BIOASSAY AND PHARMACOLOGICAL SCREENINGS IMPORTANCE IN DRUG RESEARCH

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BIOASSAY AND PHARMACOLOGICAL SCREENINGS-
IMPORTANCE IN DRUG RESEARCH

• Muhammad Iqbal Choudhary
• Ahsana Dar
• Shabana U. Simji

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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

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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.

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

• Selection of Disease Target
• Discovery of Lead Molecules by Bioassay
• 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 experiment. Bioassays are typically
  conducted to measure the effects of a substance
  on a living oraganism.

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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.

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Compound Bank at the PCMD Over 2,500 compounds,
and 1,500 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
• Cell based Bioassays
• Tissue based Bioassays
• In Vivo Bioassays
• Animal-based Assays/Preclinical Studies
• Human trial/Clinical Trials

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Broad Categories of Bioassays

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

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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

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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

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

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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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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 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

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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-throughput screening (HTS), wherein
  large libraries of chemicals are tested for their
  ability to modify the target.

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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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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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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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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
• Phase II
• Phase III
• Phase IV (Post Approval Studies)
• Randomized, Double-blind, Placebo

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VARIOUS STAGES IN DRUG DEVELOPMENT
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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.