Fragment Based Approaches to
Drugging Proteases
4th RSC-BMCS Fragment Based Drug Discovery Meeting
STFC Rutherford Appleton Laboratory, Harwell, Oxfordshire UK
Steven J. Taylor
Agenda
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
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Evolution of Fragment Based Drug Discovery at
BIPI
Skeptics
Believers
2000
2013
-Small Focused group,
separate from project
teams
-Chemistry and
structural research
FTE’s embedded
1-2 Projects Max
-No Dedicated FTE’s
-Efforts Driven entirely
by project team
-All projects that are
structurally enabled.
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Fragment Hit to Lead
Three Possible Strategies
A fragment hit having high “ligand efficiency” can be leveraged to drive
chemistry using several strategies
Grow
Extend the fragment
hit into adjacent
pockets to gain
potency
Fragment-Based Screening
Confidential
Link
Join adjacent fragment
hits to gain potency
Replace
Exchange regions of
a lead associated
with a liability (e.g.
PK) with fragment
hit
Jhoti Nat. Biotech. 2005 23 184
A fragment hit will generally not be sufficiently potent to be considered a
“lead”
Chymase
Chymase as a Target for Heart Failure and
Fibrosis
• 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 TPC806 identifies a new chemical series and non-covalent
inhibitor; specificity against Cathepsin G is desired
O
OH
O
S
N
N
N
Scaffold Hop
S
Chymase IC50 22nM
Cat G IC50 50nM
S
N
OH
O
Chymase IC50 70 nM
Cat G IC50 2030 nM
6
Lead has undesirable drug properties
O
Crystal structure of Inhibitor bound Chymase
N
N
S
OH
O
Chymase IC50 70 nM
Cat G IC50 2030 nM
LogP = 4.3
tPSA = 64
Forms reactive metabolites
Odds of in vivo toxicity at 10 M
tPSA < 75
tPSA > 75
logP > 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.
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8
Jhoti Nat. Biotech. 2005 23 184
Fragment Hit to Lead Strategy
Grow
Extend the fragment
hit into adjacent
pockets to gain
potency
Link
Join adjacent fragment
hits to gain potency
Replace
Exchange regions of
a lead associated
with a liability (e.g.
PK) with fragment
hit
•Strong correlation between logP of P1
substituent and potency against the
protease.
Fragment-Based Screening
Confidential
•Can this trend be disrupted by FBS?
Chymase Fragment Based Screening
Screening and Hit Triaging Summary
770 fragments screened
206 Total Fragment Hits
95/206 hits screened by X-Ray
41/95 fragments yield co-structure
Overlay of fragment structures
bound to Chymase
NMR
9/35
NMR
80
S1
20
11
24
SEC-MS
12
46
FA
13
7/11
4/5
13/16
FA
SEC-MS
3/7
3/10
4/16
• 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
10
Fragment Hit and Analoging
Crystal structure of fragment bound
Chymase
Overlay of fragment analogs bound
Chymase
His57
Asp102
Ser195
S1
Ser214
S1
Lys192
Val227
Arg217
O
N
H
Cl
Chymase IC50 470
M
LE 0.42
CatG IC50 > 1000
M
HN
Cl
Chymase IC50
>500 M
O
• 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
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Structure Based Inhibitor Design
The “Replace” Approach to Fragment HTL
O
O
N
N
HN
N
S
O
N
H
O
N
N
O
O H
O
O
O
H
Cl
Chymase IC50 3,800 nM
CatG IC50 > 10,000 nM
Asp102
H
Br
O
Cl
O
Chymase IC50 70 nM
Chymase IC50 470,000 nM CatG IC50 2,030 nM
N
HN
O
Chymase IC50 50 nM
CatG IC50 >10,000 nM
Asp102
His57
His57
Lys40
Ser195
Lys40
Ser195
S1
S1
3.2Å
1.3Å
2.7Å
Lys192
Lys192
• 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.
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• Selectivity over Cathepsin G is achieved via polar substituent on P1 moiety
Understanding of Unanticipated Cathepsin G
Selectivity
Overlay of Chymase inhibitor with its
calculated water dipole structure from apo
Docking of Chymase inhibitor to CatG and CatG
water dipoles
• 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
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MMP-13
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Pierre-Auguste
Renoir
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MMP-13 as an Rheumatoid Arthritis Target
Rationale
• 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.
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MMP-13 Structural Biology
Program Starting Point
F
Selectivity loop
H
N
S1´ pocket
F
N
H
N
O
O
Aventis
1XUD
O
O
O
N
H
N
H
N
O
O
O
Pfizer
EX 75,470
S1´ *
O
HO
O
N
N
O
Pfizer
EX 75,484
Catalytic
Zinc
N
HO
Literature crystal structures of both zincchelating and non-zinc chelating inhibitors
available
N
O
F
N
H
N
O
H
N
O
Alantos
BI chemistry focused on developing non zinc-binding inhibitors
accessing the S1`* pocket of MMP13 to gain selectivity over
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Fragment Based Screening
Primary Screening Summary
Functional Assay
NMR STD Binding
SEC
MS
Prioritization for Fragment Crystallography
Starting points for
a medicinal
chemistry
optimization
campaign
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I. Mugge, A. Padyana, B. Co
MMP-13 Indole series initial FBS “Hit”
Co-Structure: Initial SAR
Co-Structure of MMP-13 with Indole
O
H2N
H
N
O
G237
G237
O
MMP-13 IC50 = 42 M
MMP-14/MMP-2 IC50 = 500/60 M
LE = 0.35
N
H
N
H
O
F241
W
MMP13
OH
O
Specificity
N
LoopH
N
W
T247 H
H
O
T247
T245
H
N
O
F241
O
Zn
O
E228
O
H
N
O
W
-O
E223
OH T245
O
NH
Key Issues &
Context
• 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
Strategy to increase potency of initial fragment hit
O
H2N
H
N
O
O
MMP-13 IC50 = <0.001M
Jhoti Nat. Biotech. 2005 23 184
MMP-13 IC50 = 42 M
?
Grow
Extend the fragment
hit into adjacent
pockets to gain
potency
PLEASE INSERT Presentation title
Link
Join adjacent fragment
hits to gain potency
Replace
Exchange regions of
a lead associated
with a liability (e.g.
PK) with fragment
hit
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Co- Crylstallography provides roadmap for
optimization
O
H2N
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F
H
N
O
O
F
N
H
N
O
H
N
O
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Are hybrid literature/fragment inhibitors
possible?
Key Issue: potency & selectivity
H
N
H2N
H
N
65°
O
O
P252
Pocket
O
MMP-13
IC50 42 M
O
H
N
R
42°
N
MMP-13
IC50 82 M
60°
N
MMP-13 IC50
9.7 (+/- 2) M
O
O
N
<2X selective over
MMP-2
N
H
MMP-13
IC50 56 M
N
N
N
>10X selective
over MMP-2
N
H
MMP-13 IC50 MMP-13 IC50
3.9 (+/- 3) M 2.5 (+/- 0.5) M
Hydrogen bonding: opportunities for
heterocycles: H20-M253, or T247
•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
Comparison of cores and their effect on potency
of elaborated molecules
Fragments
H
N
H
N
O
O
N
O
N
NH
pyridyl
MMP-13 IC50 (nM): 2600
O
O
O
O
N
N
pyrazole
O
H
N
O
MMP-13 IC50 (nM): 2800
O
O
O
imidazole
O
MMP-13 IC50 (nM): 40
N
OH
Elaboration
H
N
H
N
N
N
O
O
O
Elaborated pyrazole
22X improvement
over fragment
O
N
O
N
N
HN
MMP-13 IC50 (nM): 120
H
N
O
H
N
N
THR245
O
NH
O
H
N
N
O
Elaborated pyridyl
Elaborated imidazole
MMP-13 IC50 (nM): 120
MMP-13 IC50 (nM): 1.9
20X improvement
over fragment
21X improvement
over fragment,
•Structure guided fragment elaboration leads to low nanomolar, potent selective MMP13 inhibitors
•Flexibility in the core heterocycle provides opportunities for adjusting physiochemical
A. Abeywardan
RHS Ester Replacements
Key Issue: Potency and Microsomal Stability
Ester, although potent presents a potential metabolic alert via oxidative metabolism or plasma
esterase activity:
O
O
O
O
OH
N N
H
N
H
N
HO
R
O
O
OH
H
In Vitro Metabolite ID of Ester
O
HO
H
N
H
N
O

N N
Ester
1 nM 80 % Qh
Acid
1,650 nM <24% Qh
Ether
220 nM 75 %
Qh
Alcohol
2,100 nM 37 % Qh
Unsubstituted
26,000 nM 25 % Qh
98% of metabolism is ester
hydrolysis
O
O
•Replacement of ester with a moiety that retains potency but is stable to esterase
activity should significantly increase half life of this series
Strategy to remove metabolic liability
O
N N
HO
H
N
O
N N
H
N
HO
O
O
O
R
O
Potent
High metabolic stability
Jhoti Nat. Biotech. 2005 23 184
Potent
Low metabolic stability
H
N
H
N
Grow
Extend the fragment
hit into adjacent
pockets to gain
potency
PLEASE INSERT Presentation title
Link
Join adjacent fragment
hits to gain potency
Replace
Exchange regions of
a lead associated
with a liability (e.g.
PK) with fragment
hit
01 March 2013
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Opportunities for ester replacements by
Fragment Merging
H
N
N
O
O
N
Indole analog
MMP13 IC50: 2,500 nM
N N
S
N
DI 603,051
MMP13 IC50: 190 M
O
HO
N N
H
N
O
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
Fragment-Based Screening
Confidential
provided
by the binding
mode of BI 644,577
Hybrid
MMP13 IC50:<1 nM
H
N
N
N. Farrow, A. Abeywardane, Z. X
MMP-13 Potency and Metabolic Stability Strategy
Methods to identify an ester replacement
N N
1. Replacement from fragment “merging”
S
H
N
N
O
Co-Structure of DI 603,051 overlaid
with Co-structure of BI 661,404
N
N
O
190,000 nM
2,900 nM
O
N N
H
N
HO
O
O
2. Replacement from fragment SAR
H
N
O
O
R
>20k nM 57,000 nM
N
H
O
HO
H
N N
H
N
NH
O
>20k nM
0.8 nM
H
N
<1 nM
N
N
N
N
O
O
160,000 nM 150,000 nM
0.13 nM
6.3 nM
R
•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
H
N
Further optimization of potency
O N
N
N
NH
H
N
R
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O
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From Fragment Hit to Prospective Lead
Series
Elaboration of ALI hit
H
N
H2N
H
N
O
H
N
O
O
H
N
O
O
O
N
N
O
N
MMP13 IC50: 42,000 nM (LE 0.35)MMP13 IC50: 56,000 nM (LE 0.31) MMP13 IC50: 2,500 nM (LE 0.38)
MMP-2/14 IC50: >500/>500 M
MMP-2/14 IC50: 77 />500 M
MMP-2/14 IC 50: 68/>500 M
Fragment virtual screening hit (IM)
Provides defined Trajectory to S1`*MMP- 1/8/9 IC50: >500/500/64 M
Accesses Pro pocket, provides potency
and selectivity
H
N
N
N
H
N
O
O
O
N
H
N
O
N
O
N
NH
O
H
N
O
HO
MMP13 IC50: 120 nM (LE 0.32)
MMP-2/14 IC50: >500 M
First fully elaborated fragment
that access S1`*
O
N
N
NH
H
N
N
H
N
O
MMP13 IC5o: 0.27 nM (LE 0.40)
MMP13 IC50: 1.8 nM (LE 0.39)
MMP-2/14 IC50: >250 M
MMP-2/14 IC50: >250 M
Core change increase potency 20xBioisosteric ester replacements identified
>150,000 fold potency improvement over starting point - Increased ligand
Fragment-Based Screening
Confidential
efficiency
32
MMP-13 Chemistry
Comparative Progression of HTS and Fragment Hit series
NH2
Number of Co-Structures
Indole
HTS Series 2
IHTS Sereis1
O
O
N
H
O
8
4
3
Fragment Series
IC50 41µM LE 0.35
HTS Series 1
IC50 10µM LE 0.24
HTS Series 2
(Best Potency)
IC503nM LE 0.35
Lowest Kd(nM)
HTS Series 2
IC50 230nM LE 0.23
HTS Series 1
(Best Potency)
IC50: 2.4nM LE: 0.32
N
Compound Count
H
N
H
N
O
N
O N
N
H
N
Fragment Series
IC500.45nM LE 0.41
Fragment-Based Screening
Confidential
Synthesis of key indole intermediates
O
O
+
O
O
Br
50%
OH
O
N N
POBr3
N N
NH2 1. 0 C, 1 h
NH 2. 120 C 72%
O
O
O
H
N
H
N
H
N
R1
O
B
N
H
N
O
O
HO
O
1.CDI
H 2N
O
R2
O
O
N
H
N
96%
2. Microwave 98%
OH
Br
N
N
15-75%
O
H
N
R1
R1
N
1. Pd Catalyst
Br
O N
1. Pd, pinacol Borane 96%
N
2. (BOC)2O 80 %
O N
N
O
N
B
O
O
SEM
HN
N
Br 1. SEM-Cl, 90%
2. LDA ethylchloroformate 70%
N
Br
O
N
O
Fragment-Based Screening
Confidential
SEM
Pd Catalyst
45-60%
N
N
O
O
N
O
N
O
N
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In Vivo Proof of Concept for MMP-13 Inhibition
Murine Collagen Induced Arthritis Model
NATURE PROTOCOLS |VOL. NO.52007 1269
Fragment-Based Screening
Confidential
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AbCIA
BID Dosing Groups – AbCIA Response
1%CMC, 0.015%Tween 80, 10ml/kgbid
EX00075470 BS1, 100mpk bid
BI00644394 SE3, 100mpk bid
BI00644569 BS, 100mpk bid
EX00075490 SE2, 100mpk bid
16
14
14
12
12
10
10
8
8
6
2
2
0
0
3
4
5
6
BI 644,569 dosed from day4,
all others dosed
prophylactically
7
8
9
10
Experiment Day
11
12
13
14
Top BI compound showed 69% inhibition (Mann-Whitney non-parametric test on
AUC).
Competitor
2
4
Top BI
Compound
4
Competitor
1
Early BI
Compound
6
Vehicle
Mean Arthritic Score (+/- SE)
16
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.
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Acknowledgements
Medicinal Chemistry
Chuck Cywin
Amy Gao
Dan Goldberg
Alexander Heim-Riether
Ken Meyers
Neil Moss
Anthony Prokopowicz
Lana Keenan-Smith
Hidenori Takahashi
Zhaoming Xiong
Yang Yu
Michael Zhang
Fragment Based Screening
Asitha Abeywardane
Brandon Collins
Sandy Farmer
Kathy Haverty
Xiang Li
Shuang Liang
Anil Padyana
John Proudfoot
Steven Taylor
Inflammation and Immunology
Laura Amodeo
Jun Li
Jerry Nabozny
Mark Panzenbeck
Don Souza
John Xiang Li
Lily Zuvela-Jelaska
High Throiughput Chemistry
Juergen Mack
Dieter Wiedenmeyer
Bernd Wellenzohn
Drug Discovery Support
Walt Cao
Ryan Fryer
Paul Harrison
Suzanne-Nodop Mazurek
Raj Nagaraja
Hani Zaher
Toxicology
Ray Kemper
James Tarca
Structural Research
Ingo Mügge
Qiang Zhang
38
Backup Slides
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Difference between Fragment hits and HTS hits
VS Drugs
HTS Hit
Fragment Hit
Lower MW fragment hits provide more room for SAR optimization
Nature Reviews Drug Discovery 3, 660-672 (August 2004)
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Fragment Based Screening
Comparison to uHTS
Typical uHTS campaigns screen 106 drug like compounds against a target
Typically, 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 satisfying
≤ 270;
Nacc ≤ 3; Nrot ≤ 4; Nfused_rings ≤ 3; 3/2.75 ≤ X/ClogP ≥ 0;
Fragment-BasedMW
Screening
Confidential
N
≤2
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 Confidential
properties (e.g. selectivity) while maintaining LE.
Fragment-Based Screening