Pharmacological Chaperone Therapy · PC ER Misfolded Protein ERAD Proteasome Degradation Transport to Cellular Location Golgi Apparatus Ubiquitination Pharmacological Chaperone Assisted

Pharmacological ChaperoneTherapy

Brandon WilcockMarch 27, 2008

Protein Folding and Structure

Wolynes, P.; Luthey-Schulten, Z.; Onuchic, J. Chemistry & Biology 1996, 3, 425-432.http://parasol.tamu.edu/foldingserver/images/landscape.gif

E

Endoplasmic Reticulum-Associated Degradation

Proteasome Ubiquitination

Sec61pComplexRibosome

Molecular Chaperone Recognition

ER

Hsp90

Ali, M.; Roe, S.; Vaughan, C.; Meyer, P.; Panaretou, B.; Piper, P.; Prodromou, C.; Pearl, L. Nature 2006, 440, 1013-1017.

Quality Control

Anelli, T.; Sitia, R. EMBO Journal 2008, 27, 315-327.

PC

ER

MisfoldedProtein

ERAD

ProteasomeDegradation

Transport to Cellular Location

Golgi ApparatusUbiquitination

Pharmacological Chaperone Assisted Rescue

Active-site Chaperone

Specific-site Chaperone

Identification and Characterization

• High-throughput screening

• Rational Design

• Thermal stability

• Mature protein quantification

• Toxicity

Exploring the mechanism

• ERAD and regulatory targets

• Direct interactions

• Specificity

Lysosomal Storage Diseases

Common Symptoms

Neurodegenerative states

anemia

skeletal dysplasia

Approved Treatments

Enzyme replacement therapy

Substrate reduction therapy Cell

ERT

AccumulationSRT

α-Galactosidase A and Fabry Disease

α-Galactosidase A(α-GalA)

Globotriaosylceramide(Gb3)

-GalA

NHOHO

O

O

OHO

HOOH

O

HO

HOOH

OHO

HO

HOOH

OH

NHOHO

O

O

OHO

HOOH

O

HO

HOOH

O

Garman, S.; Garboczi, D.; J. Mol. Biol. 2004, 337, 319-335.

DGJ Enhances R301Q-α-GalA Activity

NH

OHHO

OHHO1-deoxygalactonojirimycin

(DGJ)

Fan, J.; Ishii, S.; Asano, N.; Suzuki, Y. Nat. Med. 1999, 5, 112-115.

4-methylumbelliferyl-α-galactoside

(4-Mu-α-Gal)O

OHHO

HOHO

O

O

O

Me

The Inhibitory Effect

DGJ inhibits R301Q-α-GalA activity at 20 μM

Fan, J.; Ishii, S.; Asano, N.; Suzuki, Y. Nat. Med. 1999, 5, 112-115.

R301Q-α-GalA Activity

ER

Lysosome

ERAD

PC

ER

proteasomedegradation

golgi apparatus, transport

DGJ Increases Mature Protein Levels

Fan, J.; Ishii, S.; Asano, N.; Suzuki, Y. Nat. Med. 1999, 5, 112-115.

Mature α-GalA 46 kD

Immature α-GalA 50 kD

Protein level enhancement is DGJ concentration Dependent

The Stability of R301Q-α-GalA

Fan, J.; Ishii, S.; Asano, N.; Suzuki, Y. Nat. Med. 1999, 5, 112-115.

Rel

ativ

e E

nzym

e A

ctiv

ity (%

)

Rel

ativ

e E

nzym

e A

ctiv

ity (%

)

Time (min) Time (min)

R301Q-α-GalA is unstable at ER pH

pH 5 pH 6 pH 7

DGJ stabilizes R301Q-α-GalA

0.01 μM0.03 μMNone

1 μM[DGJ]

Structure and Inhibitor Strength

NHHO

HO

OHHO

manno-DNJNI

NHHO

OHHO

2-deoxy-DGJ250

Asano, N.; Ishii, S.; Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.; Martin, O.; Fan, J. Eur. J. Biochem. 2000, 267, 4179-4186.Ishii, S.; Chang, H.; Kawasaki, K.; Yasuda, K.; Wu, H.; Garman, S.; Fan, J. Biochem. J. 2007, 406, 285-295.

Structure and Inhibitor Strength

N

OHHO

OHHOMe

N-ethyl-DGJ306

N

OHHO

OHHO

OH

N-hydroxyethyl-DGJ520

Asano, N.; Ishii, S.; Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.; Martin, O.; Fan, J. Eur. J. Biochem. 2000, 267, 4179-4186.

N-alkylation decreases binding affinity

NH

HOHO

HOOH

-HNJNI

OH

NHHO

HO

OHHO

OH

-manno-HNJ464

NH

HO

HOOH

OHOH

-allo-HNJ4.3

NH

HOHO

OHHO

OH

-galacto-HNJ0.21

Structure and Activity Enhancement

Binding affinity

Cell and ER permeability

Rate of metabolism

Asano, N.; Ishii, S.; Kizu, H.; Ikeda, K.; Yasuda, K.; Kato, A.; Martin, O.; Fan, J. Eur. J. Biochem. 2000, 267, 4179-4186.

Cell ER

R301Q-α-GalA Localization

Yam, G.; Zuber, C.; Roth, J. FASEB J. 2005, 19, 12-18.

Wild Type R301Q DGJ treated R301Q

α-GalA

LAMP1

Overlay

DGJ increases R301Q-α-GalA levels in lysosomes

Yam, G.; Zuber, C.; Roth, J. FASEB J. 2005, 19, 12-18.

Reducing the Accumulated Gb3

Gb3

LAMP1

Overlay

Untreated R301Q Fibroblasts DGJ treated R301Q Fibroblasts

Gb3 accumulation is decreased in DGJ-treated R301Q fibroblasts

In Vivo Mice Studies

Ishii, S.; Yoshioka, H.; Mannen, K.; Kulkarni, A.; Fan, J. Biochim. Biophys. Acta 2004, 1690, 250-257.

TissueR301Q-α-GalA

Activity Increase

Heart

Kidney

Spleen

Liver

13.8 fold

3.3 fold

3.9 fold

2.6 fold

3 mg/kg DGJ per day

No toxicity observed

Expansion to Other Mutations

Shin, S.; Murray, G.; Kluepfel-Stahl, S.; Cooney, A.; Quirk, J.; Schiffmann, R.; Brady, R.; Kaneski, C. Biochem. Biophys. Res. Comm. 2007, 359, 168-173.

A97V

R301Q

R112H

R112C

A143T

L300P

R356W

G132R

R220X

A143P

30ΔG

Rescued Not Rescued

DGJ treatment success is mutation dependent

Neg. C

ontrolW

ild Type

A20PE59K

E66QM72V

I91TR112H

A97VF113L

P146S

Expansion to Other Mutations

Ishii, S.; Chang, H.; Kawasaki, K.; Yasuda, K.; Wu, H.; Garman, S.; Fan, J. Biochem. J. 2007, 406, 285-295.

DGJ can be therapeutic for several mutants

Application in Cystic Fibrosis

NBD2NBD1

TMD1 TMD2

RD

ATP

P

TMD1 TMD2

NBD2NBD1

RD

PKA ATP

ATP

Cystic fibrosis transmembrane conductance regulator protein (CFTR)

http://www.cystic-fibrosis-symptom.com/cystic_fibrosis_pictures.htm

A Common Mutation ∆F508

Lung infections and inflammation

ΔF508

F508A

F508

90 % of CF patients

Folding defect corrected at 27 °C

Loo, T.; Bartlett, M.; Clarke, D. J. Bioenerg. Biomembr. 2005, 37, 501-507. Lewis, H.; et al. J. Biol. Chem. 2005, 280, 1346-1353.

High-Throughput Screening

YFPYFP

I-

Halide sensitive fluorescent proteins

Cl-

DiSBAC2(3)

Coumarin Coumarin

Voltage sensitive dye

Pedemonte, N.; Lukacs, G.; Du, K.; Caci, E.; Zegarra-Moran, O.; Galietta, L.; Verkman, A.; J. Clin. Invest. 2005, 115, 2564-2571.Van Goor, F.; et al. Am. J. Physiol. Lung. Cell. Mol. Physiol. 2006, 290, 1117-1130.

Potential Pharmacological Chaperones

Lewis, H.; et al. J. Biol. Chem. 2005, 280, 1346-1353.Van Goor, F.; et al. Am. J. Physiol. Lung Cell. Mol. Physiol. 2006, 290, 1117-1130.

CFcor-325 and ∆F508-CFTR

CFcor-325 increases ∆F508-CFTR activity and

mature protein levels

Loo, T.; Bartlett, C.; Clarke, D. Molecular Pharmaceutics 2005, 2, 407-413.

CFcor-325

Further Studies With CFcor-325

Loo, T.; Bartlett, C.; Wang, Y.; Clarke, D. Biochem J. 2006, 395, 537-542.Varga, K.; Goldstein, R.; Jurkuvenaite, A.; Chen, L.; Matalon, S.; Sorscher, E.; Bebok, Z.; Collawn, J. Biochem J. 2008, 410, 555-564.

CFcor-325 extends half-life of ∆F508-CFTR

Treatment is mutation dependent

ControlCFcor-325

P-Glycoprotein and Specificity

Wang, Y.; Bartlett, C.; Loo, T., Clarke, D. Molecular Pharmacology 2006, 70, 297-302.

CFcor-325 inhibits P-Glycoprotein

CFcor-325 Rescues P-Glycoprotein Mutant

P-Glycoprotein Activity

CFpot-532 and corr-2b are more specific for CFTR

Summary

Mutant protein needs to be retained in the ER

Mutant protein must demonstrate some activity

Treatment is mutation specific

Applicable to many genetic diseases characterized by misfolded proteins

High-throughput screening can identify potential pharmacological chaperones

Low impact on other cellular pathways

Limitations

Future

Advantages

More general method for identification

Acknowledgements

Professor Silverman

Chem 575 Class

Professor Burke

Burke Group