The document discusses high-throughput screening (HTS), which enables rapid screening of numerous compounds for active substances using bio-microassays on well plates. It outlines the principles, types of microassays like biochemical and whole cell assays, and the process for identifying potential 'hits', as well as considerations for validity of the assays. The importance of defining hit criteria and understanding hit rates for assay evaluation is emphasized.

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. Flow of Contents
Introduction
General principles of HTS
Types of microassays in HTS
Biochemical Assays
Whole Cell Assays
Hits and hit rates
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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.
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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.
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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.
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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.
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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.
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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.
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 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. 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.
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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.

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 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.

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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.
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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.
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