Choline dendrimers as versatile tools for the development of new antibiotics and tissue imaging

Protein Biotechnology LaboratoryInstituto de Biología Molecular y Celular

Universidad Miguel HernándezElche (Alicante)

Jesús M. Sanz

Málaga, March 26th, 2010

• 10-25% pneumonia cases in developed countries

• 25% mortality

• 4th cause of death in the world (World Health Organization)

• Responsible of bacterial child meningitis

• 4 million deaths in children under 5 (yearly)Maestro & Sanz, Recent Patents Anti-Infect Drug Disc, 2007, 2, 188-196

Streptococcus pneumoniae (pneumococcus)

Streptococcus pneumoniae

Choline-binding proteins (CBPs)

Functional domain Choline-binding domain

Peptidoglycan hydrolasesPhosphorylcholinesterasesAdhesion to hostToxin release

Choline

Structure of C-LytA (15 kDa)

- The choline-binding repeats (CBRs) are β-hairpins

- Two consecutive CBRs configure a binding site- The hairpins conform an extended superhelix - the ββ-3-solenoid

N-terminal

C-terminal

ChBR6(dimerization)

ChBR5

ChBR4

ChBR3

ChBR2

ChBR1

C-LytA

Choline-binding proteins do not exclusively recognize choline

Protein purification/immobilization

Sanz et al., FEBS lett. 232, 308-312 (1988)

Choline-binding moduleNH2- Protein -COOH

Purification of fusion proteins in DEAE

O-CH2-CH2-N-CH2-CH3

CH2-CH3

H+

Diethylaminoethanol (DEAE)

DEAE-cellulose

HO-CH2-CH2-N-CH3

CH3

CH3

+

Choline

Sanchez-Puelles et al., Eur. J. Biochem. 203, 153-159 (1992)Patents ES 2 032 717, ES 200700281

Proteins containing choline-binding domainsare easily immobilized

Choline analogCholine analogCholine analog

C-LYT-lacZ

lacZ

extracto

Biosensors

Aqueous two-phase systems

Binding of C-LytA-GFP to DEAE-functionalized magnetic nanoparticles (≈750 nm and ≈ 200 nm)

Protein + particles

1.5M NaClwashes

Choline elution

1940 1960 1980 2000

Sulph ‘36β-lactams ‘40

Chloramphenicol,Tetracyclin ‘49Aminoglycosides ‘50

Macrolides ‘52

Glycopeptides‘58

Quinolones, Streptogramines ‘62

Oxazolydinones ‘00

Discovery of antibiotics

Mortality caused by Streptococcus pneumoniae in the world

OMS, The Lancet, 374, 893-902 (2009)

Fighting pneumococcusthe direct way

Why CBPs?

•Increasing levels of resistance to traditional antibiotics acting on the cell wall (penicillins) (Spain > 50%)

•Efficiency of current vaccines is lower than desired (90 serotypes to encompass)

•CBPs are common to all pneumococcal serotypes

•CBPs are involved in essential, physiological and virulence processes

•There are many CBPs: the same compound might inhibit all of them: Resistance becomes more difficult

•CBPs may recognize (and be inhibited by) many different molecules (choline analogs)

The subtle way: targeting choline-binding proteins

Searching for CBP inhibitors

Control + 50 mM CholineMore easily fagocitableLess infective

Choline cannot be used as therapy:High (millimolar) concentrations required Side interactions with muscarinic receptors

Searching for CBP inhibitors

N

NN

N

N

NN

N

N

N

N

N

N

N

N

N

N

NN

N

NN

N

N

NN

N

N

N

N

N

N

N N

N

N

N

N

N

N

N

N

NN

N

NN

NN

N

N N

N

N

N

NN

N

N

NN

N

N

NN

N

N

N

N

N

N N

N

N

N

N

N

NN

N

NN

N

N

g1-cho

g3-cho

g5-cho

g2-cho

ON

NH

O

Cl=N

NN

N

N

NN

N

N

N

N

N

N

N

N

N

N

NN

N

NN

N

N

NN

N

N

N

N

N

N

N N

N

N

N

N

N

N

N

N

NN

N

NN

NN

N

N N

N

N

N

NN

N

N

NN

N

N

NN

N

N

NN

N

N

N

N

N

N

N

N

N

N

NN

N

NN

N

N

NN

N

N

N

N

N

N

N N

N

N

N

N

N

N

N

N

NN

N

NN

NN

N

N N

N

N

N

NN

N

N

NN

N

N

NN

N

N

N

N

NN

N

N

N

N

N

N

N

N N

N

N

N

N

N

NN

N

NN

N

N

N N

N

N

N

N

N

NN

N

NN

N

N

N N

N

N

N

N

N

NN

N

NN

N

N

g1-cho

g3-cho

g5-cho

g2-cho

ON

NH

O

Cl= ON

NH

O

Cl=

Peptidoglycan

Cytoplasmicmembrane

Cell wall teichoic acid

Membrane teichoic acid

Phospho-choline

Peptidoglycan

Cytoplasmicmembrane

Cell wall teichoic acid

Membrane teichoic acid

Phospho-choline

Searching for CBP inhibitors: Dendrimers

Cell wall

Dendrimers asmimics of cell wall

Hernández-Rocamora et al. Angew. Chem. Int. Ed. 48, 948-951 (2009)

Surface plasmon resonance (immobilized C-LytA)

Choline 1M

Analyte Ligand Kd1 (μM) Kd2 (μM) analyte : ligand g1-cho C-Lyt-GFP 4.1 ± 1.0 263 ± 52 2.3g2-cho C-Lyt-GFP 0.15 ± 0.05 23 ± 5 2.3g3-cho C-Lyt-GFP 0.05 ± 0.01 2.5 ± 0.8 1.7g5-cho C-Lyt-GFP 0.023 ± 0.004 3.0 ± 0.4 1.1

G1G2G3G5colina

LytC lysozyme

[Ligand] (μM)

0.01 0.1 1 10 100 1000 10000100000

Act

ivity

(%)

0

20

40

60

80

100

120Pce phosphorylcholinesterase

[Ligand] (μM)

0.01 0.1 1 10 100 1000 10000 100000

Activ

ity (%

)

0

20

40

60

80

100

LytA amidase

[Ligand] (μM)

0.01 0.1 1 10 100 1000 10000 100000

Act

ivity

(%)

0

20

40

60

80

100

120

140

160LytB glucosaminidase

[Ligand] (μM)

0.01 0.1 1 10 100 1000 10000 100000

Act

ivity

(%)

0

20

40

60

80

100

120

Searching for CBP inhibitors: Dendrimers

Searching for CBP inhibitors: Dendrimers

Only micromolar concentrations required...Dendrimers increase the affinity of choline by 1000-10000 times

Development of new dendrimers

Core:• PAMAM (Total positive charge is reduced)• PEG (less toxicity)

Functionalization:• Choline• Tertiary amines (DEAE) (modulación of charge by pH)

Visualization of tissues: collagen fibers

N

NN

N

N

NN

N

N

N

N

N

N

N

N

N

N

N N

N

NN

NN

NNN

N

N

N

N

N

N N

N

N

N

N

NH

N

NN

N

NN

N

NN

NN

NN N

N

N

N

NN

N

N

N N

N

O

O

O

OHO

NH

S

C

g5-cho/FITC

Collagen

CNA35 (collagen binding protein)fused to C-LytA

Solid-phase binding assays demonstrate specificity of interaction

+ 0.5M NaCl

+ 0.5 M Choline

Surface plasmon resonance on immobilized collagen

+ dendrimersKD= 0.92 ± 0.07 μM

- dendrimersKD = 7.4 ± 0.7 μM

Visualization of collagen in porcine pericardium

(b) (c)(a)

5 μM C-LytCNA35 0.5 μM g5-cho/FITC

5 μM C-LytA 0.5 μM g5-cho/FITC

No protein0.5 μM g5-cho/FITC

Human kidney

Centro de Investigaciones Biológicas - CSIC (Madrid, Spain)•Ernesto García•Pedro García

- Microbiological effects of antimicrobials

Eindhoven University of Technology(Eindhoven, The Netherlands) •Maarten Merkx- Synthesis of dendrimers- Collagen binding assays

External collaborations

Unidad de Química Combinatoria(Parque Científico de Barcelona)•Miriam Royo•Fernando Albericio

-PEG dendrimers