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Chapter

PROTEIN‐LIGANDINTERACTIONS

GeoffHoldgate

Contents 4.1.  INTRODUCTION......................................................................................................................................167 

4.2.  OVERVIEWOFDRUGDISCOVERY..................................................................................................168 

4.3.  PROTEIN–LIGANDBINDINGEQUILIBRIA,THERMODYNAMICSANDKINETICS.....169 

4.4.  NON‐COVALENTINTERACTIONSINPROTEIN–LIGANDBINDING.................................172 4.4.1.  Hydrogenbonding...................................................................................................................173 4.4.2.  Ionicorelectrostaticinteractions.....................................................................................173 4.4.3.  VanderWaalsinteractions..................................................................................................173 4.4.4.  Hydrophobicinteractions.....................................................................................................173 

4.5.  BIOPHYSICALMETHODSFORCHARACTERISINGPROTEIN–LIGANDINTERACTIONS.......................................................................................................................................174 4.5.1.  Isothermaltitrationcalorimetry(ITC)...........................................................................174 4.5.2.  SurfacePlasmonResonance(SPR)...................................................................................175 4.5.3.  Opticalwaveguidegrating(OWG)....................................................................................178 4.5.4.  Spectroscopicmethods–NuclearMagneticResonance(NMR)..........................178 

4.5.4.1.  2DNMRchemicalshiftmapping......................................................................179 4.5.4.2.  1DNMRdirectbindingandreferencedisplacement..............................179 

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4.5.5.  Spectrometricmethods–MassSpectrometry(MS).................................................180 4.5.5.1.  Directdetectionoftheligand............................................................................180 4.5.5.2.  Detectionoftheprotein‐ligandcomplex.....................................................181 

4.6.  BRIEFCASESTUDIESEXEMPLIFYINGTHEUSEOFBIOPHYSICALMETHODS.........181 4.6.1.  DNAgyrase.................................................................................................................................181 4.6.2.  Beta‐siteAPPcleavingenzyme1(BACE‐1).................................................................183 4.6.3.  MEKproteinkinase.................................................................................................................184 

4.7.  FUTURETRENDSFORBIOPHYSICALAPPROACHES.............................................................185 

4.8.  SUMMARY.................................................................................................................................................186 

REFERENCES......................................................................................................................................................186 

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4.1. INTRODUCTION

The quest to discover or develop new medicines to facilitate healthcaremanagement for a growing and ageing global population is a long and difficulttask.Althoughknowledgeof thehumangenomeandtheproteinsencodedby ithashelped to identifynewdrug targets,ourunderstandingofproteins, ligandsandtheircomplexesisstillrelativelysuperficial.Proteinsarethemostcommonmolecular target of drugs, since they play an essential role in cellular activity.Understanding protein‐ligand interactions, whether the protein is an enzyme,transporter, ion channel, or G‐protein coupled receptor (GPCR), orwhether itsligandisanaturalligand,substrateordrugmoleculeisthereforeoffundamentalimportanceacrossmanybiological,biochemical,andbiophysicaldisciplines.

Asrecognised in the late19th [1]andearly20th [2,3]centuries, inorder for theeffectiveoccurrenceofcellularprocessesanddrug‐mediatedinterventions,theseprocessesrelyuponligandbindingtotheproteintargettoformacomplex.Thesecellularproteins,whicharethetargetsfordrugmoleculesaretermedreceptors.Thetermreceptorsisnowmorecommonlyusedforasubsetofproteinsthatactasintracellularbindingpartnersforchemicalmessengers,andforclaritywithinthis chapter we will continue to refer to protein‐ligand interactions (althoughthesemaybe interchangedwith terminology suchas receptor‐ligand, receptor‐drug,etc.).

The process termed molecular recognition by which the protein and ligandinteract is dependent upon factors such as surface complementarity,hydrophobicity, andelectrostatics.Adetailedunderstandingof theseprinciplesat a microscopic and macroscopic level is required in order to begin tounderstandhowtorationallydesignnewdrugmolecules.

Thus, to fully understand protein–ligand interactions and to utilise thisinformation tomakean impact ondrugdiscoveryandhealthcare requires thatthebiophysicalpropertiesoftheprotein,ligandandtheprotein–ligandcomplexbeinvestigated.

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4.2. OVERVIEWOFDRUGDISCOVERY

Thegoalofdrugdiscovery is toutilisetheknowledgeofandinterplaybetweenchemistry, biology and pharmacology in order to design and develop newmedicines.Understandingandmodifyinginteractionsbetweenligands,designedduring the drug discovery process, and both target and other proteins arecriticallyimportantstepsthatmusttakeplaceinordertoprogressprojects.

Historically, the approach to discovering drug molecules targeting proteinsinvolved in disease mechanisms was to investigate compounds isolated fromvariousnaturalsources.Overall,thesecompoundsareinvolvedinthetreatmentofmanyhumandiseases [4]. This important startingpoint has provided a richsource of drugs including chemotherapy agents (e.g. paclitaxel), immuno‐suppressive drugs (e.g. cyclosporins) and cholesterol lowering drugs (e.g. thestatins).Moderndrugdiscovery,however,utilisesboththeskillofthemedicinalchemist to provide creative solutions to improve compound properties, alongwith genomics and proteomics approaches (5) to identify proteins involved inrelevantbiologicalpathways.

Targetselectionisachievedfromacombinationoftheunderstandingofbiologicalprocesses and molecular approaches aimed at identifying protein targetmodulationwhichcouldprovideclinicalbenefitsintherelevantdiseasesetting[6].

Most targets are proteins, with most drugs being small molecular compoundswhich interact physically with the target and alter its biological function. Bio‐logicalunderstandingwastraditionallythemaintargetdiscoverystrategy,anditremains important for diseases in which the relevant phenotype can only bedetectedat thewholebody level, suchasatherosclerosis,obesity,heart failure,hypertensionandneurodegenerativedisorders.Molecularapproachesaredrivenbytechnologiesthatattempttocorrelatemolecularchangeswithhumandisease,suchaschangesingeneexpression(genomics),proteinexpression(proteomics)orgeneticvariation.Theabsenceormutationof aparticulargenecanresult inserious disease or the risk of contracting a disease, examples include the linkbetween apolipoprotein A and cardiovascular disease, or BRCA 1 and 2 andbreastandovariancancer.Followingtheselectionof theproteintarget,severalapproaches are taken for hit and lead identification. These include in silicomethods, combinatorial chemistry and high‐throughput screening utilising theresultantcorporatecompoundlibraries.Hitsare identifiedasthosecompoundsshowing activity in the assay. The lead identification process is designed toidentify several structurally distinct chemical series that produce the desiredpharmacologicaleffects,haveacceptabledruglikepropertiesorthepotentialforoptimisation and are patentable. Another valuable source of leads is fromcompetitor compounds, where the aim is to identify groups from those com‐pounds,whichmay bemodified to improve physicochemical properties,whilstavoiding competitor patent restrictions. This approach can lead to dramaticimprovementsinbiologicalandpharmacokineticprofiles,andisreflectedinthe

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quotefromNobelPrizewinnerSirJamesBlack(1924–2010):“Themostfruitfulbasisforthediscoveryofanewdrugistostartwithanolddrug”.

It is during this lead optimisation stage that structure‐activity relationships(SARs)aredevelopedwithrespect topotency for the targetprotein.Selectivityconsiderations,detailingwhichotherproteinsthe ligandmaybindto,andtheirrelative affinities, also are investigated. It is also at this stage that drugmetabolismandpharmacokinetics(DMPK)investigationsbegin.

During lead optimisation, 2‐4 compounds that meet the candidate drug targetprofileareidentifiedbyapplyingmedicinalchemistrytoproduceanaloguesofalead series [7]. It is thus here, that these lead compounds are optimised withrespecttopotency,selectivity,DMPKandchemistryscaleupisbegun.

Atthesametime,theclinicalstrategyisdefined,andmarkersofdrugactionanddiseaseeffectarecharacterised.

Onceasuitabledrugcandidateisfoundpreclinicaltestingbegins,wheretheaimis to evaluate safety, toxicology, metabolism and pharmacokinetics of thedeveloped compound. This will involve in vitro, in vivo and ex vivo testing asrequiredbytheregulatoryauthorities.

Clinical trialsarethenrequired,butbeforeuseadrugneedstobeapprovedbyeithertheFoodandDrugAdministration(FDA)ortheEuropeanAgencyfortheEvaluationofmedicinalProducts(EMEA).Duringthefirstpartofthesetrials,thecandidatedrugisadministeredtohealthyvolunteers(PhaseI),inordertoassessdrug safety, possible side effects and to gather basic pharmacokineticinformation.Duringthenextstage(Phase II), thedrug isgiventopatientswiththedisorderforwhichthecandidatedrugisaproposedtreatment.Itisherethatthe dosage will be evaluated and preliminary data on efficacy in patientscollected. Subsequently, the drug will be given to a larger number of patients(PhaseIII),allowingthedrugtobecomparedtoexistingtherapiesandenablingstatisticstobecollectedonanyadversereactions[8].

4.3. PROTEIN–LIGANDBINDINGEQUILIBRIA,THERMODYNAMICSANDKINETICS

Assuming that theprotein,P,hasasinglebindingsite for the ligand,L,and thebindinginteractionisareversiblebimolecularreaction:

on

offP+L PL

k

k

We can describe the total protein, [P]t, and total ligand, [L]t, concentrations intermsofapairofmassconservationequations:

[P]t=[PL]+[P]f

[L]t=[PL]+[L]f

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where, [P]f, and, [L]f, are the concentrations of free protein and ligand,respectively.

Wealsocanthendescribethechange inGibbsfreeenergy(G) forthebindingreactionbytheequation:

f f

[PL]ln[P] [L]

G G RT

where G° is the standard free energy change, R is the gas constant(8.31Jmol‐1K‐1), and T is the temperature in Kelvin. At equilibrium, whereG=0,thentheequationbecomes:

f f

[PL]ln[P] [L]

G RT

If we represent the ratio of the reactants and product concentrations as theequilibriumdissociationconstantKd=[P]f[L]f/[PL],thenweobtain:

dlnG RT K

Theequilibriumdissociationconstant ismostoftenusedinbiochemicalstudiesbecauseithasunitsofmolarity,andsomayberelatedtotheligandconcentrationleadingtohalfmaximalsaturationoftheprotein.Itdescribesthestrengthoftheinteraction betweenprotein and ligand,with lower values representing tighterbinding.

BecausetheG°maybedescribedbythechangeinstandardenthalpy(H°)andstandardentropy(S°),theseparameterscanalsoberelatedtoKd:

ln dG H T S RT K

The enthalpy represents the energy reduced by so called volume work of areactionatconstantpressure,andtheentropyrepresentsthedegreeofdisorderintroduced by the energy being distributed over the number of accessibledegreesoffreedom.Wecanseethatforaspontaneousbindinginteraction,whereG° is negative, that a decrease in enthalpy and an increase in entropy arefavourablecontributionstoanincreaseinbindingaffinity.

Thus, it can be seen that the equilibriumdissociation constant is derived fromthermodynamic principles and can be related to fundamental thermodynamicparameters. A Kd value of 1 nM, which is a value commonly observed foroptimised leadsorevencandidatedrugs,correspondstoaGibbs freeenergyofbindingof ‐53.4kJmol‐1atbodytemperature,witha ten foldchange inaffinityrepresentingachangeof5.9kJmol‐1

The binding interaction may also be described in kinetic terms, because theequilibrium between free partners and the protein‐ligand complex will begoverned by the rate of complex formation and rate of its dissociation. The

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secondorderrateconstantforassociationistermedkon,andthefirstorderrateconstantfordissociationiskoff,asshownabove.

TheKdisgivenbytheratiooftherateconstants:

offd

on

kK

k

Fordiffusioncontrolledbinding,wheretheassociationrateconstant(atleastforenzyme–substrateinteractions)canbeupto109M‐1s‐1[9]–1010M‐1s‐1[10],whilethedissociationrateconstantforaKdvalueof1nMwillbe1–10s‐1.

Inthesituationwherealargeexcessofligandisrequiredtoproduceasignificantdegreeofbindingtotheprotein,thenbindingoccurswithlittlechangeinthefreeligand concentration, and so the association reaction occurs with pseudo‐firstorderkinetics,sothat:

[PL]t=[PL]eq(1‐exp(‐kobst))

where[PL]tistheconcentrationoftheprotein‐ligandcomplexattimet,[PL]eqistheconcentrationofthecomplexatequilibriumandkobsisthepseudo‐firstorderrateconstant.

Thevalueofkobsforareversibleinteractionisgivenby:

kobs=koff+kon[L]f

Hence,aplotofkobsversus[L]fwillallowtherateconstantsandhencetheKdtobeevaluated.

For this single step reversible equilibrium the residence time,, (the length oftimetheligandoccupiestheproteinbindingsite)isgivenby1/koff.

Althoughit ispossibletodeterminetheKdvaluefromkineticmeasurements,asshownabove,thesevaluescanoftenbedifficulttomeasure,becausetheyoccuronashorttimescale,andsoKdvaluesareoftenmeasuredatequilibrium.

Many protein–inhibitor binding interactions of importance in drug discoveryproceedmore slowly than thediffusion controlled limit, andoftenexhibit slowbindinginhibition.

Thistypicallyoccursviaatwostepmechanism,

1 3

2 4P+L PL PL

k k

k k

where the enzyme encounters the ligand and subsequently a tightening of theinitial interaction occurs, for example via a conformational change, allowing atighterbindingsteadystatecomplexPL*toform.

This situation is described by two dissociation (or, more correctly, inhibition)constants,KiandKi*.

Thesecondinhibitionconstantisgivenby:

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43

4ii * kk

kKK

Forthistypeofinteractionthekobsisgivenbyamorecomplicatedcombinationofrateconstants:

3obs 4 '

i1[ ]

kk k

KL

whereKi’istheapparentKiwhichwilldependuponthemechanismofinhibitionand the substrate concentration, and may be related to the true Ki valueaccordingtotheCheng‐Prusoffequation[11]:

' is ii mi

m ii m

( [ ])[ ]

K K K SK

K K S K

whereKisandKiiaretheinhibitionconstantsat[S]<<Kmand[S]>>Km.

Theresidencetimeforthetwostepbindingmechanismisgivenby:

2 3 4

3 4

k k kk k

Twostepmechanismssuchasthisappeartobecommonindrugdiscovery,withmany inhibitors displaying slow kinetics according to this mechanism. Thesecondstepisslowwhichisoftenattributedtoconformationalrearrangementsorevenirreversibleinhibitionsuchasinthecasewherek2ork4tendtozero.

Investigationintothekineticsandthermodynamicsofthebindinginteractionisaninvaluabletoolindrugdiscoveryasitmayhelptoidentifycompoundswhichactbypreferredoralternativemechanisms.

4.4. NON‐COVALENTINTERACTIONSINPROTEIN–LIGANDBINDING

Moleculescaninteractwitheachotherviaanumberofnon‐covalentinteractionsthataremediatedbysurfacecomplemenatritybetweentheproteinbindingsiteand the ligand. These weak attractive forces are important in molecularrecognition occurring in biochemical binding reactions because they occurreversibly.

As these interactions are weak in nature, multiple interactions are usuallyrequired for the formation of a stable binding complex, and the interactinggroupstendtobeincloseproximity.

The four typesofnon‐covalent interactions important inprotein‐ligandbindingare hydrogen bonding, ionic or electrostatic interactions, van derWaals forcesandhydrophobicinteractions.

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4.4.1. Hydrogenbonding

Hydrogen bonds are electrostatic attractions between two dipoles and formwhenahydrogenatomissharedbytwoelectronegativeatoms,referredtoasthedonor (to which the hydrogen is covalently bonded), and the acceptor. Inproteinstheelectronegativeheteroatomsarepredominantlyoxygenornitrogen.

Hydrogenbondsareweak,contributingaround12.5–21kJmol‐1,withtheactualstrengthbeingdependentuponseveralfactorsincludingthelocalmedium,butinparticularthedistanceanddirectionbetweenthehydrogenandtheacceptor[12].

Although individuallyweak, hydrogenbonding is the largest contributor to thebindingenergyof ligands,aswellastothestructuralelementswithinaproteinstructure.

4.4.2. Ionicorelectrostaticinteractions

These interactions occurwhen two oppositely charged groups are attracted tooneanotherthroughaColoumbicforce.Ithasbeensuggestedthattheseinterac‐tionsarelikelytoberesponsiblefortheinitialrecognitionofproteinandligand.

Thestrengthoftheinteractionisdependentuponthechargesonthetwoatoms,aswellasthedistancebetweenthemandthedielectricconstantofthemedium.

Many ligands rely on ionic interactions for specific binding, with the greatestattractive force occurring in the low dielectric constant environment of thehydrophobicinterioroftheprotein.

4.4.3. VanderWaalsinteractions

VanderWaalsinteractionsoccurasaresultoftheattractionbetweentemporarydipoles that arise due to the uneven distribution of electrons betweenneighbouringatoms.

This attractive force is much weaker than ionic or hydrogen bonds, with anenergy of no more than around 4 kJ mol‐1. However, due to high surfacecomplementarity between protein and ligand, the number of van der Waalsinteractions can be large, with the collective force contributing significantly tocomplexstability.

4.4.4. Hydrophobicinteractions

Hydrophobic interactions occur between two non‐polar groups, due to thereduction in the unfavourable organisation of water around the non‐polargroups,whenthegroupsassociatewithoneanother[13].

Forligandbinding,thehydrophobicregionofthebindingsitetendstostabilisethebindingofhydrophobicligands.Thishydrophobicportioningofligandsfromsolu‐tiontotheproteinactivesitecanbeastrongcontributortothebindingenergy.

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4.5. BIOPHYSICALMETHODSFORCHARACTERISINGPROTEIN–LIGANDINTERACTIONS

4.5.1. Isothermaltitrationcalorimetry(ITC)

The ITC experiment involves the monitoring of the heat change during thebindingreaction(foracomprehensiveprotocolsee[14]).Theligandsolutionisusually titrated from the injection syringe into the protein solution containedwithin the calorimeter cell. The instrument measures the heat change using apower compensation process whereby the difference in the variable power,proportional to the binding heat, applied to the sample cell and the constantpowerappliedtothethermalreferencecellismonitoredbytheinstrument.

Dependinguponthebindingaffinityandtheamountsofavailablereagents, it isoftenpossibletoarrangetheexperimentalconditionssothatasingleexperimentcan provide precise estimates of the affinity (Kd), the enthalpy (H) and thestoichiometry (n) of the binding interaction. This also allows calculation of theentropy(S)fromtheGibbs‐Helmholtzequation,above.

Duringthetitration,inwhichsmallaliquotsoftheligandsolutionareadded,thefirst injections generate the largest heat change as most of the added ligandbecomesbound to theprotein.As the titrationprogresses theproteinbecomesincreasinglysaturatedwithligand,andtheheatdifferentialheatchangebecomessmaller. Finally, following complete saturation, no further heat change isdetected. However, sometimes significant, non‐zero heats following saturationareobserved,whichareoftenattributabletotheheatassociatedwithdilutionofthe ligand, and can be corrected for by control titrations. Figure 4.1 shows atypicalITCthermogram.

Understanding the thermodynamic components of molecular interaction isimportant in drug discovery, as it allows optimisation of test compounds in amore meaningful way. The thermodynamic measurements that are madeaccessible throughthe ITCexperimentare fundamental in tryingtounderstandmolecularinteraction,andinapplyingthatlearninginthepursuitofcompounds,not only with higher affinity, but with the appropriate thermodynamic andkineticprofilesfortheirbiologicalfunction[15].

As shown above, the binding affinity of a test compound is related to the freeenergy of the interaction, which itself is dependent upon the enthalpic andentropic components. The situation is complicated by factors such as theinfluence of solvent water on the binding thermodynamics and the change indynamics and conformation of the ligand and protein between the free andbound states. Thismakes the individual thermodynamic parameters incrediblydifficulttopredict.

BecauseITCallowsmeasurementofaffinityandenthalpyinasingleexperiment,itcanbeusefulindeterminingdiscontinuitiesinSARwhichmaybemissedfromKdvaluesalone.

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0.0 0.5 1.0 1.5 2.0-66.94

-58.58

-50.21

-41.84

-33.47

-25.10

-16.74

-8.37

0.00

8.37

-3.35

-2.51

-1.67

-0.84

0.00

0 10 20 30 40

Time, min

µW

Molar Ratio

kJ /

Mol

e of

Inj

ecta

nt

Figure4.1.RepresentativeITCthermogramforacompoundbindingtoatargetprotein

Changes in enthalpy may provide a valuable approach for the selection ofcompounds during lead identification and in helping to guide the leadoptimisation process towards compounds of higher quality. Enthalpic optimi‐sation,wheretheenthalpyofinteractionisincreasedmovingfromearlytolatermarketeddrugs,hasbeensuggestedasausefulapproachtoobtainadvantagesintheclinic[16,17].Theapproachsuggeststhatmoreefficientoptimisationcanbeachieved if the contributions of both enthalpy and entropy are improvedsimultaneously[18].

Byproactivelyusing ITC tohelpguidemedicinal chemistry in thisway,wewillinevitably initiate the exploration of the relationship between thermodynamicsand structure in a more coherent manner that will enable us to increase andexploitourknowledgeofmolecularinteraction.

4.5.2. SurfacePlasmonResonance(SPR)

Optical biosensorsmeasure changes in some characteristic of light, coupled tochanges (often in mass) at the sensor surface by making use of the electro‐magnetic evanescent‐wave formed at the gold interface of the sensor chipsurface. Biosensors employing SPR, such as BIAcore (GE Healthcare), are the

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best‐knownopticalbiosensors,utilisingthisphenomenontoenabledetectionofprotein‐ligandinteractionsinrealtime.

At total internal reflection, theelectromagnetic field componentof the incidentlight(theso‐calledevanescentwave)penetratesintothegoldfilmonthesensorchipandinteractswithfreeelectronsorplasmons.Photonsofincidentlightareabsorbed, the evanescent field strength amplified, and the light is no longerreflected. This decrease in the reflected intensity occurs at an angle whichdependsontherefractiveindexofthematerialintheflowcell,andiscalledtheSPRangle.Therefractiveindexoftheflowcellchangesconcomitantlywithmassonthesurfaceandthusallowsdetectionofligandbindingevents,Figure4.2(forareviewoftheuseofSPRindrugdiscoverysee[19]).Thispotentiallyallowsthekineticandequilibriumconstantsforagiveninteractiontobedetermined.

Figure4.2.RepresentativeSPRsensorgramforacompoundbindingtoan

immobilisedtargetprotein

Inordertomonitorprotein‐ligandbinding,oneofthepartnershastobeimmobi‐lisedontothesensorsurface.Indirectbindingassays(DBA)thetargetproteiniscoupled,withoutcompromisingtheactivityorfunctionalityoftheproteinortheaccessibilitytothebindingsite.Severaldifferentmethodsareavailableinordertoachievethis,withthemostcommonbeingdirectimmobilisationviaaccessibleprimaryaminesexposedontheproteinsurface.Thisisachievedbyactivatingthecarboxymethyl‐dextranmatrixsurfacewithamixtureof1‐ethyl‐3‐(3‐dimethyl‐aminopropyl)carbodiimide(EDC)andN‐hydroxysuccinimide(NHS)tointroducereactive succinimide esters that can react spontaneouslywith protein primaryamines. Other immobilisationmethods are available employing either covalentattachment using different chemistry strategies, or by employing non‐covalent

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captureusing affinitymethods, employing tags specifically incorporatedwithintheprotein.

TheSPRsignalisdependentonseveralfactorsassociatedwiththetargetprotein,including the molecular weight (MW) of the attached protein, the amountcapturedonthesurfaceandthebindingcapacityoftheprotein,orthestoichio‐metryofthebindingreaction.FactorsassociatedwiththeligandarealsotheMW,thetotalconcentration,[L]tandtheequilibriumdissociationKd.ThedependenceuponMWandKdforthesmallmoleculeligandrepresentsaparticularchallengewhenworkingwithfragments,wherebothsizeandaffinityarelow.

Issuesthatariseduetoligandmolecularsizemaybecircumventedbytheuseofalternative assay formats such as the surface competition assay (SCA) orinhibitioninsolutionassay(ISA).TheISAovercomestheseissuesbymonitoringbindingofthemacromolecule,wherebyincreasingtestcompoundconcentrationresultsinadecreaseinobservedsignalasthetestcompoundcompetesforbind‐ing to the targetprotein,and thusmakessystemsamenable forsmallmoleculeworkthatmaybeoutofthescopeofdirectbinding.

The ISA format requires coupling of a tool compound, or so called targetdefinition compound (TDC), to the biosensor surface which then serves as aprobe for the binding site. The binding interaction between the TDC and theproteingeneratesacontrolsignalwhichwillbeloweredinthepresenceofcom‐peting test compounds. Only compounds showing kinetic competitionwith theTDC for binding to the target protein will reduce the amount of free proteinavailable at equilibrium to bind to the TDC. This approach thus allows affinityinformationforthetestcompoundtobederived[20].

AnessentialpartoftheinitialdevelopmentofanISAistheidentificationofasuit‐abletoolcompoundtobeusedasaTDC.IdealpropertiesforasuitableTDCwo‐uldbebindingwhichblocksthewholebindingsite,withrapidassociationkine‐tics andhighpotency (slowdissociation). This situation serves to ensuremasstransportlimitedkinetics,sothattheobservedsignalisproportionaltothecon‐centration of free target protein, with the biosensor essentially measuring thefreeproteinconcentrationresulting from incubationwithdifferentconcentrati‐onsoftestcompound.AttachmentoftheTDCviaprimaryamineswithoutcom‐promisingbindingtothetargetproteinisalsothepreferredattachmentapproach.

In addition to an absence ofMW‐limitations for proteins or ligands in the ISA,another major advantage is that the interaction of interest occurs in solution,alleviatinganyissueswithpotentialchangesinaffinitycausedbyimmobilisation.Theprotocols for immobilisation and regeneration areusually straightforward,as conditions usually too harsh to be used with immobilised protein can beroutinely used with the small molecule attached to the surface. However, themain disadvantage compared to the DBA is that determination of the kineticparametersisprecludedbytheISAmethod.

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4.5.3. Opticalwaveguidegrating(OWG)

Biosensor instruments using optical waveguide‐based systems have beengrowinginuserecently.BothCorningandSRUBiosystems,manufactureinstru‐ments(EPICandBIND,respectively)thatallowincreasedsamplethroughputbyusingplate‐basedplatforms.

These instruments make use of the evanescent waves formed when lighttravellingthroughaboundarybetweentwomediaofdifferentrefractiveindicesundergoes total internal reflection.Wave coupling is then achieved by placingelectromagnetic elements, such as optical waveguides, close together enablingpropagation of awave fromone element to the next. Changes in the refractiveindex at the interface between the sensor surface and the solution phase,occurring as a consequence of ligand binding,modifies thewave coupling andtriggers a change in the reflected or transmitted light. This allows changes inmass at the sensor surface, caused by binding to proteins attached to sensorsurface,tobemonitored(forareviewofwaveguide‐basedbiosensorssee[21]).

ThemainpracticaldifferencebetweentheEPICandBINDOWGsystemstoSPR‐basedsystemsistheabsenceofsolutionflow.Thus,theOWGsystemsareunabletomeasure accurate kinetics. However, this apparent disadvantage is compen‐satedforby(1) increasedthroughputand(2)theabilitytoaddseveralcompo‐nents either simultaneously or sequentially into the same well of the plate inordertostudytheeffectofaddition.

AswithSPR‐basedplatforms,ISAformatscanbeusefulonOWGplatforms.Manyof the limitations described above for SPR are also problematic for OWGplatforms.Limitationsinsensitivityforsmallmoleculebindingrepresentamajorchallenge.Often,toovercometheseissues,proteinsarecoupledathighdensity,resulting in high local concentrations. This leads to an increased risk of non‐specific binding due to the potential increase in aggregated, unfolded, or func‐tionallycompromisedproteinscoupledtothesensorsurface.

Thus, the immobilisationofatoolcompoundorTDCandmakinguseof theISAapproachwillcircumventtheseissues.

Theuseofthesetechnologiesisexpectedtoexpandasissueswithproteinimmo‐bilisation are overcome, perhaps via expansion of the available immobilisationchemistriesand/orthesensitivityofdetectionimproves.

4.5.4. Spectroscopicmethods–NuclearMagneticResonance(NMR)

FollowingtheintroductionofSARbyNMRbyFesikin1996[22],NMRhasbeenanimportanttool fordetectingprotein‐ligandbinding inthe leadgenerationphaseofdrugdiscovery.Infact,thisligandscreeningapplicationhasnowalmostcompletelyreplaced theuseofNMR for structuresolutions,which ispredominantlyachievedusingX‐raycrystallography.

Protein‐ligand interactions formost soluble targetproteins canbe studiedbyNMR,andtheassaysaresimpleandrobustgeneratingfewfalsenegativesorfalsepositives.

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ThemainstrengthofNMRisthatbindingoflowaffinityligandswithKdvaluesintheμMtomMrangecanbereadilydetected,allowingNMRtobeusedasafront‐line fragment screeningmethod. In fact,NMR isoften themethodof choice forbiophysicalfragmentscreening.

ThreemainmethodstomonitorligandbindingbyNMRareused:2DNMRchemi‐calshiftmapping,1DNMRdirectbindingand1DNMRreferencedisplacement.

4.5.4.1. 2DNMRchemicalshiftmapping

Protein‐observe2DNMRchemicalshiftmappingexperimentscanbeconductedeitherusing15N‐1Hor13C‐1H(requiringuniformoraminoacidspecific15Nor13Cisotope labelling) correlations in Heteronuclear Single Quantum Coherence(HSQC)experiments[23],toprovideafingerprintofamideormethylgroupsintheprotein, respectively.Specificchanges in individualresiduesare thenmoni‐toredupontheadditionofbindingligands.Residuepeakassignmentsalsocanbeobtainedthroughaseriesofadditional3DNMRexperiments,allowinganalysisofthebindingmode.

The approach has a wide dynamic range, identifying binders from mM to nMrange,butquantitativeKdmeasurementsaremostreliableinthelowaffinity(μMtomM)range,under fast ligandexchange.Non‐specificbinding isgenerallynotproblematic in2DNMRexperimentssincesuchbindersareeithernotdetectedortendtoproducenon‐specificlinebroadening.

Therequirementforanisotopicallylabelledproteinandthesizerestrictionuptoaround40 kDa (80 kDa if uniformdeuteration of theprotein canbe achieved)placessomelimitationontheapplicationof2DNMR.

4.5.4.2. 1DNMRdirectbindingandreferencedisplacement

In 1D NMR ligand signals are observed directly. This can be advantageous inofferingabuilt‐inqualitycontrolandsolubilitymeasuresforthetestcompoundsstudied.There isno limitonprotein size,with largerproteinsgiving increasedrelaxation effects, resulting in magnified ligand binding effects. Generally, 1Hdetectionisusedfor1Dligand‐observeNMR,withthemostcommontechniquesbeingwaterLOGSY,saturationtransferdifference(STD),andT1ρandT2filteredexperiments[24,25].

1DNMRscreeningcanberuneitherindirectbindingmodewhereligandbindingis observed directly, or by monitoring the competition with an establishedreferenceligand,oftentermedaspymoleculeorreporterligand.

Thedirectbindingmethodisextremelysensitive,withbindingeffectsobservedevenat ligandconcentrationswellbelowKd,althoughcompetitionwitha ligandthatisknowntobindtothesiteofinterestisrequiredtoensurethatthebindinginteractionisspecific.

Amajorlimitationofthe1DNMRdirectbindingapproachisthatthemagnitudeofthebindingeffectdecreaseswhenthebindingapproacheshighaffinity(nM)or

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there isaslowligandexchangeregime,althoughthis isnotproblematic for thecompetitionmethod.

Typically, NMR approaches use compound mixtures, in order to increase thethroughput of the techniques. Mixtures can be screened in direct 1D NMRwithoutsubsequentdeconvolution,ifreferencespectrafortheligandsinthemix‐ture have been previouslymeasured. Reference displacementmethods requiredeconvolution to identify hits present in the mixture. Thus, typical screenscomprisinguptoseveralthousandcompoundsareoftencompletedinatimescalecoveringafewdaystoseveralweeksdependinguponthescreeningmode.

4.5.5. Spectrometricmethods–MassSpectrometry(MS)

MSapproachesforassessingprotein‐ligandbindingmaybegroupedinto2maintypes: (1) direct detection of the ligand and (2) detection of theprotein‐ligandcomplex.

4.5.5.1. Directdetectionoftheligand

MSanalysisofsmallmoleculesisaroutinetechnology.Itsstrengthisthathitscanbeidentifiedbymass,allowingmixturesofcompounds(withdifferentMW)tobeutilisedduringstudy.This,however,requirestheseparationofbindersfromnon‐binding compounds directly before the MS analysis step. This separation ofboundfromunboundcompoundscanbeachievedinavarietyofwaysincludingsize exclusion chromatography, ultrafiltration, equilibrium dialysis, or frontalaffinitychromatography.Theseapproachesaresuitedtoliquidchromatographyelectrospray ionisation MS (LC‐ESI‐MS) which facilitates protein denaturationandthereleaseoftheboundligand.

Forsizeexclusionbasedseparations,thetargetproteinisincubatedwithoneoramixtureofpotential ligandsbefore chromatography.The chromatographic steprapidly separatesunboundcompounds from thosebound to theproteinbeforeanalysisbyLC‐ESI‐MS.Thisapproachisamenabletohighthroughputscreening(HTS).

Usingtheultrafiltrationapproach,thetargetproteinisincubatedwithpotentialligandsbeforebeingconcentratedinacentrifugaldevicewithasemi‐permeablemembrane. Unbound compounds pass through the membrane and becomesignificantly diluted, beyondMSdetection limits.Bound ligands are retained intheprotein‐containingsample,andareidentifiedbyLC‐ESI‐MS.

Inequilibriumdialysis(ED)compoundmixturesareallowedtofullyequilibratebetween two chambers separated by a membrane permeable to only thecompounds,withonechambercontainingthetargetprotein.Thecontentsofthetwo chambers are analyzed quantitatively by LC‐ESI‐MS; with bound ligandsidentified as the total concentration in the protein‐containing chamberwill behigherthanthatontheproteinfreeside[26].

Afundamentallydifferentapproachis frontalaffinitychromatography, inwhichthe target protein is immobilized onto a liquid chromatography column. Com‐

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poundsareintroducedanddetectedafterthecolumnstepbyESI‐MS.Changesinretentionvolume,indicatingbinding,allowsboundligandstobeidentified[27].With the introduction of tool ligands with known affinity, competitionexperimentscanbeperformedtoestimatebindingaffinityfortestcompounds.

4.5.5.2. Detectionoftheprotein‐ligandcomplex

Nativemassspectrometry[28]hasbeenshowntoidentifynon‐covalentprotein‐ligandcomplexes,astheconditionsallowtheproteintoretainanativefoldandretain bound ligands in the gas phase [29]. This screening approach has beenemployed for standard libraries [30] and also has been used for screeningfragments[31].

4.6. BRIEFCASESTUDIESEXEMPLIFYINGTHEUSEOFBIOPHYSICALMETHODS

Themost powerful use of biophysicalmethods is in the combinationof appro‐aches,eitheracrossthebiophysicalmethodologyrange,orcoupledwithbioche‐micaldataaimedataddressingspecificproblemsduringthedrugdiscoverypro‐cess.TheexamplesbelowrefertodrugdiscoveryprojectscarriedoutatAstraZe‐neca,wheretheuseofbiophysicalmethodswasintegraltoprojectprogression.

4.6.1. DNAgyrase

DNAgyrase is a bacterial topoisomerase, essential forDNA replication and cellviability.TopoisomerasesaretheonlyDNAreplicationtargetsvalidatedbydrugsin the clinical setting, and DNA gyrase is well conserved across all bacterialpathogensofinterest.

The DNA gyrase enzyme relieves the supercoiling of DNA generated duringreplicationandtranscription,andusestheenergyfromATPtocatalysetheDNAtopologychanges.Itisatetramericenzymecomposedofadimerofdimers,withthe A subunits involved with DNA cleavage and resealing and the B subunit(GyrB)responsiblefortheATPaseactivity.

24 kDa and 43 kDa fragments of the DNA gyrase B subunit were used forstructuralbiologyeffortsandwithboth ITCandNMRmethods inorder tohelpidentifyandcharacterisegyrasebindingcompounds.ITCwasusedtoinvestigatethebindingaffinityandthermodynamicsofcompoundsbindingtotheseproteinfragments. The utility of the ITCmethod can be emphasised in this project, asthesefragmentsoftheBsubunitlackthetopoisomerase‐linkedATPaseactivityofthe intact tetramer, and so traditional biochemicalmethods following catalyticactivitycouldnotbeusedtomeasuretestcompoundaffinity.

Inasubsequentfragmentbasedapproach,2DNMRwasusedtoscreenalibraryoflowmolecularweightfragmentcompoundscontainingbothgenericfragmentsand fragments of known gyrase inhibitors. The 2D NMRmethod allowed bothaffinity and binding site information to be obtained. The fragment hits were

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foundtohavehighligandefficiency(G/heavyatom)ofupto0.46,andsharedahydrogenbonddonor/acceptormotifsimilartotheadeninemoietyofATP.

Having identified a naphthol fragment as an efficient binder to the primaryfragment site, a secondary screen was undertaken using the GyrB:naphtholcomplex. Following identification of aweak (5mM)binding quinoline, dockingmethods suggested likelyorientationsof the2 fragmentsandalloweda linkingstrategytobefollowedtoimprovepotency,Figure4.3a.

(a)

(b)

Figure4.3.(a)Dockingsuggestinglikelybindingposesofpyrroleandquinolinefragmentsinthe24kDafragmentofDNAGyraseBsubunit;(b)Crystalstructure

demonstratinghowpyrrolesubstitutionswereoptimisedtofillsite1,whilstadditionalH‐bondswereformedwiththeamideoccupyingsite2

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Improvedpotencywasdrivenbyoptimisationof theGyrB interactions,makingsubstitutions on a pyrrole moiety to fill site 1. Additional hydrogen bondinginteractionswere incorporated into site2,which extended into thenovobiocinbindingpocket,whilstmaintainingtheligandefficiencyat0.39,Figure4.3b.

Thepyrrolamideseries[32]ofestablishedcompoundsmaintainpotencyagainsta range of drug resistant bacterial strains, are bactericidalwith low resistancefrequencyanddemonstratepotencyagainstabroadspectrumofbacteria.

4.6.2. Beta‐siteAPPcleavingenzyme1(BACE‐1)

Combinations of NMR and SPR methods have been applied in the search forinhibitorsofBACE‐1.

BACE‐1, also known as beta‐secretase 1, is an aspartic acid protease whichcleaves amyloidprecursorprotein (APP)extracellularly.This cleavage step is aprerequisite for the subsequent cleavage by gamma‐secretase to the 40 or 42amino acid amyloid peptides. The aggregation of these peptides in the brainleadstoAlzheimer'sdisease.

Figure4.4.Discoveryofisocytosinesanddihydroisocytosinesfrominitialfragment

screeninghitsversusBACE‐1.

1DNMR screeningwas undertakenwith BACE‐1 protein expressed as a C‐ter‐minal fusion of the Fc part of human IgG1. Due to the large size of this fusionprotein, which causes increased relaxation effects, enhanced ligand bindingeffects occur and low micromolar concentrations of protein could be used tocarryoutthescreening.Afragmentlibraryof2000genericfragmentswasscre‐enedusingwaterLOGSYcompetitionexperiments.Thefragmentlibrarywasscre‐enedincompoundmixturescontaining6compounds,andresultedinahitrateofaround 0.5%. Following the initial fragment screen, an inhibition in solutionassay was established on the BIAcore platform, using a TDC peptide. The P1(S)‐statinsubstitutedsubstrateanalogue(KTEEISEVNstatin‐DAEF)wascapturedusing amine coupling, and displays nM affinity to BACE‐1. A reference surface

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was also preparedwith a scrambled version of the peptide (KFES‐statin‐ETIA‐EVENV).InitialbindingofthefragmenthitsfromtheNMRscreenweretestedat1mM,withsubsequentconcentrationresponsescarriedoutoverthe0.1–5mMrange. This fragment based NMR screening approach coupled with BIAcorefollow up confirmation and analogue testing resulted in the identification of arangeof6‐substitutedisocytosinesasnovelwarheadsforBACE‐1inhibition,withligand efficiencies of around 0.3, that were subsequently developed into inhi‐bitorswithnanomolarpotencyandcellularactivity,Figure4.4[33].

4.6.3. MEKproteinkinase

MEKproteinkinaseisalsoknownasMKKandMAPKK.Itexistsastwoisoforms,MEK1andMEK2,whichhave a79% sequence identity and eachhas a similarabilitytophosphorylateERK1andERK2.MEK1isregulatedbyactivatingphos‐phorylationsatSer217andSer221.CompoundsthataffectMEKmayactby2dif‐ferentmechanisms:(1)preventionofactivation(PoA),wherebindingtothenon‐activatedenzymeinterfereswithitsphosphorylationbyRAFisoformssuchasB‐RAF,or(2)byinhibitionofcatalysis(IoC),wherebindingtoactivatedMEKpre‐vents the phosphorylation of substrates such as ERK. Targeting non‐activatedkinases via the PoA mechanism offers some advantages over the traditionalapproachofIoC,suchasgreaterselectivityduetoamorediverserangeofproteinstructuresasthecatalyticmachineryisnotrequiredtobeinplace,andreducedcompetitionbytheATPsubstrateasthenon‐activatedformstendtohavealowerintrinsicaffinityforATP.

BiophysicalmethodshaveausefulapplicationinidentifyingpotentialPoAcom‐pounds, since they offer simpler assay set‐up than the biochemical PoA assayswhich involve upstream, activated kinase phosporylation of the non‐activatedtarget kinase. BIAcore inhibition in solution assays allows the detection ofbindingtoboththeenzymeandsubstratekinasesindividuallywithouttheneedfor substrate turnover. ISAs were established for both B‐RAF and MEK1 byimmobilising ureidoquinazoline and cyanoquinoline as target definition com‐pounds, respectively. These assays allowed comparisons betweenKd values fortheindividualproteinsfromISAandIC50valuesat[ATP]=KminbothIoCandPoAassays.KinasedrugdiscoveryofteninvolvestherankingofcompoundpotencyinassaysthatfollowIoCwhenATPispresentarounditsKmconcentration,anditisoftenusedtoshowselectivity.However,itisclearfromthebiochemicalandbio‐physicalstudieshere, thatasinglecompoundgivesdifferentIC50valuesagainstthesametargetproteinindifferentassays,whichintroduceschallengesintotheevaluationofpotencyandselectivity.ITCwasthereforeemployedtoinvestigatethedependenceofcompoundKdvaluesupon[ATP],andshowedthattheMEK‐1inhibitorU‐0126demonstratesnoncompetitivekinetics,whereasanalkylamidecompound showeduncompetitivekinetics.Thiswork illustrates that thedeter‐minationof themechanismofactionandmeasurementofequilibriumdissocia‐tionandinhibitionconstantshelpsintheunderstandingofthestructure–activityrelationshipsofMEKinhibitors[34].

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4.7. FUTURETRENDSFORBIOPHYSICALAPPROACHES

Thefutureuseofbiophysicalmethodstocharacteriseprotein‐ligandinteractionswill undoubtedly be facilitated by improvements in 2 key areas: (1) increasedthroughput and/or sensitivity of current instrumentation and (2) increasedapplication to currently intractable targets by emerging target preparationmethodsornewemergingtechnology,orboth.

Successful improvements in throughputhavebeenexemplifiedover the last20yearsorsobythetransformationofSPRfromasinglemoleculecharacterisationtool to a method capable of driving fragment based lead generation projects,wherethousandsofpotentialligandsaretestedinasinglescreen.

ThethroughputofOWGinstrumentshasalsoprogressedfromearlyinstrumentsto384–wellplatebased(EPIC)andeven1536‐wellplates(BIND),andtheAdvionnanomateholdsa96or384rackofconductivetipsaswellasa96or384‐wellsampleplate,allowinghighthroughputMS.

These methods will be increasingly used as orthogonal methods to verify thevalidityofhitsderivedfromtraditionalHTS.

The possibility of increased throughput for these biophysical technologies to1536‐wellbasedmaybetechnicallyfeasible,allowinganevengreaterimpactondrugdiscoveryinthefuture.

Recent efforts in building enthalpy arrays offer the potential to applythermodynamic profiling to both a wider number and range of protein‐ligandinteractionsandsystems[35].Theseinstrumentsarearraysofnanocalorimetersthat enable label‐free detection of molecular interactions using small samplevolumesandshortmeasurementtimescomparedtotraditionalinstruments.

Intermsofanewlyemergingtechnology,anapproachthathasgainedininterestrecentlyisback‐scatteringinterferometry(BSI),whichisabletoprovidealabel‐free,homogenousandmass‐independentdetectionof ligandbindinginsolutionwithmuchlowerrequirementsonsamplequantityandpuritythanOWGandSPRmethods[36].BSIuses light interactionwithamicrofluidicchannel tomeasuretemporal changes in refractive index (RI), brought about upon ligand binding.Studies of unlabeled proteinswith test compounds can be carried out, both insolutionandfollowingtargetimmobilisationtothesurface.Laserilluminationofthe microfluidic channel produces a highly modulated fringe patternperpendicular to the channel. The bright and dark features shift positionwithchanges intheRIof thesampleandmonitoringthisshift formsthebasisof theBSImeasurement.

New technology such as this, alongside novel methods to prepare stabilisedmembraneproteins,suchasGPCRs,usingasmallnumberofpointmutations,forexample the STaR technology fromHeptares [37]will allow the application ofbiophysical methods to membrane protein systems that have been difficult totacklepreviously.

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Extending measurements to whole cells is also something that has beenbeginningtooccur,particularlywithOWGinstruments,owingtotheabilityoftheinstrumenttodetectthedynamicmassredistributionthatoccurswithchangesincellmorphologyuponadditionofactiveligands.

The use of in‐cell NMR makes it possible to study biological macromoleculeswhiletheyremaininlivingcells.Thefirstin‐cellproteinNMRexperimentswereperformed inyeastby labellingwith 19F,although in‐cell 15N‐NMRand 13C‐NMRexperimentshavesincebeenconductedinEscherichiacoli[38].

The future for in‐cell NMRwill be the routine application of thesemethods toeukaryotic cells with the highly desirable opportunity to study large proteincomplexesandmembranes.

4.8. SUMMARY

Detecting, characterisingandmodifying ligandbinding interactions isof funda‐mentalimportancetodrugdiscovery.Recently,awiderrangeofleadgenerationapproaches,employedalongsidetraditionalHTS,havebeenadoptedbythephar‐maceutical industry. Themost important of thesemethods have been the bio‐physicaltechniquesusedeitherasfrontlinescreeningmethods,especiallyinrela‐tiontofragmentbasedleadgeneration,orasorthogonalmethodstovalidatehitsfromhighthroughputorhighconcentrationscreens.

Thesebiophysicalapproachesincludetechniquesoperatingviaanumberofdif‐ferentphysicalprocessesthattogetherofferthepossibilityofmeasuringnotonlyaffinity,butalso the thermodynamicandkinetic contributions to thatobservedaffinity.

This powerful combination of kinetic and thermodynamic data, coupled withstructural information, allows a more detailed understanding of the bindinginteraction and facilitates decision making during the hit to lead and leadoptimisationprocesses.

Furtherrefinement,bothintermsofnewmethodologythatwillallowthestudyofsystemscurrentlynotamenabletobiophysicalmethodsandintheapplicationofincreasedthroughputorincreasedsensitivityextensionstoexistingmethods,or both, will ensure that biophysical methods will remain embedded in highqualityleadgenerationprogrammesfortheforeseeablefuture.

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