- Assay development is the process of creating biological and compound screening assays to identify compounds, called "hits", that have desired activity at drug targets. This involves developing biochemical and cell-based assays. - Key factors in assay development include relevance, reproducibility, quality as measured by Z'-factor, and avoiding interference. High throughput screening uses automation to test tens of thousands of compounds against targets daily using miniaturized assays. - Biochemical assays use purified protein or enzyme targets, while cell-based assays examine responses at transcriptional, proliferation, or second messenger levels. Automation and robotics are important for achieving desired screening rates in high throughput screening.

1. Seminar presentation on
Assay Development for hit
Identification
Presented by: Soheila Ahanjan
M.Pharm 2nd semester
Department of Pharmacology
SPER, JAMIA HAMDARD
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2. Content
• Introduction
• Assay Development
• Factors important in Assay Development
• High Throughput Screening
• Biochemical assays
• Cell based assays
• Automation and Robotics in HTS
• Applications
• References
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3. Introduction
• Hit identification is the most critical step to identify compounds able to interact with
the fully validated target.
• There are different strategies available to identify good hits, devoted to maximizing the
number of initial hits and hit series.
• At the same time, the strategy must ensure it reduces the amount of potential false
positives, thereby reducing the risk of identifying a series of compounds that are
suboptimal for development, spending resources on the wrong compounds.
• A successful hit identification relies on the use of a high-quality, diverse library of
potential compounds to be screened, but also capitalizing on other complementary
techniques, such as virtual screening and fragment-based screening.
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4. 4

5. Assay Development
• Assays are investigative procedures that qualitatively assess a compound or
examine a compound’s effects on identified molecular, cellular, or
biochemical targets.
• In the following stage of drug development, biological assays and
compound screening assays are created. These assays are used to identify
compounds that have a desired activity at the drug target. These compounds
are referred to as “hit” molecules.
• During the initial phase of hit compound identification, termed high
throughput screening (HTS), a compound library that contains many
potential hit molecules is tested to identify any compounds with the desired
activity towards the target.
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6. Cont.
• Further assays are required to retest the hit molecule’s activity at the
target.
• Finally, cell-based assays are used to examine a drug’s toxicity, safety
profile, and efficacy.
• Every drug that is developed undergoes a unique series of assays that
are specifically designed and organized for the drug target and
compound in question.
• This process is termed assay development.
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7. Factors important in Assay Development
FACTOR IMPORTANCE IN ASSAY DEVELOPMENT
Relevance Research should be conducted to examine the ability of the assay to:
1.Predict the specific disease state.
2.Identify compounds that exhibit an appropriate mechanism of action and
strength.
Reproducibility In a compound screening environment, an assay must be reproducible. This
means that feasible reproducibility exists across assay plates, screen days
and the full duration of the specific drug discovery program.
Quality Z’-factor is calculated to measure the power or quality of an HTS assay. The
signal window and variance of negative and positive signals is used in this
calculation.
The Z’-factor must be >0.4 to be considered acceptable for use, with some
researchers preferring a Z’-factor of >0.6.
Interference Assays must be designed that consider the effects of compounds found in
the assay, such as the solvents used.
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8. High Throughput Screening
• HTS is a process to accelerate drug discovery, which involves a brute
force approach where tens of thousands of compounds (Compound
libraries) are tested against a particular target daily.
• This method is very valuable to early drug discovery.
• Compounds are being tested using a quantitative bioassay via the use
of automation, miniaturized assays, micro fluidic chips, sub nano litre
dispensing, fluorescence, large-scale data analysis.
• High-throughput screening methods are also used to characterize
metabolic, pharmacokinetic and toxicological data about new drugs.
• This HIT compound can be generated into LEAD compound.
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9. • High throughput screening (HTS) involves the screening of the entire
compound library directly against the drug target or in a more complex
assay system, such as a cell-based assay, whose activity is dependent
upon the target but which would then also require secondary assays to
confirm the site of action of compounds.
• Basically HTS is a process of screening and assaying large number of
biological modulators and effectors against selected and specific
targets.
Principle of High Throughput Screening:
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10. Methods which are commonly followed
are:
1) Target selection:
Currently there are about 500 targets being
used by companies.
cell membranes receptors, mostly G-protein
coupled receptors make up the largest group
(45% of the total), Enzymes make up the next
largest group (28%), followed by hormones
(11%), unknowns (7%), ion-channels (5%),
nuclear receptors (2%), and finally DNA
(2%).
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11. Cont.
2) HTS library to be screened:
i) they usually consist of microtiter plates with frozen or dried samples
of compounds to be screened.
ii) Initially the assays were carried out in 96-well plates but with
advancement now there are also 1586-well plates available.
iii) Typical HTS programs have potentials to screening up to 10000
compounds per day, while some laboratories with Ultra High
Throughput Screening (UHTS) can perform100,000 assays per day.
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12. Cont.
3) Assay design:
• Assays mainly divided into biochemical and cell based assays.
• Biochemical assay are further divided into two homogenous and
heterogenous assays.
3.1) Homogenous assay:
i) Measurement are based on the distinct physical/chemical properties of
analyte, or interaction between analyte and surrounding environment.
ii) It is a single step process; reagent may be added at single stage or in
multiple steps.
iii) It only involves usual steps like fluid addition, incubation and reading.
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13. Cont.
• It can be coupled with different detection technique fluorescence, radiometric
for HTS.
• Advantage:
i) Simplicity(Mix and read)
ii) Reduction of cost and robotic complexity.
3.2) Heterogenous assay:
i) Heterogeneous assays involves additional steps like filtration centrifugation.
that separates component(s) to be measured from the rest of component which
may interfere in assay.
ii) contributes to the high signal to background ratio.
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14. Biochemical assays:
i) Biochemical assays are receptor, protein or enzyme based assays uses
the particular target in a purified form.
ii) Biochemical assays are most frequently carried out using scintillation
proximity assay(SPA), radiometric, colorimetric fluorescence detection
techniques.
iii) Scintillation Proximity Assay is a technology whereby binding
reactions can be assayed without the washing or filtration procedures
normally used to separate bound from free fractions.
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15. Some techniques of biochemical assays are:
i) Fluorescence resonance energy
transfer(FRET):
a) In this process, donor fluorophore
absorbs the energy from incident light
and transfer this energy to nearby
acceptor molecule.
b) One frequent pair fluorophores is a
cyan fluorescent protein (CFP)- yellow
fluorescent protein(YFP) pair,
spectrally distinct variants of green
fluorescent protein. (a) Graph of FRET
(b) Jablonski diagram illustrating the FRET process.
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16. ii) Fluorescence polarization:
a) When fluorophore is irradiated with light, it
gets exited and if remain steady throughout
excitation state, it emits light in same
polarized plane.
b) While if it rotate and tumbles during
excitation state, it emits light in different
plane(depolarized).
c) Larger molecule shows little movement
while small molecule rotates quickly and
gives high and low polarization value
respectively FP is widely used in HTS.
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17. Cell Based Assays
• Cell-based assays for HTS can be classified under following classes:
1) Second Messenger Assay:
a) It monitors signal transduction from activated cell-surface receptors.
b) Second messenger assays typically measure fast, transient fluorescent
signals that occur in matter of seconds or milliseconds.
c) Many fluorescent molecules are known to respond to changes in
intracellular Calcium ion concentration, membrane potential and various
other parameters, hence they are used in development second messenger
assays for receptor stimulation and ion channel activation.
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18. Cont.
2) Reporter gene assays:
a) It monitors cellular responses at transcription/translation level.
b) It indicates the presence or absence of a gene product that in turn
reflects changes in signal transduction pathway.
c) The quantification of the reporter is usually carried out by
biochemical methods viz by measuring the enzymatic activity.
3) Cell proliferation assays:
a) It monitors overall growth/no growth responses of the cell to external
stimuli.
b) Quick and easy to be employed for automation.
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19. 4) Reagents in HTS:
• In any chemical synthesis or testing and screening, reagents play a
major role, HTS is no exception to this.
• Examples of reagents used are:
i) Aptamers
ii) Enzymes (e.q: Tyrosine kinase)
iii) Biotinylated Deoxy uridine Triphosphate
iv) Streptavidin- allophycocyanin
v) Dimethyl sulfoxide (DMSO)
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20. Automation and Robotics in HTS:
• The union of robotics and HTS has been important to achieve the
desired screening rates, as well as relieving scientific staff from
tedious work.
• Problems associated with screening robotics have included long design
and implementation time, long manual to automated method transfer
time, non-stable robotic operation, and limited error recovery abilities.
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• The presently used robot-
centric HTS systems have a
central robot with a gripper
that can pick and place
microplates around a
platform.
• They typically process
between 40 and 100
microplates in a single run
(the duration of the run
depends on the assay type).

22. Applications
• Selection of compounds from a vast number synthesized by
combinatorial chemistry and other methods.
• Efficient tool in studying biomolecular interactions and pathways.
• Highly efficient, fast, accurate and dependable in compound screening.
• Used in DNA sequencing.
• Useful in toxicology.
• Used to study drug-drug interactions.
• Useful in genotoxicity assays.
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23. References
• https://www.irbm.com/drug-discovery/hit-
identification/#:~:text=Hit%20identification%20is%20the%20most,ini
tial%20hits%20and%20hit%20series.
• https://www.technologynetworks.com/drug-discovery/articles/assay-
development-329953
• Hughes JP, et.al . Principles of early drug Discovery. British Journal of
Pharmacology 2011; (162) :1239–1249.
• Armstrong, J.W. A review of high-throughput screening approaches
for drug discovery. Am. Biotechnol. Lab 2018; 17: 26-28
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