1. Safety in drug research:Safety in drug research:
hERG channel blockade,hERG channel blockade,
k ?k ?a known enemy?a known enemy?
P.M. GrimaP.M. Grima--PovedaPoveda
Medicinal Chemistry Department OssMedicinal Chemistry Department Oss
N.V.OrganonN.V.Organongg
Nuevas fronteras en el desarrollo de fNuevas fronteras en el desarrollo de fáármacosrmacos
VII Jornadas de la SEQT, Sitges (SPN), October 19VII Jornadas de la SEQT, Sitges (SPN), October 19--20, 200620, 2006

2. ContentsContents
Potential side effectsPotential side effectsPotential side effectsPotential side effects
Biological roleBiological role
StructureStructure SARSARStructureStructure SARSAR
“Promiscuous” target“Promiscuous” target
Strategies proposedStrategies proposed
to avoidto avoid hERGhERG

3. Early assessment of cardioEarly assessment of cardio--safety: why?safety: why?
8
ZomaxZomax
OraflexOraflex
MeritalMerital
SafetySafety--related withdrawals (1980related withdrawals (1980--2005, U.S. market)2005, U.S. market)
6
MeritalMerital
SuprolSuprol
SeldaneSeldane
EnkaidEnkaid
HismanalHismanal
2
4 OmnifloxOmniflox
ManoplaxManoplax
PropulsidPropulsid
DuractDuract
P iP i
0
PosicorPosicor
RaxarRaxar
TrovanTrovan
RezulinRezulin
BaycolBaycolCardioCardio--toxicitytoxicity HepatoHepato toxicitytoxicity RenalRenal toxicity Otherstoxicity Others BaycolBaycol
RaplonRaplon
VioxxVioxx
LotronexLotronex
BextraBextra
•• Cardiac safety is theCardiac safety is the most important reasonmost important reason for post market withdrawalfor post market withdrawal
ExtremelyExtremely low incidencelow incidence (up to 1:20 000 doses)(up to 1:20 000 doses)
CardioCardio--toxicitytoxicity HepatoHepato--toxicitytoxicity RenalRenal--toxicity Otherstoxicity Others
•• ExtremelyExtremely low incidencelow incidence (up to 1:20.000 doses)(up to 1:20.000 doses)
•• ProPro--arrhythmic risk veryarrhythmic risk very difficult to assessdifficult to assess during clinical trials ( << 20.000 patients)during clinical trials ( << 20.000 patients)
•• Early evaluationEarly evaluation of proof pro--arrhythmic potential of NCEs is a priority of both Drug Regulatoryarrhythmic potential of NCEs is a priority of both Drug Regulatory
Tuffs CSDD Impact report, Vol7(5),2005
Fermini et al, Nature Reviews in Drug Discovery, 2,439-447 (2003)
Early evaluationEarly evaluation of proof pro arrhythmic potential of NCEs is a priority of both Drug Regulatoryarrhythmic potential of NCEs is a priority of both Drug Regulatory
Authorities and Pharmaceutical IndustriesAuthorities and Pharmaceutical Industries

4. Early assessment of cardioEarly assessment of cardio--safety: how?safety: how?
•• No preclinical method is fully predictive for proNo preclinical method is fully predictive for pro--arrhythmic potential in humansarrhythmic potential in humans
hERG channel inhibitionhERG channel inhibition as cornerstone for earlyas cornerstone for early in vitroin vitro cardiocardio safety assessmentsafety assessment
•• Integrated risk assessment of all relevant data is recommended by ICHIntegrated risk assessment of all relevant data is recommended by ICH (S7b draft guideline)(S7b draft guideline)
•• hERG channel inhibitionhERG channel inhibition as cornerstone for earlyas cornerstone for early in vitroin vitro cardiocardio--safety assessmentsafety assessment
•• QTQT--interval prolongationinterval prolongation as cornerstone for earlyas cornerstone for early in vivoin vivo cardiocardio--safety assessmentsafety assessment
V i lV i l Ab lAb lAb l i lAb l i l
ArrhythmiaArrhythmia Long QTLong QT--syndromesyndrome
hERG channelhERG channel
inhibitioninhibition
VentricularVentricular
arrhythmiaarrhythmia
AbnormalAbnormal
KK++ fluxflux
Abnormal ventricularAbnormal ventricular
functionfunction
•• Most (but not all) withdrawnMost (but not all) withdrawn drugs causing arrhythmiasdrugs causing arrhythmias (like TdP) induce(like TdP) induce long QT syndromelong QT syndrome
•• Most (but not all) drugs inducingMost (but not all) drugs inducing longlong--QT syndromeQT syndrome in man alsoin man also inhibit hERG channelinhibit hERG channel
Redfern elt al, Cardiovascular Research, 58, 32-45 (2003)
Hoffmann et al, J.Pharmacological and Toxicological Methods, 53,87-105,(2006)

5. Pivotal role of hERG channel in QT prolongationPivotal role of hERG channel in QT prolongation
•• Normal conditionsNormal conditions
RECG QTQT--intervalinterval•• Electrocardiogram (ECG)Electrocardiogram (ECG)
P =P = Auricular depolarisationAuricular depolarisation
P
QQ
S
TT
QTQT intervalinterval
P =P = Auricular depolarisationAuricular depolarisation
QQRS =RS = Ventricular depolarisationVentricular depolarisation
TT == Ventricular reVentricular re--polarisationpolarisation
•• Ventricular myocyte action potential (AP)Ventricular myocyte action potential (AP)
0 =0 = depolarisationdepolarisation NaNa++ inin
1
2
AP
QTQT--intervalinterval
0 =0 = depolarisationdepolarisation NaNa inin
1 = transition phase1 = transition phase
2 =2 = slow reslow re--polarisationpolarisation CaCa2+2+ in Kin K++ outout
3=3= rapid rerapid re--polarisationpolarisation KK++ out (out (hERGhERG)) 200200
0
33
2
K+ flow (hERG mediated)
•• Outward KOutward K++ flow mediated by hERG channelflow mediated by hERG channel
33 rapid rerapid re polarisationpolarisation KK out (out (hERGhERG)) ~ 200 ms~ 200 ms
Stansfeld et al, Expert Opin.Drug.Metab.Toxicol.2(1), 81-94, (2006)

6. Pivotal role of hERG channel in QT prolongationPivotal role of hERG channel in QT prolongation
•• Although it is not the only ion channel involved, hERG channel is the most important factor inAlthough it is not the only ion channel involved, hERG channel is the most important factor in
QT prolongation events since it plays a key role during reQT prolongation events since it plays a key role during re--polarisationpolarisation
RECG
•• Electrocardiogram (ECG)Electrocardiogram (ECG)
QT prolongation events, since it plays a key role during reQT prolongation events, since it plays a key role during re--polarisationpolarisation
P
QQ
S
TT
QTQT--intervalinterval
Increase in QT interval (long QT syndromeIncrease in QT interval (long QT syndrome --> Δ > 10 %)> Δ > 10 %)
•• Ventricular myocyte action potential (AP)Ventricular myocyte action potential (AP)
3=3= “not so rapid”“not so rapid” rapid rerapid re polarisationpolarisation
1
2
AP
3=3= not so rapidnot so rapid rapid rerapid re--polarisationpolarisation,,
due to the lack of contribution of hERG to Kdue to the lack of contribution of hERG to K++ rapid effluxrapid efflux
200200
0
33
2
~ 200 ms~ 200 ms
K+ flow (hERG mediated)
•• Outward KOutward K++ flow mediated by hERG channelflow mediated by hERG channel•• Outward KOutward K++ flow mediated by hERG channelflow mediated by hERG channel K+ flow
Stansfeld et al, Expert Opin.Drug.Metab.Toxicol.2(1), 81-94, (2006)
Drug induced inhibition reduces hERG mediated KDrug induced inhibition reduces hERG mediated K++ effluxefflux

7. Unusual promiscuous targetUnusual promiscuous target
Room to accommodateRoom to accommodate
large moleculeslarge molecules
Large cylindricalLarge cylindrical
inner cavityinner cavity
High density of aromaticHigh density of aromatic Highly symmetricHighly symmetric
and polar residuesand polar residues arrangementarrangement
Multiple simultaneousMultiple simultaneous
interactions feasibleinteractions feasible
Several bindingSeveral binding
orientations feasibleorientations feasible

8. hERG: human Ether ahERG: human Ether a--gogo--go Related Genego Related Gene
•• hERG gene isolated by Dr. Barry Ganetzky in 1994hERG gene isolated by Dr. Barry Ganetzky in 1994
•• hERG encodes thehERG encodes the --subunit ofsubunit of humanhuman IKr channelIKr channel
•• hERG named inhERG named in relationrelation to its counterpart in fruit flies (EAG)to its counterpart in fruit flies (EAG)
Dr. Barry Ganetzky•• EAG (ether aEAG (ether a--gogo--go gene) discovered in the 60s by Dr. Kaplango gene) discovered in the 60s by Dr. Kaplan
•• Flies with mutations in thisFlies with mutations in this genegene start to shake their legsstart to shake their legsgg gg
when anaesthetised withwhen anaesthetised with etherether
•• This shaking movement was pretty similar to a danceThis shaking movement was pretty similar to a dance
performed in the late 60s at the popular nightperformed in the late 60s at the popular night--club in Westclub in West
Hollywood theHollywood the WhiskyWhisky a Goa Go--GoGo
Whisky a Go-Go in the late 60s
Hollywood, theHollywood, the WhiskyWhisky a Goa Go--GoGo

9. Topology of hERG channel α subunitsTopology of hERG channel α subunits
Voltage sensor Pore domain
Turret segment
- +
-
-
Out
Voltage sensor Pore domain
Pore helix
Selectivity filter
- - - +
+
+
-
S1 S6S5S4
S5 Helix
S6 Helix
+
N-terminal C-terminal
In
Single α sub unit of hERG channel
Homology model of the pore domain
•• KK++ Channel formed by 4 identical subunitsChannel formed by 4 identical subunits
•• Each subunit consists of:Each subunit consists of:
•• Intracellular terminal domainsIntracellular terminal domains:: modulation of channel state by interaction with cytosolic messengersmodulation of channel state by interaction with cytosolic messengers
•• 6 TM domain:6 TM domain:
S1S1 S4 lt d iS4 lt d i d l ti f h l t t b h i d tid l ti f h l t t b h i d ti•• S1S1--S4 voltage sensor domain:S4 voltage sensor domain: modulation of channel state by changes in conductionmodulation of channel state by changes in conduction
•• S5S5--S6 Pore domain:S6 Pore domain: formation of the actual pore and selectivity for Kformation of the actual pore and selectivity for K++
Homology model based on KcsA (closed state of pore domain)

10. Topology of pore domain of hERG channel (closed state)Topology of pore domain of hERG channel (closed state)
Selectivity
filter
S5
S6
Single subunit
Homology model (top view) Homology model (side view)
•• Each subunit contributes with S5Each subunit contributes with S5--S6 pore domainS6 pore domain
F ti lF ti l i t t ith C4 ti t t ith C4 t
Single subunit
S5-S6 domain
•• Functional poreFunctional pore is a tetramer with C4 symmetryis a tetramer with C4 symmetry
•• Selectivity filters facing each other generate a ~6Å pore thatSelectivity filters facing each other generate a ~6Å pore that mimics the hydration shell of Kmimics the hydration shell of K++ inin
the open state of the channelthe open state of the channel
Homology model based on KcsA (closed state of pore domain)

11. Topology of pore domain of hERG channel (closed state)Topology of pore domain of hERG channel (closed state)
S5
S6
Single subunit
Homology model (top view) Homology model (side view)
•• Each subunit contributes with S5Each subunit contributes with S5--S6 pore domainS6 pore domain
F ti lF ti l i t t ith C4 ti t t ith C4 t
Single subunit
S5-S6 domain
•• Functional poreFunctional pore is a tetramer with C4 symmetryis a tetramer with C4 symmetry
•• Selectivity filters facing each other generate a ~6Å pore that mimics the hydration shell of KSelectivity filters facing each other generate a ~6Å pore that mimics the hydration shell of K++ inin
the open state of the channelthe open state of the channel
•• Space between S6 helixes generatedSpace between S6 helixes generated a central cavity below that porea central cavity below that pore
Homology model based on KcsA (closed state of pore domain)

12. Topology of hERG: KTopology of hERG: K++ efflux in open and closed statesefflux in open and closed states
KK++
Repolarisation
Depolarisation
KK++
KK++ KK++
KK++
KK++
Homology model closed state (side view) Homology model open state (side view)
KK++
KK
•• ClosedClosed (inactive) state: C(inactive) state: C--terminus ends ofterminus ends of S6 cross overS6 cross over limiting the movement of Klimiting the movement of K++ ionsions
•• OpenOpen (active) state:(active) state: GlyGly648648 hingehinge bending results in an increase of aperture sizebending results in an increase of aperture size
Homology model based on KcsA (closed state of pore domain)
Homology model based on KvAP (open state of pore domain)
Morais-Cabral et al, Nature,414, 37-42 (2001)
KK++ gain access to selectivity filtergain access to selectivity filter

13. Topology of hERG: blockade in open and close statesTopology of hERG: blockade in open and close states
Repolarisation
Depolarisation
KK++
KK++
Homology model closed state (side view) Homology model open state (side view)
•• Access to the inner cavity is gained from theAccess to the inner cavity is gained from the intracellularintracellular side of the poreside of the pore
•• As a result, most inhibitors only block hERG channel when the activationAs a result, most inhibitors only block hERG channel when the activation gate is opengate is open
•• Nevertheless, the inner cavity is big enough toNevertheless, the inner cavity is big enough to trap inhibitorstrap inhibitors after deactivation, process thatafter deactivation, process thatNevertheless, the inner cavity is big enough toNevertheless, the inner cavity is big enough to trap inhibitorstrap inhibitors after deactivation, process thatafter deactivation, process that
may increase the apparent binding affinity (lower Kmay increase the apparent binding affinity (lower Koffoff))
Homology model based on KcsA (closed state of pore domain)
Homology model based on KvAP (open state of pore domain)

14. “Promiscuous” K“Promiscuous” K++ channel: larger inner cavity?channel: larger inner cavity?
Channel S6 sequenceChannel S6 sequence
hERGhERG IIFFGG
bEAGbEAG IIFFGG
KvAPKvAP LLIIGGKvAPKvAP LLIIGG
KcSAKcSA VVTTAA
Kv1.1Kv1.1 PPVVPP
Kv1.2Kv1.2 PPVVPPKv1.2Kv1.2 PPVVPP
Kv1.5Kv1.5 PPVVPP
Kv2.1Kv2.1 PPVVPP
Kv3.1Kv3.1 PPVVPP hERG KV1.2
S l h l t iS l h l t i PP XX PP t t th d f S6 ti ki k S6 dt t th d f S6 ti ki k S6 d
Kv4.1Kv4.1 PPVVPPhERG Homology model
(2 subunits shown)
(homology model) (crystal structure)
S5-S6 domains
•• Several channels contain aSeveral channels contain a ProPro--XX--ProPro segment at the end of S6, creating a kink on S6 andsegment at the end of S6, creating a kink on S6 and
reducing the volume of the inner cavityreducing the volume of the inner cavity
•• The lack of this motif partially explains the large scope of drugs able to interact with hERGThe lack of this motif partially explains the large scope of drugs able to interact with hERG
•• PVPPVP--hERG mutant insensitive to known hERG blockershERG mutant insensitive to known hERG blockers Del Camino et al, Nature, 403,321(2000)
Long et al, Science, 309, 897-903 (2005)

15. “Promiscuous” K“Promiscuous” K++ channel: Y652 and F656 residueschannel: Y652 and F656 residues
Scheme of Y652 and F656 crownsScheme of Y652 and F656 crowns
Channel S6 sequenceChannel S6 sequence
hERGhERG YYASIASIFF
bEAGbEAG YYATIATIFF
K APK AP LLTLLTLLII
Open stateOpen state Closed stateClosed state
KvAPKvAP LLTLLTLLII
KcSAKcSA FFGLVGLVTT
Kv1.1Kv1.1 IIALPALPVV
Kv1 2Kv1 2 IIALPALPVVKv1.2Kv1.2 IIALPALPVV
Kv1.5Kv1.5 IIALPALPVV
Kv2.1Kv2.1 IIALPALPVV
Kv3.1Kv3.1 IIAMPAMPVV
Kv4.1Kv4.1 IIALPALPVVhERG Homology model
(2 subunits shown)
•• Only in hERG channel both residuesOnly in hERG channel both residues face the inner cavityface the inner cavity
•• TyrTyr652652 residues able to establish either πresidues able to establish either π--π orπ or ππ--cation interactionscation interactions with drugswith drugs
•• PhePhe656656 residues generate aresidues generate a hydrophobichydrophobic crown at the bottom (entrance) of the inner cavitycrown at the bottom (entrance) of the inner cavity•• PhePhe residues generate aresidues generate a hydrophobichydrophobic crown at the bottom (entrance) of the inner cavity.crown at the bottom (entrance) of the inner cavity.
•• In the closed state this crown closes, trapping the drug molecule inside the cavityIn the closed state this crown closes, trapping the drug molecule inside the cavity
Sanguinetti et al, J.Biol.Chem.,279 (11)10120-10127 (2004)
Mitsheson et al, Curr. Opin. Drug Discov. Devel., 6, 667 (2003)

16. Additional interactions: Thr623 and Ser624 residuesAdditional interactions: Thr623 and Ser624 residues
Channel S6 sequenceChannel S6 sequence
hERGhERG TSTSVV
bEAGbEAG TSTSVV
K APK AP TTTTVV
Open stateOpen state Closed stateClosed state
KvAPKvAP TTTTVV
KcSAKcSA TTTTVV
Kv1.2Kv1.2 TTTTVV
hERG Homology model
(2 subunits shown)
•• Polar character of these residuesPolar character of these residues conservedconserved among Kamong K++ channelschannels
•• Crown of polar side chains may establishCrown of polar side chains may establish HH--bond interactionsbond interactions with drugs trapped in inner cavitywith drugs trapped in inner cavity
•• Novel hypothesis suggests ThrNovel hypothesis suggests Thr623623 backbone carbonyl may also be involved in Hbackbone carbonyl may also be involved in H--bond formationbond formation
Sanguinetti, Mol. Pharmacol.69,1709-1716 (2006)
Mitcheson et al, Proc.Natl.Acad.Sci. USA, 97 12329-12333 (2000)
Choe et al, Biochem. & Biophys. Research Comm. 344,72 (2006)

17. General pharmacophore model of hERG blockersGeneral pharmacophore model of hERG blockers
HH--bond donor/acceptorbond donor/acceptor
Positive charge / AromaticPositive charge / Aromatic
Hydrophobic groupHydrophobic group
hERG Homology model
(2 subunits shown)
•• Partially explains the large scope of drugs able to interact with hERG channelPartially explains the large scope of drugs able to interact with hERG channel
•• Volume, symmetry and number of combinations explainVolume, symmetry and number of combinations explain the lack of a universal predictive modelthe lack of a universal predictive model
•• “Local”“Local” pharmacophores show predictive valuepharmacophores show predictive value
Aronov, Drug Disc. Today, 10(2), 149 (2005)

18. “Local” pharmacophores: Amine containing blockers“Local” pharmacophores: Amine containing blockers
N OHOH
NN++Ekins’ PharmacophoreEkins’ Pharmacophore
N
OH
OHN
OH
OH
HydrophobicHydrophobic
HydrophobicHydrophobic
HydrophobicHydrophobic
HydrophobicHydrophobic
Terfenadine
HH
NN++
Cavalli’s PharmacophoreCavalli’s Pharmacophore
N
F N
N
H
N
ON
F N
N
H
N
O
AromaticAromatic
AromaticAromatic
(CO)(CO)
AromaticAromatic
(C2)(C2)
Astemizole
(C1)(C1)
Pearlstein’s PharmacophorePearlstein’s Pharmacophore
NN++
AromaticAromatic Arom/polarArom/polar
N
N
F
N NH
O
N
N
F
N NH
OSertindole
NN++
AromaticAromatic
(“Handle”)(“Handle”)
(“Handle”)(“Handle”)
pp
(“Tail”)(“Tail”)
ClCl
Ekins et al, J. Pharmacol.Exp. Ther., 301, 427-434 (2002)
Cavalli, J. Med. Chem., 45, 3844-3853 (2002)
Pearlstein, J. Med. Chem., 46, 2017-2022 (2003)

19. Strategies to avoid hERG inhibitory activityStrategies to avoid hERG inhibitory activity
Formation of zwitterionsFormation of zwitterions
Avoid access toAvoid access toAvoid access toAvoid access to
the targetthe target
Avoid keyAvoid key
interactionsinteractions
FineFine--tune generaltune general
featuresfeaturesinteractionsinteractions
D l ti / di ti fD l ti / di ti f
featuresfeatures
P di t t t lP di t t t lDeletion / disruption ofDeletion / disruption of
-- oror --NN++ interactionsinteractions
Pursue discrete structuralPursue discrete structural
modificationsmodifications
Aronov, Drug Disc. Today, 10(2), 149 (2005)
Jameison et al, J.Med.Chem., 49(17),5029-5046 (2006)

20. Avoid access to the target: Corpora non agunt nisi fixateAvoid access to the target: Corpora non agunt nisi fixate
Most inhibitors are believed to gain access to theMost inhibitors are believed to gain access to the NRR
inner cavity after entering the cellinner cavity after entering the cell
ByBy blocking cell permeabilityblocking cell permeability the inhibitory activitythe inhibitory activityByBy blocking cell permeabilityblocking cell permeability, the inhibitory activity, the inhibitory activity
is drastically reduced.is drastically reduced.
Physical limitation of membrane permeability isPhysical limitation of membrane permeability is
ll li h d bll li h d b f ti f itt if ti f itt i
NRR
usually accomplished byusually accomplished by formation of zwitterionsformation of zwitterions
(introduction of carboxylic acid groups in amine(introduction of carboxylic acid groups in amine
containing blockers)containing blockers)
NHRRCOO
- +
Pro’sPro’s On / off effect on hERGOn / off effect on hERG
ti it f COti it f CO H i t tH i t tconnectivity of COconnectivity of CO22H group unimportantH group unimportant
Con’sCon’s Detrimental effect on:Detrimental effect on: target activity (intracellular targets)target activity (intracellular targets)
Oral BioavailabilityOral Bioavailability
CNS penetrationCNS penetration

21. Targeting (physical) access to the binding siteTargeting (physical) access to the binding site
OCFOH
HO2C
NK1 antagonistsNK1 antagonists
N
NH
OCF3
H
N
OH
NH
OCF3
H
N
NH
OCF3
H
2
x 0.75 x 13
OcPrOcPr OcPr
FhNK1 IC50 = 0.1 nM
hERG Ki = 360 nM
ED50 =2 6 mg/kg
hNK1 IC50 = 0.09 nM
hERG Ki = 270 nM
ED50 =1 1 mg/kg
hNK1 IC50 = 1 nM
hERG Ki = 4800 nM
Not active in vivoED50 2.6 mg/kgED50 =1.1 mg/kg Not active in vivo
No brain penetration
DPPDPP--IV inhibitorsIV inhibitors
O OO
NH2
N
O
F
NH2
N
O
F
NH2
N
O
F
HO2
C
x 69 > x 90
HO2
C
DPP-IV IC50 = 64 nM
hERG Ki = 1100 nM
DPP-IV IC50 = 7 nM
hERG Ki = 76000 nM
DPP-IV IC50 = 56 nM
hERG Ki > 100000 nM
x 69 > x 90
Thomson et al, Bioorg. Med. Chem. Letters,16,811-814 (2006)
Xu et al, Bioorg. Med. Chem. Letters, 15, 2533-36 (2005)
FF
F = 85 %F = 16 % F = 2.3 %

22. Deletion of key interactionsDeletion of key interactions
Deletion of aromatic ringDeletion of aromatic ring
Reduction of binding affinity by lack ofReduction of binding affinity by lack of
interaction with Pheinteraction with Phe656656 and/or Tyrand/or Tyr654654yy
Pro’sPro’s Drastic effect on binding affinity to hERGDrastic effect on binding affinity to hERG
Positive effect on physicoPositive effect on physico--chemical properties of the molecule:chemical properties of the molecule:yy
reduction of log Preduction of log P
increase in solubilityincrease in solubility
C ’C ’ P b bl l t i t t ffi itP b bl l t i t t ffi itCon’sCon’s Probable lost in target affinityProbable lost in target affinity

23. Deletion of key interactionsDeletion of key interactions
Comparison of set of 69 blockers based on the number of interactions with hERGComparison of set of 69 blockers based on the number of interactions with hERG
•• 30 blockers able to establish two interactions30 blockers able to establish two interactions
•• 38 blockers able to establish three interactions38 blockers able to establish three interactions
15
38 blockers able to establish three interactions38 blockers able to establish three interactions
% compounds
10
55
hERG pIC
0
3.5-4 4-4.5 4.5-5 5-5.5 5.5-6 6-6.5 6.5-7 7-7.5 7.5-8 8-8.5 8.5-9 9-9.5
Choe et al, Biochem. Piophys. Research Comm., 344, 72-78 (2006)
hERG pIC50

24. Deletion of key interactionsDeletion of key interactions
VEGFRVEGFR--2 inhibitors2 inhibitors
S
N
N
N N
O
H
S
N
N
N N
O
HNC
x 44
N
N
N N
N
N
VEGFR-2 IC50 = 13 nM
hERG IP = 10600 nM
VEGFR-2 IC50 = 8 nM
hERG IP = 240 nM
H5H5--HTHT2A2A receptor antagonistsreceptor antagonists
N H N HN H x 17 x 33
N NN
H
h5HT2A Ki = 20 nM h5HT2A Ki = 4.7 nMh5HT2A Ki = 0.99 nM
Bilodeau et al, J. Med. Chem., 47, 6363 (2004)
Rowley et al, J. Med. Chem., 44,1063 (2001)
hERG Ki = 290 nM hERG Ki = 9600 nMhERG Ki = 4900 nM

25. Deletion of key interactionsDeletion of key interactions
Deletion of amine groupDeletion of amine group
Reduction of binding affinity by lack of keyReduction of binding affinity by lack of key
interaction with Tyrinteraction with Tyr654654yy
Pro’sPro’s Drastic effect on binding affinity to hERGDrastic effect on binding affinity to hERGg yg y
Con’sCon’s Generally amine is a key warhead in target interactionGenerally amine is a key warhead in target interaction
Generally amine is use as tool to improve solubility of intrinsic insoluble seriesGenerally amine is use as tool to improve solubility of intrinsic insoluble series

26. Deletion of key interactionsDeletion of key interactions
KSP inhibitorsKSP inhibitors
F F
N
F
O
H
N
F
O
H
O> x 7.7
N
O
NH
N
O
N
KSP IC50 = 2.6 nM
hERG IC50 = 1300 nM
l 10 / L
KSP IC50 = 50 nM
hERG IC50 > 10000 nM
l H5 0 0
F
F
OHF
aq.sol.pH=5 > 10 mg/mL aq.sol.pH5.0 ~ 0
N
F
O
H
N
F
O
H
N
F H
(I)
x 3.1 x 1.6
O
OH
O
OH
O
NH2
KSP IC50 = 11 nM KSP IC50 = 2.9 nM
hERG IC50 18000 nM
KSP IC50 = 2.0 nM
(I)
Fraley et al, Bioorg. Med. Chem. Letters, 16, 1775 (2006)
Garbaccio et al, Bioorg. Med. Chem. Letters,16,1780 (2006)
hERG IC50 = 11000 nM
aq.sol.pH5.0 ~ 0
hERG IC50 = 18000 nM
aq.sol.pH5.0 ~ 0
hERG IC50 = 3500 nM

27. Disruption of key interactionsDisruption of key interactions
•• Disruption ofDisruption of -- aromatic interactionsaromatic interactions
Replace EDG by EWG. Increase steric hindranceReplace EDG by EWG. Increase steric hindrance
•• Disruption ofDisruption of --cation interactionscation interactionsDisruption ofDisruption of  cation interactionscation interactions
Reduce pKa of amine groupReduce pKa of amine group
•• Reduce log PReduce log P
In average reduction 1 log PIn average reduction 1 log P --> reduce 0.8 log Ec> reduce 0.8 log Ec5050
hERGhERG
•• Discrete Structural ModificationsDiscrete Structural Modifications
Rigidity, sterochemistry,….Rigidity, sterochemistry,….
Pro’sPro’s minimal change on molecular propertiesminimal change on molecular properties
Con’sCon’s little impact on hERG (DSM: not always predictable)little impact on hERG (DSM: not always predictable)

28. Reduction of hERG inhibitory activity by modulation of amineReduction of hERG inhibitory activity by modulation of amine
55--HTHT2A2A antagonistsantagonists
N S
F
F O
O
N S
F
F O
O
O
N S
F
F O
O
x 6.1 x 2.2N SN S N S
F
h5HT2A Ki = 0.31 nM
hERG Ki = 1100 nM
pKa = 7 3
h5HT2A Ki = 2.4 nM
hERG Ki = 6800 nM
pKa = 6 3
h5HT2A Ki = 0.68 nM
hERG Ki = 2446 nM
F F
pKa 7.3pKa 6.3
N S
F O
O
N S
F O
O
F
x 7.8
F F
h5HT2A Ki = 0.42 nM
hERG Ki = 710 nM
pKa = 7.2
h5HT2A Ki = 0.39 nM
hERG Ki = 5561 nM
pKa = 6.3
Humphries et al, Bioorg. Med. Chem. Letters, 15, 3665 (2005)

29. Reduction of hERG inhibitory activity by reducing log PReduction of hERG inhibitory activity by reducing log P
VEGFRVEGFR--2 inhibitors2 inhibitors
O
N
N
N N
O
S
N
N
O O
N N
x 3.1
H O H
H O H
VEGFR-2 IC50 = 10 nM
hERG IP = 3440 nM
log P = 3.0
VEGFR-2 IC50 = 5 nM
hERG IP = 10800 nM
log P = 2.6
N
O
S
N
O O
> x 4.1
N
N
H
NH
O H
N
N
N
H
N
O H
H
VEGFR-2 IC50 = 11 nM
hERG IP 2450 M
VEGFR-2 IC50 = 5 nM
Reduction of log P may be an additional effect of former strategies:Reduction of log P may be an additional effect of former strategies:
hERG IP = 2450 nM
log P = 2.3
hERG IP > 10000 nM
log P = 1.7
Deletion of aromatic groupsDeletion of aromatic groups
“De“De--shielding” of amine groupsshielding” of amine groups
Fraley, Bioorg. Med. Chem. Letters, 14,351 (2004)

30. Identification of the appropriate strategyIdentification of the appropriate strategy
Series clogP ?Series clogP ?
< 3  3
Series clogP ?Series clogP ?
< 3  3
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
Reduce polarityReduce polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Increase polarityIncrease polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Jameison et al, J.Med.Chem., 49(17),5029-5046 (2006)
Reduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activity

31. Identification of the appropriate strategyIdentification of the appropriate strategy
Series clogP ?Series clogP ?
< 3  3
Series clogP ?Series clogP ?
< 3  3
Series clogP ?Series clogP ?
< 3  3
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
Reduce polarityReduce polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Reduce polarityReduce polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Increase polarityIncrease polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Jameison et al, J.Med.Chem., 49(17),5029-5046 (2006)
Reduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activity

32. Identification of the appropriate strategyIdentification of the appropriate strategy
Series clogP ?Series clogP ?
< 3  3
Series clogP ?Series clogP ?
< 3  3
Series clogP ?Series clogP ?
< 3  3
Series clogP ?Series clogP ?
< 3  3
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR’?Series involves NRR’?
R < 5 R  5Y
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
hERGhERG--clogP correlationclogP correlationSeries involves NRR ?Series involves NRR ?
N
Reduce polarityReduce polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Reduce polarityReduce polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Reduce polarityReduce polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Increase polarityIncrease polarityReduce pKaReduce pKa
Replace by other solubilizersReplace by other solubilizers
Include EWGInclude EWG
Delete peripheral arylDelete peripheral aryl
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Pursue Discrete StructuralPursue Discrete Structural
ModificationsModifications
Jameison et al, J.Med.Chem., 49(17),5029-5046 (2006)
Reduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activityReduction on hERG inhibitory activity

33. Final remarksFinal remarks
•• DoDo not underestimatenot underestimate the value of discrete structural changesthe value of discrete structural changes
•• Test aTest a significant numbersignificant number of analogues within a series to build hERG SARof analogues within a series to build hERG SAR
•• DoDo not overestimatenot overestimate the value of hERG testingthe value of hERG testing•• DoDo not overestimatenot overestimate the value of hERG testingthe value of hERG testing
•• Test as early as possible key candidates inTest as early as possible key candidates in ex vivoex vivo // in vivoin vivo QT prolongation modelsQT prolongation models
•• Avoid “easy optimisations of desired target”: gain potency by introducing lipophilicityAvoid “easy optimisations of desired target”: gain potency by introducing lipophilicity
gain solubility by introducing amine solubilizersgain solubility by introducing amine solubilizers
Jameison et al, J.Med.Chem., 49(17),5029-5046 (2006)

34. Do not forget.... There is always an exception for every ruleDo not forget.... There is always an exception for every rule
•• Amine deletion .... still 21 nM!Amine deletion .... still 21 nM!
MeSO NHMeSO2
NH
N
O
NHSO2
Me
MeSO2
NH
N
O
NHSO2
MeO
hERG IC50 = 21 nM
•• Decrease pKa ... and retain hERG affinityDecrease pKa ... and retain hERG affinity
hERG IC50 = 11 nM hERG IC50 = 21 nM
O O
O
N NN
N NN
O
•• Increase log D ... and decrease hERG affinityIncrease log D ... and decrease hERG affinity
hERG 80% inh @ 300nM
measured pKa = 7.8
hERG 70% inh @ 300nM
measured pKa = 6.0
N NN
O
N
Ph
N NN
O
N
PhF
F
Price et al, Bioor.Med.Chem.Letters,16 (17),4633 (2006)
N
hERG 30% inh @ 300nM
logD = 1.6
hERG 0% inh @ 300nM
logD = 2.1

35. AcknowledgmentsAcknowledgments
The speaker is greatly indebted to his colleagues:The speaker is greatly indebted to his colleagues:
Dr. Jac WijkmansDr. Jac Wijkmans
Dr. Thijs StockDr. Thijs Stock
Dr. Craig Jamieson*Dr. Craig Jamieson*
Dr. Elizabeth M. Moir*Dr. Elizabeth M. Moir*
Dr. Zoran Rankovic*Dr. Zoran Rankovic*
Dr Grant Wishart*Dr Grant Wishart*
** Authors of:Authors of: “Medicinal Chemistry of hERG Optimizations: Highlights and Hang“Medicinal Chemistry of hERG Optimizations: Highlights and Hang--Ups”, J.Med.Chem.,Ups”, J.Med.Chem.,49(17),pp 502949(17),pp 5029--46 (2006)46 (2006)