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Infectious Diseases Drug Discovery: An AstraZeneca Perspective

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Title: Infectious Diseases Drug Discovery: An AstraZeneca Perspective


1
Infectious Diseases Drug DiscoveryAn
AstraZeneca Perspective
  • Tomas Lundqvist
  • GSC LG-DECS
  • AstraZeneca RD Mölndal
  • Stewart L. Fisher
  • Infection Discovery
  • AstraZeneca RD Boston

2
AstraZeneca RD Boston
  • History
  • AZs 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

3
Why Focus on Infectious Disease?
  • Medical Need
  • Business Opportunity
  • Social Responsibility

4
Causes of Death
Percentage of all deaths worldwide
Ref. WHO Data
5
A Major Issue for All
6
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

7
Target Based Approaches
  • 1990s 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?

8
Cell Based Approaches
  • 1990s 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

9
Look Back Programs
  • Revisiting past discoveries, finding new value
  • Ramoplanin, Tiacumicin B value of C. difficile
    in 1980s?
  • Daptomycin value of MRSA in 1980s
  • Advances in chemistry make intractable scaffolds
    amenable
  • ADEPs
  • Anisomycin
  • Moiramide

10
Target-Based Approaches Pipeline
Target Identification
HitIdentification
Lead Identification
Lead Optimisation
Preclinical/ Clinical
Peptide Deformylase GyrB/ParE
H. pylori MurI FabI/K Phe-tRNAS Ile-tRNAS GyrB
MurA MurB MurC MurD MurE MurF MurG MurA-F
pathway MurG MraY-PBPII pathway DdlB FtsZ FtsZ/Zip
A LpxC RNA Polymerase (RNAP) DNA Polymerase
(DNAP) DnaB Phe-tRNAS Trp-tRNAS Met-tRNAS GyrB Pan
K
many (100s) see genomic patents
FabDFGAI pathway FabI AcpS FtsZ Mur Pathway
11
First Step Define the Problem
Target Product Profile
Target Identification
HitIdentification
Lead Identification
Lead Optimisation
Preclinical/ Clinical
  • Definition of a Target Product Profile
  • Define the disease unmet medical need
  • Set the requirements for the drug
  • Find targets that fit the requirements

12
Therapy for Helicobacter pylori Infections
13
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 (General Utility)
  • No threat to current antibiotic regimens
    (Cross-Resistance)
  • No target based toxicity issues (Patient
    Safety)

14
Phases of Target-Based Approach Target
Identification
15
Glutamate Racemase (MurI)
UDP-GlcNAc
Fosfomycin
UDP-MurNAc
MurC
  • Attributes
  • Novel target for drug discovery
  • Essential target
  • Pathway is specific to bacteria
  • Clinically validated
  • Cons
  • Cytoplasmic target (Drug penetration?)
  • Bacterial kingdom conservation (Selectivity?)

UDP-MurNAc-(L) Ala
MurI
L-Glu
D-Glu
MurD
UDP-MurNAc-(L) Ala-(D) Glu
B-lactam classes glycopeptides
peptidoglycan
16
Genomic-based Hypotheses for Selectivity
  • 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
    (gt99 pure)

17
Phases of Target-Based Approach Hit
Identification
Target Identification
HitIdentification
  • 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

18
H. pylori MurI an Enigma
  • Novel Enzyme Crystal Structure Solved 1998
  • Crystal Structure Features
  • Dimeric enzyme
  • Active sites occluded from solvent
  • Selective binding of D-Glu

19
Enzyme Mechanism and Assays
Carbanion intermediate
Coupled Assay with L-Glutamate
dehydrogenase Measure NADH Preferred HTS Assay
Coupled Assay with MurD Measure Pi or
ADP Resource intensive, Expensive
20
Kinetic Analysis of Native H. pylori MurI
D-Glu L-Glu
L-Glu D-Glu
D-Glu KM 63 mM kcat 12 min-1 KIS 5.8 mM
L-Glu KM 700 mM kcat 88 min-1
kcat/KM 185 mM-1 min-1
kcat/KM 126 mM-1 min-1
21
Glutamate Racemases Biochemistry
22
Implications of Unique Biochemical Profile
  • Screening unlikely to identify substrate-competiti
    ve inhibitors
  • EnzymeSubstrate 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

HTS of corporate collection using novel assay
23
Suicide Substrate HTS Assay
x4000
x1
  • HTS Assay
  • All reagents commercially available
  • Linear time course (irreversible)
  • Excellent Assay Window
  • Amenable to 384-well HTS format

Screened corporate collection for inhibitors
(150,000 cpds)
24
Pyrimidinediones Features of the Hit Cluster
  • Hit Attributes
  • 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 mM MIC 8 mg/mL
25
Phases of Target-Based Approaches Lead
Identification
26
Mechanism of Inhibition?
?
Substrate
Inhibitor
27
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
28
Protein NMR Demonstrates Substrate Dependence
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
29
InhibitorEnzyme 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
30
Cryptic Binding Site Detailed View
MurI D-Glutamate
MurI D-Glutamate Inhibitor
Unexpected allosteric inhibition mechanism
impact of HTS
31
Biochemical Confirmation of Inhibition Mode
  • Binding mode confirmed in multiple formats
  • Intrinsic Protein Fluorescence Quenching
  • Isothermal Titration Calorimetry
  • Kinetic Mechanism Consistent with Uncompetitive
    Inhibition

32
Mode of Inhibition The How
  • Catalytic activity dependent on hinge movement
  • Compounds bind at domain interface lock hinge
    movement

33
Bacterial Growth Inhibition Mode of Action
Confirmation
Peptidoglycan Biosynthesis
Pentapeptide
UDP-MurNAc
UDP-MurNAc-(L) Ala
MurC
UDP-MurNAc-(L) Ala
MurI
L-Glu
MurD
D-Glu
A254nm
UDP-MurNAc-(L) Ala-(D) Glu
MurE
Inhibitor
UDP-MurNAc-(L) Ala-(D) Glu-mDap
MurF
UDP-MurNAc-(L) Ala-(D) Glu-mDap-(D) Ala-(D) Ala

Growth inhibition through MurI inhibition
34
Phases of Target-Based Approaches Lead
Optimization
35
Trojan Horse or Goldmine?
Can we improve potency? What is the potential
for resistance? Can we achieve the desired
selectivity margin?
36
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)
R4
Exposed to solvent
R1
Deep large hydrophobic pocket
R3
R2
Site mainly surrounded by hydrophobic groups with
a polar terminus (His, Lys)
37
SAR - Highlights
IC50 2200 nM
IC50 103 nM
Potent inhibitors used to assess resistance
38
Novel Pocket Concerns Resistance Rates
Compound Condition ARHp55 ARHp80 ARHp206
Inhibitor A 8x MIC lt1.4 x10-9 lt4.9 x10-9 lt2.7 x10-9
Inhibitor B 8x MIC lt1.2 x10-9 lt8.3 x10-10 lt2.9 x10-9
Inhibitor C 8x MIC ND lt1.7 x10-9 lt3.3 x10-9
Inhibitor D 8x MIC lt3.9 x10-9 lt1.9 x10-9 lt2.3 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 Inhibito
    r 2 x MIC

39
Biochemical Analysis of Resistance Mutants
- 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)
40
A75T H. pylori MurI Kinetic Profile
D-Glu L-Glu
L-Glu D-Glu
D-Glu KM 275 mM (63 mM) kcat 4 min-1 (12
min-1) KIS 660 mM (5.8 mM)
L-Glu KM 7400 mM (700 mM) kcat 106
min-1 (88 min-1)
kcat/KM 14.5 mM-1 min-1
kcat/KM 14.3 mM-1 min-1
Inhibition elevation (IC50A75T/IC50wt) 9
fold MIC elevation 4 8 fold
41
E151K H. pylori MurI Kinetic Profile
D-Glu L-Glu
L-Glu D-Glu
D-Glu KM 280 mM (63 mM) kcat 5 min-1 (12
min-1) (5.8 mM)
L-Glu KM 7300 mM (700 mM) kcat 136
min-1 (88 min-1)
kcat/KM 18 mM-1 min-1
kcat/KM 18 mM-1 min-1
Inhibition elevation (IC50E151K/IC50wt) 15
fold MIC elevation 8 - 16 fold
42
Destabilization of ES Complex
43
Resistance Mechanism
D-Glu
(MurID-Glu)
MurI
MurI
Substrate inhibited
L-Glu
D-Glu
(MurIL-Glu)
(MurID-Glu)
Resistance mutants disfavor ES/FS species
- Higher Km - Reduced/Eliminated Substrate
Inhibition
Reduced ES less inhibition! But increased
potency can overcome effect
44
Direct Binding Measurements with Inhibitors
10000
8000
6000
?RFU
4000
2000
0
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Inhibitor uM
Dissociation Constant (Kd)
MurI Enzyme
Native
A75T Mutant
45
Bacterial Selectivity Requirement
What about the selectivity profile?
46
Selectivity Profile
  • Excellent selectivity profile observed in series
  • in vitro (IC50) gt 50,000-fold
  • Whole cell gt 128-fold
  • Basis for selectivity understood variations in
    inhibitor binding pocket
  • Binding pocket sequence divergence
  • Limited flexibility to form pocket across species

47
Trojan Horse or Goldmine?
Can we improve potency? YES! What is the
potential for resistance? Low Can we achieve
the desired selectivity margin? YES!
So, wheres the drug?
48
Target Inhibitor ? Drug
  • biochemical properties
  • bona fide enzyme inhibition
  • potency, spectrum
  • physical properties
  • molecular size
  • lipophilicity
  • solubility
  • in-vivo properties
  • plasma protein binding
  • absorption
  • metabolism
  • excretion
  • pharmacokinetics
  • safety
  • microbiological properties
  • potency, spectrum
  • bona fide inhibition of bacterial growth (MOA)
  • resistance frequency
  • population MICs (MIC90)

49
Pharmacokinetic Profiles in Mouse
in vivo
Drug Levels in Mouse Plasma
10
iv 5 mg/kg
8
po 40 mg/kg
Cl 14 µl/min/kg t½ 0.7 hr
F 76
6
Concentration (mg/ml)
4
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

50
Requirements for Efficacy Free Fraction
in vivo
Drug Levels in Mouse Plasma
10
po 40 mg/kg, free
8
po 40 mg/kg, total
Cl 14 µl/min/kg t½ 0.7 hr
F 76 fu lt 3
6
Concentration (mg/ml)
4
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

51
The Agony of Defeat
52
Phases of Target-Based Approaches Preclinical
53
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
  • Dont be satisfied with your best lead series
    keep looking!

54
More reading
55
Acknowledgments
  • AZ Boston
  • Richard Alm Beth Andrews
  • Barbara Arsenault Greg Basarab
  • April Blodgett Gloria Breault
  • Ken Coleman Janelle Comita
  • Boudewijn deJonge Gejing Deng
  • Joe Eyermann Tatyana Friedman
  • Ning Gao Bolin Geng
  • Madhu Gowravaram Oluyinka Green
  • Lena Grosser Laurel Hajec
  • Pamela Hill Sussie Hopkins
  • Janette Jones Camil Joubran
  • Thomas Keating Gunther Kern
  • Amy Kutschke Stephania Livchak
  • Jim Loch Kathleen McCormack
  • Larry MacPherson John Manchester
  • Cynthia Mascolo Scott Mills
  • Marshall Morningstar Trevor Newton
  • Brian Noonan Linda Otterson
  • AZ Mölndal
  • Marie Andersen Rutger Folmer
  • Tomas Lundqvist Yafeng Xue
  • Nan Albertson Mark Divers
  • Bo Xu

56
Supporting Slides
57
Biochemical Studies on MurI Isozymes
Species Biochemical data Biochemical data Biochemical data UNAM-Ala Activation
Species L-Glu ? D-Glu D-Glu ? L-Glu UNAM-Ala Activation
Escherichia coli KM 1200 ? 140 µM kcat 730 ? 20 min-1 KM 2100 ? 140 µM kcat 2600 ? 44 min-1 Monomer Yes
Enterococcus faecalis KM 1200 ? 12 µM kcat 1500 ? 40 min-1 KM 250 ? 20 µM kcat 704 ? 14 min-1 Dimer No
Enterococcus faecium KM 1100 ? 100 µM kcat 2200 ? 50 min-1 KM 240 ? 23 µM kcat 900 ? 32 min-1 Dimer No
Staphylococcus aureus 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

58
Physiology Resistance vs. D-Glutamate Regulation
UDP-Mur
Catabolic Energy Source
MurC
UDP-Mur-(L) Ala
MurI
L-Glu
MurD
Nitrogen Fixation
D-Glu
UDP-Mur-(L) Ala-(D) Glu
Amino Acid Biosynthesis
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

59
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 Q248R
60
Clinical Resistance Potential?
  • Sequenced murI from 16 clinical strains
  • Selection criteria
  • Global distribution
  • Disease state progression
  • Based on sequence conservation, low probability
    of naturally occurring resistant strains
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