BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT
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Transcript of BIOASSAY TECHNIQUES FOR DRUG DISCOVERY AND DEVELOPMENT
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
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
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
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 12,500 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 BioassaysBioassays•Animal-based Assays/Preclinical StudiesAnimal-based Assays/Preclinical Studies•Human 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-high-throughput screening (HTS)throughput screening (HTS), wherein , wherein large libraries of chemicals are tested large libraries of chemicals are tested for their ability to modify the target. for their ability 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
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High-throughput Screening Strategy for Enzyme Inhibition Assays
SOME EXAMPLES OF ASSAYS SOME EXAMPLES OF ASSAYS AT THE ICCBSAT THE ICCBS
Examples of Primary AssaysExamples of Primary Assays• Antioxidant AssaysAntioxidant Assays• Enzyme Inhibition AssaysEnzyme Inhibition Assays• Cytotoxicty BioassaysCytotoxicty Bioassays• Anti-cancer Bioassays (Cancer Cell Lines)Anti-cancer Bioassays (Cancer Cell Lines)• Brine Shrimp Lethality BioassaysBrine Shrimp Lethality Bioassays• In Vitro Antiparasitic BioassaysIn Vitro Antiparasitic Bioassays• Anti-bacterial BioassaysAnti-bacterial Bioassays• Antifungal BioassaysAntifungal Bioassays• Insecticidal BioassaysInsecticidal Bioassays• Phytotoxicity BioassaysPhytotoxicity Bioassays
• Etc.Etc.
Examples of Examples of In Vivo In Vivo AssaysAssays Metabolic Disorders Metabolic Disorders (Diabetes, IGT, etc)(Diabetes, IGT, etc) CardiovascularCardiovascular CNS Assays CNS Assays (Anti-depressant. Anti-anxiety, (Anti-depressant. Anti-anxiety,
Anti-epilepsy, memory, etc)Anti-epilepsy, memory, etc) Anti cancerAnti cancer Drug MetabolismDrug Metabolism Anti-parasiticAnti-parasitic Anti-obesityAnti-obesity ToxicityToxicity
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.
Enzyme Inhibition- Key Tool in Drug Development
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
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Medium-throughput Screening Strategy for Enzyme Inhibition Assays
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Example: Urease InhibitionExample: Urease Inhibition
• Urease catalyzes the hydrolysis of urea into carbon Urease catalyzes the hydrolysis of urea into carbon dioxide and ammonia. dioxide and ammonia.
• The reaction occurs as follows:The reaction occurs as follows:
(NH(NH22))22CO+ HCO+ H22O →COO →CO22 + 2NH + 2NH33
• Ammonia in water forms ammonium hydroxide, a base.Ammonia in water forms ammonium hydroxide, a base.
• Urease inhibition is a successful approach towards the Urease inhibition is a successful approach towards the treatment of diseases caused by ureolytic bacteria.treatment of diseases caused by ureolytic bacteria.
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4444
Inhibition of Urease- Inhibitors TypeInhibition 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
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4545
O
NNH
NO2
NO2
O2N
Standard: ThioureaIC50 (Jack bean) = 21±0.11 µM
IC50 = 1.25±0.021 µM
Letters in Drug Design & Discovery, Volume 5, Number 6, September 2008, pp. 401-405(5)
Substrate like inhibition mechanism -structurally similar to the natural substrate urea.
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Urea Derivatives- Novel Urea Derivatives- Novel UreaseUrease Inhibitors Inhibitors
4646
Cl
HN
HN
S
CH3
NH
NH
H3C S
O
EtO
Br
IC50 = 5.36±0.027 µM
N
NN
SH
OCH3
Br
DihydropyrimidinesTriazoles
IC50 = 10.66±0.16 µM
1,2,4-Triazole-3-thiones
NN
HN S
OH
IC50 = 16.7±0.178 µM
N
O
N HN
OH
IC50 = 16.1±0.12 µMOxadiazoles
IC50 = 15.03±0.02 µMThioureas
Standard (Thiourea) IC50 = 21 ± 0.11 µM
Substrate Like –Novel Urease InhibitorsSubstrate Like –Novel Urease Inhibitors
GlycationGlycation
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
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.
Inhibitor (1 mM) +
HSA (10mg/mL) +
Fructose (500 mM)
+ Sodium
Phosphate Buffer
Activity was monitored at Excitation: 330 nm
Emission: 440 nm.
In Vitro Assay for Inhibition of Protein Glycation
Incubation 7 days at 37° C
O
OH
OH
OH
O
O
O
OHHO
HO
HO
OH
H3CO
IC50 58.02 ± 0.813
Plant Name Tagetus patula
Anti-glycation Activity of Some Natural Compounds (Flavonoid glycoside)
Rutin IC50 = 294.50
Benzimidazoles
NH
N
O2N
HO OH
3-(6-Nitro-1H-benzimidazol-2-yl)-1,2-benzenediol
NH
N
O2N
HO
OH
2- (6-Nitro-1H-benzimidazol-2-yl)-1,4-benzenediol
IC50 17.7± 0.001 µM IC50 48.7± 0.006 µM
Rutin IC50 = 70 ± 0.5 µM
Oxidation and Human HealthOxidation and Human Health
One of the paradoxes of life on this One of the paradoxes of life on this planet is that the molecule that planet is that the molecule that sustain aerobic life, oxygen, is not sustain aerobic life, oxygen, is not only fundamentally essential for only fundamentally essential for energy metabolism and energy metabolism and respiration, but implicated in many respiration, but implicated in many diseases and degenerative diseases and degenerative conditions.conditions.
Marx, Marx, ScienceScience, , 235235, 529-531 , 529-531 (1985). (1985).
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Methods Used to Determine Antioxidant Methods Used to Determine Antioxidant PotentialPotential
For quantitative determination of electron For quantitative determination of electron donation.donation.
The molecule of 1, 1-diphenyl-2-The molecule of 1, 1-diphenyl-2-picrylhydrazyl (DPPH) is a long-lived organic picrylhydrazyl (DPPH) is a long-lived organic nitrogen radical.nitrogen radical.
DPPH Radical Scavenging AssayDPPH Radical Scavenging Assay
5454
PrinciplePrinciple
N N
O2N
O2N
NO2
R-H
N N
O2N
O2N
NO2
H
R
1,1-Diphenyl-2-picrylhydrazyl (Free radical form, deep voilet in color)
1,1-Diphenyl-2-picrylhydrazine (Nonradical, pale yellow in color)
•The delocalization gives rise to the deep violet color.The delocalization gives rise to the deep violet color.•Characteristic absorption band in ethanol solution at Characteristic absorption band in ethanol solution at
515 nm.515 nm.•When a solution of DPPH is mixed with that of a substance (RH) When a solution of DPPH is mixed with that of a substance (RH) which can donate a hydrogen atom, then pale yellow reduced form which can donate a hydrogen atom, then pale yellow reduced form
of DPPH is formed.of DPPH is formed.
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ProtocolProtocol
Pre read at Pre read at 515nm515nm
DPPH in EthanolDPPH in Ethanol(300 μM(300 μM))
Normal read atNormal read at 515nm515nm
Micro plate reader (Spectra MAX-340)
Sample in DMSOSample in DMSO
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Superoxide Anion Scavenging Assay Superoxide Anion Scavenging Assay
The assay involves a non-enzymatic generation of The assay involves a non-enzymatic generation of superoxide anion radicals. The superoxide anion superoxide anion radicals. The superoxide anion scavenging activity was determined by measuring the scavenging activity was determined by measuring the reduction in rate of formation of blue colored reduction in rate of formation of blue colored formazan dye which absorbs at 560 nm. formazan dye which absorbs at 560 nm.
A sample with antioxidant potential scavenge the A sample with antioxidant potential scavenge the super oxide anion radicals and eventually reduces the super oxide anion radicals and eventually reduces the rate of formation of formazan dye, which can be rate of formation of formazan dye, which can be monitored by means of decrease in the absorbance.monitored by means of decrease in the absorbance.
5757
Superoxide Anion Scavenging Assay Superoxide Anion Scavenging Assay
N
NH2
O
OO
PO
O
O
PO O
O
ON
N
N
N
NH2OHHO
OHHO
NADH
N
N
PMS
N
NH2
O
OO
PO
O
O
PO O
O
ON
N
N
N
NH2OHHO
OHHO
NAD+
NH
N
PMSH
5858
N
N N
N
NO2
H3CO
H3CO NN N
N
NO2
PMSH NAD+
O2
O2 O2
O2
H
2Cl
NADH
N
N NH
N
H3CO NN N
NH
NO2
NO2
H3CO
PMS+
NBTLight Yellow Color
FormazanReduced NBT, Blue Color
Superoxide Anion Scavenging Assay Superoxide Anion Scavenging Assay
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OHO
OH O
OH
OH
Kaempferol (9)(3,5,7-Trihydroxy-4'-hydroxy f lavone)
Source: Whole plant of Iris unguicularis Isolated by Dr. Sumaira Hareem
Source: Whole plant of Iris tenuifolia Pall.
Studied in DPPHBHA= 44.0±2.70PG= 30.0±0.27
IC50 M= 40.26±1.04
IC50 M= 159.153±4.49
IC50 M= >500
FlavonesFlavones
6060
N
O
O2N
O
F
2-(3-f luorophenyl)-7-nitro-4H -benzo[1,3]oxazin-4-one (30)
N
O
O2N
O
F
2-(4-f luorostyryl)-7-nitro-4H -benzo[1,3]oxazin-4-one (29)
DPPH= InactiveSuperoxide anion= 40.35 M ±1.87
DPPH= InactiveSuperoxide anion= 30.98 M ±1.65
DPPH= InactiveSuperoxide anion= 97.99 M ±2.65
Studied at 500 M in DPPH and SOBHA= 44.0±2.70 /97.0±3.0PG= 30.0±0.27 /104.0±2.4
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Source: Iris unguicularisIsolated by: Dr. Sumaira Hareem
ICIC5050 M= M= 22.45 ± 0.3522.45 ± 0.35
Studied at 500 M in DPPHBHA= 44.0±2.70PG= 30.0±0.27
Xanthone glycosideXanthone glycoside
Studied in DPPHBHA= 44.0±2.70PG= 30.0±0.27
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
04/21/2304/21/23
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 with 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.
Fragment based discoveryFragment based discovery Target identificationTarget identification Lead optimizationLead 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