Infectious Diseases Drug Discovery: An AstraZeneca Perspective Tomas Lundqvist GSC LG-DECS AstraZeneca R&D Mölndal Stewart L. Fisher Infection Discovery

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

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

AstraZeneca R&D Boston

Why Focus on Infectious Disease?

Medical Need

Business Opportunity

Social Responsibility

0%

5%

10%

15%

20%

25%

30%

35%

RespiratoryDisease

Cancers CirculatoryDisease

InfectiousDisease Ref. WHO Data

Causes of Death

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

Percentage of all deaths worldwide

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 Identification

Target-Based Approaches: Pipeline

HitIdentification

Lead Identification

Lead Optimisation

Preclinical/Clinical

many (100’s) see genomic patents

Peptide DeformylaseGyrB/ParE

H. pylori MurIFabI/KPhe-tRNASIle-tRNASGyrB

MurAMurBMurCMurDMurEMurFMurGMurA-F pathwayMurGMraY-PBPII pathwayDdlBFtsZFtsZ/ZipALpxCRNA Polymerase (RNAP)DNA Polymerase (DNAP)DnaBPhe-tRNASTrp-tRNASMet-tRNASGyrBPanK

FabDFGAI pathwayFabIAcpSFtsZMur Pathway

• Definition of a Target Product Profile – Define the disease & unmet medical need– Set the requirements for the drug– Find targets that fit the requirements

First Step: Define the Problem


HitIdentification

Lead Identification

Lead Optimisation

Preclinical/Clinical

Target Product Profile

Therapy for Helicobacter pylori Infections

Need for New Therapeutic Strategies

Proton pump inhibitor (O) + two antibiotics: Clarithromycin (C), Amoxicillin (A), Metronidazole (M)

• Current therapy effective (~ 90%) if properly completed

• Poor patient compliance due to complicated regimen and side effects

• Resistance• Metronidazole 20 - 60%, Clarithromycin 10 -15%

• Causative agent for stomach ulcers

• Implicated in gastric cancer

Target Product Profile (H. pylori TPP)

• 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)

Deliver a candidate drug with this profile:


• Target Identification– Genomics-based selection– Validation of essentiality in relevant organisms– Cloning and expression of target proteins– Production of target proteins

Phases of Target-Based Approach: Target Identification

peptidoglycan

Glutamate Racemase (MurI)

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

UDP-MurNAc-(L) Ala

UDP-MurNAc-(L) Ala-(D) Glu

MurC

MurDL-Glu

MurID-Glu

NH2

OOH

OH

O

NH2

OOH

OH

O

UDP-GlcNAc

B-lactam classesglycopeptides

Fosfomycin

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 (>99% pure)

Aquifex aeolicusHelicobacter pyloriCampylobacter jejuni

Porphyromonas gingivalisPseudomonas aeruginosaDeinococcus radioduransBorrelia burgdorferi

Treponema pallidumVibrio choleraeShewanella putrefaciensEscherichia coliHaemophilus influenzae

Mycobacterium tuberculosisMycobacterium lepraeLactobacillus fermentum

Lactobacillus brevisPediococcus pentosaceus

Enterococcus faecalisStreptococcus pyogenesStreptococcus pneumoniaeBacillus sphaericus

Staphylococcus aureusStaphylococcus haemolyticus

Bacillus subtilisBacillus anthracis

Gram +ve

Gram -ve

H. pylori


Phases of Target-Based Approach: Hit 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

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

Results from Biochemical and Biophysical Characterization:• Active protein is a dimer• No cofactors required for activity• Kinetic analysis of enzyme reaction indicates an unusual profile

• Assays required for forward and reverse reaction

Coupled Assaywith MurDMeasure Pi or ADP

Resource intensive,Expensive

Coupled Assay with L-Glutamate dehydrogenaseMeasure NADH

Preferred HTS Assay

Cys 181Cys 70

70 181

O

O

OH

O

NH3+O

O

ONH3+

O

O

O

ONH3+

O

H

SH -S SH HS S- HS

L-Glutamate D-Glutamate

70 181 70 181

Enzyme Mechanism and Assays

Carbanionintermediate

Kinetic Analysis of Native H. pylori MurI

D-Glu (M)

0 20 40 60 80 100 120 140 160

Ra

te (

RF

U/m

in)

0

20

40

D-Glu KM = 63 M kcat = 12 min-1

KIS = 5.8 M

kcat/KM = 185 mM-1 min-1

D-Glu L-Glu

[L-Glu] (mM)

0 20 40 60

Ra

te (

/min

)

0

20

40

60

80

100

L-Glu D-Glu

L-Glu KM = 700 M kcat = 88 min-1


Glutamate Racemases: Biochemistry

E+L-glu

E.L-glu E.D-glu

E+D-glu

En

erg

yReaction Coordinate

70 181

O

O

OH

O

NH3+O

O

ONH3+

O

O

O

ONH3+

O

H

SH -S SH HS S- HS

L-Glutamate D-Glutamate

70 181 70 181

E+L-glu

E.L-glu

E.D-glu

E+D-glu

En

erg

yReaction Coordinate

H. pylori MurI

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

time (min)0 10 20 30 40 50

rel.

Fluo

resc

ence

0

10

20

30

40

blank

0.2mM S

0.5mM S

2.0mM S

NH2

O

O

OH

SO3

NH2

OH

O

NH3

NH2

OH

O

O

OH

O

+

kinact

kcat

pyruvate

+MurI

NADH NAD+

LDH

Lactate

inactive

k release

MurI

x1

x4000

Suicide Substrate HTS Assay

• 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)

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 M

MIC = 8 g/mL

NNN

N

O

N

O



Phases of Target-Based Approaches: Lead Identification

HitIdentification

• Hit Identification– Biophysical and biochemical characterization of targets– Development of primary HTS assay and secondary

assays for evaluation of hits– Kinetic mechanism studies for enzyme targets– HTS Screening and chem-informatic analysis– Limited SAR generation

Lead Identification

• Lead Identification– Biochemical mode of inhibition understood– Facile synthetic strategies in-place (combichem, MPS)– Whole-cell activity– Confirmed target-mediated mode of action in cells– Early drug metabolism/pharmacokinetics (DMPK) studies

Mechanism of Inhibition?

NNN

N

O

N

ONH2

OOH

OH

O

Inhibitor Substrate

≠

Protein NMR – Foundational Work

• 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

glutamate free 1.8 mM D-Glutamate

NMR indicates multiple conformations at room temperature

D-Glutamate stabilizes protein – consistent with kinetic profile

Black = D-Glu + MurIRed = D-Glu + MurI + Inh

N

O

O

NN

N

N

Protein NMR Demonstrates Substrate Dependence

• 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 substrateBinding 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

• Binding mode confirmed in multiple formats:

– Intrinsic Protein Fluorescence Quenching

– Isothermal Titration Calorimetry

• Kinetic Mechanism Consistent with Uncompetitive Inhibition

E + S

ES FP

F+P

FS

ESI

FSI

DSOS

0.1 1 10

RF

U/8

0m

in

0

5

10

15

20

25

30

[D-Glu] (μM)

Rat

e (R

FU

/min

)

Wavelength (nM)

280 300 320 340 360 380 400 420 440 460

RF

U

0

20000

40000

60000

80000

100000

120000

0 0.05 0.1 0.15 0.2 0.25

0

5000

10000

15000

ΔR

FU

[Inhibitor] μM

Increasing[Inh]

KI = IC50

Mode of Inhibition: The “How”

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

HingeInhibitor

Time (min)0 10 20 30 40 50

0

50

100

150

200

0

50

100

UD

P-M

urN

Ac

-(L

) A

la

Pe

nta

pe

pti

de

Bacterial Growth Inhibition Mode of Action Confirmation

+ Inhibitor

A25

4nmL-Glu

UDP-MurNAc

UDP-MurNAc-(L) Ala

UDP-MurNAc-(L) Ala-(D) Glu

UDP-MurNAc-(L) Ala-(D) Glu-mDap

UDP-MurNAc-(L) Ala-(D) Glu-mDap-(D) Ala-(D) Ala

MurC

MurD

MurE

MurF

MurI D-Glu

Peptidoglycan Biosynthesis

Growth inhibition through MurI inhibition

*



Phases of Target-Based Approaches: Lead Optimization

HitIdentification



Lead Identification

• Lead Identification– Facile synthetic strategies in-place (combichem, MPS)– Biochemical mode of inhibition understood– Whole-cell activity– Confirmed target-mediated mode of action in cells– Early drug metabolism/pharmacokinetics (DMPK) studies

Lead Optimization

• Lead Optimization– Focus on analogs of central scaffold(s)– Activity in animal disease-state model– Assess potential for resistance– in vivo DMPK studies for human dosing estimation– in vitro toxicological studies– Scale up 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

N

N NN

O

O

N

R1

R2

R4

R3

Exposed to solvent

Site mainly surrounded by hydrophobicgroups with a polar terminus (His, Lys)

Site partially open to solvent but has potential for specific H-bond interactions (Glu, Ser, H2O)

Deep large hydrophobic pocket

N

N

O

O NN

N

S

H

N

N

O

O NN

N

S

O

NH

IC50 = 67 nM

Glu150

IC50 = 503 nM

N

N

O

O NN

NN

NH

HN

N

O

O NN

NN

NH

Cl

IC50 = 2200 nM

Cl

IC50 = 103 nM

SAR - Highlights

• Combination of best R3 and R4 resulted in 250-fold improvement in potency from Hit

N

N

O

O NN

N

NH

O

NH

Cl

IC50 = 6 nM

Potent inhibitors used to assess resistance

Novel Pocket Concerns: Resistance Rates

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

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

Biochemical Analysis of Resistance Mutants

- Two were chosen for biochemical characterization:

A75T (most prevalent) E151K (most dramatic)

- Mapping onto crystal structure did not yield an obvious answer:

Not in the substrate binding pocket

Not in the inhibitor binding pocket (L186F)

A35T

A75T

A75V

E151K

C162Y

I178T

G180S

L186F

L206P

Q248R

A75T H. pylori MurI Kinetic Profile

[L-Glu] mM0 20 40 60

Ra

te (

M/m

in)

0

20

40

60

80

100

[D-Glu] M

0 2000 4000 6000 8000 10000

Ra

te

0

200

400

600

D-Glu L-Glu L-Glu D-Glu

D-Glu KM = 275 M (63 M)kcat = 4 min-1 (12 min-1)KIS = 660 M (5.8 M)

kcat/KM = 14.5 mM-1 min-1

L-Glu KM = 7400 M (700 M)kcat = 106 min-1 (88 min-1)

kcat/KM = 14.3 mM-1 min-1

Inhibition elevation: (IC50A75T/IC50wt) ~9 foldMIC elevation: ~4 – 8 fold

E151K H. pylori MurI Kinetic Profile

D-Glu L-Glu L-Glu D-Glu

D-Glu KM = 280 M (63 M) kcat = 5 min-1 (12 min-1)

(5.8 M) kcat/KM = 18 mM-1 min-1

L-Glu KM = 7300 M (700 M) kcat = 136 min-1 (88 min-1)


[D-Glu] M

0 2000 4000 6000 8000 10000

Ra

te (

RF

U/m

in)

0

10

20

30

[L-Glu] M0 20 40 60

Ra

te

0

20

40

60

80

100

120

Inhibition elevation: (IC50E151K/IC50wt) ~15 foldMIC elevation: ~8 - 16 fold

Destabilization of ES Complex

WT

A75T

E151K

Reaction Coordinate

En

erg

y

E

ES

Dec

reas

ed S

tab

ilit

y

Res

ista

nce

im

pac

t

Resistance Mechanism

MurI MurI*

(MurI•D-Glu) (MurI*•L-Glu)

(MurI*•D-Glu)

D-Glu L-Glu

D-Glu

Substrateinhibited

Resistance mutants disfavor [ES]/[FS] species:

- Higher Km

- Reduced/Eliminated Substrate Inhibition

Reduced [ES] = less inhibition!But…

increased potency can overcome effect

MurI

Direct Binding Measurements with Inhibitors

Native

A75T Mutant

MurI Enzyme

[Inhibitor] uM

0 0.2 0.4 0.6 0.8 1 1.2 1.4

ΔR

FU

0

2000

4000

6000

8000

10000

Wavelength (nM)

280 300 320 340 360 380 400 420 440 460

RF

U

0

20000

40000

60000

80000

100000

120000

High D-Glu (5mM)

26 nM

31 nM

Low D-Glu (50M)

23 nM

170 nM

Dissociation Constant (Kd)

Bacterial Selectivity Requirement

What about the selectivity profile?

Selectivity Profile

• 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

Organism IC50 (nM) MIC (g/mL)

H. pylori 9.2 0.5

E. coli >400000 >64H. influenzae >64M. catarrhalis >64P. aeruginosa >64

S. aureus >400000 >64S. pneumoniae >64S. pyogenes >64E. faecalis >400000

C. albicans >64

N

N

N

O

O NN

CH3

N

Cl

CN

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, where’s the drug?

Target Inhibitor Drug

• microbiological properties– potency, spectrum

– bona fide inhibition of bacterial growth (MOA)

– resistance frequency

– population MICs (MIC90)

• physical properties– molecular size

– lipophilicity

– solubility

• biochemical properties– bona fide enzyme inhibition

– potency, spectrum

• in-vivo properties– plasma protein binding

– absorption

– metabolism

– excretion

– pharmacokinetics

– safety

N

N NN

N

O

O

Cl

NN

Cl = 14 µl/min/kg t½ = 0.7 hr F = 76 %

Drug Levels in Mouse Plasma

0

2

4

6

8

10

0 1 2 3 4 5 6Time (h)

Co

nce

ntr

atio

n (g

/ml)

iv 5 mg/kg

po 40 mg/kg

MIC

• Improved PK in dogs

• Total drug levels above MIC for extended period of time

Pharmacokinetic Profiles in Mouse

in vivo

N

N NN

N

O

O

Cl

NN

Cl = 14 µl/min/kg t½ = 0.7 hr F = 76 % fu < 3 %

0

2

4

6

8

10

0 1 2 3 4 5 6Time (h)

Co

nce

ntr

atio

n (g

/ml)

po 40 mg/kg, total

MIC

• Free drug levels in plasma below MIC

• Difficult to achieve balance between protein binding and potency

Requirements for Efficacy: Free Fraction

in vivo

po 40 mg/kg, freeDrug Levels in Mouse Plasma

The Agony of Defeat

Microbiology

MICMBC

Killing Kinetics

DMPK

ClearanceBioavailabilityPermeability

Vss

Physical Properties

Protein BindingSolubility

Decrease LogD - basesHigh Efflux

Low metabolismLow protein binding

Increase logDLow EffluxHigh metabolismHigh protein binding

Decrease logD - AcidsHigh Efflux

Low metabolismHigh protein binding

Zwitterions



Phases of Target-Based Approaches: Preclinical

HitIdentification



Lead Identification

• Lead Identification– Facile synthetic strategies in-place (combichem, MPS)– Biochemical mode of inhibition understood– Whole-cell activity– Confirmed target-mediated mode of action in cells– Early drug metabolism/pharmacokinetics (DMPK) studies

Lead Optimisation

• Lead Optimization– Focus on analogs of central scaffold(s)– Activity in animal disease-state model– in vivo DMPK studies for human dosing estimation– in vitro toxicological studies– Scale up synthesis; process chemistry

Preclinical

• Preclinical– Several compounds – Documentation for FDA filing– Toxicological studies to support human dosing

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 BostonRichard Alm Beth AndrewsBarbara Arsenault Greg BasarabApril Blodgett Gloria BreaultKen Coleman Janelle ComitaBoudewijn deJonge Gejing DengJoe Eyermann Tatyana FriedmanNing Gao Bolin GengMadhu Gowravaram Oluyinka GreenLena Grosser Laurel HajecPamela Hill Sussie HopkinsJanette Jones Camil JoubranThomas Keating Gunther KernAmy Kutschke Stephania LivchakJim Loch Kathleen McCormackLarry MacPherson John ManchesterCynthia Mascolo Scott MillsMarshall Morningstar Trevor NewtonBrian Noonan Linda OttersonOlga Rivin Mike RooneyMaria Uria-Nickelsen Jim WhiteakerJonny Yang Wei YangMark Zambrowski

Christer Cederberg Paul ManningJohn Primeau Gautam SanyalTrevor Trust Peter WebbornMark Wuonola

• AZ MölndalMarie Andersen Rutger

Folmer

Tomas Lundqvist Yafeng Xue

Nan Albertson Mark Divers

Bo Xu

Supporting Slides

Biochemical Studies on MurI Isozymes

• Various pathogens represented

• Gram negative enzymes = activated• Gram positive enzymes = high catalytic turnover

Species Biochemical data UNAM-Ala ActivationL-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

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

Physiology: Resistance vs. D-Glutamate Regulation

• 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

L-GluL-Glu

UDP-Mur

UDP-Mur-(L) Ala

UDP-Mur-(L) Ala-(D) Glu

MurC

MurDMurI

D-Glu

Peptidoglycan

CatabolicEnergy Source

Nitrogen Fixation

Amino AcidBiosynthesis

Genomic DNA from representative strains from a variety of disease states and geographical locations was screened for resistance mutations.

Strain Country Year of

Isolation Disease state Identity

to J99 MurI(%)

UA861 Canada 1991 Duodenal ulcer 95.359 ARHp18 Canada 1989 94.531 ARHp25 Australia 1989 94.922 ARHp64 Argentina 1996 Nonulcer

dyspepsia 95.703

ARHp65 Argentina 1996 Nonulcer dyspepsia

93.359

ARHP55 United States

1996 Duodenal ulcer 92.188

ARHp124 Bangladesh 1996 Hiatus hernia and gastritis

93.75

ARHp54 United States

1996 Duodenal ulcer 92.578

ARHp43 Australia 1984 94.531 ARHp246 Kuala

Lumpur 1998 Duodenal

ulcer, gastritis 93.359

ARHp241 Kuala Lumpur

1998 Duodenal ulcer, erosive gastritis

93.359

ARHp243 France 1998 Duodenal ulcer 94.141 ARHp244 France 1998 Nonulcer

dyspepsia 93.75

A35T

A75T

A75V

E151K

C162Y

I178T

G180S

L186F

L206P

Q248R

Sampling Diverse H. pylori Strains

160 170 180 190 200 AH244 ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA UA861 ENILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA SS1_206_ ESILGGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA ARHP65 ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA ARHP18 ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IESYFMGHFA ARHp243 ENILEGELLE TCMRYYFTPL EILPEVIILG CTHFPLIAQK IEGYFMGHFA ARHP246 ENILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAHQ IEGYFMEHFA ARHP241 ENILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAHQ IEGYFMEHFA ARHP55 ENILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAHQ IEGYFMEHFA 26695 ESILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAQK IEGYFMEHFA ARHp244 ESILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAQK IEGYFMEHFA ARHP124 ESILEGELLE TCMRYYFTPL EILPEVVILG CTHFPLIAQK IEGYFMEHFA ARHP54 ESILEGELLE TCMRYYFTPL KILPKVIILG CTHFPLIAHQ IKGYFMGHFA ARHP43 ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA ARHP25 ESILEGELLE TCMRYYFTPL EILPEVIILG CTHFPLIAQK IESYFMEHFA J99 ESILEGELLE TCMHYYFTPL EILPEVIILG CTHFPLIAQK IEGYFMGHFA ARHP64 ESILEGELLE TCMRYYFTPL KILPEVIILG CTHFPLIAQK IEGYFMEHFA Clustal Co *.** ***** ***:****** :***:*:*** ********:: *:.*** ***

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

Documents

Infectious Diseases Drug Discovery: An AstraZeneca Perspective Tomas Lundqvist GSC LG-DECS AstraZeneca R&D Mölndal Stewart L. Fisher Infection Discovery