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.