1. BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT
Dr. Muhammad Iqbal Choudhary
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. 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.

3. 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= years of R&D, and regulatory approval
People= scientists of multi-disciplinary expertise
Chemical Diversity: Screening of 100, ,000 compounds
Global Approval= Lots of paper works, based on often ill-planned studies, and malpractices

4. 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

5. A Book Worth Reading Bioassay Techniques for Drug Research By
Atta-ur-Rahman, M. Iqbal Choudhary and William J. Thomsen
Harwood Academic Press, London

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 KhanI

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. Over 11,500 compounds, and 6,000 Plant Extracts
Molecular Bank at the PCMD
Over 11,500 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 Silico Screenings 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 Sada Bahar

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
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

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

32. HIGH-THROUGHPUT BIOLOGICAL SCREENINGS
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. NMR-BASED SCREENING IN
DRUG DISCOVERY

39. NMR-A Versatile Tool in Drug Discovery
Ligand
Binding
Dynamics
Metabolic
Profiling
Folding
Unfolding
Structure

40. 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
4/19/2017

41. Fragment Based Drug Discovery
Thrombin Inhibitor
HIV Protease Inhibitor

42. Fragment Based Drug Discovery
C. Acetylcholinesterase Inhibitor

43. Substrate Binding Specificity
Geometric Complementarity
Electronic (electrostatic) Complementarity
“Induced fit” vs. “Lock & Key”
Stereospecific (enzymes and substrates are chiral)

44. NMR for Drug Research
1. Detect the weakest ligand–target interactions even millimolar binding constants.
2. Enables a determination of binding constants.
4. Allows direct screening and deconvolution of mixtures from natural sources or combinatorial chemistry.
5. Provide structural information for both target and ligand with atomic resolution.

45. NMR for Drug Research NMR is used for fragment based discovery
NMR is used for target identification
NMR is used for lead optimization

46. NMR for Drug Research Promising new method in drug discovery
Unmatched screening sensitivity.
Abundance of information about the structure and nature of molecular interaction and recognition.

47. Basic Development of NMR Spectroscopy for Drug Research
Cryoprobe technology which increase signal-to-noise ratio and lower accessible binging affinities.
Flow probe alleviating the need for NMR tubes and time-consuming handling.
Micro-coil tubes (micro- and nano-probes) reduce the required sample volumes and also superior Rf field homogeneity. Thus facilitating difference based NMR screening methods.

48. Experiments Commonly Used in NMR-Based Drug Discovery
Acronyms
Full Name
Brief Definition
HSQC/HMQC
Heteronuclear Single Quantum Correlation/ Heteronuclear Multiple Quantum Correlation
2D experiments which correlate proton and heteronuclear resonances. Very useful for protein binding studies and central to the chemical shift perturbation method.
TROSY
Transverse Relaxation-Optimized Spectroscopy
Technique that, by taking advantage of the interference of different relaxation mechanisms, allows for a significant increase in the molecular weight limit of biomolecular NMR.
SEA-TROSY
Solvent-Exposed Amides Transverse Relaxation-Optimized Spectroscopy
TROSY-based experiment that, by detecting only solvent-exposed amides, greatly simplifies the spectrum of high molecular weight proteins. This facilitates the use of chemical shift perturbation methods for screening.
INEPT
Insensitive Nuclei Enhanced by Polarization Transfer
Technique which uses heteronuclear coupling constants to transfer magnetization to insensitive nuclei, allowing their detection.
CRIPT
Cross Relaxation-Induced Polarization Transfer
Alternative to the INEPT technique where magnetization is transferred using cross-correlated relaxation.

49. Experiments Commonly Used in NMR-Based Drug Discovery
Acronyms
Full Name
Brief Definition
NOE/NOESY
Nuclear Overhauser Effect/NOE Spectroscopy
Effect that can be used to measure approximate through-space proton to proton distances. NOEs are the main NMR parameter used for conformational analysis and protein structure determination by NMR. NOEs are in general measured using a 2-D NMR experiment termed NOESY.
STD
Saturation Transfer Difference
Technique that allows the identification of ligands from a mixture of low molecular weight compounds by transferring saturation from the macromolecular target to the ligands.
Water-LOGSY
Water-ligand observed via gradient spectroscopy
Technique which uses water molecules to mediate the transfer of magnetization from the macromolecular target to the ligand.
SAR by NMR
Structure Activity Relationships by NMR
Structure-based NMR approach for the discovery of high affinity protein ligands based on chemical shift perturbation.
NMR-DOC
NMR docking of compounds
Structure-based NMR approach well-suited for very high molecular weight proteins which relies on selective isotope enrichment and requires no previous knowledge of the chemical shift assignments of the protein.

50. FRAGMENT-BASED DRUG DISCOVERY
Target- or Receptor-Based Screening- Does ligand interact with the target by following the changes in the chemical shifts of target protons?. It observe and compare the chemical shifts of targets in the absence and presence of ligand
Ligand-Based-Screening- Does ligand is interacting with the target by following the changes in the NMR parameters of ligand after the addition of the target

51. Receptor Based Screening by Chemical Shift Mapping
Identification of high affinity ligands by mapping the chemical shifts changes in the receptor spectrum (1H-15N- HSQC)
Require more quantities of receptor (proteins)

52. RECEPTOR-BASED SCREENING FOR DRUG DISCOVERY

53. Receptor Based HSQC/HMQC
2D [1H, 15N] or [1H, 13C]-HSQC are used in the absence and presence of ligand.
The affinity constant between the ligand and the target can be accurately measured by determining the chemical shift changes as a function of ligand concentration.
[1H, 15N]-HSQC experiment use to monitor changes in the amide protons and nitrogen nuclei of the backbone and Asn and Gln side chains (it requires the protein sample to be enriched in 15N).
[1H, 13C]-HSQC experiment gives information about the chemical shift changes in all side chains.
Drug-discovery programs usually deals with very large proteins. Using traditional method very long correlation time of protein (MW >30 kDa) causes their NMR resonances to be too wide to be detected.

54. 2D [1H–15N]-HSQC Experiment (Chemical shift perturbation method)
1H (ppm)
The black contours correspond to FKBP (family of enzymes that function as protein folding cheprons), the macromolecular target, whereas the red contours correspond to the complex formed by FKBP and phenylimidazole.

55. SAR by NMR
The first step of the method is the screening of libraries of low molecular weight compounds (fragments) using the [1H,15N]-HSQC spectrum.
Once a hit is identified, the binding site can be discovered by deconvolution of the mixture, identification of the actual binding compound and mapping the chemical shift changes on the surface of the target .
This information is used to guide the combinatorial search for modified ligands of higher affinity.

56. SAR by NMR
Various step are involved in the screening of different libraries in search of compounds that will cause chemical shift perturbation in a different second site at the surface of the protein.

57. SAR by NMR
This step requires the screening to be carried out on a protein solution in the presence of saturating amounts of the first ligand so that the first binding site is fully occupied. When a hit for the second binding site is obtained the chemical shift changes are again mapped on the surface of the protein in order to ascertain the relative positions of the two binding sites.
After optimization of the affinity of the second hit, the two compounds (fragments?) can be covalently linked, yielding a lead compound of high affinity due to the chelating effect.

58. Structure-Activity-Relationship
(SAR) by NMR
Identification of ligands with high binding affinity from library of compounds by using 2D 1H-15N- HSQC
Optimization of ligands by chemical modification
Identification of ligand (optimized) binding by again recoding 2D 1H-15N- HSQC
Re-optimization of ligand by chemical modifications
Lining two ligands with appropriate linkers and checking the affinity again

59. SAR by NMR

60. SAR by NMR
Use of the SAR by NMR approach for the discovery of inhibitors of Stromelysins (matrix metaloproteineases).

61. 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.

62. Clinical Trials Human Trial/Clinical Trials
Phase I (Safety Volunteers)
Phase II (Efficacy/Safety patients)
Phase III (Efficacy/Safety patients)
Phase IV (Post Approval/Marketing Studies)
Randomized, Double-blind, Placebo

63. VARIOUS STAGES IN DRUG DEVELOPMENT

66. BIOASSAY-GUIDED FRACTIONATION (BGF)
Bioassay-guided fractionation (BGF) of Isolation is the process in which natural product extract or mixtures of synthetic products is chromatographically fractionated and re-fractionated until a pure biologically active constituent(s) is isolated.
At every stage of chromatographic separation, every fraction is subjected to a specific bioassay to identify the most active fraction(s).
Only those fraction(s) which are active are further processed.

67. Thank You Very Much