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Fragment Based Approaches to Drugging Proteases4th RSC-BMCS Fragment Based Drug Discovery MeetingSTFC Rutherford Appleton Laboratory, Harwell, Oxfordshire UK
Steven J. Taylor
Agenda
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1. Overview of 3 Fragment Based Strategies the Boehringer Ingelheim Leverages for Identification and Optimization of Chemical Matter
2. Vignettes
• Chymase• MMP-13
3. Summary and Conclusions
Evolution of Fragment Based Drug Discovery at BIPI
01 March 2013PLEASE INSERT Presentation title 3
2000
-Small Focused group, separate from project
teams -Chemistry and
structural research FTE’s embedded1-2 Projects Max
2013
-No Dedicated FTE’s-Efforts Driven entirely
by project team-All projects that are structurally enabled.
Skeptics Believers
Fragment Hit to LeadThree Possible Strategies
GrowExtend the fragment
hit into adjacent pockets to gain
potency
Jhot
i Nat
. Bio
tech
. 200
5 23
184
LinkJoin adjacent fragment
hits to gain potency
ReplaceExchange regions of
a lead associated with a liability (e.g. PK) with fragment
hit
A fragment hit will generally not be sufficiently potent to be considered a “lead”
A fragment hit having high “ligand efficiency” can be leveraged to drive chemistry using several strategies
Fragment-Based Screening Confidential
Chymase
Chymase as a Target for Heart Failure and Fibrosis
6
• Chymase is a serine protease that catalyzes the peptide cleavage and conversion of angiotensin I to active angiotensin II independent of ACE
• Chymase contributes to heart failure by —Inducing fibrosis through enhancement of collagen and
ECM deposition in key cells—Stimulating remodeling via MMP activation—Activating inflammatory mediators• Chymase inhibitors demonstrated efficacy in heart
failure animal models • Lead ID campaign around the literature compound TPC-
806 identifies a new chemical series and non-covalent inhibitor; specificity against Cathepsin G is desired
Chymase IC50 70 nMCat G IC50 2030 nM
Chymase IC50 22nMCat G IC50 50nM
NN
O
S O
OHNN
S
S
O
OH
Scaffold Hop
Lead has undesirable drug properties
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tPSA < 75 tPSA > 75logP > 3 2.4 (85) 0.41 (38)logP < 3 1.1 (27) 0.39 (57)
• tPSA < 75 and logP >3 space is 6-times as likely to have in vivo tox signal @ 10 M.
Odds of in vivo toxicity at 10 M
Crystal structure of Inhibitor bound Chymase
Chymase IC50 70 nMCat G IC50 2030 nMLogP = 4.3tPSA = 64Forms reactive metabolites
NN
O
S O
OH
8
Fragment Hit to Lead Strategy
GrowExtend the fragment
hit into adjacent pockets to gain
potency
Jhot
i Nat
. Bio
tech
. 200
5 23
184
LinkJoin adjacent fragment
hits to gain potency
ReplaceExchange regions of
a lead associated with a liability (e.g. PK) with fragment
hit
Fragment-Based Screening Confidential
•Strong correlation between logP of P1 substituent and potency against the protease.
•Can this trend be disrupted by FBS?
Chymase Fragment Based ScreeningScreening and Hit Triaging Summary
10
NMR80
FA13
SEC-MS46
11
12
2024
770 fragments screened206 Total Fragment Hits
NMR9/35
FA3/10
SEC-MS4/16
4/5
3/7
7/1113/16
95/206 hits screened by X-Ray41/95 fragments yield co-structure
• X-ray success rate is low (~25-30%) when hits unique to a single screening method is pursued.
• Hit confirmation by at least two techniques consistently improves the X-Ray success rate.
• Overlap hits from all the three primary techniques have high probability to yield co-structures and used in prioritizing fragments for X-Ray follow up.Most fragments bind to the S1 ‘hot spot’ site of Chymase
Overlay of fragment structures bound to Chymase
S1
Fragment Hit and Analoging
11
S1
Asp102
His57
Lys192
Val227
Ser214
Arg217
Ser195
NH
O
Cl
Chymase IC50 470 M LE 0.42CatG IC50 > 1000 M
S1
• Polar fragment binds to lipophilic S1 pocket and hosts water mediated interactions to network with protein
• Fragment SAR is used to probe the nature of interactions and the stability of binding mode
Crystal structure of fragment bound Chymase
Overlay of fragment analogs bound Chymase
NH
ClO
Chymase IC50>500 M
12
Structure Based Inhibitor DesignThe “Replace” Approach to Fragment HTL
1.3Å
Ser195
S1
Asp102
His57
Lys40
Lys192
NH
O
Cl
Chymase IC50 470,000 nM
NN
OS O
O
H
Chymase IC50 70 nMCatG IC50 2,030 nM
Ser195S1
Asp102
His57 Lys40
Lys192
2.7Å
3.2Å
• Co-structure overlay of fragment with inhibitor shows 4-position to be most suitable for linking.
• Polar substituent is allowed in the S1 Pocket and binds as deep as the fragment hit.• Selectivity over Cathepsin G is achieved via polar substituent on P1 moiety
NH
O
Cl
NN
O
O
OH
Chymase IC50 3,800 nMCatG IC50 > 10,000 nM
NH
O
Br
NN
O
O
OH
Chymase IC50 50 nMCatG IC50 >10,000 nM
13
Understanding of Unanticipated Cathepsin G Selectivity
• Understanding role of waters in the binding site is key for modulating potency and selectivity
• Gain of selectivity is conferred to the negative interaction with E226 and polar P1 substituent
Overlay of Chymase inhibitor with its calculated water dipole structure from apo
Docking of Chymase inhibitor to CatG and CatG water dipoles
MMP-13
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Pierre-Auguste Renoir
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MMP-13 as an Rheumatoid Arthritis TargetRationale
• Inhibition of MMP-13 (the most proficient catalyst of collagen II) predicted to reduce cartilage degradation associated with the progression of RA. Reduced inflammation response predicted as a secondary effect.
• MMP-13 associated with osteoclast attachment and maturation on bone surfaces leading to bone erosion.
• MMP-13 implicated in invasion of synovial fibroblast cells.
• Adenovirus over expression of MMP-13 in joints produces an RA-like phenotype.
• MMP-13 -/- mouse shows ~40-50% AbCIA efficacy (Poster report – Takaishi/D’Armiento Groups)
Non-selective MMP programs have failed in the clinic principally due to MSS
LI program goals: Generate two series having required potency, selectivity and drug-like properties. Demonstrate support of drug concept. 18
MMP-13 Structural BiologyProgram Starting Point
Selectivity loop S1´ pocket
S1´ *
CatalyticZinc
N
O
NH
O
NH
O
O O
O
O
NH
O
NH
N
NN FO
OH
Alantos
O
N
NOO
OH
PfizerEX 75,470
PfizerEX 75,484
N
O
NH
O
NH
F F
Aventis1XUD
BI chemistry focused on developing non zinc-binding inhibitors accessing the S1`* pocket of MMP13 to gain selectivity over
Literature crystal structures of both zinc-chelating and non-zinc chelating inhibitors
available
19
Fragment Based ScreeningPrimary Screening Summary
Functional Assay NMR STD Binding SEC MS
Prioritization for Fragment Crystallography
20
Starting points for a medicinal chemistry
optimization campaign
NH
O
O
NH2
O
MMP-13 IC50 = 42 MMMP-14/MMP-2 IC50 = 500/60 MLE = 0.35
MMP-13 Indole series initial FBS “Hit”Co-Structure: Initial SAR
• Low potency and no selectivity, can this be elaborated into potent selective inhibitor?• Can the ester be replaced?• Novel interactions as well as chemical motif and LE make indole fragment an attractive SP
Key Issues & Context
ONH
ONH
OH
ONH
W
ONH
O
-O
N
O
OO
N
H
HH
W
WO
NH
OH
F241
G237
E223
T245
T247 T245
F241 Zn
G237
T247
E228MMP13 Specificity Loop
Co-Structure of MMP-13 with Indole
I. Mugge, A. Padyana, B. Co
Strategy to increase potency of initial fragment hit
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GrowExtend the fragment
hit into adjacent pockets to gain
potency
Jhot
i Nat
. Bio
tech
. 200
5 23
184
LinkJoin adjacent fragment
hits to gain potency
ReplaceExchange regions of
a lead associated with a liability (e.g. PK) with fragment
hit
NH
O
O
NH2
O
MMP-13 IC50 = 42 M MMP-13 IC50 = <0.001M
?
Co- Crylstallography provides roadmap for optimization
01 March 2013PLEASE INSERT Presentation title 23
NH
O
O
NH2
O N
O
NH
O
NH
F F
Are hybrid literature/fragment inhibitors possible?Key Issue: potency & selectivity
NH
N
Hydrogen bonding: opportunities for heterocycles: H20-M253, or T247
N
NH
NN
•Multiple methyl-substituted heterocycles can be used to gain potency and selectivity by accessing the P252 pocket which is specific for MMP-13•Heterocycles as opposed to other linkers, provides a defined trajectory for accessing the S1` * pocket
42°
60°
NH
O
ONH2
O
N
N
NH
O
O
NH
OR
MMP-13 IC50 42 M
65°
MMP-13 IC50 82 M
MMP-13 IC509.7 (+/- 2) M
MMP-13 IC50 56 M
MMP-13 IC503.9 (+/- 3) M
MMP-13 IC502.5 (+/- 0.5) M
P252PocketP252Pocket
<2X selective over MMP-2
>10X selective over MMP-2
Comparison of cores and their effect on potency of elaborated molecules
NH
O
O
NN
NH
ON
NH O
NH
O
O
N
N
NH
O
O
NNH
NH
ON
NH
O
O
NN
OO
O O
NH
O
O
N
NH
O
O
NNH
OO
pyridylMMP-13 IC50 (nM): 2800
imidazoleMMP-13 IC50 (nM): 40
pyrazoleMMP-13 IC50 (nM): 2600
Elaboration
Fragments
Elaborated imidazoleMMP-13 IC50 (nM): 1.9
Elaborated pyridylMMP-13 IC50 (nM): 120
Elaborated pyrazoleMMP-13 IC50 (nM): 12022X improvementover fragment
20X improvementover fragment
21X improvementover fragment,
O
OH
N
THR245
•Structure guided fragment elaboration leads to low nanomolar, potent selective MMP-13 inhibitors•Flexibility in the core heterocycle provides opportunities for adjusting physiochemical
RHS Ester ReplacementsKey Issue: Potency and Microsomal Stability
Ester, although potent presents a potential metabolic alert via oxidative metabolism or plasma esterase activity:
NNNH
O
O
OH NH
R
O
O
Ether 220 nM 75 % Qh
Ester1 nM 80 % Qh
Alcohol2,100 nM 37 % Qh
Unsubstituted26,000 nM 25 % Qh
O
OHAcid1,650 nM <24% Qh
•Replacement of ester with a moiety that retains potency but is stable to esterase activity should significantly increase half life of this series
NNNH
O
O
OH NH
O
O
In Vitro Metabolite ID of Ester
98% of metabolism is ester hydrolysis
O
H
OH
A. Abeywardan
Strategy to remove metabolic liability
01 March 2013PLEASE INSERT Presentation title 27
GrowExtend the fragment
hit into adjacent pockets to gain
potency
Jhot
i Nat
. Bio
tech
. 200
5 23
184
LinkJoin adjacent fragment
hits to gain potency
ReplaceExchange regions of
a lead associated with a liability (e.g. PK) with fragment
hit
NNNH
O
O
OH NH
O
O
NNNH
O
O
OH NH
RPotent
Low metabolic stabilityPotent
High metabolic stability
Opportunities for ester replacements by Fragment Merging
NH
O
O
N
N
Indole analogMMP13 IC50: 2,500 nM
NNNH
O
O
OH NH
N
HybridMMP13 IC50:<1 nM
N
NNS
DI 603,051 MMP13 IC50: 190 M
A challenge for the team was to remove the “metabolic liability” of the ethyl ester of the original hit.Proof that this could be accomplished was provided by the binding mode of BI 644,577Fragment-Based Screening Confidential
NNNH
O
O
OH NH
N
MMP-13 Potency and Metabolic Stability Strategy Methods to identify an ester replacement
1. Replacement from fragment “merging”
N
NNS
190,000 nM 2,900 nM
0.13 nM
N O
N
6.3 nM
O
N2. Replacement from fragment SAR
<1 nM
NH
NH
RO
0.8 nM
57,000 nM 160,000 nM 150,000 nM
H
>20k nM
>20k nM
•Despite steep SAR, equipotent ester replacements can be identified from fragment merging and from SAR done on fragment starting points, independent of the elaborated molecule
NH
R
NNNH
O
O
OH
NH
O
ON
N
O
O
Co-Structure of DI 603,051 overlaid with Co-structure of BI 661,404
N. Farrow, A. Abeywardane, Z. X
Further optimization of potency
01 March 2013PLEASE INSERT Presentation title 30
NH
N
NO
NNH
NH
OR
From Fragment Hit to Prospective Lead SeriesElaboration of ALI hit
MMP13 IC50: 42,000 nM (LE 0.35)MMP-2/14 IC50: 77 />500 M
Fragment virtual screening hit (IM)
MMP13 IC50: 120 nM (LE 0.32)MMP-2/14 IC50: >500 M
First fully elaborated fragment that access S1`*
NH
O
O
NH
N
NH
O
O
N
N
MMP13 IC50: 56,000 nM (LE 0.31)MMP-2/14 IC 50: 68/>500 M
Provides defined Trajectory to S1`*
NH
O
O
NH2
O
NH
O
O
NN
NH
ON
MMP13 IC50: 2,500 nM (LE 0.38)MMP-2/14 IC50: >500/>500 M
MMP- 1/8/9 IC50: >500/500/64 MAccesses Pro pocket, provides potency
and selectivity
NH
O
O
NNH
NH
ON
NH
NNH
NH
O
N
NO
O
OHMMP13 IC50: 1.8 nM (LE 0.39)
MMP-2/14 IC50: >250 MCore change increase potency 20x
MMP13 IC5o: 0.27 nM (LE 0.40)MMP-2/14 IC50: >250 M
Bioisosteric ester replacements identified
>150,000 fold potency improvement over starting point - Increased ligand efficiencyFragment-Based Screening Confidential
32
MMP-13 ChemistryComparative Progression of HTS and Fragment Hit series
Compound Count
Low
est
K d(n
M)
Number of Co-StructuresIndole 8HTS Series 2 4IHTS Sereis1 3
HTS Series 1IC50 10µM LE 0.24
HTS Series 1(Best Potency)
IC50: 2.4nM LE: 0.32
HTS Series 2IC50 230nM LE 0.23
HTS Series 2(Best Potency)
IC503nM LE 0.35
NH
O
O
NH2
O
NH
NH
N
NH
O
N
NNO
Fragment SeriesIC50 41µM LE 0.35
Fragment SeriesIC500.45nM LE 0.41
Fragment-Based Screening Confidential
Synthesis of key indole intermediates
01 March 2013Fragment-Based Screening Confidential 34
OO
OO
NH2
NH
NN
OHO
O
NN
BrO
O
POBr3
NH
BO
O
R1
NH
NN
OO
R1NH
NN
NH
OR2
R1
+1. 0 C, 1 h 2. 120 C 72% 50%
1. Pd Catalyst15-75%
NH
OH
O
Br
NH
Br N
NO N
B N
NO
O
O
OON
NH2
OH
1.CDI 96%2. Microwave 98%
1. Pd, pinacol Borane 96%2. (BOC)2O 80 %
N
NHBr
N
NBr
SEM
O
O
N
NN
O
O
ON
NO
O
SEM1. SEM-Cl, 90%2. LDA ethylchloroformate 70%
Pd Catalyst
45-60%
In Vivo Proof of Concept for MMP-13 InhibitionMurine Collagen Induced Arthritis Model
01 March 2013Fragment-Based Screening Confidential 35
NATURE PROTOCOLS |VOL. NO.52007 1269
AbCIA BID Dosing Groups – AbCIA Response
0
2
4
6
8
10
12
14
16
3 4 5 6 7 8 9 10 11 12 13 14
Experiment Day
Mea
n A
rthr
itic
Scor
e (+
/- SE
)
1%CMC, 0.015%Tween 80, 10ml/kg bidEX00075470 BS 1, 100mpk bidBI00644394 SE 3, 100mpk bidBI00644569 BS, 100mpk bidEX00075490 SE 2, 100mpk bid
0
2
4
6
8
10
12
14
16
Top BI compound showed 69% inhibition (Mann-Whitney non-parametric test on AUC).
BI 644,569 dosed from day4,all others dosed
prophylactically Veh
icle
Com
petit
or 1
Com
petit
or 2
Top
BI
Com
poun
d
Ear
ly B
I C
ompo
und
Summary
Fragment based screening and optimization can provide a complementary method to HTS for identifying attractive chemical matter
LE should be tracked and used to help asses progress of an optimization campaign in parallel to potency and physicochemical properties
It is important to keep a focus on the Patients and why we as Scientists got into this business, after all we are saving and improving lives of those with few options.
01 March 2013PLEASE INSERT Presentation title 37
Acknowledgements
Medicinal ChemistryChuck CywinAmy GaoDan GoldbergAlexander Heim-RietherKen MeyersNeil MossAnthony ProkopowiczLana Keenan-SmithHidenori TakahashiZhaoming XiongYang YuMichael Zhang
Fragment Based ScreeningAsitha AbeywardaneBrandon CollinsSandy FarmerKathy HavertyXiang LiShuang LiangAnil PadyanaJohn ProudfootSteven Taylor
Inflammation and ImmunologyLaura AmodeoJun LiJerry NaboznyMark PanzenbeckDon SouzaJohn Xiang LiLily Zuvela-Jelaska
Drug Discovery SupportWalt CaoRyan FryerPaul HarrisonSuzanne-Nodop MazurekRaj NagarajaHani Zaher
High Throiughput ChemistryJuergen MackDieter WiedenmeyerBernd Wellenzohn
Structural Research Ingo MüggeQiang Zhang
ToxicologyRay KemperJames Tarca
38
Backup Slides
01 March 2013PLEASE INSERT Presentation title 39
Difference between Fragment hits and HTS hits VS Drugs
01 March 2013Nature Reviews Drug Discovery 3, 660-672 (August 2004) 40
HTS Hit
Fragment Hit
Lower MW fragment hits provide more room for SAR optimization
Fragment Based ScreeningComparison to uHTS
Typical uHTS campaigns screen 106 drug like compounds against a targetTypically, hits are defined as having IC50s of < 20 M
Typical Fragment based campaigns run 103-104 MW <270 compounds against a target Typically, hits are defined as having IC50s in the M - mM range
Makeup of Screening Libraries
HTS Library
A collection on ~1 million compounds collected from multiple sources MW ≤ 700 purity > 50%
Generic Fragment Library
A collection of ~1,500 highly characterized compounds satisfyingMW ≤ 270; Nacc ≤ 3; Nrot ≤ 4; Nfused_rings ≤ 3; 3/2.75 ≤ X/ClogP ≥ 0;
N ≤ 2Fragment-Based Screening Confidential
Ligand Efficiency Definition
Ligand Efficiency is a measure of how effective a compound is at binding its target.
Generally, it is possible to increase binding by increasing the MW of a compound – however Lipinski’s Rules shows that a desirable MW for a drug is < 500
Ideally a HTL program would like to start with a small highly potent compound – a compound with high Ligand Efficiency (LE)
LE = -RT log(IC50)/N
N = Number of heavy atoms (rule of thumb N = MW/13.1)
The nature of fragments is such that even though they bind in the mM-M range they are highly ligand efficient as a result of their low MW
A Fragment Hit to Lead (FHTL) program will seek to increase potency and build in other properties (e.g. selectivity) while maintaining LE.
Fragment-Based Screening Confidential