BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT

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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
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Drug 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.

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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 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

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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

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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.p
  df

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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.

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Courtesy of Prof. Dr. Azad Khan
I
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Main Stages in Drug Discovery and Development

• Selection of Disease Target/Designing of
  Bioassay
• Discovery and Optimization of Lead Molecules
• Preclinical Studies
• Clinical Studies

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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.

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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.

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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)

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Molecular Bank at the PCMD Over 11,500 compounds,
and 6,000 Plant Extracts
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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

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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

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Predicting 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

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Broad Categories of Bioassays

• Virtual Screenings
• Primary Bioassays
• Secondary Bioassays
• Preclinical Trials
• Clinical Trials

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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

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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

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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.

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Salient Features of Primary Bioassay Screenings

• Predictive Potential
• General in nature
• Tolerant of impurities
• Unbiased
• High-throughput
• Reproducible
• Fast
• Cost-effective
• Compatible with DMSO

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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

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Secondary Bioassays

• Animal-based assays (In Vivo)
• Toxicological Assessments in whole animals
• ADME Studies
• Behavioral Studies
• Preclinical Studies

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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.

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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

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VINBLASTINE- A Novel Anticancer Drug from Flowers
of Sada Bahar
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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

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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

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In Vivo Screenings or Pharmacological Screenings

• In Vivo Test performed in a living system such
  as antidiabetic assays, CNS assays,
  antihypertensive assays, etc.

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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

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High-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.

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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
  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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High-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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NMR-BASED SCREENING IN DRUG DISCOVERY
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NMR-A Versatile Tool in Drug Discovery
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ON-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
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Fragment Based Drug Discovery
Thrombin Inhibitor
HIV Protease Inhibitor
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Fragment Based Drug Discovery
C. Acetylcholinesterase Inhibitor
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Substrate Binding Specificity

• Geometric Complementarity
• Electronic (electrostatic) Complementarity
• Induced fit vs. Lock Key
• Stereospecific (enzymes and substrates are chiral)

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NMR 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.
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NMR for Drug Research

• NMR is used for fragment based discovery
• NMR is used for target identification
• NMR is used for lead optimization

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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.

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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 nano-probes) reduce
  the required sample volumes and also superior Rf
  field homogeneity. Thus facilitating difference
  based NMR screening methods.

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Experiments 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.
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Experiments 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.
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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

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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)

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RECEPTOR-BASED SCREENING FOR DRUG DISCOVERY
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Receptor 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.

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2D 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.
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SAR 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.

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SAR 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.

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SAR 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.

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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

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SAR by NMR
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SAR by NMR
Use of the SAR by NMR approach for the discovery
of inhibitors of Stromelysins (matrix
metaloproteineases).
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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.

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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

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VARIOUS STAGES IN DRUG DEVELOPMENT
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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
  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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• Thank You Very Much