ASSAY DEVELOPMENT IN

DRUG DISCOVERY
 The Drug Discovery Process: TheAssay Development
Stage Chemistry
Structure-Activity Pre-
“Hit” Relationship (SAR) clinical
validatio bioavailability (PK, GLP-Tox
n ADME),
toxicity Clinical
In vivo efficacy

Assay Lead Lead

Target Target
HT Identification Validatio Development
HTS Identification Optimization Development

S n

Assay development
Primary assays
Secondary assays

Target Target Assay Lead

Non- Identificati Validatio Developm Optimizatio Development
on n ent n
HTS
 Assay development: A critical part of the “hit”
discovery process

“ HITS”
A “hit” is a compound which has the desired
activity in a compound screen and whose
activity is confirmed upon retesting
 Assay
Development

Why is this a
bottleneck?
Assay Development=months
HTS=weeks

Roadblocks to faster assay

development
 Assay Development
The first steps in drug development are the
identification and validation of potential drug
targets involved in human disease. These targets
are typically either a cellular structure or specific
protein. Targets include:

•          Receptors
•         Enzymes
•         Hormones and factors
•         Nuclear receptors
•         DNA
•         Ion channels
Target-in Drug Discovery

 Enzymes:
• Kinases
Receptor Tyrosine Kinase
Non-receptor tyrosine
kinase
Serine Threonine kinase
• Phosphatases
•Proteases:
Serine proteases
Zinc proteases
 Receptors:
Ion channel receptors
GPCRs(G-Protein
Coupled recepterc)
Nuclear receptors
 Assay Development

 Target Assay HT
identified and Development S
Validated

Assays are investigative procedures that

qualitatively assess a compound or examine
a compound’s effects on identified molecular,
cellular, or biochemical targets
 Assay Development
In the 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. 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.
 Assay Development
Assay types and technologies
Assays in drug discovery fall into two main categories: biochemical
assays and cell-based assays. Biochemical assays are often the first
type of assay used.
• Biochemical assays are valuable for evaluating and examining
the target protein and identifying the compounds that possess
the desired activity at the target
•  Can be applied to enzyme or receptor targets
• are consistent and reliable
• are simpler than cell-based assays
Cell-based assays elucidate compound activity via a functional read-
out of said activity. Cell-based assays often follow biochemical
assays. These assays:(assays that take place within a living cell can
be grouped into cell profilation,cell deats cell signal

  Can be applied to ion channels, nuclear receptors or membrane

receptors
•  Report on the toxicity, efficacy and other properties of the hit
compound
•  Are more complex than biochemical assays
 Types of
Assays
Assays in Drug
Discovery
Biochemical assays Cell-based assays
Target-based Phenotype-based
 Measure function of a purified target  Measure function of the target in
Activity assays: Enzymes (e.g. kinases, proteases) the context of the cell
Binding assays: Receptors -Transcriptional read-outs, second messenger levels,
(e.g. Nuclear receptors, Kinase receptors, ion cell viability (cell death/apoptosis), proliferation
channels, GPCRs)
 Measure expression of the target
Identify compounds that modulate activity / mRNA levels, protein expression and localization
binding of the target protein
 Provide a functional read-out of
Recombinant (engineered) proteins, proteins isolated compound activity (as a functional consequence
from crude cell lysates of target engagement)
Monitor a surrogate read-out
 Examples: reporter assays, viability assays,
 Examples: Kinase/ATPase assays, GPCR and ion channel assays, qPCR
protease assays, protein interaction
assays
 Biochemical versus Cell-based
assays
Biochemical assays Cell-based assays
Advantages:
-Simple  Advantages:
-More consistency -More physiological, amenable to systems approach
-Direct measurement of target engagement -Can simultaneously assay for compound properties
-Can measure compound (membrane permeability, toxicity, off-target effects)
characteristics such as Kd, Ki etc.
-Increased specificity of compounds
Disadvantages:
-May be non-physiological  Disadvantages:
-Not possible to determine compound -Complex
properties such as membrane -High rate of noise
permeability, toxicity, off-target effects
-Exclusion of less soluble/permeable compounds
 Causes of Assay
Variation
Biochemical Assays Cell Based Assays
Same as for biochemical
assays Plus:
• pH
• Temperature • Cell culture plastics
• Ion concentration • Culture media
• Reagent Solubility • Culture conditions
• Reagent Stability • Serum
• Reagent Aggregation • Cell cycle
• Order of reagent • Passage number
addition
• Instrumentation
 Assay Development

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 although in recent years there has been an
increase in the number of reports describing the use of primary
cell systems for compound screening..
 Assay Devolpment

Generally, cell-based assays have been applied to target

classes such as membrane receptors, ion channels and
nuclear receptors and typically generate a functional read-
out as a consequence of compound activity 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
relative merits of biochemical and cell-based assays have
been debated extensively and have been reviewed elsewhere
(). Both assay paradigms have been used successfully to
identify hit and candidate molecules
 Assay Development

A plethora of assay formats have been enabled to

support compound screening. 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.
 Assay Devolpment

• For example compound screening assays at GPCRs have been

configured
• To measurethe 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 or
• To measure the activation of downstream reporter genes.
 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.
 Parametrs

Source of the molecule

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

• It will alsodetermine the

concentration of the molecule
of
interest and may profoundly influence its stability.
 Parametrs
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.
 Parametrs
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

Factors
Key Considerations in Assay
Development
The three “Rs”
-Relevance
-Robustness
-Reliability/Reproducibility

Practicality/Feasibility

Cost

Automati

on
 Assay Development factors
Whatever the assay format that is selected, it is a requirement that the
following factors are considered :
1.Pharmacological relevance of the assay. If available, studies should be
performed using known ligands with activity at the target under study,
to determine if the assay pharmacology is predictive of the disease state
and to show that the assay is capable of identifying compounds with the
desired potency and mechanism of action.

2.Reproducibility of the assay. Within a compound screening

environment it is a requirement that the assay is reproducible across
assay plates, across screen days and, within a programme that may run
for several years, across the duration of the entire drug discovery
programme.
 Assay Development factors

3 Assay costs: Compound screening assays are typically

performed in microtitre plates. Within academia or for
relatively small numbers of compounds assays are typically
formatted in 96-well or 384-well microtitre plates whereas in
industry or in HTS applications assays are formatted in 384-well
or 1536-well microtire plates in assay volumes as small as a few
microlitires. In each case assay reagents and assay volumes are
selected to minimize the costs of the assay.
 Assay Development factors

4 Assay quality. Assay quality is typically determined according to the Z'

factor This is a statistical parameter that in addition to considering the signal
window in the assay also considers the variance around both the high and low
signals in the assay. The Z factor has become the industry standard means of
measuring assay quality on a plate bases. The Z factor has a range of 0 to 1;
an assay with a Z factor of greater than 0.4 is considered appropriately robust
for compound screening although many groups prefer to work with assays
with a Z factor of greater than 0.6.
In addition to the Z factor assay quality is also monitored through the
inclusion of pharmacological controls within each assay. Assays are deemed
acceptable if the pharmacology of the standard compound(s) falls within
predefined limits.
Assay quality is affected by many factors. Generally, high-quality assays are
created through implementing simple assay protocols with few steps,
minimizing wash steps or plate to plate reagent transfers within the assay,
through the use of stable reagents and biologicals, and through ensuring that
all the instrumentation used to perform the assay is performing optimally.
Assay Development factors
 Assay Development factors

5Effects of compounds in the assay. Chemical libraries are typically stored

in organic solvents such as ethanol or dimethyl sulphoxide (DMSO).
Thus, assays need to be configured that are not sensitive to the
concentrations of solvents used in the assay. Typically, cell-based
assays are intolerant to solvent concentrations of greater than 1%
DMSO whereas biochemical assays can be performed in solvent
concentrations of up to 10% DMSO. Studies are also performed to
establish the false negative and false positive hit rates in the assay. If
these are unacceptably high then the assay will need to be
reconfigured. Finally some consideration should be made to the
screening concentration. Compound screening assays for hit
discovery are typically run at 1–10 µM compound concentration. At
these concentrations compounds with activities of up to 40 µM can be
identified. The test concentration can be varied to identify
compounds with higher or lower activity.
 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,
the selection of effective chemical/biological controls,
and positive and negative

(iii) the development of effective QC metrics to measure the

degree of differentiation so that assays with inferior data
quality can be identified.
 Common Assay
formats
• Fluorescence
• Luminescenc
e
 Fluorescence-based
assays
 Based on excitation of a fluorophore 1 to 10 nano
 Variety of assays using fluorescence secs

- Simple assays where protein of interest is conjugated

to fluorophore, or where the protein of interest
generates a fluorescent product
- Reporter assays

- Advantages: High sensitivity, ease of set- up and

operation
- Disadvantages: Prone to false positives due to
auto fluorescence of compound

Examples:
- FRET, TR-FRET
- Fluorescence polarization
(FP)
 Luminescence-based
Example:
assays Luciferase reporter
assay:
• Chemical reaction produces light
• Bioluminescence is the
production and emission of light
by a living organism (e.g.
luciferase by firefly)

Luciferas
Luciferin + ATP e Luciferyl
adenylate+PPi
Luciferyl + O2 Oxyluciferin +AMP +Light
Adenylate
Examples of biochemical
assays
 Assay for kinase activity

ADP Hunter
assay

Fluorescent
product
Kinase target
Substrate Phosphorylated Substrate+ ADP

Inhibitors

Kinase target Fluorescent

Substrate Phosphorylated Substrate+ ADP product

Reduced signal
Examples of cell-based
assays
 Cell based
• Proliferation
assays
Cell Proliferation
Fluorescent dyes (non-specific dye)

• Viability
-Assays that measure ATP Influenza CPE assay - Preliminary CV Plate

content (Cell Titer Glo), viral CPE 140000

(bioluminescence-based)
120000

100000 Cell viability

-Apoptosis assays 80000 (CPE assay)
(bioluminescence/fluorescence) 60000

• Migration
40000

20000 1

-Scratch assay
8
0
15
S

S5

S7
1 S9
S
1

S15
3

S1

S1

S19
S1

S2
3

S2
7

3
• Reporter Gene
Cell migration
assays
“scratch assay”
-Transcriptional
activity/expression
Assay development: From the bench to
HTS
When you
From go To
This This

THE RULES
CHANGE