and hit rates 2
  3. 3. Introduction  High-throughput screening (HTS) is the name given to rapid semi- automated simultaneous primary screening of large numbers of compounds, mixtures or extracts for active compounds.  The process is based on the use of bio-microassays that are rapid to carry out and require very small quantities of the reagents and test compound.  These assays are carried out on 96- and bigger-well plates using specialised handling equipment. 3
  4. 4. Contd…  They are based on the test compound interacting with a target, such as an enzyme, a cell membrane receptor, hormone, nuclear receptors and DNA, that is related to the disease state under investigation.  Consequently, it may be necessary to identify, purify and isolate this target before a library can be screened.  With the ability of rapid screening of diverse compounds to identify active compounds, HTS has led to an explosion in the rate of data generated in recent years. 4
  5. 5. General Principles of HTS  The assays involving biological targets are generally carried out in aqueous media.  Consequently, an assay will only be effective if a significant amount of the compound under test dissolves in water.  Dimethylsulphoxide (DMSO) is often added to assay mixtures in order to improve the water solubility of the test compound. 5
  6. 6. Contd…  The microassays used in HTS may be classified for convenience as either biochemical or cell-based assays.  Biochemical assays are those that are based on the interaction of the test compound with defined chemical entities isolated from cells such as an enzyme, hormone or receptor, whereas whole cell assays are based on the use of intact cells.  Any active compounds (hits) that appear worthy of further investigation must be subjected to a wider range of activity tests before they could be considered for clinical development. 6
  7. 7. Types of Microassays in HTS 1. Biochemical Assays  Biochemical assays are also referred to as mechanism-based assays.  They are usually based on the binding of a ligand to a receptor or the inhibition of an enzyme-catalysed reaction using a target that has been identified as being relevant to a specified disease state.  This target has usually been isolated from a cell and is no longer part of a cell. 7
  8. 8. Contd…  The binding of the test compound to the target is measured by the use of radioactive isotopes and/or traditional analytical methods such as spectroscopy using a variety of protocols, such as by measuring fluorescence in scintillation proximity assays (SPA).  Scintillation proximity assays use resin beads (SPA beads) whose surface has been engineered so it is capable of binding to a wide variety of substances.  The bead also contains a scintillant that only fluoresces when a low energy radioactive source comes within about 20 mm of the surface of the beads. 8
  9. 9. Contd… 9  The radioactive isotopes used in SPA assays emit low energy emissions that have very short pathways in aqueous media (given in the following table). Many SPA enzyme-based assays are carried out using radio labelled substrates or ligands.
  10. 10. Contd….  Consider, for example, an enzyme inhibition assay, based on the use of a substrate A-B for the enzyme where B is the part of the substrate that contains the radioactive isotope and A contains a so-called capture group, which contains structures that bind to the SPA beads.  B does not contain a capture group. Treatment of the substrate A-B with the enzyme and any essential co-enzymes in the absence of the inhibitor results in cleavage of all of the substrate A-B.  When the SPA beads are added no fluorescence is observed as only the non-radioactive A binds to the beads. The radioactive B remains in solution too far from the beads to cause fluorescence. 10
  11. 11. 11
  12. 12. 2. Whole Cell Assays 12  Whole cell assays are preferred when the nature of the steps in the mechanism of the disease state have not been well defined.  They also offer a number of other advantages over biochemical tests.  For example, whole cell tests may identify compounds that act at sites other than the target site. They are usually conducted under conditions that are more like those encountered if the test compound was used in a patient.
  13. 13. Contd… 13  Consequently, test compounds that are either too hydrophobic, and as a result bind too strongly to serum albumin or will not cross cell membranes will not usually be active.  Therefore it is relatively easy to identify these compounds and eliminate them from the investigation.  Furthermore, test compounds that are toxic are often readily identified because of their effect on the cells used in the test.  These advantages mean that many medicinal chemists prefer whole cell assays to biochemical assays. A wide range of different types of whole cell assays are in use.
  14. 14. 14
  15. 15. Hits and Hit Rates  A hit occurs when the activity of a test compound has a value greater than an arbitrary minimum value set by the investigators using that assay.  For example, in an enzyme inhibition assay a hit may be scored when the activity of the enzyme is inhibited by a pre-agreed value of 50 per cent.  It is important to set the criteria for a hit before carrying out the assay since hit rates are often used as a measure of the validity of an assay procedure. 15
  16. 16. Contd…  Hit rates are defined as the number of active samples discovered by an assay expressed as a percentage of the total samples used in that screen.  Assays with values of about 0.1–1per cent hits are normally regarded as being valid.  Higher values may occur for a number of reasons: for example, a series of compounds with similar structures and hence similar activities is being tested, the assay is not specific enough or the hit criteria are not rigorous enough.  However, high hit rates may be of use when developing an assay. 16
  17. 17. 17

×