REVIEWbph_1127 1239..1249Principles of early drugdiscoveryJP
Hughes1, S Rees2, SB Kalindjian3and KL Philpott31MedImmune Inc,
Granta Park, Cambridge, UK,2GlaxoSmithKline, Gunnels Wood
Road,Stevenage, Hertfordshire, UK, and3Kings College, Guys Campus,
London, UKCorrespondenceDr Karen Philpott, HodgkinBuilding, Kings
College, GuysCampus, London SE1 1UL, UK.E-mail:
karen.philpott@kcl.ac.uk----------------------------------------------------------------Keywordsdrug
discovery; high throughputscreening; target identication;target
validation; hit series; assaydevelopment; screening cascade;lead
optimization----------------------------------------------------------------Received2
August 2010Revised7 October 2010Accepted8 November 2010Developing a
new drug from original idea to the launch of a nished product is a
complex process which can take 1215years and cost in excess of $1
billion. The idea for a target can come from a variety of sources
including academic and clinicalresearch and from the commercial
sector. It may take many years to build up a body of supporting
evidence before selectinga target for a costly drug discovery
programme. Once a target has been chosen, the pharmaceutical
industry and morerecently some academic centres have streamlined a
number of early processes to identify molecules which possess
suitablecharacteristics to make acceptable drugs. This review will
look at key preclinical stages of the drug discovery process,
frominitial target identication and validation, through assay
development, high throughput screening, hit identication,
leadoptimization and nally the selection of a candidate molecule
for clinical development.AbbreviationsADME, absorption,
distribution, metabolism and excretion; DMPK, drug metabolism
pharmacokinetics; DMSO,dimethyl sulphoxide; GPCRs,
G-protein-coupled receptors; HTS, high throughput screening; mAbs,
human monoclonalantibodies; PD, pharmacodynamic; PK,
pharmacokinetic; SAR, structureactivity
relationshipIntroductionAdrug discovery programme initiates because
there is adiseaseorclinicalconditionwithoutsuitablemedicalprod-ucts
available and it is this unmet clinical need which is
theunderlyingdrivingmotivationfor the project. The initialresearch,
often occurring in academia, generates data todevelopa hypothesis
that the inhibitionor
activationofaproteinorpathwaywillresultinatherapeuticeffectinadisease
state. The outcome of this activity is the selection of atarget
which may require further validation prior to progres-sion into the
lead discovery phase in order to justify a
drugdiscoveryeffort(Figure 1). Duringleaddiscovery, aninten-sive
searchensues to nd a drug-like small molecule
orbiologicaltherapeutic,typicallytermedadevelopmentcan-didate,
thatwill progressintopreclinical, andifsuccessful,into clinical
development (Figure 2) and ultimately be a mar-keted
medicine.Target identicationDrugs fail in the clinic for two main
reasons; the rst is thatthey do not work and the second is that
they are not safe.
Assuch,oneofthemostimportantstepsindevelopinganewdrug is target
identication and validation. A target is a
broadtermwhichcanbeappliedtoarangeofbiologicalentitieswhich may
include for example proteins, genes and RNA. Agoodtarget
needstobeefcacious, safe, meet clinical andcommercial needs and,
aboveall, bedruggable. Adrug-gable
targetisaccessibletotheputativedrugmolecule, bethat a small
molecule or larger biologicals and upon
binding,elicitabiologicalresponsewhichmaybemeasuredbothinvitroandinvivo.Itisnowknownthatcertaintargetclassesare
more amenable tosmall molecule drugdiscovery, forexample,
G-protein-coupled receptors (GPCRs), whereas anti-bodies are good
at blocking
protein/proteininteractions.GoodtargetidenticationandvalidationenablesincreasedBJPBritish
Journal
ofPharmacologyDOI:10.1111/j.1476-5381.2010.01127.xwww.brjpharmacol.orgBritish
Journal of Pharmacology (2011) 162 12391249 1239 2011 The
AuthorsBritish Journal of Pharmacology 2011 The British
Pharmacological Societycondence in the relationship between target
and disease
andallowsustoexplorewhethertargetmodulationwillleadtomechanism-based
side effects.Dataminingof availablebiomedical datahas
ledtoasignicantincreaseintargetidentication. Inthiscontext,data
mining refers to the use of a bioinformatics approach tonot only
help in identifying but also selecting and prioritiz-ing potential
disease targets (Yang et al., 2009). The datawhich are available
come froma variety of sources butincludepublications andpatent
information, geneexpres-sion data, proteomics data, transgenic
phenotyping and com-pound proling data. Identication approaches
also includeexaminingmRNA/proteinlevelstodeterminewhethertheyare
expressed in disease and if they are correlated with
diseaseexacerbation or progression. Another powerful approach is
tolookfor genetic associations, for example, is there a
linkbetweenageneticpolymorphismandtheriskofdiseaseordiseaseprogressionoristhepolymorphismfunctional.
Forexample, familial Alzheimers Disease (AD) patients
com-monlyhavemutationsintheamyloidprecursorproteinorpresenilin
genes which lead to the production and depositionin the brain of
increased amounts of the Abeta peptide, char-acteristicof
AD(BertramandTanzi, 2008). Therearealsoexamples of phenotypes
inhumans wheremutations cannullify or overactivate the receptor,
for example, the voltage-gated sodium channel NaV1.7, both
mutations incur a painphenotype, insensitivity or oversensitivity
respectively (Yanget al., 2004; Cox et al., 2006).An alternative
approach is to use phenotypic screening toidentifydiseaserelevant
targets. Inanelegant experiment,Kurosawa et al. (2008) used a
phage-display antibody libraryto isolate human monoclonal
antibodies (mAbs) that bind tothe surface of tumour cells. Clones
were individually screenedby immunostaining and those that
preferentially and stronglystained the malignant cells were chosen.
The antigens recog-nized by those clones were isolated by
immunoprecipitationandidentiedby mass spectroscopy. Of 2114 mAbs
withunique sequences they identied 21 distinct antigens
highlyexpressed on several carcinomas, some of which may be
usefultargets for the corresponding carcinoma therapy and
severalmAbs which may become therapeutic agents.Target
validationOnce identied, the target then needs to be fully
prosecuted.Validationtechniquesrangefrominvitrotoolsthroughtheuse
of whole animal models, to modulation of a desired targetFigure
1Drug discovery process from target ID and validation through to
ling of a compound and the approximate timescale for these
processes. FDA,Food and Drug Administration; IND, Investigational
New Drug; NDA, New Drug Application.in vitro & ex vivosecondary
assays(mechanistic)Selectivity & liabilityassaysHTS &
selectivelibrary screens;structure based designReiterative
directedcompound synthesis toimprove compoundpropertiesGenetic,
cellularand in vivoexperimentalmodels to identifyand validate
targetCompoundpharmacologyDisease efficacymodelsEarly safety
&toxicity studiesCandidatesecondary assays In vivo analysis
Compound screeningTargetvalidationPreclinicalsafety &
toxicitypackageEnzyme/Receptor% inhibitionFigure 2Overview of drug
discovery screening assays.BJPJP Hughes et al.1240 British Journal
of Pharmacology (2011) 162
12391249indiseasepatients.Whileeachapproachisvalidinitsownright,
condenceintheobservedoutcomeis signicantlyincreased by a
multi-validation approach (Figure 3).Antisense technology is a
potentially powerful techniquewhich utilizes RNA-like chemically
modied oligonucleotideswhicharedesignedtobecomplimentarytoaregionof
atarget mRNA molecule (Henning and Beste, 2002). Binding
oftheantisenseoligonucleotidetothetargetmRNApreventsbinding of the
translational machinery thereby blocking syn-thesis of the encoded
protein. A prime example of the
powerofantisensetechnologywasdemonstratedbyresearchersatAbbottLaboratorieswhodevelopedantisenseprobestotherat
P2X3 receptor (Honore et al., 2002). When given byintrathecal
minipump, toavoidtoxicities associatedwithbolus injection, the
phosphorothioate antisense P2X3
oligo-nucleonucleotideshadmarkedanti-hyperalgesicactivityintheCompleteFreundsAdjuvant
model, demonstratinganunambiguous role for this receptor in chronic
inammatorystates. Interestingly, after administration of the
antisense oli-gonucleonucleotides was discontinued, receptor
functionand algesic responses returned. Therefore, in contrast to
thegene knockout approach, antisense oligonucleotide
effectsarereversibleandacontinuedpresenceof theantisenseisrequired
for target protein inhibition (Peet, 2003).
However,thechemistryassociatedwithcreatingoligonucleotideshasresultedinmoleculeswithlimitedbioavailabilityandpro-nounced
toxicity, making their in vivo use problematic. Thishas
beencompounded by non-specic actions, problemswithcontrols for
these tools anda lackof
diversityandvarietyinselectingappropriatenucleotideprobes(Henningand
Beste, 2002).Incontrast,
transgenicanimalsareanattractivevalida-tiontoolastheyinvolvewholeanimalsandallowobserva-tionof
phenotypic endpoints toelucidate the functionalconsequence of gene
manipulation. In the early days of
genetargetinganimalsweregeneratedthatlackedagivengenesfunctionfrominceptionandthroughout
their lives. Thiswork yielded great insights into the in vivo
functions of a widerange of genes. One such example is through use
of the P2X7knockout mouse to conrm a role for this ion channel in
thedevelopmentandmaintenanceof
neuropathicandinam-matorypain(Chessell et al., 2005). Inmice
lackingP2X7receptors,inammatoryandneuropathichypersensitivityiscompletelyabsenttobothmechanicalandthermalstimuli,while
normal nociceptive processing is preserved. Thesetransgenic animals
were also used to conrm the mechanismof action for this ablation in
vivo as the transgenic mice wereunable to release the mature
pro-inammatory cytokineIL-1beta from cells although there was no
decit in IL-1betamRNA expression. An alternative to gene knockouts
are geneknock-ins,
whereanon-enzymaticallyfunctioningproteinreplacestheendogenousprotein.Theseanimalscanhaveadifferent
phenotypetoaknockout, for
examplewhentheproteinhasstructuralaswellasenzymaticfunctions(Abellet
al., 2005)
andthesemiceshouldostensiblymimicmorecloselywhathappensduringtreatmentwithdrugs,
thatis,the protein is there but functionally inhibited.More
recently, the desire to be able to make
tissue-restrictedand/orinducibleknockoutshasgrown. Althoughthese
approaches are technically challenging, the
mostobviousreasonforthisistheneedtoovercomeembryoniclethality of
the homozygous null animals. Other reasonsinclude avoidance of
compensatory mechanisms due tochronic absence of a gene-encoded
function and avoidance ofdevelopmental phenotypes. However,
theuseoftransgenicanimals is expensive andtime-consuming. Soinorder
tocircumvent some of these issues, the use of small interferingRNA
(siRNA) has become increasingly popular for target vali-dation.
Double-stranded RNA (dsRNA) specic to the gene tobe silenced is
introduced into a cell or organism, where it
isrecognizedasexogenousgeneticmaterial andactivatestheRNAi pathway.
TheribonucleaseproteinDicer is activatedwhich binds and cleaves
dsRNAs to produce double-strandedfragments of 2125 base pairs with
a few unpaired overhangDiseaseassociation:genetics andexpression
dataOver-expression,transgenics,RNAi,
antisenseRNAComparativegeneticsExpressionprofile: taqman,IHC,
westernblottingTarget id andvalidationLiterature survey&
competitorinformationTool compounds&
bioactivemoleculesMolecularpharmacology ofvariantsAnalysis
ofmolecularsign allingpath waysInteractions:immunoprecip;yeast 2
hybridCell-based and invivo diseasemodelsFigure 3Target ID and
validation is a multifunctional process. IHC,
immunohistochemistry.BJPPrinciples of early drug discoveryBritish
Journal of Pharmacology (2011) 162 12391249 1241bases on each end.
These short double-stranded fragments arecalledsiRNAs.
ThesesiRNAsarethenseparatedintosinglestrands and integrated into an
active RNA-induced silencingcomplex (RISC). After integration into
the RISC, siRNAs base-pair to their target mRNA and induce cleavage
of the mRNA,therebypreventingitfrombeingusedasatranslationtem-plate
(reviewedinCastanottoandRossi, 2009).
However,RNAitechnologystillhasthemajorproblemofdeliverytothe target
cell, but many viral and non-viral delivery
systemsarecurrentlyunderinvestigation(forreviewseeWhiteheadet al.,
2009).Monoclonal antibodies are an excellent target
validationtoolastheyinteractwithalargerregionofthetargetmol-ecule
surface, allowing for better discriminationbetweeneven closely
related targets and often providing higher afn-ity. Incontrast,
small molecules aredisadvantagedbytheneed to interact with the
often more conserved active site
ofatarget,whileantibodiescanbeselectedtobindtouniqueepitopes. This
exquisite specicity is the basis for their lack ofnon-mechanistic
(or off-target) toxicity a major advantageover small-molecule
drugs.However, antibodies cannot cross cell membranes
restrict-ingthetargetclassmainlytocellsurfaceandsecretedpro-teins.
One impressive example of the efcacy of a mAb in vivois that of the
function neutralizing anti-TrkA antibodyMNAC13, which has been
shown to reduce both neuropathicpain and inammatory
hypersensitivity (Ugolini et al.,
2007),therebyimplicatingNGFintheinitiationandmaintenanceofchronicpain.
Finally, theclassictargetvalidationtoolisthesmall
bioactivemoleculethat interactswithandfunc-tionally modulates
effector proteins.More recently, chemical genomics, a systemic
applicationof tool molecules to target identication and validation
hasemerged. Chemical genomics can be dened as the study
ofgenomicresponsestochemicalcompounds.Thegoalistherapid
identication of novel drugs and drug targets embrac-ing multiple
early phase drug discovery technologies rangingfromtarget
identicationandvalidation, over compounddesign and chemical
synthesis to biological testing.
Chemicalgenomicsbringstogetherdiversity-orientedchemicallibrar-ies
and high-information-content cellular assays, along
withtheinformaticsandminingtoolsnecessaryforstoringandanalysing the
data generated (reviewed inZanders et al.,2002). The ultimate goal
of this approach is to provide chemi-cal tools against every
protein encoded by the genome. Theaim is to use these tools to
evaluate cellular function prior tofull investment in the target
and commitment to a screeningcampaignThe hit discovery
processFollowing the process of target validation, it is during the
hitidentication and lead discovery phase of the drug
discoveryprocess that compoundscreeningassays aredeveloped. Ahit
moleculecanvaryinmeaningtodifferentresearchersbutinthisinreviewwedeneahitasbeingacompoundwhichhas
thedesiredactivityinacompoundscreenandwhose activity is conrmed
uponretesting. Avariety ofscreening paradigms exist to identify hit
molecules (seeTable 1). High throughput screening (HTS) involves
thescreening of the entire compound library directly against
thedrugtarget or inamorecomplexassaysystem, suchas acell-based
assay, whose activity is dependent upon the targetbut which would
then also require secondary assays
toconrmthesiteofactionofcompounds(Foxet al., 2006).This screening
paradigm involves the use of complex labora-tory automationbut
assumes noprior knowledge of thenature of the chemotype likely to
have activity at the targetprotein. Focused or knowledge-based
screening involvesselectingfromthechemical librarysmallersubsetsof
mol-eculesthat arelikelytohaveactivityat thetarget
proteinbasedonknowledgeof thetargetproteinandliteratureorpatent
precedents for the chemical classes likely to have activ-ityat
thedrugtarget (Boppanaet al., 2009). This
typeofknowledgehasgivenrise, morerecently,
toearlydiscoveryparadigmsusingpharmacophoresandmolecularmodellingto
conduct virtual screens of compound databases (McInnes,2007).
Fragment screeninginvolvesthegenerationof verysmall molecular
weight compound libraries which arescreened at high concentrations
and is typically accompaniedby the generation of protein structures
to enable compoundprogression(Lawet al., 2009). Finally, a more
specializedfocused screening approach can also be taken,
physiologicalscreening. This is atissue-basedapproachandlooks for
aresponse more aligned with the nal desired in vivo effect
asopposed to targeting one specic molecular component.High
throughput and other compound screens are
devel-opedandruntoidentifymoleculesthat interact withthedrug
target, chemistry programmes are run to improvethe potency,
selectivity and physiochemical properties of themolecule, and data
continue to be developed to support thehypothesis that
interventionat the drugtarget will haveefcacy in the disease state.
It is this series of activities that arethe subject of intense
activity withinthe pharmaceuticalindustry and increasingly within
academia to identify candi-date molecules for clinical development.
Pharmaceuticalcompanies have built large organizations with the
objectiveof identifying targets, assembling compound collections
andtheassociatedinfrastructuretoscreenthosecompoundstoidentifyinitiallyhitmoleculesfromHTSorotherscreeningparadigms
and to optimize those screening hits into
clinicalcandidates.Inrecentyearstheacademicsectorhasbecomeincreasingly
interested in the activities traditionally per-formed within the
lead discovery phase in the pharmaceuti-cal industry.
Academicscientistsarenowformattingassaysfor drug discovery whichare
passedontoacademic drugdiscovery centres for compound screening.
These centres, asexemplied by the NIH Roadmap initiative in the USA
(Frear-son and Collie, 2009), have established compound
libraries,screeninginfrastructureandtheappropriateexpertisetradi-tionallyfoundwithintheindustrial
sectortoscreentargetproteins to identify so-called chemical probes
for use in targetvalidationanddiseasebiologystudies
andincreasinglytoidentify chemical start points for drug discovery
programmes.The success of these efforts has been facilitated by the
transferof skills between the industrial and academic sectors.A
typical programme critical path within the lead discov-ery phase
consists of a number of activities and begins withthe development
of biological assays to be used for the iden-tication of molecules
with activity at the drug target. Oncedeveloped, such assays are
used to screen compound librariesBJPJP Hughes et al.1242 British
Journal of Pharmacology (2011) 162 12391249to identify molecules of
interest. The output of a compoundscreenis typicallytermedahit
molecule, whichhas
beendemonstratedtohavespecicactivityatthetargetprotein.Screening
hits form the basis of a lead optimization chemistryprogramme to
increase potency of the chemical series at theprimary drug target
protein. During the lead discovery, phasemolecules are also
screened in cell-based assays predictive ofthe disease state and in
animal models of disease to charac-terize both the efcacy of the
compound and its likely safetyprole (Figure 2). The following
paragraphs describe in moredetail the requirements and application
of compound screen-ing assays within hit and lead discovery.Assay
developmentInthe recombinant era the majority of assays inuse
withintheindustry rely upon the creation of stable mammalian cell
linesover-expressing the target of interest or upon the
over-expression and purication of recombinant protein to
estab-lishso-calledbiochemical assays althoughinrecent yearsthere
has been an increase in the number of reports describingthe use of
primary cell systems for compoundscreening(Dunneet al., 2009).
Generally, cell-basedassayshavebeenappliedtotarget classes suchas
membranereceptors, ionchannels and nuclear receptors and typically
generate a func-tional read-out as a consequence of compound
activity (Mich-elini et al., 2010). In contrast, biochemical
assays, which havebeenappliedtobothreceptor andenzyme targets,
oftensimply measure the afnity of the test compound for the
targetprotein. Therelativemerits of biochemical andcell-basedassays
have been debated extensively and have been reviewedelsewhere
(Moore and Rees, 2001). Both assay paradigms
havebeenusedsuccessfully to identify hit andcandidate molecules.A
plethora of assay formats have been enabled to
supportcompoundscreening. Thechoiceofassayformatisdepen-dent upon
the biology of the drug target protein, the equip-ment
infrastructure in the host laboratory, the experience ofthe
scientists inthat laboratory, whether aninhibitor oractivator
molecule is sought and the scale of the compoundTable 1Screening
strategiesScreen Description CommentsHigh throughput Large numbers
of compounds analysed in a assaygenerally designed to run in plates
of 384 wellsand aboveLarge compound collections often run by big
pharma butsmaller compound banks can also be run in eitherpharma or
academia which can help reduce costs.Companies also now trying to
provide coverage across awide chemical space using computer
assisted analysis toreduce the numbers of compounds
screened.Focused screen Compounds previously identied as
hittingspecic classes of targets (e.g. kinases) andcompounds with
similar structuresCan provide a cheaper avenue to nding a hit
molecule butcompletely novel structures may not be discovered
andthere may be difculties obtaining a patent position in
awell-covered IP areaFragment screen Soak small compounds into
crystals to obtaincompounds with low mM activity which canthen be
used as building blocks for largermoleculesCan join selected
fragments together to t into thechemical space to increase potency.
Requires a crystalstructure to be availableStructural aided
drugdesignUse of crystal structures to help design molecules Often
used as an adjunct to other screening strategieswithin big pharma.
In this case usually have docked acompound into the crystal and use
this to help predictwhere modications could be added to provide
increasedpotency or selectivityVirtual screen Docking models:
interogation of a virtualcompound library with the X-ray structure
ofthe protein or, if have a known ligand, as abase to develop
further compounds onCan provide the starting structures for a
focused screenwithout the need to use expensive large library
screens.Can also be used to look for novel patent space
aroundexisting compound structuresPhysiological screen A
tissue-based approach fordetermination of the effects of a drug at
thetissue rather than the cellular or subcellularlevel, for
example, muscle contractilityBespoke screens of lower throughput.
Aim to more closelymimic the complexity of tissue rather than just
looking atsingle readouts. May appeal to academic experts indisease
area to screen smaller number of compounds togive a more disease
relevant readoutNMR screen Screen small compounds (fragments) by
soakinginto protein targets of known crystal or NMRstructure to
look for hits with low mM activitywhich can then be used as
building blocks forlarger moleculesUse of NMR as a structure
determining toolNMR, nuclear magnetic resonance.BJPPrinciples of
early drug discoveryBritish Journal of Pharmacology (2011) 162
12391249 1243screen. For examplecompoundscreeningassays at
GPCRshavebeenconguredtomeasurethebindingafnityof aradio- or
uorescently labelledligandtothe receptor, tomeasure guanine
nucleotide exchange at the level of theG-protein,
tomeasurecompound-mediatedchangesinoneof a number of second
messenger metabolites includingcalcium, cAMPor inositiol phosphates
or tomeasure theactivation of downstream reporter genes. Whatever
the assayformat that is selected, it is a requirement that the
followingfactors are considered:1. Pharmacological relevance of the
assay. If available, studiesshould be performed using known ligands
with activity atthe target under study, to determine if the assay
pharma-cology is predictive of the disease state and to show
thattheassayiscapableof identifyingcompoundswiththedesired potency
and mechanism of action.2.
Reproducibilityoftheassay.Withinacompoundscreen-ing environment it
is a requirement that the assay is repro-ducible across assay
plates, across screen days and, withinaprogrammethat
mayrunforseveral years, acrosstheduration of the entire drug
discovery programme.3. Assay costs. Compound screening assays are
typically
per-formedinmicrotitreplates.Withinacademiaorforrela-tivelysmall
numbersof compoundsassaysaretypicallyformatted in 96-well or
384-well microtitre plates whereasin industry or in HTS
applications assays are formatted in384-well or 1536-well microtire
plates in assay volumes assmall as a few microlitires. In each case
assay reagents andassayvolumesareselectedtominimizethecostsof
theassay.4. Assay quality. Assay quality is typically determined
accord-ing to the Z factor (Zhang et al., 1999). This is a
statisticalparameter that in addition to considering the
signalwindowintheassayalsoconsidersthevariancearoundboth the high
and low signals in the assay. The Z factor
hasbecometheindustrystandardmeansofmeasuringassayquality on a plate
bases. The Z factor has a range of 0 to 1;an assay with a Z factor
of greater than 0.4 is consideredappropriatelyrobust for
compoundscreeningalthoughmany groups prefer to work with assays
with a Z factor ofgreater than 0.6. In addition to the Z factor
assay quality isalso monitored through the inclusion of
pharmacologicalcontrols within each assay. Assays are deemed
acceptable ifthe pharmacology of the standard compound(s)
fallswithin predened limits. Assay quality is affected by
manyfactors. Generally, high-quality assays are created
throughimplementing simple assay protocols with few steps,
mini-mizing wash steps or plate to plate reagent transfers
withinthe assay, through the use of stable reagents and
biologi-cals, andthroughensuringthat all theinstrumentationused to
perform the assay is performing optimally. This istypically
achieved through developing quality controlpractices for all items
of laboratory automation (see
http://www.ncgc.nih.gov/guidance/section2.html#replicate-experiment-study-summary-acceptance).5.
Effectsofcompoundsintheassay.Chemicallibrariesaretypicallystoredinorganic
solvents suchas ethanol ordimethyl sulphoxide (DMSO). Thus, assays
needtobecongured that are not sensitive to the concentrations
ofsolvents used in the assay. Typically, cell-based assays
areintolerant tosolvent concentrationsof greater
than1%DMSOwhereasbiochemical assayscanbeperformedinsolvent
concentrations of up to 10%DMSO. Studies are alsoperformed to
establish the false negative and false positivehit rates inthe
assay. If these are unacceptably highthentheassay will need to be
recongured. Finally some consider-ationshouldbe made tothe
screening concentration. Com-pound screening assays for hit
discovery are typically runat110 mMcompoundconcentration. At
theseconcentra-tionscompoundswithactivitiesof upto40
mMcanbeidentied. The test concentration can be varied to
identifycompounds with higher or lower activity.One example of an
HTS technology implemented for theidentication of hit molecules
with activity at GPCRs is theaequorinassay(Stableset al., 2000).
Aequorinisacalcium-sensitivebioluminescent
proteinclonedfromthejellyshAequoreavictorea. Stablemammaliancell
lines havebeencreated transfected to express the GPCR drug target
and theaequorinbiosensoer. For receptors capable of
couplingtoheterotrimeric G-proteins of the Gaq/11 family, ligand
activa-tion results in an increase in intracellular calcium
concentra-tion. Whenaequorinis expressedinthe same cells,
thisincrease in intracellular calciumconcentration is detected as
aconsequenceofcalciumbindingtotheaequorinphotopro-tein,
whichinthepresenceof thecofactorcoelenterazine,results in the
generation of a ash of light that can be detectedwithina microtitre
plate-basedluminometer suchas the Lumi-luxplatform(PerkinElmer,
Waltham, MA, USA). The aequo-rinassay has a very simple protocol
andhas beendevelopedforHTS in 1536-well plate format in assay
volumes of 6 mL and forcompound proling activities in 384-well
plate format.When developing any HTS assay, which can involve
thescreening of several million molecules over several weeks, it
isbestpracticetoscreentrainingsetsofcompoundstoverifythattheassayisperformingacceptably.
Figure 4showsthescreening of a 12 000compoundtraining set against
thehistamineH1receptorexpressedinChinesehamsterovarycells in a
1536-well format HTS assay. The training set is typi-cally run on
two or three occasions to identify the hit rate inthe assay, the
reproducibility of the assay and the false positiveand false
negative hit rates in the assay. Typically,
statisticalpackageshavebeendevelopedtoidentifytheseparameters.When
screened to detect agonist ligands the hit rates in
theaequorinassayaretypicallyless than0.5%of
compoundsscreenedwithastatisticalassaycut-offof5%orlessoftheagonist
signal seenwitha standard agonist ligand. Inthis assayformat false
positive and false negative hit rates are very low.For antagonist
screening the hit rate in the aequorin assay istypicallyof 23%of
compoundsscreenedwithanactivitycut-off of greater
than25%inhibition. This is acommonphenomenon of all screening
assays. Hit rates in antagonist
orinhibitorformattendtobehigherthanhitratesinagonistassays as
antagonist assays, which are dened by detection
ofadecreaseinassaysignal, will alsodetectcompoundsthatinterfere
insignal generation. Following completionof robust-ness testing an
assay moves into HTS. During HTS, up to 200assay plates are
screened each day, often using complex
labo-ratoryautomation.Duringthescreen,assayperformanceismeasuredaccordingtotheZ
ontheassayplateandthevariance in the pharmacology of a standard
compound, withBJPJP Hughes et al.1244 British Journal of
Pharmacology (2011) 162 12391249assay plates being failed and
rescreened if these quality controlmeasures fall outside predened
limits (Figure 5).Dening a hit
seriesCompoundlibrarieshavebeenassembledtocontainsmallmolecularweightmoleculesthatobeychemicalparameterssuchastheLipinskiRuleofFive(Lipinskiet
al.,2001),andmore oftenhave molecular weights of less
than400andclogP(ameasureof lipophilicitywhichaffects
absorptionintothebody)oflessthan4.Moleculeswiththesefeatureshave
been termed drug-like, in recognition of the fact
thatthemajorityofclinicallymarketeddrugshaveamolecularweight of
less than350andacLogPof less than3. It
iscriticallyimportanttoinitiateadrugdiscoveryprogrammewith a small
simple molecule as lead optimization, toimprove potency and
selectivity, typically involves anincrease in molecular weight
which in turn can lead to safetyandtolerabilityissues.Figure
4Aequorin high throughput screening: validation testing GPCR
antagonist assay (1536-well). Assay validation of a GPCR drug
screening assay forthe identication of agonist and antagonist
ligands. Cells expressing the histamine H1 receptor and the
calcium-sensitive photoprotein aequorinwere dispensed into
1536-well microtitre plates. A total of 12 000 compounds were
screened in duplicate to detect agonist ligands (left
panel)andantagonist ligands(right panel). Intheagonist assay(left
panel), nodrugresponseisrepresentedinred,
theresponsetoamaximalconcentration of the ligand histamine in blue
and compound data in yellow. As is typically seen in agonist
assays, the hit rate is very low due tothe absence of false
positives. In the antagonist assay (right panel), the response to
histamine in the absence of test compound is represented inred
(basal response), the response to a maximal concentration of a
histamine antagonist in blue (100% inhibition) and compound data in
yellow.As is typically seen in a cell-based inhibitor assay, there
is signicant spread of the compound data due to a combination of
assay interference andcompound activity. True actives correlate in
the range 40% to 100% inhibition. Both assays have excellent Z.
GPCR, G-protein-coupled receptor.Figure 5Quality control (QC) in
high throughput screening. To ensure the control of screening data
in compound screening campaigns each assay platetypically contains
a number of pharmacological control compounds. (A) Each 384-well
plate contains 16 wells containing a low control and afurther 16
wells containing an EC100 concentration of a pharmacological
standard which are used to calculate the Z factor (reference
Zhanget al., 1999). PlatesthatgenerateaZ
factorbelow0.4arerescreened. (B)Eachplatealsocontains16wellsof
anEC50concentrationof apharmacological standard to monitor the
variance in the assay (diamonds). (C) A heat map is generated for
all plates that pass the pharmacologicalstandard QC to monitor the
distribution of activity across the assay plate. One would expect
to see a random distribution of activity across thescreening plate.
A plate such as the one presented would be failed and rescreened
due to the active wells clustering in the centre of the
plate.BJPPrinciples of early drug discoveryBritish Journal of
Pharmacology (2011) 162 12391249
1245Onceanumberofhitshavebeenobtainedfromvirtualscreening or HTS,
the rst role for the drug discovery team
istotrytodenewhichcompoundsarethebesttoworkon.Thistriagingprocessisessential
as, fromalargelibrary,
ateamwilllikelybeleftwithmanypossiblehitswhichtheywill need to
reduce, conrm and cluster into series. There areseveral steps to
achieving this. First, although this is less of aproblemas the
quality of libraries have improved,
com-poundsthatareknownbythelibrarycuratorstobetobefrequent hitters
in HTS campaigns need to be removed fromfurther consideration.
Second, anumber of computationalchemistry algorithms have
beendevelopedto grouphitsbased on structural similarity to ensure
that a broad spectrumof chemical classes are represented on the
list of compoundstaken forward. Analysis of the compound hit list
using thesealgorithms allows the selection of hits for progression
basedon chemical cluster, potency and factors such as ligand
ef-ciency which gives an idea of how well a compound binds forits
size (log potency divided by number of heavy atoms i.e.non-hydrogen
atoms, in a molecule).Thenext phaseintheinitial renement process is
togenerate doseresponse curves in the primary assay for eachhit,
preferably with a fresh sample of the compound.Showing normal
competitive behaviour in hits is
important.Compoundswhichgiveanallornothingresponsearenotacting in a
reversible manner and indeed may not be bindingto the target
protein at all, with the activity at high concen-trations arising
from an interaction between the sample andanother component of the
assay system. Reversible com-pounds are favoured because their
effects can be more
easilywashed-outfollowingdrugwithdrawal,animportantcon-sideration
when using in patients. Obtaining a doseresponsecurve allows the
generationof ahalf maximal inhibitoryconcentration which is used to
compare of the potencies
ofcandidatecompounds.Sourcingandusingfreshsamplesofcompounds for
this exerciseis highlydesirable.
NearlyallHTSlibrariesarestoredasfrozenDMSOsolutionswiththeresult
that, after some time, the compound canbecomedegraded or modied.
Virtually anyone who has worked withlibraries of this typehas got
anecdotes about howpotentactivity has disappeared when the compound
was resynthe-sizedandusedinre-testing,
althoughoccasionallyidenti-cation of potent impurities has allowed
progress to be made.With reliable doseresponse curves generated in
theprimaryassayforthetarget,thestageissettoexaminethesurvivinghitsinasecondaryassay,
if oneisavailable, forthetarget of choice. This neednot
beanassayinahighthroughputformatbutwillinvolvelookingattheaffectofthecompoundsinafunctional
response, forexampleinasecond messenger assay or in a tissue-or
cell-based bioassay.Activity in this setting will give reassurance
that compoundsare able to modulate more intact systems rather than
simplyinteractingwiththeisolatedandoftenengineeredproteinused inthe
primary assay. Throughout the conrmationprocess, medicinal chemists
would be looking to cluster com-pounds into groups which could form
the basis of lead series.As part of this process, considerationwill
begiventotheproperties of each cluster such as whether there is an
identi-ablestructureactivityrelationship(SAR) evolvingover
anumberofcompounds, thatis,
identicationofagroupofcompoundswhichhavesomesectionorchemicalmotifincommonandtheadditionof
different chemical groupstothis core structure results indifferent
potencies. Issues ofchemical synthesis wouldalsobeexamined. Thus,
easeofpreparation, potentialamenabilitytoparallelsynthesisandthe
ability to generate diversity from late-stage intermediateswould be
assessed.Withdenedclustersinplaceanexercisecannowtakeplace on
several groups of compounds in parallel. This phasewill
includetherapidgenerationof rudimentarySARdataanddeningtheessential
elementsinthestructureassoci-ated with activity. At the same time,
representative examplesof each of these mini-series will be
subjected to various in vitroassays designed to provide important
information withregard to absorption, distribution, metabolism and
excretion(ADME)propertiesaswellasphysicochemicalandpharma-cokinetic
(PK) measurements (see Table 2). Selectivity
prol-ing,especiallyagainstthetypesoftargets,ifany,forwhichthe
compounds were originally made, is also useful to carryout at this
time. For example you may want to inhibit kinaseX but avoid kinase
Y to reduce unwanted in vivo side effects.This exercise will reveal
the strengths and aws of each
seriesandallowadecisiontobetakenaboutthemostpromisingseries of
compounds to be progressed. The numbers of seriestaken forward at
this stage will depend on the resource
avail-ablebutideallyseveralshouldbetakenintothehit-to-leadstage to
allow for attrition in the coming
phase.Whateverthescreeningparadigm,theoutputofthehitdiscovery phase
of a lead identication programme is aso-called hit molecule,
typically with a potency of 100 nM5 mM at the drug target. A
chemistry programme is initiatedto improve the potency of this
molecule.Hit-to-lead phaseThe aim of this stage of the work is to
rene each hit series totry to produce more potent and selective
compounds whichpossessPKpropertiesadequatetoexaminetheirefcacyinany
in vivo models that are
available.Typically,theworknowconsistsofintensiveSARinves-tigationsaroundeachcorecompoundstructure,
withmea-surements being made to establish the magnitude of
activityandselectivityofeachcompound.
Thisneedstobecarriedoutsystematicallyand, wherestructural
informationaboutthetargetisknown,structure-baseddrugdesigntechniquesusing
molecular modelling and methodologies such as X-raycrystallography
and NMR can be applied to develop the
SARfasterandinamorefocusedway. Thistypeofactivitywillalso often
give rise to the discovery of new binding sites onthe target
proteins.Ascreeningcascadeatthistimewouldgenerallyconsistof a
relatively high throughput assay establishing the activityof
eachmolecule onthe molecular target, together withassays in the
same format for sites where selectivity might
beknown,orexpectedtobe,anissue(Figure 6).Acompoundmeeting basic
criteria at this stage would be escalated into afurther bankof
assays. Theseshouldincludehigher orderfunctional investigations
against themolecular target andalso whether the compounds were
active in primary assays indifferentspecies.
TheHTSassayisgenerallycarriedoutonproteinencodedbyhumanDNAsequences
but as animalBJPJP Hughes et al.1246 British Journal of
Pharmacology (2011) 162 12391249models are used to validate the
activity of compounds in invivo disease models, in pharmacodynamic
(PD)/PK modellingand in preclinical toxicity studies, it is
important to have dataonactivityinvitroonorthologues.
Thisisalsoparticularlyimportant as it will assist
inminimizingdosinglevels intoxicology studies which are chosen on
the basis multiples ofthe pharmacologically effective
doses.Attention in this phase has to also turn to more
detailedprolingof physicochemical
andinvitroADMEpropertiesandthisseriesofstudiesiscarriedoutinparallel,withkeycompounds
being selected for assessment. The sort of assaystobeconsidered,
withtargetsthat havebeenfoundtobeappropriate are shown in Table
2.Solubility and permeability assessments are crucial
inrulinginoroutthepotentialofacompoundtobeadrug,thatis,drugsubstanceoftenneedsaccesstoapatientscir-culation
and therefore may be injected or more generally hasto be adsorbed
in the digestive system. Deciency in one
orotherparameterinamoleculecansometimesbeputright.Forexampleformulationstrategiescanbeusedtodesignatablet
such that it dissolves in a particular region of the gut ata pH in
which the compound is more soluble. A compoundthat lacks both these
properties is very unlikely to become adrugnomatter howpotent it is
intheprimaryscreeningassay. Microsomal stability is a useful
measure of the ability
ofinvivometabolizingenzymestomodifyandthenremoveacompound.
Hepatocytesaresometimesusedinthissortofstudyinsteadandthesewillgivemoreextensiveresultsbutare
not used routinely as they need to be prepared freshly onaregular
basis. CYP450inhibitionis examinedas,
amongotherthings,itisanimportantpredictorofwhetheranewcompound
might have an inuence on the metabolism of anexisting drug with
which it may be co-administered.If one or more of these properties
is less than ideal, thenit might be necessary to screen many more
compounds spe-cicallyfor thoseproperties. Eachprogrammewill
endupsubtlydifferent inthis regard. For exampleinonerecentproject
toidentifynovel GPCRantagonists, a number ofsub-micromolar hit
compounds wereidentied. Themainissues associated with these
molecules was that they showedsome speciationwithpoorer receptor
afnities inrodentTable 2Key in vitro assays in early drug
discoveryAssays Targetvalue CommentsAqueous solubility >100 mM
Important for running in vitro assays and for in vivo delivery of
drugLog D7.403 (for BBB penetration ca 2) A measure of
lipophilicity hence movement across membranesMicrosomal stability
Clint10 mM Main enzymes in body which metabolize drugs and their
inhibitioncan cause toxicityCaco-2 permeability Papp>1
10-6cm-1(asymmetry 10 10-6cm-1(asymmetry 50% inhibition at10 mMat
30out of 63GPCRsandtransporterstestedinacross-screening panel as
well as
broadCYP450inhibitoryactivity.Itwasfeltthatanumberofthesedeciencieswereassociatedwiththenatureof
thebasecommontoall theinitial structures. Modication of the basic
residue resulted ina number of compounds which were as potent as
the initialhits at the principal receptor but which were more
selective intheir actions. In common with many programmes,
aspotency at the principal target improved selectivity issues
inthis series were left
behind.Keycompoundswhicharebeginningtomeetthetargetpotency and
selectivity, as well as most of the physicochemi-cal and ADME
targets, should be assessed for PK in rats. Hereone would normally
be aiming for a half-life of >60 min whenthe compound is
administered intravenously and a fraction inexcess of 20% absorbed
following oral dosing although some-times,
differenttargetsrequireverydifferentPKproles. Inlarge pharma with
inhouse drug metabolism pharmacokinet-ics (DMPK) departments
numerous compounds might be pro-led while in academic environments
there may be funds
foronlyapredenednumberoftheseexpensiveinvestigationsAs the receptor
antagonist programme, described
above,advancedthroughthehit-to-leadphase, anumberofcom-pounds were
prepared which had potency in the nanomolarrange and a benign
selectivity prole except for some potencyat the hERG channel, a
potassium voltage-gated ion
channelimportantforcardiacfunctionandinhibitionatwhichcancause
cardiac liability. Ideally for hERG we were aiming for anactivity
over 30 uM or at least a 1000-fold selectivity for thetarget. A
number of these compounds were examined in PKstudies and were found
to have a reasonable half-life follow-ingintravenousdosingbut poor
plasmalevelswerenotedwhen the compound was given orally to rats. It
was felt thatsome of these compounds, representing the end of the
hit-to-lead phase of the project were, although not likely
themselvestobeprogressed,
capableofansweringquestionsindiseasemodels. Thus, compounds were
administered intra-peritoneally and results from the experiments
gave substan-tial credence to the developing programme.Lead
optimization
phaseTheobjectofthisnaldrugdiscoveryphaseistomaintainfavourable
properties in lead compounds while improving
ondecienciesintheleadstructure.Continuingwithexampleabove, theaimof
theprogrammewasnowtomodifythestructure tominimize
hERGliabilityandtoimprove theabsorptionofthecompound. Thus,
moreregularchecksofhERG afnity and CACO2 permeation were undertaken
andcompounds were soon available which maintained theirpotency and
selectivity at the principal target but which hada much reduced
hERG afnity and a better apparent
perme-ationthaninitialleadcompounds. WhenexaminedforPKproperties in
rat one of these compounds, with 8 nM afnityat the receptor of
interest, had an oral bioavailability of over40% in rats and about
80% in dogs.Compounds at this stage may be deemed to have met
theinitial goals of the lead optimization phase and are ready
fornal characterization before being declared as preclinical
can-didates. Discovery work does not cease at this stage. The
teamhas to continue to explore synthetically in order to
producepotential back up molecules, in case the compound
undergo-ingfurtherpreclinical orclinical
characterizationfailsand,more strategically, to look for follow-up
series.Thestageatwhichthevariouselementsthatconstitutefurther
characterization are carried out will vary fromcompany to company
and parts of this process may be incor-porated into the lead
optimization phase. However,
ingeneralmoleculesneedtobeexaminedinmodelsofgeno-toxicity such as
the Ames test and in in vivo models of generalbehaviour such as the
Irwins test. High-dose pharmacology,PK/PD studies, dose linearity
and repeat dosing PK looking fordrug-induced metabolism and
metabolic proling all need tobecarriedoutbytheendof thisstage.
Considerationalsoneedstobegiventochemicalstabilityissuesandsaltselec-tion
for the putative drug
substance.Alltheinformationgatheredaboutthemoleculeatthisstage will
allowfor the preparationof a target candidateprole which with
together with toxicological and chemicalmanufacture and control
considerations will form the basis
ofaregulatorysubmissiontoallowhumanadministrationtobegin.The
process of hit generationtopreclinical candidateselection often
takes a long time and cannot in any way beconsidered a routine
activity. There are rarely any short cutsand signicant,
intellectual input is required from scientistsfrom a variety of
disciplines and backgrounds. The quality
ofthehit-to-leadstartingpointandtheexpertiseoftheavail-able team
are the key determinants of a successful outcome ofthis phase of
work. Typically, within industry for each project200 000 to
>106compounds might be screened initially
andduringthefollowinghit-to-leadandleadoptimizationpro-grammes 100s
of compounds are screened to hone down toone or two candidate
molecules, usually fromdifferentchemical series. In academia
screens are more likely to be ofa focused nature due to the high
cost of an extensive HTS orcompounds are derived froma
structure-based approach.Only 10% of small molecule projects within
industry
mightmakethetransitiontocandidate,failingatmultiplestages.These
caninclude the (i) inabilitytocongure a reliableassay; (ii)
nodevelopablehitsobtainedfromtheHTS; (iii)compounds do not behave
as desired in secondary or nativetissue assays; (iv) compounds are
toxic in vitro or in vivo;
(v)compoundshaveundesirablesideeffectswhichcannot beeasily screened
out or separated from the mode of action ofthe target; (vi)
inability to obtain a good PK or PD prole inline with the dosing
regeme required in man, for example, ifrequire a once a day tablet
then need the compound to havea half-life in vivo suitable to
achieve this; and (vii) inability tocross the blood brain barrier
for compounds whose target lieswithinthe central nervous system.
The attritionrate forproteintherapeutics,
oncethetargethasbeenidentied, ismuch lower due to less off target
selectivity and prior expe-rience of PK of some proteins, for
example, antibodies.Although relatively less costly than many
processescarriedout later oninthedrugdevelopment andclinicalphases,
preclinical activity is sufciently high risk and remotefromnancial
returntooftenmakefundingit aproblem.Ensuring transparency of the
cost of each stage/assay withinlarge pharma may help reduce some of
their costs and thereBJPJP Hughes et al.1248 British Journal of
Pharmacology (2011) 162
12391249aresomemovementstowardsthisascompaniesinstigateabiotech
mentality and accountability for costs.Onceacandidateisselected,
theattritionrateof com-poundsenteringtheclinical phaseisalsohigh,
againonlyone in 10 candidates reaching the market but at this stage
thenancial consequences of failure are much higher. There
hasbeenconsiderabledebateinindustryastohowtoimprovethe success
rate, by failing fast and cheap. Once a candidatereaches the
clinical stage, it can become increasingly difculttokill
theproject, asat thisstagetheproject hasbecomepublic knowledge and
thus termination can inuence
con-denceinthecompanyandshareholdervalue.Carryingoutmore studies
prior to clinical development such as improvedtoxicology screens
(using failed drugs to inform these assays),establishing predictive
translational models based on a thor-ough disease understanding and
identifying biomarkers mayhelpinthisendeavour.
Itisparticularlyintheselatertwoareas where academic-industry
partnerships could really
addvaluepreclinicallyandeventuallyhelpbringmoreeffectivedrugs to
patients.AcknowledgementsKaren Philpott is supported by the Medical
Research Counciland Guys and St Thomas Charity.S. Barret Kalindjian
is supported by a Seeding Drug DiscoveryWellcome Trust grant.Conict
of interestJane Hughes is employed by MedImmune, Steve Rees
isemployed by GSK and Karen Philpott was previouslyemployed by
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