BIOASSAY TECHNIQUES FOR
DRUG DISCOVERY AND
DEVELOPMENT
Dr. Muhammad Iqbal Choudhary
Distinguished National Professor
International Center for Chemical and Biological Sciences
(H. E. J. Research Institute of Chemistry
Dr. Panjwani Center for Molecular Medicine and Drug Research)
University of Karachi, Karachi-75270
CONTENT
Molecular basis of diseases
Stages in drug development
Why Bioassays?
Different types/classes of bioassays
Difference
between
bioassay
and
pharmacological screenings?
Various types of bioassays?
High-throughput
bioassays-Definitions,
advantages and disadvantages
Bioactivity
directed isolation of natural
products- Strategies
Bioassay-guided fractionation (BGF) and
isolation

Drug DiscoveryPast and Present
In the past, most drugs were either
discovered by trial and error (traditional
remedies) or by serendipitous discoveries.

Today efforts are made to understand the
molecular basis of different diseases and
then to use this knowledge to design and
develop specific drugs.

In modern drug discovery process, bioassay
screenings play an extremely important role.

What is Required to Develop a Modern
Drug (NME)?
• Decision= Corporate decision to invest in
specific therapeutic area, based on “economic
feasibility”
• Cost= $ 1.4 billion- 1.8 billion
• Duration= 10-12 years of R&D, and regulatory
approval
• People=
600-800
scientists
of
multidisciplinary expertise
• Chemical Diversity: Screening of 100,000200,000 compounds
• Global Approval= Lots of paper works, based
on often ill-planned studies, and malpractices
A Book Worth Reading

Bioassay Techniques for Drug
Research
By
Atta-ur-Rahman, M. Iqbal Choudhary
and William J. Thomsen
Harwood Academic Press, London
http://nadjeeb.wordpress.com/2009/
05/9058230511.pdf
Diseases- Molecular Basis
Overwhelming majority of diseases are caused by change in
biochemistry and molecular genetics of human body
(Molecular Pathology)
 Over- and under-expression of catalytic proteins (enzymes)
 Toxins produced by microorganisms
 Viruses (wild DNA/molecular organisms) cause cancers, AIDS,
influenza, Dengue fever, etc.
 Mutation in DNA cause cancers
 Malfunction of signaling pathways cause various disorders
 Congenital diseases due to genetic malfunctions
 Oxidation of biomolecules (proteins, carbohydrates, lipids, nucleic
acid), degenerative diseases and ageing
 Deficiency of essential elements, vitamin, nutrients, etc.
I
Courtesy of Prof. Dr. Azad Khan
Main Stages in Drug
Discovery and Development
Selection of Disease Target/Designing
of Bioassay

Discovery and Optimization of Lead
Molecules


Preclinical Studies

Clinical Studies
Why we Need to Perform
Bioassay?
To predict some type of therapeutic potential,
either directly or by analogy, of test compounds.
Bioassay is a shorthand commonly used term
for biological assay and is usually a type of in
vitro experiments

Bioassays are typically conducted to measure
the effects of a substance on a living organism
or other living samples.

What is Bioassay?
Bioassay
or
biological
assay/screening is any qualitative
or quantitative analysis of a
substances that uses a living
system, such as an intact cell, as a
component.

Essential Components of
Bioassays/Assays
Stimulus
(Test
sample,
drug
candidate, potential agrochemical, etc)

Subject (Animal, Tissues, Cells, Subcellular orgenlles, Biochemicals, etc.)

Response (Response of the subject to
various doses of stimulus)

Molecular Bank at the PCMD
Over 12,500 compounds, and 6,000 Plant Extracts
Bioassays/Assays
Whole animals
Isolated organs of vertebrates
Lower organisms e.g. fungi, bacteria,
insects, molluscs, lower plants, etc.
Cultured cells such as cancer cells and
tissues of human or animal organs
Isolated sub-cellular systems, such as
enzymes, receptors, etc

Types of Bioassays?
In Silico Screenings
Non- physiological Assays
Biochemical or Mechanisms-Based Assays
In Vitro Assays
 Assays on Sub-cellular Organelles
Cell based Bioassays
Ex-Vivo Assays
Tissue based Bioassays
NMR Based Drug Discovery
In Vivo Bioassays
•Animal-based Assays/Preclinical Studies
•Human trial/Clinical Trials
Predicting Drug Like BehaviorLipinski “Rule of Five”

Molecular weight about 500 a. m. u.
(Optimum 350)

Number of hydrogen bond accepter ~ 10
(Optimum 5)

Number of hydrogen bond donor ~ 5
(Optimum 2)

Number of rotatable bonds ~5
(Conformational Flexibility)

1-Octanol/water partition coefficient between
2-4 range
Broad Categories of
Bioassays
Virtual Screenings
Primary Bioassays
Secondary Bioassays
Preclinical Trials
Clinical Trials

Virtual and In Silico
Screenings







Ligand based or Target based
Target Selection
Data Mining (Chemical space of over 1060
conceivable compounds)
Screening of Libraries of Compounds
Virtually
Lead Optimization
Prediction of Structure-Activity
Relationships
It Save, Time, Money and Efforts
Primary “Bioassay/Assays”
Screenings
Non- physiological Assays
Biochemical or Mechanism-Based
Assays
Microorganism-based bioassays
Cell-based Bioassays
Tissue-based Bioassays
Many other In Vitro bioassays/assays

Examples of Primary Assays
Antioxidant Assays
Enzyme Inhibition Assays
Cytotoxicty Bioassays
Anti-cancer Bioassays (Cancer Cell Lines)
Brine Shrimp Lethality Bioassays
In Vitro Antiparasitic Bioassays
Anti-bacterial Bioassays
Antifungal Bioassays
Insecticidal Bioassays
Phytotoxicity Bioassays
Etc.

Salient Features of Primary
Bioassay Screenings
Predictive Potential
General in nature
Tolerant of impurities
Unbiased
High-throughput
Reproducible
Fast
Cost-effective
Compatible with DMSO

Hit Rate of Primary Bioassay
Screenings
A hit rate of 1% or less is generally
considered a reasonable
False positive are acceptable
False negative are discouraged

Secondary Bioassays
Animal-based assays (In Vivo)
Toxicological Assessments in whole
animals
ADME Studies
Behavioral Studies
Preclinical Studies

Importance of Standards in
Bioassays/Assays


The results of the assay/bioassay need to
validated by monitoring the effect of an
available known compound (Standard).
Without judicious choice of standard and
its reproducible results in an assay
system, no screening can be claimed
credible.
Importance of Reproducibility
and Dose Dependency


Without reproducible results (within the
margin of error or esd), an assay has any
value. It is a share loss of time and efforts.
Dose dependency is the key to a successful
outcome of study.
Without reproducibility and dose dependency,
it can be magic, but not science
VINBLASTINE- A Novel Anticancer
Drug from Flowers of Sada Bahar
In Vitro Bioassays
In Vitro: In experimental situation
outside the organisms. Biological or
chemical work done in the test tube( in
vitro is Latin for “in glass”) rather than
in living systems
Examples
include
antifungal,
antibacterial,
organ-based
assays,
cellular assays, etc

Examples of In Vitro Bioassays

Activity Assays
•DPPH assay
•Xanthine oxidase inhibition assays
•Superoxide scavenging assay
•Antiglycation assay

Bioassays (cell-based)
•DNA Level
•Protein Level
•RNA Level
•Immunology assay

Toxicity Assays
•MTT assay
•Cancer cell line assays
In Vivo Screenings or
Pharmacological Screenings
In Vivo: Test performed in a living
system such as antidiabetic assays,
CNS assays, antihypertensive assays,
etc.

Examples of In Vivo Bioassays

Animal Toxicity
•Acute toxicity
•Chronic toxicity

Animals Study
•Animal model with induced disease
•Animal model with induced injury


Pre-Clinical Trials
Clinical Trials
High-throughput Assays
The process of finding a new drug
against a chosen target for a particular
disease
usually
involves
highthroughput screening (HTS), wherein
large libraries of chemicals are tested
for their ability to modify the target.

HIGH-THROUGHPUT BIOLOGICAL
SCREENINGS




96-384 Well plates (medium throughput) and
more (high-throughput).
Development of straight-forward in-vitro
biological assays (enzyme-based, cellular and
microbiological assays) into automated highthroughput screens (HTS).
Rapid assays of thousands or hundreds of
thousands of compounds (upto 200,000 samples
per day).
Specifically suitable for the isolation of bioactive
constituents from complex plant extracts or
complex combinatorial library.
High-throughput Screening Strategy
for Enzyme Inhibition Assays
Enzyme + Buffer
+ Potential inhibitor
% Inhibition = [(E-S)/E]  100
E = Activity of enzyme without test material
S = Activity of enzyme with test material
Substrate
Incubation
Measurement of absorbance
96-well plate
12
SOME EXAMPLES OF ASSAYS
AT THE ICCBS
Examples of Primary Assays
•
Antioxidant Assays
Enzyme Inhibition Assays
Cytotoxicty Bioassays
Anti-cancer Bioassays (Cancer Cell Lines)
Brine Shrimp Lethality Bioassays
In Vitro Antiparasitic Bioassays
Anti-bacterial Bioassays
Antifungal Bioassays
Insecticidal Bioassays
Phytotoxicity Bioassays
•
Etc.
•
•
•
•
•
•
•
•
•
Examples of In Vivo Assays



Metabolic Disorders (Diabetes, IGT, etc)
Cardiovascular
CNS Assays (Anti-depressant. Antianxiety, Anti-epilepsy, memory, etc)





Anti cancer
Drug Metabolism
Anti-parasitic
Anti-obesity
Toxicity
Enzyme Inhibition- Key Tool in Drug
Development
A wide range of diseases are enzyme related.
More than 30% of the drugs in clinical use are
enzyme inhibitors.
Many pesticides and insecticides (chemical
weapons!) also work as enzyme inhibitors in the
target organisms.
Plants and other living sources, as well as medicinal
chemistry can provide novel and potent enzyme
inhibitors.
Medium-throughput Screening Strategy
for Enzyme Inhibition Assays
Enzyme + Buffer
+ Potential inhibitor
% Inhibition = [(E-S)/E]  100
E = Activity of enzyme without test material
S = Activity of enzyme with test material
Substrate
Incubation
Measurement of absorbance
96-well plate
12
Example: Urease Inhibition
•
•
Urease catalyzes the hydrolysis of urea into carbon
dioxide and ammonia.
The reaction occurs as follows:
(NH2)2CO+ H2O →CO2 + 2NH3
•
Ammonia in water forms ammonium hydroxide, a base.
•
Urease inhibition is a successful approach towards the
treatment of diseases caused by ureolytic bacteria.
43
43
Inhibition of Urease- Inhibitors Type
• Substrate Like Inhibitors: Inhibitors which
bind in a substrate or active-site directed
mode.
• Mechanism Based Inhibitors: Inhibitors
which bind in a non-substrate like manner
or in mechanism-based directed mode
44
44
Urea Derivatives- Novel Urease Inhibitors
IC50 = 1.25±0.021 µM
Substrate like inhibition mechanism -structurally similar to
the natural substrate urea.
Standard: Thiourea
IC50 (Jack bean) = 21±0.11 µM
Letters in Drug Design & Discovery, Volume 5, Number 6,
September 2008, pp. 401-405(5)45
45
Substrate Like –Novel Urease Inhibitors
Thioureas
IC50 = 15.03±0.02 µM
1,2,4-Triazole-3-thiones
IC50 = 16.7±0.178 µM
Oxadiazoles
IC50 = 16.1±0.12 µM
Dihydropyrimidine
IC50 = 5.36±0.027 µM
Triazoles
IC50 = 10.66±0.16 µM
46
Standard (Thiourea) IC50 = 21 ± 0.11 µM
Glycation
Occurs in everyone, but at a faster rate in
diabetics
AGEs formation effect the molecular functioning
of the body and cause various diseases
Activate RAGEs (Receptors of AGEs) which
contribute in triggering a number of diseasecausing inflammatory response
Prevention of Non-enzymatic Glycation



Inhibition of AGEs formation can lead
to the prevention of diabetic
complications by suppressing or
delaying the formation of AGEs .
Various inhibitors have been discovered,
such as Aminogunadine, Aspirin, Rutin,
Antioxidants, AGE breakers, etc.
Aminoguanidine (AG), a potent AGEs
inhibitor also underwent the clinical
trials.Idealy AGEs inhbitors should be
able to reverse the process of
glycation, and repair the damage.
In Vitro Assay for Inhibition of Protein Glycation
Inhibitor (1 mM)
+
HSA (10mg/mL)
+
Fructose (500
mM)
+
Sodium
Phosphate Buffer
Activity was monitored
at Excitation: 330 nm
Emission: 440 nm.
Incubation 7 days at 37° C
Anti-glycation Activity of Some Natural
Compounds (Flavonoid glycoside)
Plant Name Tagetus patula
HO
OH
O
HO
HO
O
OH
O
OH
OH
H3CO
OH
O
IC50 58.02 ± 0.813 
Rutin IC50 = 294.50 
Benzimidazoles
3-(6-Nitro-1H-benzimidazol-2-yl)1,2-benzenediol
2- (6-Nitro-1H-benzimidazol-2-yl)1,4-benzenediol
IC50 17.7± 0.001 µM
IC50 48.7± 0.006 µM
Rutin IC50 = 70 ± 0.5 µM
Oxidation and Human Health
One of the paradoxes of life on this
planet is that the molecule that
sustain aerobic life, oxygen, is not
only fundamentally essential for
energy metabolism and respiration,
but implicated in many diseases
and degenerative conditions.
Marx, Science, 235, 529-531
(1985).
Methods Used to Determine Antioxidant
Potential
DPPH Radical Scavenging Assay


For quantitative determination of electron
donation.
The molecule of 1, 1-diphenyl-2picrylhydrazyl (DPPH) is a long-lived organic
nitrogen radical.
53
Principle
•The delocalization gives rise to the deep violet color.
•Characteristic absorption band in ethanol solution at
515 nm.
•When a solution of DPPH is mixed with that of a substance (RH)
which can donate a hydrogen atom, then pale yellow reduced form
of DPPH is formed.
54
Protocol
Pre read at
515nm
Sample in DMSO
Normal read at
515nm
DPPH in Ethanol
(300 μM)
Micro plate reader
(Spectra MAX-340)
55
Superoxide Anion Scavenging Assay
The assay involves a non-enzymatic generation of
superoxide anion radicals. The superoxide anion
scavenging activity was determined by measuring the
reduction in rate of formation of blue colored
formazan dye which absorbs at 560 nm.
A sample with antioxidant potential scavenge the
super oxide anion radicals and eventually reduces the
rate of formation of formazan dye, which can be
monitored by means of decrease in the absorbance.
56
Superoxide Anion Scavenging Assay
57
Superoxide Anion Scavenging Assay
58
Flavones
Studied in DPPH
BHA= 44.0±2.70
PG= 30.0±0.27
IC50 M= 40.26±1.04
Source: Whole plant of Iris tenuifolia Pall.
IC50 M= 159.153±4.49
IC50 M= >500
Source: Whole plant of Iris unguicularis
59
Isolated by Dr. Sumaira Hareem
Studied at 500 M in DPPH and SO
BHA= 44.0±2.70 /97.0±3.0
PG= 30.0±0.27 /104.0±2.4
DPPH= Inactive
Superoxide anion= 40.35 M ±1.87
DPPH= Inactive
Superoxide anion= 97.99 M ±2.65
DPPH= Inactive
Superoxide anion= 30.98 M ±1.65
60
Xanthone glycoside
Studied at 500 M in DPPH
BHA= 44.0±2.70
PG= 30.0±0.27
Source: Iris unguicularis
Isolated by: Dr. Sumaira Hareem
Studied in DPPH
BHA= 44.0±2.70
PG= 30.0±0.27
IC50 M= 22.45 ± 0.35
61
NMR-BASED SCREENING IN
DRUG DISCOVERY
NMR-A Versatile Tool in Drug
Discovery
Ligand
Binding
Structure
Folding
Unfolding
NMR
Metabolic
Profiling
Dynamics
ON-LINE ISOLATION AND BIOASSAY
SCREENING
UV/VIS DETECTOR
(Photodiode Array Detector)
Sample
CHROMATOGRAPHIC
METHODS
FRACTION COLLECTOR
ON-LINE SPECTROMETERS
SPECTRAL AND
STRUCTURAL
DATABASES
Dictionary of Natural Products,
Bioactive Natural Products
Database, DEREP,
NAPRALERT, MARINLIT,
Marine Natural Products
Database, STN Files
3/14/2016
-NMR
-MASS
-IR
-ICP
96-well plates
or
384-well microplate
SPLITER
BIOASSAYS
Fragment Based Drug
Discovery
Thrombin Inhibitor
HIV Protease
Inhibitor
Fragment Based Drug
Discovery
C. Acetylcholinesterase Inhibitor
Substrate Binding Specificity
Geometric
Complementarity
Electronic (electrostatic)
Complementarity
“Induced fit” vs.
“Lock & Key”
Stereospecific (enzymes
and substrates are chiral)

NMR for Drug Research
1.
Detect
the
interactions
binding constants.
2.
Enables a
constants.
4.
Allows
deconvolution
sources or
weakest
ligand–target
even
with
millimolar
determination
of
binding
direct
screening
and
of
mixtures from natural
combinatorial chemistry.
5.
Provide structural information for both
target and ligand with atomic resolution.
NMR for Drug Research



Fragment based discovery
Target identification
Lead optimization
NMR for Drug Research
•Promising new method in drug
discovery
•Unmatched screening sensitivity.
•Abundance of information about
the structure and nature of
molecular
interaction
and
recognition.
Basic Development of NMR
Spectroscopy for Drug Research
• Cryoprobe technology which increase
signal-to-noise
ratio
and
lower
accessible binging affinities.
•Flow probe alleviating the need for NMR
tubes and time-consuming handling.
•Micro-coil tubes (micro- and nanoprobes) reduce the required sample
volumes and also superior Rf field
homogeneity. Thus facilitating difference
based NMR screening methods.
FRAGMENT-BASED DRUG
DISCOVERY
•Target- or Receptor-Based Screening- Does ligand
interact with the target by following the changes in
the chemical shifts of target protons?. It observe
and compare the chemical shifts of targets in the
absence and presence of ligand
• Ligand-Based-Screening- Does ligand is
interacting with the target by following the changes
in the NMR parameters of ligand after the addition of
the target
Receptor Based Screening by
Chemical Shift Mapping


Identification of high affinity ligands
by mapping the chemical shifts
changes in the receptor spectrum
(1H-15N- HSQC)
Require more quantities of receptor
(proteins)
RECEPTOR-BASED SCREENING FOR
DRUG DISCOVERY
Receptor Based HSQC/HMQC

2D [1H, 15N] or [1H,
presence of ligand.
13C]-HSQC

The affinity constant between the ligand and the target can be
accurately measured by determining the chemical shift changes
as a function of ligand concentration.

[1H, 15N]-HSQC experiment use to monitor changes in the amide
protons and nitrogen nuclei of the backbone and Asn and Gln
side chains (it requires the protein sample to be enriched in 15N).

[1H, 13C]-HSQC experiment gives information about the chemical
shift changes in all side chains.

Drug-discovery programs usually deals with very large proteins.
Using traditional method very long correlation time of protein
(MW >30 kDa) causes their NMR resonances to be too wide to be
detected.
are used in the absence and
2D [1H–15N]-HSQC Experiment
(Chemical shift perturbation method)
1H
(ppm)
The black contours correspond to FKBP (family of enzymes
that
function
as
protein
folding
cheprons),
the
macromolecular target, whereas the red contours.
correspond to the complex formed by FKBP and
Structure-Activity-Relationship
(SAR) by NMR
•Identification of ligands with high binding affinity
from library of compounds by using 2D 1H-15N- HSQC
• Optimization of ligands by chemical modification
•Identification of ligand (optimized) binding by again
recoding 2D 1H-15N- HSQC
•Re-optimization of ligand by chemical modifications
•Lining two ligands with appropriate linkers and
checking the affinity again
SAR by NMR
SAR by
NMR
Use of the SAR by NMR
approach for the discovery of
inhibitors of Stromelysins (matrix
metaloproteineases).
Pre-clinical Trials
Involve in vivo (test tube) and in vivo
(animal) experiments using wide-ranging
doses of the study drug to obtain preliminary
efficacy,
toxicity
and
pharmacokinetics
information.

Assist pharmaceutical companies to decide
whether a drug candidate has scientific merit
for further development as an investigational
new drug.

Clinical Trials
Human Trial/Clinical Trials
Phase I (Safety 20-80 Volunteers)
Phase II (Efficacy/Safety 100-300 patients)
Phase
III
(Efficacy/Safety
300-3000
patients)
Phase IV (Post Approval/Marketing Studies)

Randomized, Double-blind, Placebo
VARIOUS STAGES IN DRUG
DEVELOPMENT
LEAD - Identification
Target
Identification
and Validation
Primary
Bioassay
Secondary
Bioassay
Chemical
Diversity
Selected
Chemical
In silico
Screening
LEAD -Validation
Toxicity
Assay
In vivo
Assay
LEAD -Development
Animal
Trials
Animal
Trials
Structure
elucidation
of Bioactive
compounds
Structure
Activity
Relation
DRUG-Development
Post
Marketing
Survelience
Registration
and
Marketing
Clinical
Trials
1, II, III
Pre-chinical
Trials
BIOASSAY-GUIDED
FRACTIONATION (BGF)



Bioassay-guided fractionation (BGF) of Isolation
is the process in which natural product extract
or
mixtures
of
synthetic
products
is
chromatographically
fractionated
and
refractionated until a pure biologically active
constituent(s) is isolated.
At every stage of chromatographic separation,
every fraction is subjected to a specific bioassay
to identify the most active fraction(s).
Only those fraction(s) which are active are
further processed.
Thank You Very Much