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BIOASSAY SCREENINGS

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BIOASSAY SCREENINGS Powered By Docstoc
					      BIOASSAY AND
    PHARMACOLOGICAL
SCREENINGS- IMPORTANCE IN
     DRUG RESEARCH
   Muhammad Iqbal Choudhary
         Ahsana Dar
       Shabana U. Simji
             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
     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.
    Courtesy of Prof. Dr. Azad
I
     Main Stages in Drug
        Development
 Selection of Disease Target
 Discovery    of Lead Molecules   by
Bioassay
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 experiment.
Bioassays are typically conducted to
measure the effects of a substance on
a living oraganism.
      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.
Compound Bank at the PCMD
Over 2,500 compounds, and 1,500 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

Cell based Bioassays

Tissue based Bioassays

In Vivo Bioassays

Animal-based Assays/Preclinical Studies

Human trial/Clinical Trials
Broad Categories of Bioassays
Virtual Screenings
Primary Bioassays

Secondary Bioassays

Preclinical Trials

Clinical Trials
       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
    Primary “Bioassay/Assays”
           Screenings

Non- physiological Assays
Biochemical or Mechanism-Based
Assays
In Vitro Assays

Microorganism-based bioassays

Cell-based Bioassays

Tissue-based Bioassays
    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

In Vivo Bioassays
Animal-based Assays/Preclinical
Studies
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
      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 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
       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     high-
throughput screening (HTS), wherein
large libraries of chemicals are tested
for their ability to modify the target.
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.
High-throughput Screening Strategy
   for Enzyme Inhibition Assays
                                % Inhibition = [(E-S)/E]  100
    Enzyme + Buffer
                                E = Activity of enzyme without test material
  + Potential inhibitor
                                S = Activity of enzyme with test material



                          Substrate




                          Incubation
                                            Measurement of absorbance
96-well plate

                                                                               12
ON-LINE ISOLATION AND BIOASSAY
           SCREENING
                                                     UV/VIS DETECTOR
                                                  (Photodiode Array Detector)

                     CHROMATOGRAPHIC
Sample                   METHODS


                                                                       FRACTION COLLECTOR
                                  ON-LINE SPECTROMETERS

SPECTRAL AND                           -NMR                                  96-well plates
STRUCTURAL                             -MASS                                       or
DATABASES                                                                 384-well microplate
                                       -IR
Dictionary of Natural Products,        -ICP                SPLITER
Bioactive Natural Products
Database, DEREP,
NAPRALERT, MARINLIT,
Marine Natural Products                                                         BIOASSAYS
Database, STN Files
  4/7/2010
       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

Phase II

Phase III

Phase IV (Post Approval Studies)



Randomized, Double-blind, Placebo
                          VARIOUS STAGES IN DRUG
                               DEVELOPMENT

                  LEAD - Identification                                              LEAD -Development
                                                       LEAD -Validation


    Target                            Secondary    Toxicity
 Identification
                     Primary                                        In vivo       Animal         Animal
                     Bioassay          Bioassay     Assay            Assay        Trials         Trials
and Validation




                                                   Structure
                     Chemical         Selected    elucidation
                     Diversity        Chemical    of Bioactive
                                                  compounds



                      In silico                   Structure
                     Screening                    Activity                     DRUG-Development
                                                  Relation

                                                                    Post       Registration      Clinical     Pre-chinical
                                                                 Marketing        and             Trials         Trials
                                                                 Survelience   Marketing         1, II, III
              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 Lipinski’s rule of
        five.

				
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