BIOASSAY TECHNIQUES FOR BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DRUG DISCOVERY AND

DEVELOPMENTDEVELOPMENT

International Center for Chemical and Biological Sciences

(H. E. J. Research Institute of ChemistryDr. Panjwani Center for Molecular Medicine and Drug Research)

University of Karachi, Karachi-75270

Dr. Muhammad Iqbal ChoudharyDistinguished National Professor

Drug Discovery-Drug Discovery-Past and PresentPast and Present

In the past, most drugs were either In the past, most drugs were either discovered by trial and error (traditional discovered by trial and error (traditional remedies) or by serendipitous discoveries.remedies) or by serendipitous discoveries.

Today efforts are made to understand the Today efforts are made to understand the molecular basis of different diseases and molecular basis of different diseases and then to use this knowledge to design and then to use this knowledge to design and develop specific drugs.develop specific drugs.

In modern drug discovery process, bioassay In modern drug discovery process, bioassay screenings play an extremely important role.screenings play an extremely important role.

What is Required to Develop a Modern What is Required to Develop a Modern Drug (NME)?Drug (NME)?

• Decision= Corporate decision to invest in Decision= Corporate decision to invest in

specific therapeutic area, based on specific therapeutic area, based on “economic “economic feasibility”feasibility”

• Cost= $ 1.4 billion- 1.8 billion Cost= $ 1.4 billion- 1.8 billion • Duration= 10-12 years of R&D, and regulatory Duration= 10-12 years of R&D, and regulatory

approvalapproval• People= 600-800 scientists of multi-People= 600-800 scientists of multi-

disciplinary expertisedisciplinary expertise• Chemical Diversity: Screening of 100,000- Chemical Diversity: Screening of 100,000-

200,000 compounds200,000 compounds• Global Approval= Lots of paper works, based Global Approval= Lots of paper works, based

on often ill-planned studies, and on often ill-planned studies, and malpracticesmalpractices

CONTENTCONTENTMolecular basis of diseasesMolecular basis of diseasesStages in drug developmentStages in drug developmentWhy Bioassays?Why Bioassays?Different types/classes of bioassaysDifferent types/classes of bioassaysDifference between bioassay and Difference between bioassay and pharmacological screenings?pharmacological screenings?Various types of bioassays?Various types of bioassays?High-throughput bioassays-Definitions, High-throughput bioassays-Definitions, advantages and disadvantagesadvantages and disadvantagesBioactivity directed isolation of natural Bioactivity directed isolation of natural products- Strategiesproducts- StrategiesBioassay-guided fractionation (BGF) and Bioassay-guided fractionation (BGF) and isolationisolation

A Book Worth ReadingA Book Worth Reading

Bioassay Techniques for Drug Bioassay Techniques for Drug ResearchResearch

By By

Atta-ur-Rahman, M. Iqbal Choudhary and Atta-ur-Rahman, M. Iqbal Choudhary and William J. ThomsenWilliam J. Thomsen

Harwood Academic Press, LondonHarwood Academic Press, London

http://nadjeeb.wordpress.com/http://nadjeeb.wordpress.com/2009/05/9058230511.pdf2009/05/9058230511.pdf

Diseases- Molecular BasisDiseases- Molecular Basis Overwhelming majority of diseases are caused by change in Overwhelming majority of diseases are caused by change in

biochemistry and molecular genetics of human body biochemistry and molecular genetics of human body (Molecular Pathology)(Molecular Pathology)

Over- and under-expression of catalytic proteins (enzymes) Over- and under-expression of catalytic proteins (enzymes) Toxins produced by microorganismsToxins produced by microorganisms Viruses (wild DNA/molecular organisms) cause cancers, AIDS, Viruses (wild DNA/molecular organisms) cause cancers, AIDS,

influenza, Dengue fever, etc. influenza, Dengue fever, etc. Mutation in DNA cause cancersMutation in DNA cause cancers Malfunction of signaling pathways cause various disorders Malfunction of signaling pathways cause various disorders Congenital diseases due to genetic malfunctionsCongenital diseases due to genetic malfunctions Oxidation of biomolecules (proteins, carbohydrates, lipids, nucleic Oxidation of biomolecules (proteins, carbohydrates, lipids, nucleic

acid), degenerative diseases and ageingacid), degenerative diseases and ageing Deficiency of essential elements, vitamin, nutrients, etc.Deficiency of essential elements, vitamin, nutrients, etc.

I Courtesy of Prof. Dr. Azad Khan

Main Stages in Drug Main Stages in Drug Discovery and DevelopmentDiscovery and Development Selection of Disease Target/Designing Selection of Disease Target/Designing of Bioassayof Bioassay Discovery and Optimization of Lead Discovery and Optimization of Lead Molecules Molecules

Preclinical StudiesPreclinical Studies

Clinical Studies Clinical Studies

Why we Need to Perform Why we Need to Perform Bioassay?Bioassay?

To predict some type of therapeutic potential, To predict some type of therapeutic potential, either directly or by analogy, of test compounds.either directly or by analogy, of test compounds.Bioassay is a shorthand commonly used term Bioassay is a shorthand commonly used term for for biological assaybiological assay and is usually a type of and is usually a type of in in vitro experimentsvitro experiments

Bioassays are typically conducted to measure Bioassays are typically conducted to measure the effects of a substance on a living organism the effects of a substance on a living organism or other living samples.or other living samples.

What is Bioassay?What is Bioassay?Bioassay or biological Bioassay or biological assay/screening is any qualitative assay/screening is any qualitative or quantitative analysis of a or quantitative analysis of a substances that uses a living substances that uses a living system, such as an intact cell, as a system, such as an intact cell, as a component.component.

Essential Components of Essential Components of Bioassays/AssaysBioassays/Assays

Stimulus (Test sample, drug candidate, Stimulus (Test sample, drug candidate, potential agrochemical, etc)potential agrochemical, etc)

Subject (Animal, Tissues, Cells, Sub-Subject (Animal, Tissues, Cells, Sub-cellular orgenlles, Biochemicals, etc.)cellular orgenlles, Biochemicals, etc.)

Response (Response of the subject to Response (Response of the subject to various doses of stimulus)various doses of stimulus)

Molecular Bank at the PCMDOver 11,500 compounds, and 6,000 Plant Extracts

Bioassays/AssaysBioassays/Assays

Whole animalsWhole animalsIsolated organs of vertebratesIsolated organs of vertebratesLower organisms e.g. fungi, bacteria, Lower organisms e.g. fungi, bacteria, insects, molluscs, lower plants, etc.insects, molluscs, lower plants, etc.Cultured cells such as cancer cells and Cultured cells such as cancer cells and tissues of human or animal organstissues of human or animal organsIsolated sub-cellular systems, such as Isolated sub-cellular systems, such as enzymes, receptors, etcenzymes, receptors, etc

Types of Bioassays?Types of Bioassays?In Silico In Silico ScreeningsScreeningsNon- physiological AssaysNon- physiological AssaysBiochemical or Mechanisms-Based AssaysBiochemical or Mechanisms-Based AssaysIn Vitro In Vitro AssaysAssays Assays on Sub-cellular Organelles Assays on Sub-cellular Organelles Cell based BioassaysCell based BioassaysEx-VivoEx-Vivo Assays AssaysTissue based BioassaysTissue based BioassaysNMR Based Drug DiscoveryNMR Based Drug DiscoveryIn Vivo In Vivo BioassaysBioassaysAnimal-based Assays/Preclinical StudiesAnimal-based Assays/Preclinical StudiesHuman trial/Clinical TrialsHuman trial/Clinical Trials

Predicting Drug Like Behavior- Predicting Drug Like Behavior- Lipinski “Rule of Five”Lipinski “Rule of Five”

Molecular weight about 500 a. m. u. Molecular weight about 500 a. m. u. (Optimum 350)(Optimum 350)

Number of hydrogen bond accepter ~ 10 Number of hydrogen bond accepter ~ 10 (Optimum 5)(Optimum 5)

Number of hydrogen bond donor ~ 5 Number of hydrogen bond donor ~ 5

(Optimum 2)(Optimum 2)

Number of rotatable bonds ~5 Number of rotatable bonds ~5 (Conformational Flexibility)(Conformational Flexibility)

1-Octanol/water partition coefficient between 1-Octanol/water partition coefficient between 2-4 range2-4 range

Broad Categories of Broad Categories of BioassaysBioassays

Virtual ScreeningsVirtual ScreeningsPrimary BioassaysPrimary BioassaysSecondary BioassaysSecondary BioassaysPreclinical TrialsPreclinical TrialsClinical TrialsClinical Trials

Virtual and Virtual and In SilicoIn Silico ScreeningsScreenings

Ligand based or Target basedLigand based or Target based Target SelectionTarget Selection Data Mining (Chemical space of over 10Data Mining (Chemical space of over 106060

conceivable compounds)conceivable compounds) Screening of Libraries of Compounds Screening of Libraries of Compounds

VirtuallyVirtually Lead OptimizationLead Optimization Prediction of Structure-Activity Prediction of Structure-Activity

RelationshipsRelationships It Save, Time, Money and EffortsIt Save, Time, Money and Efforts

Primary “Bioassay/Assays” Primary “Bioassay/Assays” ScreeningsScreenings

Non- physiological AssaysNon- physiological AssaysBiochemical or Mechanism-Based Biochemical or Mechanism-Based AssaysAssaysMicroorganism-based bioassaysMicroorganism-based bioassaysCell-based BioassaysCell-based BioassaysTissue-based Bioassays Tissue-based Bioassays Many other Many other In VitroIn Vitro bioassays/assays bioassays/assays

Examples of Primary AssaysExamples of Primary Assays Antioxidant AssaysAntioxidant AssaysEnzyme Inhibition AssaysEnzyme Inhibition AssaysCytotoxicty BioassaysCytotoxicty BioassaysAnti-cancer Bioassays (Cancer Cell Lines)Anti-cancer Bioassays (Cancer Cell Lines)Brine Shrimp Lethality BioassaysBrine Shrimp Lethality BioassaysIn Vitro Antiparasitic BioassaysIn Vitro Antiparasitic BioassaysAnti-bacterial BioassaysAnti-bacterial BioassaysAntifungal BioassaysAntifungal BioassaysInsecticidal BioassaysInsecticidal BioassaysPhytotoxicity BioassaysPhytotoxicity BioassaysEtc.Etc.

Salient Features of Primary Salient Features of Primary Bioassay ScreeningsBioassay Screenings

Predictive Potential Predictive Potential General in natureGeneral in natureTolerant of impuritiesTolerant of impuritiesUnbiasedUnbiasedHigh-throughputHigh-throughputReproducibleReproducibleFast Fast Cost-effectiveCost-effectiveCompatible with DMSOCompatible with DMSO

Hit Rate of Primary Bioassay Hit Rate of Primary Bioassay ScreeningsScreenings

A hit rate of 1% or less is generally A hit rate of 1% or less is generally considered a reasonableconsidered a reasonableFalse positive are acceptable False positive are acceptable False negative are discouragedFalse negative are discouraged

Secondary BioassaysSecondary Bioassays

Animal-based assays (Animal-based assays (In VivoIn Vivo))Toxicological Assessments in whole Toxicological Assessments in whole animalsanimalsADME StudiesADME StudiesBehavioral StudiesBehavioral StudiesPreclinical StudiesPreclinical Studies

Importance of Standards in Importance of Standards in Bioassays/AssaysBioassays/Assays

The results of the assay/bioassay need to The results of the assay/bioassay need to validated by monitoring the effect of an validated by monitoring the effect of an available known compound (Standard).available known compound (Standard).

Without judicious choice of standard and Without judicious choice of standard and its reproducible results in an assay system, its reproducible results in an assay system, no screening can be claimed credible.no screening can be claimed credible.

Importance of Reproducibility Importance of Reproducibility and Dose Dependencyand Dose Dependency

Without reproducible results (within the Without reproducible results (within the margin of error or esd), an assay has any margin of error or esd), an assay has any value. It is a share loss of time and efforts.value. It is a share loss of time and efforts.

Dose dependency is the key to a successful Dose dependency is the key to a successful outcome of study.outcome of study.

Without reproducibility and dose dependency, Without reproducibility and dose dependency, it can be magic, but not scienceit can be magic, but not science

VINBLASTINE- A Novel Anticancer Drug from Flowers of Sada Bahar

In VitroIn Vitro Bioassays Bioassays

In Vitro: In experimental situation In Vitro: In experimental situation outside the organisms. Biological or outside the organisms. Biological or chemical work done in the test tube( in chemical work done in the test tube( in vitro is Latin for “in glass”) rather than vitro is Latin for “in glass”) rather than in living systemsin living systemsExamples include antifungal, Examples include antifungal, antibacterial, organ-based assays, antibacterial, organ-based assays, cellular assays, etccellular assays, etc

Examples ofExamples of In Vitro In Vitro Bioassays Bioassays

Activity AssaysActivity Assays•DPPH assayDPPH assay•Xanthine oxidase inhibition assaysXanthine oxidase inhibition assays•Superoxide scavenging assaySuperoxide scavenging assay•Antiglycation assayAntiglycation assay

Bioassays (cell-based)Bioassays (cell-based)•DNA LevelDNA Level•Protein Level Protein Level •RNA LevelRNA Level•Immunology assayImmunology assay

Toxicity AssaysToxicity Assays•MTT assayMTT assay•Cancer cell line assaysCancer cell line assays

In VivoIn Vivo Screenings or Screenings or Pharmacological ScreeningsPharmacological Screenings

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

Examples ofExamples of In Vivo In Vivo Bioassays Bioassays

Animal ToxicityAnimal Toxicity•Acute toxicityAcute toxicity•Chronic toxicityChronic toxicity

Animals StudyAnimals Study•Animal model with induced diseaseAnimal model with induced disease•Animal model with induced injuryAnimal model with induced injury

Pre-Clinical TrialsPre-Clinical Trials Clinical TrialsClinical Trials

High-throughput AssaysHigh-throughput Assays

The process of finding a new drug The process of finding a new drug against a chosen target for a particular against a chosen target for a particular disease usually involves disease usually involves high-through high-through screening (HTS)screening (HTS), wherein large libraries , wherein large libraries of chemicals are tested for their ability of chemicals are tested for their ability to modify the target. to modify the target.

96-384 well plates (medium throughput) and 96-384 well plates (medium throughput) and more (high-throughput)more (high-throughput)

Development of straight-forward Development of straight-forward in-vitroin-vitro biological assays (enzyme-based, cellular and biological assays (enzyme-based, cellular and microbiological assays) into automated high-microbiological assays) into automated high-throughput screens (HTS). throughput screens (HTS).

Rapid assays of thousands or hundreds of Rapid assays of thousands or hundreds of thousands of compounds (upto 200,000 samples thousands of compounds (upto 200,000 samples per day).per day).

Specifically suitable for the isolation of bioactive Specifically suitable for the isolation of bioactive constituents from complex plant extracts or constituents from complex plant extracts or complex combinatorial library.complex combinatorial library.

HIGH-THROUGHPUT BIOLOGICAL HIGH-THROUGHPUT BIOLOGICAL SCREENINGSSCREENINGS

Enzyme + Buffer+ Potential inhibitor

96-well plate

Substrate

Incubation

Measurement of absorbance

% Inhibition = [(E-S)/E] 100E = Activity of enzyme without test materialS = Activity of enzyme with test material

12

High-throughput Screening Strategy for Enzyme Inhibition Assays

NMR-BASED SCREENING IN NMR-BASED SCREENING IN DRUG DISCOVERYDRUG DISCOVERY

NMR

LigandBinding

Dynamics

Metabolic Profiling

FoldingUnfolding

Structure

NMR-A Versatile Tool in Drug NMR-A Versatile Tool in Drug DiscoveryDiscovery

04/19/2304/19/23

ON-LINE ISOLATION AND BIOASSAY ON-LINE ISOLATION AND BIOASSAY SCREENING SCREENING

UV/VIS DETECTOR(Photodiode Array Detector)

SPLITER

CHROMATOGRAPHIC METHODS

FRACTION COLLECTOR

BIOASSAYS

-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

ON-LINE SPECTROMETERS

Sample

96-well platesor

384-well microplate

Fragment Based Drug Fragment Based Drug DiscoveryDiscovery

Thrombin Inhibitor

HIV Protease Inhibitor

Fragment Based Drug Fragment Based Drug DiscoveryDiscovery

C. Acetylcholinesterase Inhibitor

Geometric Geometric ComplementarityComplementarityElectronic (electrostatic) Electronic (electrostatic) ComplementarityComplementarity““Induced fit” vs. Induced fit” vs. “Lock & Key”“Lock & Key”Stereospecific (enzymes Stereospecific (enzymes and substrates are chiral)and substrates are chiral)

Substrate Binding SpecificitySubstrate Binding Specificity

NMR for Drug ResearchNMR 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.

NMR is used for fragment based NMR is used for fragment based discoverydiscovery

NMR is used for target identificationNMR is used for target identification NMR is used for lead optimizationNMR is used for lead optimization

NMR for Drug ResearchNMR for Drug Research

NMR for Drug ResearchNMR 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.

Basic Development of NMR Basic Development of NMR Spectroscopy for Drug ResearchSpectroscopy 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.

•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

FRAGMENT-BASED DRUG FRAGMENT-BASED DRUG DISCOVERYDISCOVERY

Receptor Based Screening by Receptor Based Screening by Chemical Shift MappingChemical Shift Mapping

Identification of high affinity ligands Identification of high affinity ligands by mapping the chemical shifts by mapping the chemical shifts changes in the receptor spectrum changes in the receptor spectrum ((11H-H-1515N- HSQC)N- HSQC)

Require more quantities of receptor Require more quantities of receptor (proteins)(proteins)

RECEPTOR-BASED SCREENING FOR RECEPTOR-BASED SCREENING FOR DRUG DISCOVERYDRUG DISCOVERY

Receptor Based HSQC/HMQCReceptor Based HSQC/HMQC 2D [2D [11H, H, 1515N] or [N] or [11H, H, 1313C]-HSQC are used in the absence and presence of C]-HSQC are used in the absence and presence of

ligand.ligand.

The affinity constant between the ligand and the target can be accurately The affinity constant between the ligand and the target can be accurately measured by determining the chemical shift changes as a function of measured by determining the chemical shift changes as a function of ligand concentration.ligand concentration.

[[11H, H, 1515N]-HSQC experiment use to monitor changes in the amide protons N]-HSQC experiment use to monitor changes in the amide protons and nitrogen nuclei of the backbone and Asn and Gln side chains (it and nitrogen nuclei of the backbone and Asn and Gln side chains (it requires the protein sample to be enriched in requires the protein sample to be enriched in 1515N).N).

[[11H, H, 1313C]-HSQC experiment gives information about the chemical shift C]-HSQC experiment gives information about the chemical shift changes in all side chains.changes in all side chains.

Drug-discovery programs usually deals with very large proteins. Using Drug-discovery programs usually deals with very large proteins. Using traditional method very long correlation time of protein (MW >30 kDa) traditional method very long correlation time of protein (MW >30 kDa) causes their NMR resonances to be too wide to be detectedcauses their NMR resonances to be too wide to be detected..

2D [1H–15N]-HSQC Experiment(Chemical shift perturbation method)

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.

1H (ppm)

•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

Structure-Activity-Relationship Structure-Activity-Relationship (SAR) by NMR(SAR) by NMR

SAR by NMRSAR by NMR

SAR by SAR by NMRNMR

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

Pre-clinical TrialsPre-clinical Trials

Involve Involve in vivoin vivo (test tube) and (test tube) and in vivoin vivo (animal) experiments using wide-ranging (animal) experiments using wide-ranging doses of the study drug to obtain preliminary doses of the study drug to obtain preliminary efficacy, toxicity and pharmacokinetics efficacy, toxicity and pharmacokinetics information. information.

Assist pharmaceutical companies to decide Assist pharmaceutical companies to decide whether a drug candidate has scientific merit whether a drug candidate has scientific merit for further development as an investigational for further development as an investigational new drug. new drug.

Clinical TrialsClinical Trials

Human Trial/Clinical TrialsHuman Trial/Clinical TrialsPhase I (Safety 20-80 Volunteers)Phase I (Safety 20-80 Volunteers)Phase II (Efficacy/Safety 100-300 patients)Phase II (Efficacy/Safety 100-300 patients)Phase III (Efficacy/Safety 300-3000 patients)Phase III (Efficacy/Safety 300-3000 patients)Phase IV (Post Approval/Marketing Studies)Phase IV (Post Approval/Marketing Studies)

Randomized, Double-blind, PlaceboRandomized, Double-blind, Placebo

SecondaryBioassay

ToxicityAssay

In vivoAssay

AnimalTrials

AnimalTrials

Pre-chinicalTrials

PrimaryBioassay

ChemicalDiversity

SelectedChemical

Structure elucidation of Bioactivecompounds

StructureActivity Relation

In silicoScreening

Post MarketingSurvelience

Registrationand

Marketing

Clinical Trials

1, II, III

LEAD - IdentificationLEAD -Validation LEAD -Development

DRUG-Development

Target Identification

and Validation

VARIOUS STAGES IN DRUG DEVELOPMENT

BIOASSAY-GUIDED BIOASSAY-GUIDED FRACTIONATION (BGF)FRACTIONATION (BGF)

Bioassay-guided fractionation (BGF) of Isolation is the Bioassay-guided fractionation (BGF) of Isolation is the

process in which natural product extract or mixtures of process in which natural product extract or mixtures of

synthetic products is chromatographically fractionated synthetic products is chromatographically fractionated

and re-fractionated until a pure biologically active and re-fractionated until a pure biologically active

constituent(s) is isolated.constituent(s) is isolated. At every stage of chromatographic separation, every At every stage of chromatographic separation, every

fraction is subjected to a specific bioassay to identify fraction is subjected to a specific bioassay to identify

the most active fraction(s).the most active fraction(s). Only those fraction(s) which are active are further Only those fraction(s) which are active are further

processed.processed.

Thank You Very MuchThank You Very Much