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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)

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infectious diseases drug discovery an astrazeneca perspective

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
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

Why Focus on Infectious Disease?
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
Causes of Death

Percentage of all deaths worldwide

Ref. WHO Data

the golden age today
The Golden Age & Today
  • 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

The Golden Age of Antibiotic Discovery was very brief, mid 1930s- early 1960s

penicillin, cephalosporin, streptomycin, erythromycin, tetracycline, vancomycin

target based approaches
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
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
“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-Based Approaches: Pipeline

Target

Identification

HitIdentification

Lead Identification

Lead Optimisation

Preclinical/

Clinical

Peptide Deformylase

GyrB/ParE

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

H. pylori MurI

FabI/K

Phe-tRNAS

Ile-tRNAS

GyrB

many (100’s)

see genomic patents

FabDFGAI pathway

FabI

AcpS

FtsZ

Mur Pathway

first step define the problem
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
slide12

Causative agent for stomach ulcers

  • Implicated in gastric cancer
  • Current therapy effective (~ 90%) if properly completed
  • Poor patient compliance due to complicated regimen and side
  • effects
  • Resistance
      • Metronidazole 20 - 60%, Clarithromycin 10 -15%

Proton pump inhibitor (O) + two antibiotics:

Clarithromycin (C), Amoxicillin (A), Metronidazole (M)

Need for New Therapeutic Strategies

Therapy for Helicobacter pylori Infections

target product profile h pylori tpp
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)
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
Phases of Target-Based Approach: Target Identification
glutamate racemase muri
Glutamate Racemase (MurI)

UDP-GlcNAc

Fosfomycin

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

MurC

UDP-MurNAc-(L) Ala

MurI

L-Glu

D-Glu

MurD

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

B-lactam classes

glycopeptides

peptidoglycan

genomic based hypotheses for selectivity

Bacillus subtilis

Bacillus anthracis

Staphylococcus haemolyticus

Staphylococcus aureus

Bacillus sphaericus

Streptococcus pneumoniae

Gram +ve

Streptococcus pyogenes

Enterococcus faecalis

Lactobacillus brevis

Pediococcus pentosaceus

Lactobacillus fermentum

Mycobacterium leprae

Mycobacterium tuberculosis

Haemophilus influenzae

Escherichia coli

Shewanella putrefaciens

Vibrio cholerae

Gram -ve

Treponema pallidum

Borrelia burgdorferi

Deinococcus radiodurans

Pseudomonas aeruginosa

Porphyromonas gingivalis

Campylobacter jejuni

Helicobacter pylori

H. pylori

Aquifex aeolicus

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)
phases of target based approach hit identification
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
h pylori muri an enigma

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
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
slide19

Enzyme Mechanism and Assays

L-Glutamate

D-Glutamate

Cys 70

Cys 181

-S

SH

S-

HS

SH

HS

70

181

70

70

181

181

Carbanion

intermediate

Coupled Assay with

L-Glutamate dehydrogenase

Measure NADH

Preferred HTS Assay

Coupled Assay

with MurD

Measure Pi or ADP

Resource intensive,

Expensive

kinetic analysis of native h pylori muri
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

glutamate racemases biochemistry

E.L-glu

Energy

E+L-glu

E+D-glu

Energy

E.D-glu

E+L-glu

E+D-glu

Reaction Coordinate

L-Glutamate

D-Glutamate

E.L-glu

E.D-glu

Reaction Coordinate

-S

SH

S-

HS

SH

HS

70

181

70

70

181

181

H. pylori MurI

Glutamate Racemases: Biochemistry
implications of unique biochemical profile

No obvious avenues

HTS Assay?

Poor Inhibition Profile

Novel Assay Format

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

HTS of corporate collection using novel assay

suicide substrate hts assay

blank

0.2mM S

0.5mM S

2.0mM S

Suicide Substrate HTS Assay
  • HTS Assay
    • All reagents commercially available
    • Linear time course (irreversible)
    • Excellent Assay Window
    • Amenable to 384-well HTS format

x4000

x1

Screened corporate collection for inhibitors (~150,000 cpds)

pyrimidinediones features of the hit cluster
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

phases of target based approaches lead identification

Lead Identification

HitIdentification

Target

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
  • 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
  • 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 Approaches: Lead Identification
mechanism of inhibition
Mechanism of Inhibition?

Substrate

Inhibitor

protein nmr foundational work
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
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

Black = D-Glu + MurI

Red = D-Glu + MurI + Inh

Compound binding requires substrate

Binding site distal from active site

inhibitor enzyme co crystal structure the where
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
Cryptic Binding Site – Detailed View

MurI + D-Glutamate

MurI + D-Glutamate + Inhibitor

Unexpected allosteric inhibition mechanism – impact of HTS

biochemical confirmation of inhibition mode

Increasing

[Inh]

Rate (RFU/min)

[D-Glu] (μM)

ΔRFU

[Inhibitor] μM

KI = IC50

Biochemical Confirmation of Inhibition Mode
  • Binding mode confirmed in multiple formats:
    • Intrinsic Protein Fluorescence Quenching
    • Isothermal Titration Calorimetry
  • Kinetic Mechanism Consistent with Uncompetitive Inhibition
mode of inhibition the how

Inhibitor

Hinge

Mode of Inhibition: The “How”
  • Catalytic activity dependent on hinge movement
  • Compounds bind at domain interface – lock hinge movement
slide33

Bacterial Growth Inhibition Mode of Action Confirmation

PeptidoglycanBiosynthesis

Pentapeptide

UDP-MurNAc

UDP-MurNAc-(L) Ala

MurC

UDP-MurNAc-(L) Ala

MurI

MurD

D-Glu

L-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

phases of target based approaches lead optimization

Lead Identification

Lead Optimization

HitIdentification

Target

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
    • 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
  • 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
  • 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 Approaches: Lead Optimization
trojan horse or goldmine
Trojan Horse or Goldmine?

Can we improve potency?

What is the potential for resistance?

Can we achieve the desired selectivity margin?

slide36

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)

slide37

IC50 = 67 nM

IC50 = 503 nM

Cl

Glu150

IC50 = 6 nM

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

SAR - Highlights

IC50 = 2200 nM

IC50 = 103 nM

Potent inhibitors used to assess resistance

novel pocket concerns resistance rates
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
slide39

A35T

A75T

A75V

E151K

C162Y

I178T

G180S

L186F

L206P

Q248R

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)

a75t h pylori muri kinetic profile
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 = 7400mM (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

e151k h pylori muri kinetic profile
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 = 7300mM (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

destabilization of es complex

E151K

Energy

Decreased Stability

Resistance impact

A75T

E

WT

ES

Reaction Coordinate

Destabilization of ES Complex
slide43

MurI

Resistance Mechanism

D-Glu

(MurI*•D-Glu)

MurI*

MurI

Substrate

inhibited

L-Glu

D-Glu

(MurI*•L-Glu)

(MurI•D-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

High D-Glu (5mM)

Low D-Glu (50mM)

26 nM

23 nM

31 nM

170 nM

Direct Binding Measurements with Inhibitors

10000

8000

6000

ΔRFU

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

bacterial selectivity requirement
Bacterial Selectivity Requirement

What about the selectivity profile?

selectivity profile

Organism IC50 (nM) MIC (mg/mL)

H. pylori 9.2 0.5

E. coli >400000 >64

H. influenzae >64

M. catarrhalis >64P. aeruginosa >64S. aureus >400000 >64

S. pneumoniae >64

S. pyogenes >64

E. faecalis >400000

C. albicans >64

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
trojan horse or goldmine47
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
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)
slide49

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
slide50

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 < 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
the agony of defeat

Increase logD

Low Efflux

High metabolism

High protein binding

Decrease logD - Acids

High Efflux

Low metabolism

High protein binding

DMPK

Clearance

Bioavailability

Permeability

Vss

Microbiology

MIC

MBC

Killing Kinetics

Zwitterions

Physical Properties

Protein Binding

Solubility

Decrease LogD - bases

High Efflux

Low metabolism

Low protein binding

The Agony of Defeat
phases of target based approaches preclinical

Lead Identification

Lead Optimisation

HitIdentification

Target

Identification

Preclinical

  • 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
    • 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
  • 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
  • Preclinical
    • Several compounds
    • Documentation for FDA filing
    • Toxicological studies to support human dosing
  • 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 Approaches: Preclinical
thoughts
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!
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

Olga Rivin Mike Rooney

Maria Uria-Nickelsen Jim Whiteaker

Jonny Yang Wei Yang

Mark Zambrowski

Christer Cederberg Paul Manning

John Primeau Gautam Sanyal

Trevor Trust Peter Webborn

Mark Wuonola

AZ Mölndal

Marie Andersen Rutger Folmer

Tomas Lundqvist Yafeng Xue

Nan Albertson Mark Divers

Bo Xu

Acknowledgments
biochemical studies on muri isozymes
Biochemical Studies on MurI Isozymes
  • Various pathogens represented
  • Gram negative enzymes = activated
  • Gram positive enzymes = high catalytic turnover
physiology resistance vs d glutamate regulation
Physiology: Resistance vs. D-Glutamate Regulation

UDP-Mur

Catabolic

Energy Source

  • 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

MurC

UDP-Mur-(L) Ala

MurI

MurD

L-Glu

Nitrogen Fixation

D-Glu

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

Amino Acid

Biosynthesis

Peptidoglycan

sampling diverse h pylori strains
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

clinical resistance potential

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