Bioassay Drug

8/17/2019 Bioassay Drug

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BIOASSAY TECHNIQUES FORBIOASSAY TECHNIQUES FOR

DRUG DISCOVERY ANDDRUG DISCOVERY AND

DEVELOPMENTDEVELOPMENT

International Center for Ce!i"al an# Biolo$i"al S"ien"e% &H' E' (' Re%ear" In%tit)te of Ce!i%tr*

Dr' Pan+,ani Center for Mole")lar Me#i"ine an# Dr)$ Re%ear"-

Uni.er%it* of /ara"i0 /ara"i123425

Dr. Muhammad Iqbal Choudhary

Distinguished National Professor


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Dr)$ Di%"o.er*1Dr)$ Di%"o.er*1

Pa%t an# Pre%entPa%t an# Pre%ent In the past, most drugs were eitherdiscovered by trial and error (traditionalremedies) or by serendipitous discoveries.

Today efforts are made to understand themolecular basis of different diseases and thento use this knowledge to design and develop

specific drugs.

In modern drug discovery process, bioassay

screenings play an extremely important role.


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6at i% Re7)ire# to De.elo8 a Mo#ern6at i% Re7)ire# to De.elo8 a Mo#ern

Dr)$ &NME-9Dr)$ &NME-9

• Decision= Corporate decision to invest in

specific therapeutic area, based on “economicfeasibility”

• Cost= $ 1.4 billion- 1.8 billion• Duration= 10-12 years of !D, and re"ulatory

approval

• #eople= 00-800 scientists of %ulti-

disciplinary e&pertise• Che%ical Diversity' (creenin" of 100,000-

200,000 co%pounds

• )lobal *pproval= +ots of paper ors, based

on often ill-planned studies, and malpractices


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CONTENTCONTENTolecular basis of diseases(ta"es in dru" develop%ent/hy ioassaysDifferent typesclasses of bioassays

Difference beteen bioassay andphar%acolo"ical screenin"s3arious types of bioassaysi"h-throu"hput bioassays-Definitions,

advanta"es and disadvanta"esioactivity directed isolation of naturalproducts- (trate"iesioassay-"uided fractionation 5)67 andisolation


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A Boo: 6ort Rea#in$A Boo: 6ort Rea#in$

ioassay echni9ues for Dru"esearch

By

tta!ur!"ahman, #. I$bal %houdhary and&illiam '. Thomsen

arwood cademic ress, *ondon

http+nad-eeb.wordpress.com//0/10/123/144.pdf


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Di%ea%e%1 Mole")lar Ba%i%Di%ea%e%1 Mole")lar Ba%i%

Overwhelming majority of diseases are caused by change inbiochemistry and molecular genetics of human body(Molecular Pathology)

Over- and under-expression of catalytic proteins (enzymes) oxins produced by microorganisms !iruses (wild "#$%molecular organisms) cause cancers& $'"&

influenza& "engue fever& etc Mutation in "#$ cause cancers Malfunction of signaling pathways cause various disorders *ongenital diseases due to genetic malfunctions Oxidation of biomolecules (proteins& carbohydrates& lipids& nucleic

acid)& degenerative diseases and ageing "eficiency of essential elements& vitamin& nutrients& etc


7/62I Courtesy of #rof. Dr. *:ad ;han


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Main Sta$e% in Dr)$Main Sta$e% in Dr)$

Di%"o.er* an# De.elo8!entDi%"o.er* an# De.elo8!ent 5election of 6isease Target6esigningof 7ioassay

6iscovery and 8ptimi9ation of *ead#olecules

reclinical 5tudies

%linical 5tudies


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6* ,e Nee# to Perfor!6* ,e Nee# to Perfor!

Bioa%%a*9Bioa%%a*9To predict some type of therapeutic potential,either directly or by analogy, of test compounds.

7ioassay is a shorthand commonly used termfor biolo"ical assay and is usually a type of invitro experiments

7ioassays are typically conducted to measurethe effects of a substance on a living organismor other living samples.


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6at i% Bioa%%a*96at i% Bioa%%a*9ioassay or biolo"icalassayscreenin" is any 9ualitativeor 9uantitative analysis of a

substances that uses a livin"syste%, such as an intact cell, as aco%ponent.


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E%%ential Co!8onent% ofE%%ential Co!8onent% of

Bioa%%a*%;A%%a*%Bioa%%a*%;A%%a*%5timulus (Test sample, drugcandidate, potential agrochemical, etc)

5ub-ect (nimal, Tissues, %ells, 5ub!cellular orgenlles, 7iochemicals, etc.)

"esponse ("esponse of the sub-ect tovarious doses of stimulus)


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Molecular Bank at the PCMDO.er


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Bioa%%a*%;A%%a*%Bioa%%a*%;A%%a*%

&hole animals

Isolated organs of vertebrates

*ower organisms e.g. fungi, bacteria,insects, molluscs, lower plants, etc.

%ultured cells such as cancer cells and

tissues of human or animal organsIsolated sub!cellular systems, such asen9ymes, receptors, etc


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T*8e% of Bioa%%a*%9T*8e% of Bioa%%a*%9

In Silico 5creenings:on! physiological ssays7iochemical or #echanisms!7ased ssaysIn Vitro ssays ssays on 5ub!cellular 8rganelles%ell based 7ioassaysEx-Vivo ssaysTissue based 7ioassays

:#" 7ased 6rug 6iscoveryIn Vivo 7ioassaysnimal!based ssaysreclinical 5tudiesuman trial%linical Trials


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Pre#i"tin$ Dr)$ Li:e Bea.ior1Pre#i"tin$ Dr)$ Li:e Bea.ior1

Li8in%:i ?R)le of Fi.e@Li8in%:i ?R)le of Fi.e@

olecular ei"ht about


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Virt)al an#Virt)al an# In SilicoIn Silico

S"reenin$%S"reenin$% *igand based or Target based Target 5election

6ata #ining (%hemical space of over 4/;/ conceivable compounds)

5creening of *ibraries of %ompounds


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Pri!ar* ?Bioa%%a*;A%%a*%@Pri!ar* ?Bioa%%a*;A%%a*%@

S"reenin$%S"reenin$%:on! physiological ssays

7iochemical or #echanism!7asedssays

#icroorganism!based bioassays

%ell!based 7ioassaysTissue!based 7ioassays

#any other In Vitro bioassaysassays


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E>a!8le% of Pri!ar* A%%a*%E>a!8le% of Pri!ar* A%%a*%

Antioxidant AssaysEnyme In!i"ition AssaysCytotoxicty Bioassays Anti-cancer Bioassays #Cancer Cell $ines%Brine S!rim& $et!ality BioassaysIn Vitro Anti&arasitic Bioassays Anti-"acterial Bioassays

Antifungal BioassaysInsecticidal BioassaysP!ytotoxicity BioassaysEtc'


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Salient Feat)re% of Pri!ar*Salient Feat)re% of Pri!ar*

Bioa%%a* S"reenin$%Bioa%%a* S"reenin$% Predictive Potential(eneral in nature

Tolerant of im&urities

)n"iased

*ig!-t!roug!&ut

+e&roduci"le

ast

Cost-effective

Com&ati"le it! .MS/


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Hit Rate of Pri!ar* Bioa%%a*Hit Rate of Pri!ar* Bioa%%a*

S"reenin$%S"reenin$% A !it rate of 01 or less is generally

considered a reasona"lealse &ositive are acce&ta"le

alse negative are discouraged


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Se"on#ar* Bioa%%a*%Se"on#ar* Bioa%%a*%

nimal!based assays (nimal!based assays (In VivoIn Vivo))

Toxicological ssessments in wholeToxicological ssessments in whole

animalsanimals

6#= 5tudies6#= 5tudies

7ehavioral 5tudies7ehavioral 5tudies

reclinical 5tudiesreclinical 5tudies


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I!8ortan"e of Stan#ar#% inI!8ortan"e of Stan#ar#% in

Bioa%%a*%;A%%a*%Bioa%%a*%;A%%a*%

The results of the assaybioassay need tovalidated by monitoring the effect of an

available known compound (5tandard).

&ithout -udicious choice of standard and

its reproducible results in an assaysystem, no screening can be claimedcredible.


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I!8ortan"e of Re8ro#)"iilit*I!8ortan"e of Re8ro#)"iilit*

an# Do%e De8en#en"*an# Do%e De8en#en"*

&ithout reproducible results (within themargin of error or esd), an assay has any

value. It is a share loss of time and efforts.

6ose dependency is the key to a successfuloutcome of study.

&ithout reproducibility and dose dependency,it can be magic, but not science


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!'#+,$'#- $ #ovel $nticancer

"rug from .lowers of ada +ahar


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In VitroIn Vitro Bioa%%a*%Bioa%%a*%

In Vitro2 In ex&erimental situationoutside t!e organisms' Biological orc!emical or3 done in t!e test tu"e# invitro is $atin for 4in glass5% rat!er t!anin living systems

Exam&les include antifungal,

anti"acterial, organ-"ased assays,cellular assays, etc


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E>a!8le% of E>a!8le% of In VitroIn Vitro Bioa%%a*%Bioa%%a*%

*ctivity *ssays•DPPH assay

• Xanthine oxidase inhibition assays

•Superoxide scavenging assay

• Antiglycation assay ioassays 5cell-based7

•DNA evel

•Protein evel

•!NA evel

• "mmunology assay

o&icity *ssays• assay

•Cancer cell line assays


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In VivoIn Vivo Screenings orScreenings or

Pharmacological ScreeningsPharmacological Screenings In Vivo2 Test &erformed in a livingIn Vivo2 Test &erformed in a living

system suc! as antidia"etic assays,system suc! as antidia"etic assays,C6S assays, anti!y&ertensive assays,C6S assays, anti!y&ertensive assays,

etc'etc'


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High-throughput AssaysHigh-throughput Assays

The process of finding a new drugThe process of finding a new drug

against a chosen target for a particularagainst a chosen target for a particular

disease usually involvesdisease usually involves high!throughhigh!through

screening (T5)screening (T5), wherein large libraries, wherein large libraries

of chemicals are tested for their abilityof chemicals are tested for their ability

to modify the target.to modify the target.


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/0-123 well plates (medium throughput) andmore (high-throughput)

"evelopment of straight-forward in-vitro

biological assays (enzyme-based& cellular andmicrobiological assays) into automated high-throughput screens (4)

5apid assays of thousands or hundreds of

thousands of compounds (upto 677&777 samplesper day) pecifically suitable for the isolation of bioactive

constituents from complex plant extracts orcomplex combinatorial library

HIGH1THROUGHPUT BIOLOGICALHIGH1THROUGHPUT BIOLOGICAL

SCREENINGSSCREENINGS


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Hi t t S i St t


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nzyme 8 +uffer

8 Potential inhibitor

/0-well plate

ubstrate

'ncubation

Measurement of absorbance

9 'nhibition : ;(-)%< =77

: $ctivity of enzyme without test material

: $ctivity of enzyme with test material

12

Hi$1tro)$8)t S"reenin$ Strate$*

for En*!e Iniition A%%a*%


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:#" 75=6 5%"==:I:> I::#" 75=6 5%"==:I:> I:


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:#"!75=6 5%"==:I:> I::#"!75=6 5%"==:I:> I:

6"?> 6I5%8 6I5%8


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/1/04;/1/04;

ON1LINE ISOLATION AND BIOASSAYON1LINE ISOLATION AND BIOASSAY

SCREENINGSCREENING>!%!' "*O5

(Photodiode $rray "etector)

P,'5

*45OM$O?5$P4'*

M4O"

.5$*'O# *O,,*O5

+'O$$@

-NMR

-MASS

-IR

-ICP

P*5$, $#"

5>*>5$,

"$$+$

Dictionary of Natural Products,

Bioactive Natural Products

Database, DRP,

NAPRA!R", MARIN!I",

Marine Natural Products

Database, S"N #iles

O#-,'# P*5OM5

Sa$%le

/0-well plates

or

123-well microplate


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Fra$!ent Ba%e# Dr)$Fra$!ent Ba%e# Dr)$

Di%"o.er*Di%"o.er*

hro%bin Anhibitor

A3 #roteaseAnhibitor


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Fra$!ent Ba%e# Dr)$Fra$!ent Ba%e# Dr)$

Di%"o.er*Di%"o.er*

C. *cetylcholinesterase Anhibitor


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>eometric%omplementarity

=lectronic (electrostatic)

%omplementarity AInduced fitB vs. A*ock C DeyB

5tereospecific (en9ymesand substrates are chiral)

5ubstrate 7inding 5pecificity5ubstrate 7inding 5pecificity

fNMR f D R


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NMR for Dr)$ Re%ear"NMR for Dr)$ Re%ear"

1. Detect the eaest li"andBtar"etinteractions even %illi%olar bindin"constants.

2. nables a deter%ination of bindin"constants.

4. *llos direct screenin" anddeconvolution of %i&tures fro% natural

sources or co%binatorial che%istry.


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? is used for fra"%ent baseddiscovery

? is used for tar"et identification ? is used for lead opti%i:ation


NMR f D R NMR f D R


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•#ro%isin" ne %ethod in dru"discovery

•n%atched screenin" sensitivity.

•*bundance of infor%ation about

the structure and nature of%olecular interaction andreco"nition.

Ba%i" De.elo8!ent of NMRBa%i" De.elo8!ent of NMR


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Ba%i" De.elo8!ent of NMRBa%i" De.elo8!ent of NMR

S8e"tro%"o8* for Dr)$ Re%ear"S8e"tro%"o8* for Dr)$ Re%ear"

• Cryoprobe technolo"y hich increasesi"nal-to-noise ratio and loer accessiblebin"in" affinities.

•6lo probe alleviatin" the need for ?tubes and ti%e-consu%in" handlin".

•icro-coil tubes 5%icro- and nano-probes7 reduce the re9uired sa%plevolu%es and also superior f fieldho%o"eneity. hus facilitatin" difference

based ? screenin" %ethods.

E">#=:T 75=6 6"?>E">#=:T!75=6 6"?>


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•Tar$et1 or Re"e8tor1Ba%e# S"reenin$1 Doe% li$an#

intera"t ,it te tar$et * follo,in$ te "an$e% in

te "e!i"al %ift% of tar$et 8roton%9' It o%er.e

an# "o!8are te "e!i"al shifts of tar$et% in tea%en"e an# 8re%en"e of li$an#

• Li$an#1Ba%e#1S"reenin$1 Doe% li$an# i%

intera"tin$ ,it te tar$et * follo,in$ te "an$e%in te NMR 8ara!eter% of li$an# after te a##ition of

te tar$et

E">#=:T!75=6 6"?>E">#=:T!75=6 6"?>

6I5%8


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SQC; QCR t B # HSQC;HMQC


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Re"e8tor Ba%e# HSQC;HMQCRe"e8tor Ba%e# HSQC;HMQC

4D


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2D [1H–15N]-HSQC Expr!m"#Chm!%al &h!'# pr#urba#!o" m#ho

he blac contours correspond to 6;# 5fa%ily ofen:y%es that function as protein foldin" cheprons7, the%acro%olecular tar"et, hereas the red contours

correspond to the co%ple& for%ed by 6;# and

1H (ppm

5t t ti it " l ti hi5t t ti it " l ti hi


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•Adentification of li"ands ith hi"h bindin" affinityfro% library of co%pounds by usin" 2D 1-1


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5" by :#"5" by :#"


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SAR *SAR *

NMRNMR

Use o t!e S"# by $M#approac! or t!edisco%ery o in!ibitors oStromelysins (matri&metaloproteineases'


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Pre-clinical #rialsPre-clinical #rials

Involve in vivo (test tube) and in vivo (animal) experiments using wide!rangingdoses of the study drug to obtain preliminary

efficacy, toxicity and pharmacokineticsinformation.

ssist pharmaceutical companies to decidewhether a drug candidate has scientific meritfor further development as an investigationalnew drug.


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Clinical #rialsClinical #rials

uman Trial%linical Trialsuman Trial%linical Trials

hase I (5afety /!2/


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econdary

+ioassay

oxicity

$ssay'n vivo

$ssay

$nimal

rials$nimal

rials

Pre-chinical

rials

Primary

+ioassay

*hemical

"iversityelected

*hemical

tructure

elucidation

of +ioactive

compounds

tructure

$ctivity

5elation

In silico

creening

Post

MarAeting

urvelience

5egistration

and

MarAeting

*linical

rials

=& ''& '''

,$" - 'dentification,$" -!alidation ,$" -"evelopment

"5>?-"evelopment

arget

'dentification

and !alidation

VARIOUS STAGES IN DRUG

DEVELOPMENT


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BIOASSAY1GUIDEDBIOASSAY1GUIDED


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BIOASSAY GUIDEDBIOASSAY GUIDED

FRACTIONATION &BGF-FRACTIONATION &BGF-

ioassay-"uided fractionation 5)67 of Asolation is the

process in hich natural product e&tract or %i&tures of

synthetic products is chro%ato"raphically fractionated

and re-fractionated until a pure biolo"ically active

constituent5s7 is isolated. *t every sta"e of chro%ato"raphic separation, every

fraction is subFected to a specific bioassay to identify

the %ost active fraction5s7.

nly those fraction5s7 hich are active are further

processed.


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han Gou 3ery uchhan Gou 3ery uch

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Bioassay Drug