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

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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

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Why Focus on Infectious Disease?

• Medical Need
• Business Opportunity
• Social Responsibility

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

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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?

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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

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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

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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
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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

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Therapy for Helicobacter pylori Infections
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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)

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Phases of Target-Based Approach Target
Identification
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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
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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)

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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

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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

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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
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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
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Glutamate Racemases Biochemistry
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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
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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)
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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
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Phases of Target-Based Approaches Lead
Identification
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Mechanism of Inhibition?
?
Substrate
Inhibitor
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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
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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
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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
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Cryptic Binding Site Detailed View
MurI D-Glutamate
MurI D-Glutamate Inhibitor
Unexpected allosteric inhibition mechanism
impact of HTS
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Biochemical Confirmation of Inhibition Mode

• Binding mode confirmed in multiple formats
• Intrinsic Protein Fluorescence Quenching
• Isothermal Titration Calorimetry
• Kinetic Mechanism Consistent with Uncompetitive
  Inhibition

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Mode of Inhibition The How

• Catalytic activity dependent on hinge movement
• Compounds bind at domain interface lock hinge
  movement

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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
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Phases of Target-Based Approaches Lead
Optimization
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Trojan Horse or Goldmine?
Can we improve potency? What is the potential
for resistance? Can we achieve the desired
selectivity margin?
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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)
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SAR - Highlights
IC50 2200 nM
IC50 103 nM
Potent inhibitors used to assess resistance
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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

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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)
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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
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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
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Destabilization of ES Complex
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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
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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
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Bacterial Selectivity Requirement
What about the selectivity profile?
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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

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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?
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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)

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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

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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

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The Agony of Defeat
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Phases of Target-Based Approaches Preclinical
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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!

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More reading
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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

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Supporting Slides
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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

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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

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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
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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