BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT

1. BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT Dr. Muhammad IqbalChoudhary 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

2. 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

3. 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.

4. 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 R&D, 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

5. A Book Worth Reading • Bioassay Techniques for Drug Research By Atta-ur-Rahman, M. IqbalChoudhary and William J. Thomsen Harwood Academic Press, London http://nadjeeb.wordpress.com/2009/05/9058230511.pdf

6. 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.

7. Courtesy of Prof. Dr. Azad Khan I

8. Main Stages in Drug Discovery and Development Selection of Disease Target/Designing of Bioassay Discovery and Optimization of Lead Molecules Preclinical Studies Clinical Studies

9. 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.

10. 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.

11. 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)

12. Molecular Bank at the PCMD Over 12,500 compounds, and 6,000 Plant Extracts

13. 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

14. Types of Bioassays? In SilicoScreenings 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

15. 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

16. Broad Categories of Bioassays Virtual Screenings Primary Bioassays Secondary Bioassays Preclinical Trials Clinical Trials

17. 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

19. 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

20. 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.

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

22. 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

23. Secondary Bioassays Animal-based assays (In Vivo) Toxicological Assessments in whole animals ADME Studies Behavioral Studies Preclinical Studies

24. 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.

25. 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

26. VINBLASTINE-A Novel Anticancer Drug from Flowers of SadaBahar

27. 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

28. Examples of In Vitro Bioassays Activity Assays DPPH assay Xanthineoxidase 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

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

30. 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

31. 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.

32. 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.

35. 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

38. Some Examples of Assays at the ICCBS

39. 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.

40. Examples of In Vivo Assays Metabolic Disorders (Diabetes, IGT, etc) Cardiovascular CNS Assays (Anti-depressant. Anti-anxiety, Anti-epilepsy, memory, etc) Anti cancer Drug Metabolism Anti-parasitic Anti-obesity Toxicity

41. Enzyme Inhibition- Key Tool in Drug Development A wide range of diseases are enzyme related. More than 30% of the drugs in clinical use are enzyme inhibitors. Many pesticides and insecticides (chemical weapons!) also work as enzyme inhibitors in the target organisms. Plants and other living sources, as well as medicinal chemistry can provide novel and potent enzyme inhibitors.

42. Medium-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

43. Example: Urease Inhibition Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia. The reaction occurs as follows: (NH2)2CO+ H2O →CO2 + 2NH3 Ammonia in water forms ammonium hydroxide, a base. Urease inhibition is a successful approach towards the treatment of diseases caused by ureolytic bacteria. 43

44. Inhibition of Urease- Inhibitors Type • Substrate Like Inhibitors: Inhibitors which bind in a substrate or active-site directed mode. • Mechanism Based Inhibitors: Inhibitors which bind in a non-substrate like manner or in mechanism-based directed mode 44

45. Urea Derivatives- Novel Urease Inhibitors IC50 = 1.25±0.021 µM Substrate like inhibition mechanism -structurally similar to the natural substrate urea. Standard: Thiourea IC50 (Jack bean) = 21±0.11 µM Letters in Drug Design & Discovery, Volume 5,  Number 6, September 2008, pp. 401-405(5) 45

46. Substrate Like –Novel Urease Inhibitors Thioureas 1,2,4-Triazole-3-thiones IC50 = 15.03±0.02 µM IC50 = 16.7±0.178 µM Oxadiazoles IC50 = 16.1±0.12 µM Dihydropyrimidines Triazoles IC50 = 5.36±0.027 µM IC50 = 10.66±0.16 µM Standard (Thiourea) IC50 = 21 ± 0.11 µM

47. Glycation Occurs in everyone, but at a faster rate in diabetics AGEs formation effect the molecular functioning of the body and cause various diseases Activate RAGEs (Receptors of AGEs) which contribute in triggering a number of disease-causing inflammatory response

48. Prevention of Non-enzymatic Glycation • Inhibition of AGEs formation can lead to the prevention of diabetic complications by suppressing or delaying the formation of AGEs . • Various inhibitors have been discovered, such as Aminogunadine, Aspirin, Rutin, Antioxidants, AGE breakers, etc. • Aminoguanidine (AG), a potent AGEs inhibitor also underwent the clinical trials.Idealy AGEs inhbitors should be able to reverse the process of glycation, and repair the damage.

49. In Vitro Assay for Inhibition of Protein Glycation Inhibitor (1 mM) + HSA (10mg/mL) + Fructose (500 mM) + Sodium Phosphate Buffer Activity was monitored at Excitation: 330 nm Emission: 440 nm. Incubation 7 days at 37° C

50. Anti-glycation Activity of Some Natural Compounds (Flavonoid glycoside) Plant Name Tagetus patula Rutin IC50 = 294.50 mM