ASSAY DEVELOPMENT FOR HIT IDENTIFICATION

Introduction
• A hit is a compound which has the desired activity in a
compound screen and whose activity is confirmed upon
retesting.

• Lead compounds are chemical compounds that show desired

biological or pharmacological activity and may initiate the
development of a new clinically relevant compound.
Process of hit identification:
Target
Compound
identification
libraries

Target
validation

Assay development

HTS

Lead optimization

Clinical trial
Hit identification:
• For hit identification, some information about either the target
protein or an active compound is necessary.

• In the case of a known structure of the protein, a virtual

screening of compound libraries leads to virtual hits which will
be synthesised and tested afterwards or tested immediately.

• If the structure of the natural substrate is known, a ligand

based approach will be accomplished.

• The computer searches for similar compounds and the

resulting hits are checked for drug like properties.
Hit Identification Assay Features

• Protein target can be membrane bound, difficult to

produce for standard biochemical assays, and
difficult to purify;
• Hit and lead compounds and focused libraries can be
screened against protein target within physiological
environment;
Cont..

• Compound cell permeability, specificity and

cytotoxicity are assessed in one process;
• Process can be upscaled to screen hundreds of
compounds in a few hours.

Hit Identification Assay Applications

• Target identification or validation for hit phenotypic
screening.
Assay development:
• Assay Development One of the first steps in drug
development and toxicity testing is creating test
systems (assays) on which to evaluate the effects of
chemical compounds on cellular, molecular or
biochemical processes of interest.

• In the recombinant era the majority of assays in

use within the industry rely upon the creation of
stable mammalian cell lines over-expressing the
target of interest,

• or upon the over-expression and purification of

recombinant protein to establish so-called
biochemical assays.
Cont..
• cell-based assays have been applied to target classes such as
membrane receptors, ion channels and nuclear receptors.

• In contrast, biochemical assays, which have been applied to

both receptor and enzyme targets, often simply measure
the affinity of the test compound for the target protein.

• The choice of assay format is dependent upon the biology

of the drug target protein, the equipment infrastructure in
the host laboratory, the experience of the scientists in that
laboratory, whether an inhibitor or activator molecule is
sought and the scale of the compound screen.
Cont…
• For example compound screening assays at
GPCRs have been configured to measure the
binding affinity of a radio- or fluorescently
labelled ligand to the receptor,
• to measure guanine nucleotide exchange at the
level of the G- protein,
• to measure compound-mediated changes in one of
a number of second messenger metabolites
including calcium, cAMP or inositiol phosphates.
 The assay format that is selected, it is a requirement that
the following factors are considered:
Points to consider
Isoform/splice variant
Total or modified (e.g. phosphorylated / acetylated/methylated)?
Soluble or membrane-bound?

Amount of molecule present?

Biological function?
Sample availability Volume of sample
Likely concentration of molecule Stability of molecule

Is semi-quantitative measurement of the molecule sufficient, or does the study require

rigorous quantitation?

10s, 100s, 1000s?

Sample-to-data streamlining Automation
Cont…
a) What molecule and parameter are to be measured?
• The starting point is to be absolutely clear as to what
molecule and precisely what property of that molecule is to
be measured.
• For example, does the researcher wish to measure the total
amount of a particular protein in a cell lysate or
• only the phosphorylated form, or both total and
phosphorylated protein ?
• In the case of a protein, whether the key parameter to
measure is the amount of the protein present or
• its biological function, such as enzymatic activity or the
effect
of a cytokine on potential cellular targets.
Cont…

b) Source of the molecule

• The source of the molecule will determine the sample
quantity and availability.

• It will also determine the concentration of the

molecule of
interest and may profoundly influence its stability.
Cont..
c) Quantitative versus semi-quantitative
• In order to develop an assay that is fit-for-purpose
it is important to decide at the outset whether a
semi-quantitative measurement of the molecule,
such as Western blot, meets the requirements of
the project or whether a rigorously quantitative
assay is required.
Cont…
d) Number of samples to be assayed
• It is also important to consider how many samples
will need to be assayed.
• If only a handful of samples will be assayed then a
labour- intensive, multi-step manual assay format
may be perfectly acceptable.
• Conversely, if thousands or tens-of-thousands of
assays are to be run, perhaps as part of a
compound profiling exercise, it will be important
to simplify, streamline and automate the assay
process as much as lab resources allow.
Assay fundamentals
Assay parameter Key considerations

Specificity Analysis of assay performance

Sensitivity Will the assay detect the levels of the

molecule in the samples of interest?
Dynamic range Will the levels of the molecule fall within
the dynamic range of the assay
Interference Will components in the assay sample
interfere with the assay?
Robustness Can the assay cope with small changes
in the assay
sample/equipment/operator?
Reproducibility Does the assay display low inter and
intra assay variability?
Accuracy (precision) Is the assay capable of accurately
determining the absolute
amount/concentration of the
molecule?
a) Specificity
• An absolutely key consideration is the specificity of the
assay.

• Having decided exactly what molecule and parameter of

that
molecule is to be measured,

• the researcher needs to establish that the assay will

measure only what they want it to measure and not
anything else.

b) Sensitivity
• This will be determined both by the levels of the molecule
of
interest in the sample and the volume of sample available.
Cont…
• This is an important consideration when deciding upon the
assay format and detection system.

• If great sensitivity is required then a fluorescence, rather than

absorbance, readout is more likely to give the desired
sensitivity.

c) Dynamic range
• It is important to determine the dynamic range of the assay.

• In other words the range over which the assay readout is

proportional to the amount of target molecule in the sample
being analysed.
Cont…
d) Reproducibility/robustness/accuracy
• The assay should be robust in that it is not unduly
affected by
changes in sample preparation and handling.

• The assay should be highly reproducible (also referred to

as precision) such that the degree of variation is as small as
possible both on an intra and inter assay basis.

• On a single assay run, replicates of both a standard and a

‘real’ sample should give very similar values, respectively.
Cont…
• If the assay is to measure the absolute (rather than relative)
amount of a molecule then it must be calibrated (see
above) against an accepted standard in order to give
accurate quantitation.
• The levels of robustness, reproducibility and accuracy that
are acceptable will depend on the purpose of the assay and
must be decided upon by the researcher for their own
particular current use.
High throughput screening:
• It is basically a process of screening and assaying
huge number of biological modulators and
effectors against selected and specific targets.

• The principles and methods of HTS find their

application for screening of combinatorial
chemistry, genomics, protein, and peptide
libraries.
Targets in HTS
Steps in HTS:
1) Assay plate preparation
• The testing vessel of HTS is the microtiter: a small
container, usually disposable and made of plastic, that
features a grid of small, open divots called wells.
• In general, modern (circa 2013) microplates for HTS
have either 384, 1536, or 3456 wells.
• Most of the wells contain test items, depending on the
nature
of the experiment.
• These could be different chemical compound dissolved e.g.
in an aqueous solution of dimethyl sulfoxide(DMSO).
• The wells could also contain cells or enzymes of some type.
(The other wells may be empty or contain pure solvent or
untreated samples, intended for use as experimental
controls.)
2) Reaction observation
• fills each well of the plate with some biological entity such
as a protein,cell or an animal embryo.
• After some incubation time, to allow the biological matter
to absorb, bind to, or
• otherwise react (or fail to react) with the compounds in the
wells, measurements are taken across all the plate's wells,
either manually or by a machine.
• Manual measurements are often necessary when the
researcher is using microscopy to (for example) seek
changes or defects in embryonic development caused by
the wells' compounds, looking for effects that a computer
could not easily determine by itself.
3) Quality control
Three important means of QC are :
i) good plate design,

ii) the selection of positive and negative

effective chemical/biological
controls, and
(iii) the development of effective QC metrics to measure the
degree of differentiation so that assays with inferior data
quality can be identified.
Types of HTS assay in drug discovery
Assay in drug
discovery

Biochemical Cell-based
assays assays

Target-based Enzymes Phenotype-based

(e.g. kinases, •Transcriptional read-outs
proteases) • Second messenger levels
Receptors (e.g. Nuclear •Protein interactions
receptors, Kinase Cell viability (cell
receptors,, GPCRs) death/apoptosis)
Hormones •Proliferation
Biochemical Assays
• Measure function of a purified target
• Identify compounds that modulate the activity of the
target protein
• Recombinant (engineered) proteins, proteins isolated
from crude cell lysates .
Examples:
Kinase/ATPase assays ,Protease assays and Protein
interaction assays
Cell-based Assays
• Provide a functional read-out of compound
activity
•Useful for follow up of biochemical assays.
Examples:
Cell proliferation: MTT etc. for oncology
Apoptosis: Caspase assays, TUNNEL
assays
GPCR activity: second messenger levels
(cAMP, Ca)
Motility/Migration: Bacterial viability
assays,
Biochemical versus Cell-based assays
Cell-based assays
Advantages:
Biochemical assay • More physiological
•Can simultaneously assay for
Advantages: compound characteristics such as
•Simple efficacy, toxicity, membrane
•More consistency permeability, off-target effects.
•High adaptability to HTS
•Increased specificity of Disadvantages:
compounds •Complex
Disadvantages: •High rate of noise and artifacts,
•May be non- variability
physiological •Less adaptable to HTS
•Not possible to •Exclusion of less
determine soluble/permeable compounds
compound
characteristics
Assay for kinase activity