High Throughput Screening

HTS is a process of drug development and automation process in drug industry.

1. HIGH THROUGHPUT
SCREENING (HTS)
By
Dr. Debasish Pradhan
Sr. Faculty Pharmacology
University Department of Pharmaceutical
Sciences
Utkal University, Bhubaneswar

2. HIGHTHROUGHPUT SCREENING(HTS)isidentificationofoneor
morepositivecandidatesextractedfromapoolofpossiblecandidates
basedonspecificcriteria.
LEADcompoundisarepresentativeofacompoundserieswithsufficient
potential(asmeasuredbypotency,selectivity,pharmacokinetics,
physicochemicalproperties,toxicityandnovelty)toprogresstoafull
drugdevelopmentprogramme.
Itisadrug-discoveryprocesswidelyusedinthepharmaceutical
industry.
Itallowsautomationtoquicklyassaythebiologicalorbiochemical
activityofalargenoofcompounds.
HTSisprocessbywhichlargenos.ofcompoundsarerapidlytestedfor
theirabilitytomodifythepropertiesofaselectedbiologicaltarget.
Goal is to identify: ‘hits’ or ‘leads’
-affect target in desired manner
-active at fairly low concentrations ( to show specificity)
-new structure

3. Itisausefulfordiscoveringligandsforreceptors,enzymes,ion-
channelsorotherpharmacologicaltargets,orpharmacologically
profilingacellularorbiochemicalpathwayofinterest.
Screening Collection
Screening libraries:
(a)Diversity Libraries
(b)Focused Libraries :
Ligand-Based Approaches
Structure-Based Approaches
Compound acquisition Purchasing commercially available
compounds is an important aspect of screening collection
enhancement

4. LIBRARIES
siRNAlibraries:providingthebestavailableRNA
interferencetechnologywithmaximumflexibilityThemouse
wholegenomecontainabout16872genetargetsandthe
Humanwhole-genomecontains18120genetargets.
microRNAlibraries:HumanlibrariesofsyntheticmicroRNA
mimicsandmicroRNAinhibitors.
SmallCompoundlibraries:Alibraryof640FDAapproved
drugs(Screen-WellFDAApprovedDrugLibrary,EnzoLife
Sciences)isalsoavailableforscreening.Thelibrarycontains
clinically-relevantpharmacophores.

5. Steps in HTS
1 ststage screening
•Test optical clarity, abrasion resistance, and adhesion
•Eliminates ~ 90% of samples
2 ndstage screening
•Test weather ability, integrity, gloss, and surface
smoothness
•~10% of the samples
Rapidly identify coating samples with desired properties
Candidates for scale up
•Test according to the customer’s specification

6. DETECTION METHODS IN HTS
•Spectroscopy
•Mass Spectrometry
•Chromatography
•Calorimetry
•X-ray diffraction
•Microscopy
•Radioactive methods

7. SPECTROSCOPY IN HTS:
•FluorescenceSpectroscopy
•Total internal reflection fluorescence(TIRF)
•Nuclear magnetic resonance(NMR)
•Absorption andluminescence
•Fourier transformedinfrared(FTIR)
•Lightscattering
CHROMATOGRAPHY INHTS:
•Gas chromatography (GC)
•Thin layerchromatography
•Liquid chromatography(HPLC)
•Ion Exchangechromatography
•Reverse phasechromatography
•Hydrophobic interactionchromatography
•Affinitychromatography

8. CALORIMETRY IN HTS:
•Isothermal Titration Calorimetry(ITC)
•Differential Scanning Calorimetry(DSC)
MICROSCOPY IN HTS:
•Scanning TunnellingMicroscopy
•Atomic ForceMicroscopy
•Confocal Microscopy
USES:
To screen Micro arrays suchas:
•DNAchips
•RNAchips
•Proteinchips
•To screen for all kind of novel biological active
compounds
•Naturalproducts

9. METHODOLOGY
TheheartoftheHTSsystemisaplate,ortray,which
consistsoftinywellswhereassayreagentsandsamplesare
deposited,andtheirreactionsmonitored
Theconfigurationoftheplatehaschangedfrom96wells
(inamatrixof8rowsby12columns)to384,andnowtoa
high-density1536-wellformat,whichenableslarge-scale
screening.
Assayreagentsmaybecoatedontotheplatesordeposited
inliquidformtogetherwithtestsamplesintothewells.
Bothsamplesandassayreagentsmaybeincubated,and
thosethatinteractshowsignals,whichcanbedetected.
TheaimofHTSiscosteffectivenessandspeedof
compoundscanning.

10. Cell-basedassayshavebecomeanimportanttestcompared
withotherinvitroassays,astheycanprovideinformationabout
bioavailability,cytotoxicityandeffectsonbiochemicalpathway
Theenzyme-basedandcell-basedassaysystemsconsistof
receptorsormimeticsofreceptors(componentsthatmimic
activepartsofreceptors)
Receptors-Normallytheassaysarelinkedtoanindicatorthat
showstheligands–receptorinteractionassomeformofsignal
Theadvantageofcell-basedassaysoverbiochemicalassaysis
thatcell-basedassaysenabletheanalysisofsamplecompound
activityinanenvironmentthatissimilartotheoneinwhicha
drugwouldact.
Italsoprovidesaplatformfortoxicitystudies.

12. CELL BASED ASSAYS
Cell-basedassaysrefertoanyofanumberofdifferent
experimentsbasedontheuseoflivecells
Thisisageneraldefinitionandcanincludeavarietyofassays
thatmeasurecellproliferation,toxicity,motility,productionof
ameasurableproductandmorphology
Cell-basedassaysofferamoreaccuraterepresentationofthe
real-lifemodelsincelivecellsareused.
ACELL-BASEDASSAYIS:onewherethefundamentalunit
ofexpressionisthecell,eithercellpopulationsorsinglecell.

13. FOURKEYELEMENTSOFCELLBASEDASSAY:
Acellularcomponente.g.acelllineoraprimarycell
population
Atarget(substrate)moleculethatrecordsthecellularresponse
AninstrumenttoconductandmonitortheassayAn
informaticscomponenttomanageandanalysedatafromthe
assay
Cell-basedreporterassaysareusedwherehumanreceptorsare
transfectedintonullcelllineseitheralone,(luciferin-
luciferase)orlighttransmission(melanophore),thatcanbe
measuredindependentlyofradioactivitywithinminutes.

14. ADVANTAGES
Assaysdonotrequirepurificationofthetargetprotein
Canimmediatelyselectagainstcompounds/potential
drugsthataregenerallycytotoxic,orthatcannot
permeatecellularmembranestoreachintracellular
sites
Hit/leadcompoundsidentifiedbycellbasedassayshave
passedimportantvalidationsteps,savingtimeandcosts
indrugdevelopment
Cell-basedassaysvisualizeallpossibledrug-target
interactionse.g.activators,targetinteractions
Highsensitivityofassays&Automation
DISADVANTAGES
HighCost
Lowdatadataquality
Localcontamination
Needofrelativelypureproduct

15. Newer method in HTS
High-Throughput, Fluorescence-Based Screening
(a)For screening, expressed proteins are labelled either as
fusions with green fluorescent protein (GFP) or through
translational incorporation of a fluorescent amino acid
derivative, BODIPY-FL-Lysine.
(a)Using fluorescence detection, the entire procedure can be
carried out in approximately 8 h.
Applications
In drug discovery
Systematic Study of Mitochondrial Toxicity of
Environmental
Chemicals High-Throughput Screening of Dendrimer-
Binding Drugs
To Fractionate Plant Natural Products for Drug Discover

16. CELLULAR COMPONENTS
Different cell lines are being used in cell based assays
Some examples are:
•HUMAN CELL LINES
•DU145, PC3, Lncap (Prostate cancer)
•MCF-7, MDA-MB-438, T47D (Breast cancer)
•THP-1 (Acute Myeloid Leukemia)

17. REFERENCES
Leach AR, Hann MM. The in silico world of virtual libraries. Drug
Discov Today 2000; 5:326–336.
J. AM. CHEM. SOC. 2010, 132, 13182–13184