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Building innovative drug discovery alliances Discovery of Selective OX2 Antagonists and Study of Orexin 1 and 2 GPCR Receptors GPCRs in Medicinal Chemistry, 18 th September 2012
Building innovative drug discovery alliances
Discovery of Selective OX2 Antagonists and Study of Orexin 1 and 2 GPCR Receptors
GPCRs in Medicinal Chemistry, 18th September 2012
PAGE
Agenda
1
Introduction
Medicinal Chemistry
Computational Chemistry
PAGE
Agenda
2
Introduction
Medicinal Chemistry HTS Campaign Indole-sulfone Profile Indole-sulfone Optimisation Conclusions
Computational Chemistry
PAGE
Orexin Receptor AntagonistsInsomnia
3
Clinically validated new mechanism for insomnia Almorexant, SB-649868, Suvorexant (MK-4305)
All 3 compounds are dual OX1/2 Antagonists
An OX2 selective compound has been reported
to have hypnotic activity*
*Dugovic et al, JPET 2009, 330(1), 142-151
Goal: discover a selective OX2 antagonist for insomnia
PAGE
Orexin Antagonist ProgrammeScreening Campaigns
250K Evotec Library
Primary screen: Ca2+ flux FLIPR in CHO cells transfected with hOX1 or hOX2
514 Primary hitsCounter-screen:
activity against CHO-endogenous ATP receptor
Synthetic tractability
Med Chem assessment
Preliminary SAR in first 10-20 cpds?
70 confirmed, specific hits
5 series, 7 singletons
1 ‘series’selected
4
OX1 HTS also produced hits (EP2161266):
NO
HN
N
O
O
HN N
EP-009-0049
EP-109-0092
PAGE
EP-009-0236 EP-009-0237
OX1 / OX2 (nM)FLIPR
3989 / 103 3704 / 50
Aq Sol (M) <20 <20
MW / tPSA 433 / 59 447 / 59
cLogP / LipEChemAxon
3.9 / 3.1 4.2 / 3.1
Microsomal ClearanceHuman / Rat High / High High / High
Hit Profiles
5
Indole-3-sulfones
Potent, OX2 selective
S
N
N
O
O
O
S
N
N
O
O
O
Metabolically unstable
PAGE
Indole-3-Sulfones
6
Optimisation
Strategy:
Block (or remove) potentially labile positions Most on benzylamine fragment
Globally reduce lipophilicity
Goals:
Establish SAR
Increase stability
Increase aqueous solubility
PAGE
NH NH
SH
Br
NH
S
NH
SO
O
NH
SO
O
N
SO
O
OH
O
HN
N
SO
O
O
N
1) I2, thiourea2) KOH
MeOH / water
(55%)
NaH
Dioxane
(55%)
OxoneNaHCO3
Acetone / Water
(87%)
1) NaH, ethyl bromoacetate, DMF2) NaOH, MeOH / water
(90%)
HOBt, HBTU
DMF
(68%)
Indole-3-sulfones
7
Synthetic Route
WO2011138265
Core and Sulfone introduced early
Late-stage variation of benzylamine
PAGE
Indole-3-sulfone Optimisation
8
Screening Cascade
hOX1 and hOX2 (FLIPR)
P2Y1 (endogenous CHO receptor Gq), rOX1 and rOX2
Non-radioactive GTPS assay Human and rat microsomes, aq solubility
Schild plot / MOA P450, PPB, Caco-2
IV / PO PK, human and rat hepatocytes
No OX1 or OX2 species differences noted
Good correlation between FLIPR and GTPS assays
PAGE
Indole-Sulfone SAR
9
Which changes are tolerated?
S
N
O
O
N
O
BenzylamineSubstitution
Electron-withdrawingindole substituents
BenzylsulfoneSubstitution
N-methyl deletion
Fluoro and chloro best
PAGE
Indole-Sulfone SAR
10
Which changes aren’t beneficial?
S
N
N
O
O
O
Conformationalrestriction
N
N
O
O
Cl
Sulfone replacement
(+ indole inversion)
S
N
N
NO
O
O
Amide replacement
S
N
N
OO
O
Shortenedlinkers
S
N
N
O
O
O
F
O
Electron-donatingindole substituents
Early SAR established
PAGE
Indole-3-sulfone Optimisation
11
Blockade of labile positions
EP-009-0236 EP-009-0249 EP-009-0312 EP-009-0372 EP-009-0339
OX1 / OX2 (nM)FLIPR
3989 / 103 1221 / 23 971 / 46 >20 M / 33 1576 / 179
Aq Sol (M) <20 - 33 <20 <20
MW / tPSA 433 / 59 451 / 59 477 / 59 485 / 59 465 / 59
cLogP / LipEChemAxon
3.9 / 3.1 4.0 / 3.6 4.5 / 2.9 4.6 / 2.9 4.4 / 2.3
S
N
N
O
O
O
F
S
N
N
O
O
O
F
S
N
N
O
O
O
Microsomal Clearance
Human / RatHigh / High High / High High / High High / High High / High
S
N
N
O
O
O
F
Cl
S
N
N
O
O
O
(R)
F
PAGE
Blocking Strategy
12
Summary
Microsomal clearance remains high
Blocking strategy unsuccessful
Would lipophilicity reduction fare any better?
PAGE
Reduced Lipophilicity
13
Benzylamine Modification
EP-009-0236 EP-009-0393 EP-009-0314 EP-009-0273
OX1 / OX2 (nM)FLIPR
3989 / 103 >20 M / >20 M >20 M / 4.1 M >20 M / 2.9 M
Aq Sol (M) <20 - - -
MW / tPSA 433 / 59 438 / 85 434 / 72 434 / 72
cLogP / LipEChemAxon
3.9 / 3.1 2.5 / - 2.7 / 2.7 2.6 / 2.9
S
N
N
O
O
O
N
S
N
N
O
O
O
N
S
N
N
O
O
O
O
N
S
N
N
O
O
O
Loss of potency on changing the benzylamine group
Revised synthetic routes needed to target different cores
PAGE
1) DBU2) Oxone
(1) 76% 2) 61%)
1) HCl2) K2CO3
(40%)
SH
NCl
SN
O O
Br
OO
N
SO
O
N
SO
O
N
SO
O
OO
O
N
SO
O
O
O
Cl
O
O
O
(61%)
1) NaBH 42) PPh 3 / I2
(1) 77% 2) 78%)
Indolizine Synthetic Route
14 WO2011138266
Core and sulfone properties fixed early on
2-Methyl group introduced for synthetic reasons
PAGE
Imidazopyridine Synthetic Route
15 WO2011138266
N
NH2
Cl
O
O
O
N
HN
O
O
O
NN
O
O
NN
O
O
NN
O
O
NN
O
O
SO
O
I S
SH
(70%)
DCM
Oxone
(77%)(66%)
Dioxane
Pd2(dba) 3XantphosiPr2NEt
MeCN
NISEt3N
(67%)
POCl3(48%)
THF
Et3N
Core properties fixed early on Sulfone introduced later
PAGE
S
N
N
O
O
O
EP-009-0466
3784 / 1.0
22
447 / 59
3.8 / 5.2
Indole-3-sulfone Optimisation
16
Reduction of Lipophilicity – core changes
EP-009-0236 EP-009-0456 EP-009-0403 EP-009-0495
OX1 / OX2 (nM)FLIPR
3989 / 103 1399 / 538 998 / 14 >20 M / 194
Aq Sol (M) <20 85 37 177
MW / tPSA 433 / 59 434 / 72 434 / 72 452 / 72
cLogP / LipEChemAxon
3.9 / 3.1 3.0 / 3.3 3.6 / 4.3 3.2 / 3.5
S
N
N
N
O
O
O
S
NN
N
O
O
O
F
S
N
N
O
O
ON
S
N
N
O
O
O
Microsomal Clearance
Human / RatHigh / High High / High High / High High / High High / High
Two orders of magnitude potency increase for 2-methylindolizine!
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Core Replacement Follow up
17
Indazole and 2-Methylindole
EP-009-0403 EP-009-0483 EP-009-0404 EP-009-0482
OX1 / OX2 (nM)FLIPR
998 / 14 >20 M / 123 278 / 1.1 >20 M / >20 M
Aq Sol (M) 37 - <20 -
MW / tPSA 434 / 72 445 / 105 465 / 59 428 / 90
cLogP / LipEChemAxon
3.6 / 4.3 3.2 / 3.7 4.2 / 4.8 2.0 / -
S
N
N
N
O
O
O
S
N
N
HN
O
O
O
N
S
N
N
HN
O
O
O
O
S
N
N
O
O
O
F
Microsomal ClearanceHuman / Rat High / High High / High High / High High / High
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LipE Progression Summary
18
From Ep-009-0236
S
N
N
O
O
O
S
N
N
O
O
O
S
N
N
HN
O
O
O
N
S
N
N
N
O
O
O
High ProbabilitySpace
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Electrostatic Complementarity
19
Useful tool for structure-base drug discovery
We developed a method1 to predict regions of the ligand that are electrostatically attracted to attracted to or repelled byrepelled by the protein, and to map / visualize it on the ligand surface
Modifications in these regions usually have a significant effect on binding affinity
We called these important regions of the ligand “hot spots”
OX2 ICOX2 IC5050 = = 195 nM195 nM OX2 ICOX2 IC5050 = = 1 nM1 nM
RepulsionRepulsion AttractionAttraction
1 Davenport and Heifetz et al ,Assay Drug Dev Technol. 2010 Dec;8(6):781
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Agenda
20
Introduction
Medicinal Chemistry
Computational Chemistry Receptors selectivity study Conclusions
PAGE
hOX1 vs. hOX2 Selectivity
21
Study Motivation
Precise determinants of antagonist binding and
selectivity were neither fully known nor rationalised Site-directed mutagenesis and domain exchange (chimera) studies
have provided important insight on key features of the OX1/2 binding
sites1,2 & 3
11 mutations were performed for OX1 and 18 for OX2
To explain the role of each mutated residue for binding
and selectivity of a set of OX1/2 antagonists
Support discovery of novel OX1/2 antagonists
1 Malherbe, et al (2010) Mol. Pharmacol. 78, 81-932 Tran, et al (2011) Eur. J. Pharmacol. 667, 120-1283 Heifetz et al Biochemistry. 2012 Apr 17;51(15):3178
PAGE
hOX1 vs. hOX2
22
What we can learn from sequence alignment?
TMD: TMD: Identity: Identity:
69.3% 69.3% Similarity: Similarity:
80.580.5
Mutations by EvotecMutations by otherMutations by Evotec and otherNot tested
PAGE
Intriguing SDM dataSmall differences in sequence can lead to large changes in IC50
Position hOX1 Almorexant3.33 A127T ↓↓7.43 Y348A ↓↓
Position hOX2 Almorexant3.33 T135A =7.43 Y354A =
? ?? ?
23
Why does the A1273.33T mutation significantly reduce binding of Almorexant to OX1, while the T1353.33A mutation in OX2 has no effect?
Why does mutating the conserved tyrosine located in the 7.43 position give opposite effects in OX1 vs OX2?mutation of Y3487.43 in OX1 eliminated Almorexant binding, the same
mutation Y3547.43 in OX2 had no effect
PAGE
Exploration of OX1/2 Receptors Method validation
24
PAGE
GPCR Modeling ProtocolThe optimized similarity routines
25
Optimized Similarity Modeling
Ab-initio Decoy Template Modeling
Hybridized Homology Modeling
Liga
nd P
ocke
t Opt
imiz
atio
n
Homology Modeling based on single templateGPCR specific seq. alignment matrix
Helical rotation alignment
MC side-chain rotamer library sim.
MD kink detection & formation
Addition of SDM data
Molecular Dynamics SimulationsMolecular Dynamics Simulations
Loop remodeling
Protein packing & complementary score
Fit to sequence alignment
Individual optimization routine scoring
Fit to SAR and SDM data
● Each tier of the modeling process assesses the need for the next stage
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MD SimulationsEstablished Protocol for GPCR Modelling
GROMACSOPLS-AA forcefield
Performance = ~1ns/day 8 cores
Aims
• Optimise homology models
• Explore structural behaviour -
OX1/OX2 and mutants/wild-types
System size ~117k atoms~31k waters
~ 327 residues~ 5200 protein atoms
~350 lipids26
PAGE
Focus on TM3
27
Focus on TM3 – important for selectivity of OX antagonists
TM2
TM4TM3
TM7 TM5C: OX2
Y2235.38
Q1874.60T1353.33
T1062.56
Q1343.32
Y3547.43
TM2TM4
TM3
A: OX1TM7 TM5
Q1263.32
Y3487.43
I982.56
Y2155.38
Q1794.60A1273.33
D: OX2
N952.45W1774.50
T1443.42
TM2 TM4TM3
TM2
TM4
TM3
N872.45
F842.42
W1694.50T1363.42
F1393.45
B: OX1
PAGE
Focus on TM3
28
OX2:TM3OX2:TM3OX1:TM3OX1:TM3
OX2:TM3OX2:TM3
T106T1062.562.56Q134Q1343.323.32
Y354Y3547.437.43
AlmorexantAlmorexant
Y223Y2235.385.38
Q187Q1874.604.60
T135T1353.333.33
OX1:TM3OX1:TM3
AlmorexantAlmorexant
Q126Q1263.323.32
Y348Y3487.437.43
N87N872.452.45
F84F842.422.42
W169W1694.504.50
T136T1363.423.42
F139F1393.453.45
AA BB CC
Long
est h
elix
axi
s of
TM
3
OX1OX1
OX2OX2
TM3TM3OX1 OX1 vs. TM3vs. TM3OX2OX2
Different interDifferent inter--helical interactions force the core of TM3helical interactions force the core of TM3OX1OX1 and TM3and TM3OX2OX2 to adopt slightly different to adopt slightly different conformations conformations
In OX2, there appeared to be a tilting of the TM3OX2 core away from its original position in OX1
PAGE
Almorexant Complex with OX1/2
29
W20645.54
H3447.39
Q1263.32F2195.42Y3116.48
Y2245.47
Y3487.43
A1273.33
Y2155.38
W21445.54
F2275.42
H3507.39
T1353.33Q1343.32
Y3176.48
Y2235.38
Y2325.47
Q1874.60T1062.56
Y3547.43
B: hOX2RhOX2R with AlmorexantA: hOX1RhOX1RWTWT with Almorexant
T135T1353.333.33
Q126Q1263.323.32
Q134Q1343.323.32
T106T1062.562.56
Y348Y3487.437.43
C: hOX1RhOX1RWTWT, TM3TM3OX1OX1, TM3TM3OX2OX2 with Almorexant
TM3TM3OX1OX1
TM3TM3OXOX22
The residue Y3487.43 does not form a direct interaction with Almorexant but it still plays a key role by stabilising Q1263.32 in a conformation in which it interacts with Almorexant.
The mutation of A1273.33 into the larger residue threonine limits the approach of the antagonists into the OX1 sub-pocket between TM3, 4 and 5
PAGE
Intriguing SDM dataSolving the mystery
Position hOX1 Almorexant3.33 A127T ↓↓7.43 Y348A ↓↓
Position hOX2 Almorexant3.33 T135A =7.43 Y354A =
? ?? ?
30
We showed that different inter-helical interactions force the conformations of TM3OX1 and TM3OX2 to be different
A127 and T135 are located in the same position in the sequences but in different locations in the structures
The mutation of A1273.33 into the larger residue threonine limits the approach of antagonists into the OX1 sub-pocket between TM3, 4 and 5, resulting in a significant decrease in the binding of certain antagonists
The OX1 residue Y3487.43 does not form a direct interaction but it still plays a key role by stabilising Q1263.32 in a conformation in which it interacts with antagonists – so mutating it into Alanine will affect antagonist binding
The OX2 residue Y3547.43 (in contrast to Y3487.43 of OX1) is “unemployed”, it neither interacts directly with antagonists nor does it stabilise Q1343.32 – so mutating it to Alanine will not have any effect
PAGE
GPCRs Modelling Conclusions
31
Minor differences in sequences can lead to significant differences in the tertiary structure of GPCRs, their
ability to bind ligands, and their selectivity
GPCR models based solely on homology modelling might not be sufficient to rationalise potency and
selectivity
MD simulations allow refinement of GPCR models to a degree that is not possible with static homology
modelling alone
The structural insights gained from this process are critical for rationalising the SDM data, and for the design of
new GPCR ligands
PAGE
Discovery of Novel OX1, -2 Selective Antagonists
32
W20645.54
Y2155.38
H3447.39
Q1263.32
F2195.42Y3116.48
Y2245.47
Y3487.43
A1273.33
a
bc
V3477.42
V1303.36
A: hOX1RhOX1R with EP-009-0049 (OX1 selective)
Q1343.32
Y3547.43
W21445.54
F2275.42
H3507.39
T1353.33Y3176.48
Y2235.38
Y2325.47
I3206.51
a
b
c
N3246.55
B: hOX2RhOX2R with EP-009-0513 (OX2 Selective)
PAGE
Acknowledgements
33
Evotec (UK) Evotec (Hamburg) University of Oxford
Tara Fryatt
Oliver Barker
Gurubaran Raju
David Hallett
Mark Whittaker
Natacha Prevost
Julia Vile
Sandeep Pal
Richard Law
Dominique Manikowski
Rita Reifegerste
Mark Slack
G. Benjamin Morris
Philip C. Biggin
In vitro DMPK Group (UK)
Evotec (India)
Royal Society, UK for Industry award
Building innovative drug discovery alliances
Appendix
PAGE 35
TMD TM3
TMD C atom RMSDs fluctuate within the same narrow range of 1.7 to 2.4 Å, which is comparable to the
values typically obtained for MD simulations of GPCR crystal structures
TM3 C atom RMSDs fluctuate within a narrow range between 1.0 and 1.5 Å for the OX1WT, OX1A127T and
OX2WT indicates that the TM3 conformation of receptors is almost “frozen” AND is not affected by the
A127T3.33 mutation.
PAGE
OX1 vs. OX2 Receptor
36
Final Models
TMD conformational differences observed in structures and binding site topology of the OX1vs. OX2 structures
OX1 is orange and OX2 is green
PAGE
Electrostatic Complementarity
37
Useful tool for structure-base drug discovery
We developed a method1 to predict regions of ligand that are electrostatically attracted to attracted to or repelled byrepelled by the protein, and to map / visualise it on the ligand surface
Modifications in these regions usually have a significant effect on binding affinity
We called these important regions of the ligand “hot spots”
OX2 ICOX2 IC5050 = = 195 nM195 nM OX2 ICOX2 IC5050 = = 1 nM1 nM
RepulsionRepulsion AttractionAttraction
1 Davenport and Heifetz et al ,Assay Drug Dev Technol. 2010 Dec;8(6):781
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Jonathan Bentley and Alex Heifetz
+44 (0)1235 861561
