BIOASSAY AND PHARMACOLOGICAL SCREENINGS IMPORTANCE IN DRUG RESEARCH - PowerPoint PPT Presentation

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BIOASSAY AND PHARMACOLOGICAL SCREENINGS IMPORTANCE IN DRUG RESEARCH

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Title: BIOASSAY AND PHARMACOLOGICAL SCREENINGS IMPORTANCE IN DRUG RESEARCH


1
BIOASSAY AND PHARMACOLOGICAL SCREENINGS-
IMPORTANCE IN DRUG RESEARCH
  • Muhammad Iqbal Choudhary
  • Ahsana Dar
  • Shabana U. Simji

2
CONTENT
  • Molecular Basis of Diseases
  • Stages in Drug Development
  • Why Bioassay?
  • Difference between bioassay and pharmacological
    screenings?
  • Various types of bioassays?
  • High-throughput bioassays-Definitions, advantages
    and disadvantages
  • Bioactivity directed isolation of natural
    products- Strategies

3
Diseases- Molecular Basis
  • Over- and under-expression of catalytic proteins
    (enzymes)
  • Toxins produced by microorganisms
  • Viruses (wild DNA/molecular organisms) cause
    cancers, AIDS, influenza etc.
  • Mutation in DNA
  • 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.

4
Courtesy of Prof. Dr. Azad
I
5
Main Stages in Drug Development
  • Selection of Disease Target
  • Discovery of Lead Molecules by Bioassay
  • Preclinical Studies
  • Clinical Studies

6
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 experiment. Bioassays are typically
    conducted to measure the effects of a substance
    on a living oraganism.

7
What is Bioassay?
  • Bioassay or biological assay is any qualitative
    or quantitative analysis of a substances that
    uses a living system, such as an intact cell, as
    a component.

8
Compound Bank at the PCMD Over 2,500 compounds,
and 1,500 Plant Extracts
9
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

10
Types of Bioassays?
  • In Silico Screenings
  • Non- physiological Assays
  • Biochemical or Mechanisms-Based Assays
  • In Vitro Assays
  • Cell based Bioassays
  • Tissue based Bioassays
  • In Vivo Bioassays
  • Animal-based Assays/Preclinical Studies
  • Human trial/Clinical Trials

11
Broad Categories of Bioassays
  • Virtual Screenings
  • Primary Bioassays
  • Secondary Bioassays
  • Preclinical Trials
  • Clinical Trials

12
Virtual Screenings
  • Target Selection
  • Data Mining (Chemical space of over 1060
    conceivable compounds)
  • Screening of Libraries of Compounds Virtually
  • lead optimization
  • Prediction of Structure-Activity Relationships

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Primary Bioassay/Assays Screenings
  • Non- physiological Assays
  • Biochemical or Mechanism-Based Assays
  • In Vitro Assays
  • Microorganism-based bioassays
  • Cell-based Bioassays
  • Tissue-based Bioassays

15
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.

16
Salient Features of Primary Bioassay Screenings
  • Predictive Potential
  • General in nature
  • Tolerant of impurities
  • Unbiased
  • High-throughput
  • Reproducible
  • Fast
  • Cost-effective
  • Compatible with DMSO

17
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

18
Secondary Bioassays
  • In Vivo Bioassays
  • Animal-based Assays/Preclinical Studies

19
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

20
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

21
Examples of In Vitro Bioassay
  • 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

22
In Vivo Screenings or Pharmacological Screenings
  • In Vivo Test performed in a living system such
    as antidiabetic assays, CNS assays,
    antihypertensive assays, etc.

23
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

24
High-throughput Assays
  • The process of finding a new drug against a
    chosen target for a particular disease usually
    involves high-throughput screening (HTS), wherein
    large libraries of chemicals are tested for their
    ability to modify the target.

25
HIGH-THROUGHPUT BIOLOGICAL SCREENINGS
  • 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 100,000 samples per
    day).
  • Specifically suitable for the isolation of
    bioactive constituents from complex plant
    extracts.

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28
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
12
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31
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
32
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.

33
Clinical Trials
  • Human Trial/Clinical Trials
  • Phase I
  • Phase II
  • Phase III
  • Phase IV (Post Approval Studies)
  • Randomized, Double-blind, Placebo

34
VARIOUS STAGES IN DRUG DEVELOPMENT
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37
Questions?
  • Q. 1 Why we perform bioassays?
  • Q. 2 What are the main difference between
    primary and secondary bioassays?
  • Q.3 What are the advantages of high- throughput
    assay screenings?
  • Q. 4 Using diabetes as the target disease, what
    are the main bioassays and pharmacological
    screenings available?
  • Q. 5 Highlight the importance of Lipinskis
    rule of five.
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