Title: BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT
1BIOASSAY 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
2Drug Discovery-Past 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.
3What 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 RD, and regulatory
approval - People 600-800 scientists of multi-disciplinary
expertise - Chemical Diversity Screening of 100,000- 200,000
compounds - Global Approval Lots of paper works, based on
often ill-planned studies, and malpractices
4CONTENT
- 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
5A 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.p
df
6Diseases- 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.
7 Courtesy of Prof. Dr. Azad Khan
I
8Main Stages in Drug Discovery and Development
- Selection of Disease Target/Designing of
Bioassay -
- Discovery and Optimization of Lead Molecules
- Preclinical Studies
- Clinical Studies
9Why 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.
10What 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.
11 Essential Components of Bioassays/Assays
- Stimulus (Test sample, drug candidate, potential
agrochemical, etc) - Subject (Animal, Tissues, Cells, Sub-cellular
orgenlles, Biochemicals, etc.) - Response (Response of the subject to various
doses of stimulus)
12Molecular Bank at the PCMD Over 11,500 compounds,
and 6,000 Plant Extracts
13 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
14Types 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
15Predicting Drug Like Behavior- Lipinski 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
16Broad Categories of Bioassays
- Virtual Screenings
- Primary Bioassays
- Secondary Bioassays
- Preclinical Trials
- Clinical Trials
17Virtual 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
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19Primary 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
20Examples 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.
21Salient Features of Primary Bioassay Screenings
- Predictive Potential
- General in nature
- Tolerant of impurities
- Unbiased
- High-throughput
- Reproducible
- Fast
- Cost-effective
- Compatible with DMSO
22Hit 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
23Secondary Bioassays
- Animal-based assays (In Vivo)
- Toxicological Assessments in whole animals
- ADME Studies
- Behavioral Studies
- Preclinical Studies
24Importance 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.
25Importance 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
26VINBLASTINE- A Novel Anticancer Drug from Flowers
of Sada Bahar
27In 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
28Examples 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
29In Vivo Screenings or Pharmacological Screenings
- In Vivo Test performed in a living system such
as antidiabetic assays, CNS assays,
antihypertensive assays, etc.
30Examples 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
31High-throughput Assays
- The process of finding a new drug against a
chosen target for a particular disease usually
involves high-through screening (HTS), wherein
large libraries of chemicals are tested for their
ability to modify the target.
32HIGH-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
high-throughput 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.
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35High-throughput Screening Strategy for Enzyme
Inhibition Assays
Inhibition (E-S)/E ? 100 E Activity of
enzyme without test material S Activity of
enzyme with test material
Enzyme Buffer Potential inhibitor
Substrate
Incubation
Measurement of absorbance
96-well plate
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38NMR-BASED SCREENING IN DRUG DISCOVERY
39NMR-A Versatile Tool in Drug Discovery
40ON-LINE ISOLATION AND BIOASSAY SCREENING
UV/VIS DETECTOR (Photodiode Array Detector)
CHROMATOGRAPHIC METHODS
Sample
FRACTION COLLECTOR
ON-LINE SPECTROMETERS
-NMR -MASS -IR -ICP
SPECTRAL AND STRUCTURAL DATABASES Dictionary of
Natural Products, Bioactive Natural Products
Database, DEREP, NAPRALERT, MARINLIT, Marine
Natural Products Database, STN Files
96-well plates or 384-well microplate
SPLITER
BIOASSAYS
41Fragment Based Drug Discovery
Thrombin Inhibitor
HIV Protease Inhibitor
42Fragment Based Drug Discovery
C. Acetylcholinesterase Inhibitor
43Substrate Binding Specificity
- Geometric Complementarity
- Electronic (electrostatic) Complementarity
- Induced fit vs. Lock Key
- Stereospecific (enzymes and substrates are chiral)
44NMR for Drug Research
1. Detect the weakest ligandtarget
interactions even millimolar binding
constants. 2. Enables a determination of
binding constants. 4. Allows direct screening
and deconvolution of mixtures from natural
sources or combinatorial chemistry. 5.
Provide structural information for both target
and ligand with atomic resolution.
45NMR for Drug Research
- NMR is used for fragment based discovery
- NMR is used for target identification
- NMR is used for lead optimization
46NMR 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.
47Basic 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 nano-probes) reduce
the required sample volumes and also superior Rf
field homogeneity. Thus facilitating difference
based NMR screening methods.
48Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery
Acronyms Full Name Brief Definition
HSQC/HMQC Heteronuclear Single Quantum Correlation/ Heteronuclear Multiple Quantum Correlation 2D experiments which correlate proton and heteronuclear resonances. Very useful for protein binding studies and central to the chemical shift perturbation method.
TROSY Transverse Relaxation-Optimized Spectroscopy Technique that, by taking advantage of the interference of different relaxation mechanisms, allows for a significant increase in the molecular weight limit of biomolecular NMR.
SEA-TROSY Solvent-Exposed Amides Transverse Relaxation-Optimized Spectroscopy TROSY-based experiment that, by detecting only solvent-exposed amides, greatly simplifies the spectrum of high molecular weight proteins. This facilitates the use of chemical shift perturbation methods for screening.
INEPT Insensitive Nuclei Enhanced by Polarization Transfer Technique which uses heteronuclear coupling constants to transfer magnetization to insensitive nuclei, allowing their detection.
CRIPT Cross Relaxation-Induced Polarization Transfer Alternative to the INEPT technique where magnetization is transferred using cross-correlated relaxation.
49Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery Experiments Commonly Used in NMR-Based Drug Discovery
Acronyms Full Name Brief Definition
NOE/NOESY Nuclear Overhauser Effect/NOE Spectroscopy Effect that can be used to measure approximate through-space proton to proton distances. NOEs are the main NMR parameter used for conformational analysis and protein structure determination by NMR. NOEs are in general measured using a 2-D NMR experiment termed NOESY.
STD Saturation Transfer Difference Technique that allows the identification of ligands from a mixture of low molecular weight compounds by transferring saturation from the macromolecular target to the ligands.
Water-LOGSY Water-ligand observed via gradient spectroscopy Technique which uses water molecules to mediate the transfer of magnetization from the macromolecular target to the ligand.
SAR by NMR Structure Activity Relationships by NMR Structure-based NMR approach for the discovery of high affinity protein ligands based on chemical shift perturbation.
NMR-DOC NMR docking of compounds Structure-based NMR approach well-suited for very high molecular weight proteins which relies on selective isotope enrichment and requires no previous knowledge of the chemical shift assignments of the protein.
50FRAGMENT-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 -
51Receptor 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)
52RECEPTOR-BASED SCREENING FOR DRUG DISCOVERY
53Receptor Based HSQC/HMQC
- 2D 1H, 15N or 1H, 13C-HSQC are used in the
absence and presence of ligand. - 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 gt30 kDa)
causes their NMR resonances to be too wide to be
detected.
542D 1H15N-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 phenylimidazole.
55SAR by NMR
- The first step of the method is the screening of
libraries of low molecular weight compounds
(fragments) using the 1H,15N-HSQC spectrum. - Once a hit is identified, the binding site can be
discovered by deconvolution of the mixture,
identification of the actual binding compound and
mapping the chemical shift changes on the surface
of the target . - This information is used to guide the
combinatorial search for modified ligands of
higher affinity.
56SAR by NMR
-
- Various step are involved in the screening of
different libraries in search of compounds that
will cause chemical shift perturbation in a
different second site at the surface of the
protein.
57SAR by NMR
- This step requires the screening to be carried
out on a protein solution in the presence of
saturating amounts of the first ligand so that
the first binding site is fully occupied. When a
hit for the second binding site is obtained the
chemical shift changes are again mapped on the
surface of the protein in order to ascertain the
relative positions of the two binding sites. - After optimization of the affinity of the second
hit, the two compounds (fragments?) can be
covalently linked, yielding a lead compound of
high affinity due to the chelating effect.
58Structure-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 -
59SAR by NMR
60SAR by NMR
Use of the SAR by NMR approach for the discovery
of inhibitors of Stromelysins (matrix
metaloproteineases).
61Pre-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.
62Clinical 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
63VARIOUS STAGES IN DRUG DEVELOPMENT
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66BIOASSAY-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
re-fractionated 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.
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