Transcript Document

Infectious Diseases Drug Discovery:
An AstraZeneca Perspective
Tomas Lundqvist
GSC LG-DECS
AstraZeneca R&D Mölndal
Stewart L. Fisher
Infection Discovery
AstraZeneca R&D Boston
AstraZeneca R&D Boston
History
•
•
•
•
AZ’s newest research facility
Construction initiated August 1998 (Astra)
Building completed March 2000 (AstraZeneca)
Three Research Areas
– Infection Discovery (Global Center)
– Oncology
– Discovery Informatics
• Building expansion completed 2003
– Increased resourcing for Oncology
• Approximately 450 employees
• Expansion underway:
– $100 mil investment in capital (buildings)
– Increased resource for Infection Research
Why Focus on Infectious Disease?
 Medical Need
 Business Opportunity
 Social Responsibility
Causes of Death
35%
30%
Percentage of
all deaths
worldwide
25%
20%
15%
10%
5%
0%
Respiratory
Disease
Cancers
Circulatory
Disease
Infectious
Disease
Ref. WHO Data
Medical need
• 41% of global disease burden is due to infection (WHO, 2002)
• Outside EU & US the disease burden from infection is greater than
the total of all other therapy areas combined
A Major Issue for All
The Golden Age & Today
The Golden Age
of Antibiotic
Discovery was
very brief, mid
1930s- early
1960s
penicillin,
cephalosporin,
streptomycin,
erythromycin,
tetracycline,
vancomycin
The pipeline for new antibacterials is drying up
Resistance to antibacterials continues to rise
There is a clear & present danger of import to
both individual patients and the public health
Target Based Approaches
• 1990’s: Dominant lead generation approach
–
–
–
–
“Genomic era”
Combinatorial/parallel chemistry = large compound libraries
Automated screening technologies provided economy of scale
Structural approaches most amenable to bacterial targets
• Soluble
• High yield overproduction/purification
• 2000-present
– Approach seen as “not delivering the pipeline”
– Many reasons for “failure”
•
•
•
•
Poor compound libraries (not as clean as envisioned)
Difficult to choose the “druggable” targets
Enzyme inhibition ≠ antimicrobial activity (efflux)
Sufficient patience in the industry?
Cell Based Approaches
• 1990’s: Diminished activity due to target-based approaches
– Hit followup appeared “messy” relative to target based
– Identification of novel antibiotics increasingly difficult
– Major efforts in combinatorial biosynthesis
• Genetic manipulation of natural product producers
• 2000-present – renewed interest
– Less faith in target based approaches (e.g. lessons from GSK FabI)
– Improvements in genomic technologies allows facile hit followup
• Regulated gene libraries
• Target identification via resistance gene mapping
– Automated screening technologies affords novel approaches
– Approach amenable to pathways and difficult targets
“Look Back” Programs
•
Revisiting past discoveries, finding new value
–
–
•
Ramoplanin, Tiacumicin B – value of C. difficile in 1980s?
Daptomycin – value of MRSA in 1980’s
Advances in chemistry make intractable scaffolds amenable
–
–
–
ADEPs
Anisomycin
Moiramide
Target-Based Approaches: Pipeline
Target
Identification
many (100’s)
see genomic patents
Hit
Identification
MurA
MurB
MurC
MurD
MurE
MurF
MurG
MurA-F pathway
MurG
MraY-PBPII pathway
DdlB
FtsZ
FtsZ/ZipA
LpxC
RNA Polymerase (RNAP)
DNA Polymerase (DNAP)
DnaB
Phe-tRNAS
Trp-tRNAS
Met-tRNAS
GyrB
PanK
Lead
Identification
Lead
Optimisation
Preclinical/
Clinical
FabDFGAI pathway
FabI
AcpS
FtsZ
Mur Pathway
H. pylori MurI
Peptide Deformylase
GyrB/ParE
FabI/K
Phe-tRNAS
Ile-tRNAS
GyrB
First Step: Define the Problem
Target Product Profile
Target
Identification
Hit
Identification
Lead
Identification
Lead
Optimisation
• Definition of a Target Product Profile
– Define the disease & unmet medical need
– Set the requirements for the drug
– Find targets that fit the requirements
Preclinical/
Clinical
Therapy for Helicobacter pylori Infections
• Causative agent for stomach ulcers
• Implicated in gastric cancer
• Current therapy effective (~ 90%) if properly completed
Proton pump inhibitor (O) + two antibiotics:
Clarithromycin (C), Amoxicillin (A), Metronidazole (M)
• Poor patient compliance due to complicated regimen and side
effects
• Resistance
• Metronidazole 20 - 60%, Clarithromycin 10 -15%
Need for New Therapeutic Strategies
Target Product Profile (H. pylori TPP)
Deliver a candidate drug with this profile:
• Monotherapy
– Oral dose, once a day
(Patient Compliance)
• High Selectivity
– Minimize gut flora disturbance
(Patient compliance)
• Novel target
– No pre-existing resistance
– No threat to current antibiotic regimens
– No target based toxicity issues
(General Utility)
(Cross-Resistance)
(Patient Safety)
Phases of Target-Based Approach: Target Identification
Target
Identification
• Target Identification
–
–
–
–
Genomics-based selection
Validation of essentiality in relevant organisms
Cloning and expression of target proteins
Production of target proteins
Glutamate Racemase (MurI)
UDP-GlcNAc
Fosfomycin
HO
Attributes
•
•
•
•
Novel target for drug discovery
Essential target
Pathway is specific to bacteria
Clinically validated
HO
O
UDP-MurNAc
O
OH
H2 N
H2 N
O
L-Glu
MurC
OH
UDP-MurNAc-(L) Ala
O
MurI
D-Glu
MurD
UDP-MurNAc-(L) Ala-(D) Glu
B-lactam classes
glycopeptides
Cons
• Cytoplasmic target
(Drug penetration?)
• Bacterial kingdom conservation
(Selectivity?)
peptidoglycan
Genomic-based Hypotheses for Selectivity
Bacillus subtilis
Bacillus anthracis
Staphylococcus haemolyticus
Staphylococcus aureus
Bacillus sphaericus
Streptococcus pneumoniae
Streptococcus pyogenes
Enterococcus faecalis
Lactobacillus brevis
Pediococcus pentosaceus
Lactobacillus fermentum
Mycobacterium leprae
Mycobacterium tuberculosis
Haemophilus influenzae
Escherichia coli
Shewanella putrefaciens
Vibrio cholerae
Treponema pallidum
Borrelia burgdorferi
Deinococcus radiodurans
Pseudomonas aeruginosa
Porphyromonas gingivalis
Campylobacter jejuni
Helicobacter pylori
Aquifex aeolicus
• Low sequence identity observed across bacterial species
– Lowest sequence identity of all mur pathway genes
– H. pylori MurI in a distinct phylogenic clade
• Facile protein expression and production
– Gram-scale quantities achieved in high purity (>99% pure)
Gram +ve
Gram -ve
H. pylori
Phases of Target-Based Approach: Hit Identification
Target
Identification
Hit
Identification
• Hit Identification
– Biophysical and biochemical characterization of targets
– Development of primary assay and secondary assays for
evaluation of hits
– Kinetic mechanism studies for enzyme targets
– Screening (e.g. HTS, virtual) and chem-informatic analysis
– Limited SAR generation
H. pylori MurI: an Enigma
• Novel Enzyme Crystal Structure Solved – 1998
Results from Biochemical and Biophysical Characterization:
Crystal
Structure
Features
• • Active
protein
is a dimer
Dimericrequired
enzyme for activity
• No –cofactors
– Active
sitesofoccluded
solvent
• Kinetic
analysis
enzymefrom
reaction
indicates an unusual profile
Selective
binding
D-Glu and reverse reaction
• – Assays
required
for of
forward
Enzyme Mechanism and Assays
Cys 70
Cys 181
L-Glutamate
O
O
O
H
SH
70
D-Glutamate
O
O
O
NH3+
O
O
O
NH3+
NH3+
-S
SH
181
O
O
-
S
HS
70
181
70
H
O
HS
181
Carbanion
intermediate
Coupled Assay with
L-Glutamate dehydrogenase
Measure NADH
Coupled Assay
with MurD
Measure Pi or ADP
Preferred HTS Assay
Resource intensive,
Expensive
Kinetic Analysis of Native H. pylori MurI
D-Glu
L-Glu
L-Glu
D-Glu
100
40
Rate (/min)
Rate (RFU/min)
80
20
60
40
20
0
0
0
20
40
60
80
100 120 140 160
0
20
[L-Glu] (mM)
D-Glu (M)
D-Glu
KM = 63 M
kcat = 12 min-1
KIS = 5.8 M
kcat/KM = 185 mM-1 min-1
40
L-Glu
KM = 700 M
kcat = 88 min-1
kcat/KM = 126 mM-1 min-1
60
Glutamate Racemases: Biochemistry
Energy
Energy
H. pylori MurI
E.L-glu
E+L-glu
E+L-glu
E+D-glu
E+D-glu
E.L-glu
E.D-glu
.
E D-glu
Reaction
ReactionCoordinate
Coordinate
L-Glutamate
O
O
O
H
SH
70
D-Glutamate
O
O
O
NH3+
O
O
O
NH3+
NH3+
-S
SH
181
70
O
O
-
S
HS
181
70
H
O
HS
181
Implications of Unique Biochemical Profile
• Screening unlikely to identify substrate-competitive inhibitors
– Enzyme:Substrate complex = dominant population
– Free Enzyme levels = very low
• Active site is not drug-friendly
– Highly charged
– Small
– Accessibility
• Options:
– Structural / Rational Design
– HTS – non-competitive or uncompetitive inhibitors?
– Suicide substrate / mechanism-based inhibitors
No obvious avenues
HTS Assay?
Poor Inhibition Profile
Novel Assay Format
HTS of corporate collection using novel assay
Suicide Substrate HTS Assay
SO3
MurI
+
O
O
OH
H2 N
kcat
OH
H2 N
x4000
O
O
x1
OH
k release
kinact
+
O
pyruvate
NH3
NAD+
NADH
LDH
MurI
Lactate
inactive
OH
H2 N
• HTS Assay
–
–
–
–
All reagents commercially available
Linear time course (irreversible)
Excellent Assay Window
Amenable to 384-well HTS format
rel. Fluorescence
O
40
blank
30
20
0.2mM S
0.5mM S
10
2.0mM S
0
0
10
20
30
40
50
time (min)
Screened corporate collection for inhibitors (~150,000 cpds)
Pyrimidinediones: Features of the Hit Cluster
N
O
N
Hit Attributes:
O
N
N
N
 in vitro inhibition confirmed in
multiple, orthogonal assay formats
 Whole cell activity in H. pylori
 Confirmed mode of action in whole cells
 Amenable to MPS routes
 Drug-Like Scaffold
Compound A
IC50 = 1.4 M
MIC = 8 g/mL
Phases of Target-Based Approaches: Lead Identification
Target
Identification
Hit
Identification
Lead
Identification
Hit
Identification
•• Target
Identification
Lead
Identification
Biochemicaland
mode
of inhibition
understood of targets
Biophysical
biochemical
characterization
–– Genomics-based
selection
Facile synthetic
in-place
(combichem,
MPS)
Development
of strategies
primaryin
HTS
assayorganisms
and secondary
–– Validation
of essentiality
relevant
assays
for evaluation
of hits
–
Whole-cell
activity
– Cloning and expression of target proteins
–
Kinetic
mechanism
studies for
enzyme
targets
–
Confirmed
target-mediated
mode
of
action
in cells
– Production of target proteins
–
and chem-informatic analysis
– HTS
EarlyScreening
drug metabolism/pharmacokinetics
(DMPK) studies
– Limited SAR generation
Mechanism of Inhibition?
N
HO
O
N
O
N
N
N
Inhibitor
≠
O
OH
H2 N
O
Substrate
Protein NMR – Foundational Work
glutamate free
1.8 mM D-Glutamate
• Double (15N, 2H) & Triple-labeled (15N, 13C, 2H) protein prepared in high yield
• D-Glutamate titration produced a highly resolved spectrum
• All backbone resonances assigned; homodimer ~ 60kD
NMR indicates multiple conformations at room temperature
D-Glutamate stabilizes protein – consistent with kinetic profile
Protein NMR Demonstrates Substrate Dependence
N
O
N
O
N
N
N
Black = D-Glu + MurI
Red = D-Glu + MurI + Inh
•
•
•
Titration of compound reveals specific shifts only when substrate present
Spectrum remains unresolved when compound titration with apo protein
Assignment of resonances allows binding site mapping
Compound binding requires substrate
Binding site distal from active site
Inhibitor:Enzyme Co-Crystal Structure: The “Where”
• Cryptic binding site identified ~7.5Å from active site
• Consistent with NMR binding studies - C-Terminal helix movement
• Catalytic residues unchanged relative to apo structure.
• Supported biochemically:
– Isothermal Titration Calorimetry
– Intrinsic Protein Fluoresence Quenching
– Uncompetitive inhibition
KI = Kd
Cryptic Binding Site – Detailed View
MurI + D-Glutamate
MurI + D-Glutamate + Inhibitor
Unexpected allosteric inhibition mechanism – impact of HTS
Biochemical Confirmation of Inhibition Mode
3 0
100000
2 5
Increasing
[Inh]
80000
2 0
1 5
60000
RFU/80min
RFU
Rate (RFU/min)
120000
1 0
40000
5
20000
0
0
0 .1
1
1 0
280 300 320 340 360 380 400 420 440 460
D
S
O
S
[D-Glu]
(μM)
Wavelength (nM)
15000
FS
FSI
ΔRFU
10000
E+S
F+P
5000
ESI
0
0
0.05
0.1
0.15
0.2
ES
FP
0.25
[Inhibitor] μM
• Binding mode confirmed in multiple formats:
– Intrinsic Protein Fluorescence Quenching
– Isothermal Titration Calorimetry
• Kinetic Mechanism Consistent with Uncompetitive Inhibition
KI = IC50
Mode of Inhibition: The “How”
Inhibitor
Hinge
• Catalytic activity dependent on hinge movement
• Compounds bind at domain interface – lock hinge movement
UDP-MurNAc-(L) Ala
100
Peptidoglycan Biosynthesis
UDP-MurNAc
50
MurC
MurD
UDP-MurNAc-(L) Ala-(D) Glu
MurE
UDP-MurNAc-(L) Ala-(D) Glu-mDap
MurF
UDP-MurNAc-(L) Ala-(D) Glu-mDap-(D) Ala-(D) Ala
A254nm
UDP-MurNAc-(L) Ala
L-Glu MurI D-Glu
Pentapeptide
Bacterial Growth Inhibition Mode of Action Confirmation
0
200
150
+ Inhibitor
100
50
*
0
0
10
20
30
Time (min)
Growth inhibition through MurI inhibition
40
50
Phases of Target-Based Approaches: Lead Optimization
Target
Identification
Hit
Identification
•
Lead
Optimization
Hit
Identification
• TargetIdentification
Identification
Lead
Identification
Lead
Optimization
– Biophysical
Focus
on analogs
of central scaffold(s)
Facile synthetic
(combichem,
MPS)
and strategies
biochemical
characterization
of targets
– Genomics-based
selection in-place
Activity
in animal
disease-state
model
Biochemical
mode
of inhibition
understood
Development
of primary
HTS
assay
and secondary
–– Validation
of essentiality
in
relevant
organisms
for
evaluation
of hits
– assays
Assess
potential
for resistance
Whole-cell
activity
– Cloning and expression of target proteins
–
Kinetic
mechanism
studies
for
enzyme
targets
in
vivo DMPK
studies
for human
dosing
estimation
–
Confirmed
target-mediated
mode
of
action
in cells
– Production of target proteins
–
Screening
and studies
chem-informatic analysis
in
vitro
toxicological
– HTS
Early
drug
metabolism/pharmacokinetics
(DMPK) studies
– Limited
generation
Scale upSAR
synthesis;
process chemistry
Trojan Horse or Goldmine?
Can we improve potency?
What is the potential for resistance?
Can we achieve the desired selectivity margin?
Potency Enhancements
• Established parallel synthesis approaches to rapidly diversify all 4 positions
• Short synthesis, clean reactions
• Amenable to MPS and readily diversified
• Compounds easily purified by preparative HPLC
• Guided by co-crystal structure
Site partially open to solvent but has potential for
specific H-bond interactions (Glu, Ser, H2O)
N
Exposed to solvent
R4
O
R1
N
O
N
N
N
R2
Site mainly surrounded by hydrophobic
groups with a polar terminus (His, Lys)
R3
Deep large hydrophobic pocket
SAR - Highlights
O
N
N
N
N
O
N
O
N
N
IC50 = 2200 nM
N
N
Cl
N
O
N
N
O
IC50 = 67 nM
IC50 = 103 nM
N
N
N
N
S
S
N
H
N
H
N
O
O
N
H
N
N
N
O
N
O
H
N
H
IC50 = 503 nM
Cl
O
Glu150
N
H
N
O
N
O
Cl
N
N
N
N
H
IC50 = 6 nM
• Combination of best R3 and R4 resulted in
250-fold improvement in potency from Hit
Potent inhibitors used to assess resistance
Novel Pocket Concerns: Resistance Rates
Compound
Condition
ARHp55
ARHp80
ARHp206
Inhibitor A
8x MIC
<1.4 x10-9
<4.9 x10-9
<2.7 x10-9
Inhibitor B
8x MIC
<1.2 x10-9
<8.3 x10-10
<2.9 x10-9
Inhibitor C
8x MIC
ND
<1.7 x10-9
<3.3 x10-9
Inhibitor D
8x MIC
<3.9 x10-9
<1.9 x10-9
<2.3 x10-9
Resistance Potential (single step selection):
•
Acceptable (very low) resistance rates observed
•
Despite the low resistance rate, mutations in murI were identified at low [Inhibitor]
[Inhibitor] ≈ 2 x MIC
Biochemical Analysis of Resistance Mutants
A35T
A75T
A75V
E151K
C162Y
I178T
G180S
L186F
L206P
Q248R
- Mapping onto crystal structure did not yield an obvious answer:
Not in the substrate binding pocket
Not in the inhibitor binding pocket (L186F)
- Two were chosen for biochemical characterization:
A75T (most prevalent)
E151K (most dramatic)
A75T H. pylori MurI Kinetic Profile
D-Glu
L-Glu
L-Glu
600
D-Glu
100
80
Rate
Rate ( M/min)
400
200
60
40
20
0
0
0
2000
4000
6000
8000
10000
0
[D-Glu] M
D-Glu
KM = 275 M
kcat = 4 min-1
KIS = 660 M
kcat/KM = 14.5 mM-1 min-1
20
40
60
[L-Glu] mM
(63 M)
(12 min-1)
(5.8 M)
L-Glu
KM = 7400 M
kcat = 106 min-1
kcat/KM = 14.3 mM-1 min-1
Inhibition elevation: (IC50A75T/IC50wt) ~9 fold
MIC elevation:
~4 – 8 fold
(700 M)
(88 min-1)
E151K H. pylori MurI Kinetic Profile
D-Glu
L-Glu
L-Glu
D-Glu
30
120
20
Rate
Rate (RFU/min)
100
10
80
60
40
20
0
0
0
2000
4000
6000
8000
10000
0
[D-Glu]  M
D-Glu
KM = 280 M
kcat = 5 min-1
kcat/KM = 18 mM-1 min-1
(63 M)
(12 min-1)
(5.8 M)
L-Glu
20
[L-Glu]  M
40
KM = 7300 M
kcat = 136 min-1
kcat/KM = 18 mM-1 min-1
Inhibition elevation: (IC50E151K/IC50wt) ~15 fold
MIC elevation:
~8 - 16 fold
60
(700 M)
(88 min-1)
A75T
E
WT
ES
Reaction Coordinate
Resistance impact
Energy
E151K
Decreased Stability
Destabilization of ES Complex
Resistance Mechanism
MurI
MurI
D-Glu
MurI*
(MurI*•D-Glu)
Substrate
inhibited
D-Glu
(MurI•D-Glu)
L-Glu
(MurI*•L-Glu)
Resistance mutants disfavor [ES]/[FS] species:
- Higher Km
- Reduced/Eliminated Substrate Inhibition
Reduced [ES] = less inhibition!
But…
increased potency can overcome effect
Direct Binding Measurements with Inhibitors
10000
RFU
120000
100000
8000
80000
6000
60000
4000
40000
2000
20000
0
0
280 300 320 340 360 380 400 420 440 460
0
Wavelength (nM)
0.2
0.4
0.6
0.8
1
1.2
[Inhibitor] uM
Dissociation Constant (Kd)
MurI Enzyme
Low D-Glu (50M)
High D-Glu (5mM)
Native
23 nM
26 nM
A75T Mutant
170 nM
31 nM
1.4
Bacterial Selectivity Requirement
What about the selectivity profile?
Selectivity Profile
Organism
IC50 (nM)
H. pylori
CN
N
O
H3 C
Cl
N
N
N
O
9.2
0.5
E. coli
H. influenzae
M. catarrhalis
P. aeruginosa
>400000
>64
>64
>64
>64
S. aureus
S. pneumoniae
S. pyogenes
E. faecalis
>400000
>64
>64
>64
N
N
>400000
C. albicans
•
MIC (g/mL)
>64
Excellent selectivity profile observed in series:
•
in vitro (IC50) > 50,000-fold
•
Whole cell > 128-fold
• Basis for selectivity understood – variations in inhibitor binding pocket
–
–
Binding pocket sequence divergence
Limited flexibility to form pocket across species
Trojan Horse or Goldmine?
Can we improve potency?
YES!
What is the potential for resistance? Low
Can we achieve the desired selectivity margin?
So, where’s the drug?
YES!
Target Inhibitor  Drug
• biochemical properties
– bona fide enzyme inhibition
– potency, spectrum
• microbiological properties
– potency, spectrum
– bona fide inhibition of
bacterial growth (MOA)
– resistance frequency
– population MICs (MIC90)
• physical properties
– molecular size
– lipophilicity
– solubility
• in-vivo properties
–
–
–
–
–
–
plasma protein binding
absorption
metabolism
excretion
pharmacokinetics
safety
Pharmacokinetic Profiles in Mouse
Concentration (g/ml)
in vivo
Drug Levels in Mouse Plasma
10
iv 5 mg/kg
po 40 mg/kg
8
6
O
Cl
N
4
O
Cl = 14 µl/min/kg
t½ = 0.7 hr
F = 76 %
N
N
N
N
N
N
2
MIC
0
0
1
2
3
4
5
6
Time (h)
• Improved PK in dogs
• Total drug levels above MIC for extended period of time
Requirements for Efficacy: Free Fraction
Concentration (g/ml)
in vivo
Drug Levels in Mouse Plasma
10
po 40 mg/kg, free
8
po 40 mg/kg, total
6
O
Cl
N
4
O
Cl = 14 µl/min/kg
t½ = 0.7 hr
F = 76 %
fu < 3 %
N
N
N
N
N
N
2
MIC
0
0
1
2
3
4
5
6
Time (h)
• Free drug levels in plasma below MIC
• Difficult to achieve balance between protein binding and potency
The Agony of Defeat
Increase logD
Low Efflux
High metabolism
High protein binding
Decrease logD - Acids
High Efflux
Low metabolism
High protein binding
Microbiology
DMPK
MIC
MBC
Killing Kinetics
Clearance
Bioavailability
Permeability
Vss
Zwitterions
Physical Properties
Protein Binding
Solubility
Decrease LogD - bases
High Efflux
Low metabolism
Low protein binding
Phases of Target-Based Approaches: Preclinical
Target
Identification
Hit
Identification
•
Lead
Optimization
•
Preclinical
Hit
Identification
• TargetIdentification
Identification
Lead
Identification
Lead
Optimisation
Preclinical
– Biophysical
Focus
oncompounds
analogs
of central scaffold(s)
Facile
Several
synthetic
(combichem,
MPS)
and strategies
biochemical
characterization
of targets
– –Genomics-based
selection in-place
Activity
in animal
disease-state
model
Biochemical
Documentation
mode
for
ofFDA
inhibition
filing
understood
Development
of primary
HTS
assay
and secondary
–––Validation
of essentiality
in
relevant
organisms
for evaluation
hits
––assays
in
vivo DMPK
studies of
for
human human
dosing estimation
Whole-cell
Toxicological
activity
to
support
– Cloning and expression of target proteinsdosing
–
Kinetic
mechanism
studies
enzyme
targets
in
vitro toxicological
studiesfor
–
Confirmed
target-mediated
mode
of
action
in cells
– Production of target proteins
–
and process
chem-informatic
analysis
Scale
up synthesis;
chemistry
– HTS
EarlyScreening
drug
metabolism/pharmacokinetics
(DMPK) studies
– Limited SAR generation
Thoughts
• MurI Specific:
– Essentiality & target conservation may be insufficient to gauge potential
– Niche opportunities may be more tractable than broad spectrum
• General:
– Understand the target:
• Mechanistic studies can clarify appropriate strategies for Hit ID
• Evaluate the physiological context of in vitro data
• Structural studies are integral
– HTS can provide novelty – with luck and persistence
– Don’t be satisfied with your best lead series – keep looking!
More reading
Acknowledgments
• AZ Boston
• AZ Mölndal
Richard Alm
Barbara Arsenault
April Blodgett
Ken Coleman
Boudewijn deJonge
Joe Eyermann
Ning Gao
Madhu Gowravaram
Lena Grosser
Pamela Hill
Janette Jones
Thomas Keating
Amy Kutschke
Jim Loch
Larry MacPherson
Cynthia Mascolo
Marshall Morningstar
Brian Noonan
Olga Rivin
Maria Uria-Nickelsen
Jonny Yang
Mark Zambrowski
Beth Andrews
Greg Basarab
Gloria Breault
Janelle Comita
Gejing Deng
Tatyana Friedman
Bolin Geng
Oluyinka Green
Laurel Hajec
Sussie Hopkins
Camil Joubran
Gunther Kern
Stephania Livchak
Kathleen McCormack
John Manchester
Scott Mills
Trevor Newton
Linda Otterson
Mike Rooney
Jim Whiteaker
Wei Yang
Marie Andersen
Tomas Lundqvist
Rutger Folmer
Yafeng Xue
Nan Albertson
Bo Xu
Mark Divers
Christer Cederberg
John Primeau
Trevor Trust
Mark Wuonola
Paul Manning
Gautam Sanyal
Peter Webborn
Supporting Slides
Biochemical Studies on MurI Isozymes
Species
Biochemical data
L-Glu → D-Glu
D-Glu → L-Glu
Escherichia coli
KM = 1200  140 μM
kcat = 730  20 min-1
KM = 2100  140 μM
kcat = 2600  44 min-1
Enterococcus
faecalis
KM = 1200  12 μM
kcat = 1500  40 min-1
Enterococcus
faecium
Staphylococcus
aureus
UNAM-Ala
Activation
Monomer
Yes
KM = 250  20 μM
kcat = 704  14 min-1
Dimer
No
KM = 1100  100 μM
kcat = 2200  50 min-1
KM = 240  23 μM
kcat = 900  32 min-1
Dimer
No
KM = 4600  270 μM
kcat = 510  90 min-1
KM = 140  10 μM
kcat = 34  3.2 min-1
Dimer
No
• Various pathogens represented
• Gram negative enzymes = activated
• Gram positive enzymes = high catalytic turnover
Physiology: Resistance vs. D-Glutamate Regulation
UDP-Mur
Catabolic
Energy Source
MurC
UDP-Mur-(L) Ala
Nitrogen Fixation
Amino Acid
Biosynthesis
L-Glu
MurI
D-Glu
MurD
UDP-Mur-(L) Ala-(D) Glu
Peptidoglycan
• Implications of biochemistry of H. pylori MurI mutants:
–
–
–
Substrate inhibition is a critical regulatory element
Resistant mutants affect enzyme regulation, not binding site
Can be overcome via potency enhancement
Sampling Diverse H. pylori Strains
Genomic DNA from representative strains from a variety of disease states and
geographical locations was screened for resistance mutations.
A35T
A75T
A75V
E151K
C162Y
I178T
G180S
L186F
L206P
Strain
Country
Year of
Disease state
Isolation
ARHP55
Identity
to J99
MurI(%)
Duodenal ulcer 95.359
94.531
94.922
Nonulcer
95.703
dyspepsia
Nonulcer
93.359
dyspepsia
Duodenal ulcer 92.188
UA861
ARHp18
ARHp25
ARHp64
Canada
Canada
Australia
Argentina
1991
1989
1989
1996
ARHp65
Argentina
1996
ARHp54
United
1996
States
ARHp124 Bangladesh 1996
United
States
ARHp43 Australia
ARHp246 Kuala
Lumpur
ARHp241 Kuala
Lumpur
1996
Hiatus hernia
93.75
and gastritis
Duodenal ulcer 92.578
1984
1998
94.531
93.359
ARHp243 France
ARHp244 France
1998
1998
1998
Q248R
Duodenal
ulcer, gastritis
Duodenal
93.359
ulcer, erosive
gastritis
Duodenal ulcer 94.141
Nonulcer
93.75
dyspepsia
Clinical Resistance Potential?
AH244
UA861
SS1_206_
ARHP65
ARHP18
ARHp243
ARHP246
ARHP241
ARHP55
26695
ARHp244
ARHP124
ARHP54
ARHP43
ARHP25
J99
ARHP64
Clustal Co
160
170
180
190
200
ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA
ENILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA
ESILGGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA
ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA
ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IESYFMGHFA
ENILEGELLE TCMRYYFTPL EILPEVIILG CTHFPLIAQK IEGYFMGHFA
ENILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAHQ IEGYFMEHFA
ENILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAHQ IEGYFMEHFA
ENILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAHQ IEGYFMEHFA
ESILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAQK IEGYFMEHFA
ESILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAQK IEGYFMEHFA
ESILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAQK IEGYFMEHFA
ESILEGELLE TCMRYYFTPL KILPKVIILG CTHFPLIAHQ IKGYFMGHFA
ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA
ESILEGELLE TCMRYYFTPL EILPEVIILG CTHFPLIAQK IESYFMEHFA
ESILEGELLE TCMHYYFTPL EILPEVIILG CTHFPLIAQK IEGYFMGHFA
ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA
*.** ***** ***:****** :***:*:*** ********:: *:.*** ***
• Sequenced murI from 16 clinical strains
• Selection criteria:
– Global distribution
– Disease state progression
• Based on sequence conservation, low probability of naturally
occurring resistant strains