UMass Dartmouth Botulinum Research Center Introduction Symposium

Insights into the mechanism of BoNT/A neuronal persistence and avenues for novel therapies

George A. Oyler MD, PhDFriday August 24, 2007

Proposed Mechanisms of Persistence

• Cleavage product of SNAP25 by BoNT/A is stable and acts as dominant negative for synaptic transmission. This requires cleavage products from different serotypes to have different recycling time.

• The catalytic subunit is stable and persists in an active form. This requires the different serotypes to have different stability.

• Differential compartmentalization of the catalytic subunits of different serotypes:

YFP-BoNT/A LC is trafficked through multiple vesicle compartments in neuronal cells

GFP-BoNT/A LC is trafficked in a polarized fashion and accumulates in specific sites of neuronal cells

YFP-BoNT/E LC is also trafficked to plasma membrane

YFP-LCE RFP-LCA Merged

N18 neuroblastoma

Differential compartmentalization alone cannot account for differences in persistence

anti-actin

anti-GFP

CHX: 0 1 2 4 6 8 0 1 2 4 6 8

YFP-LCE YFP-LCA

BoNT/A and /E LC stability in SH-SY5Y cells

Ubiquitin proteasome system

250

100

75

50

25

IB: anti-HAIP: anti-GFPIB: anti-GFP

IP: anti-GFP

YF

P

YF

P-L

CE

YF

P-L

CA

YF

P

YF

P-L

CE

YF

P-L

CA

+ HA-Ub

Ubin YFP-BoNT LC

-Ubi-YFP

YFP-LC

YFP

-

LC/A

YFP

-

LC/E

YFP

-

LC/A

YFP

-

LC/E

YFP-BoNT/E LC is ubiquitinated more extensively than YFP-BoNT/A LC in N18 cells

Myc-LC/A Myc-LC/E

HectD2 TRIM63

TRIP12 Cbl-b

E4A

TEB4

Triad3

DZIP3

ProteasomeComplex

Degraded BoNT lc

BoNT LC

Ub

Ub

UbUb

ubiquitination

Proteasomerecognition

slow BoNT degradation

Natural proteasomal turnover of BoNT LC

Designer E3 ligases that target toxins for proteasome degradation

Ub

BoNT lc

UbUb

UbTarget bindin

g domai

n

Ubiquitin E3-ligase

Cellular E3 ligase

Target bindin

g domai

n

Ubiquitin E3-ligase

“slow”

Ub

BoNT lc

UbUb

UbUb

Ub

Degraded BoNT lc

Therapeutic fusion protein

BoNT LC

Ub

Ub

UbUb

Designer E3 ligases that target toxins for proteasome degradation

Enhanced proteasomal turnover of BoNT LC

UbUbUb

Ub

“Designer E3 ligase”

“fast”

ProteasomeComplex

Ub

BoNT lc

UbUb

Ub

E3-ligase

LC bindingagent

E3-ligase

LC bindingagent

BoNT lcubiquitination

Proteasomerecognition

Accelerated BoNT

degradationUb

Ub

Antidotes that accelerate turnover of intraneuronal BoNT LC

Background:

• The concept of targeted proteolysis of cellular proteins has been demonstrated several times in the literature.

• SNAP25/nc based “proof of concept” for a designer E3-ligase strategy.

• For potential therapeutic applications, we are currently developing:

1. Camelid antibodies as more effective LC targeting domains.

2. Optimal E3-ligase domain (e.g. F-box proteins).

3. Neuronal delivery vehicle.

XIAP RING is Catalytic E3 domain

SNAP25 replaces XIAP BIR1-3 domains and recognizes BoNT as substrate for ubiquitination

SNAP-25/NC

BoNT/A and E noncleavable C-terminus of SNAP25

BIR1 BIR2 BIR3

CC

C

C

C

H

C CZn

Zn

XIAP BIR1-3 domains recognizes and binds caspase substrate for ubiquitination

CC

C

C

C

H

C CZn

Zn

SNAP-25/NC-RING

cells alone

SNAP-25/NC Control

SNAP-25/NC-RING + proteasome inhibitor (MG132)

Rel

ativ

e am

ou

nt

of

35S

la

bel

led

YF

P-L

C

Time (hours)

5 10 150 20 25

SNAP-25/NC-RING “designer E3 ligase” substantially accelerates proteasome-mediated degradation of recombinant BoNT/A in transfected neurons

Designer E3 ligase accelerates BoNT/A LC turnover in N18 cells

Spinelli S, Desmyter A, Frenken L, Verrips T, Tegoni M, Cambillau C. Domain swapping of a llama VHH. FEBS Lett. 2004;564(1- 2):35- 40.

Camelid VHH forms a compact well-folding single peptide structure

Background:

• VH domains of camelid HcAbs (VHHs) are easy to produce as recombinant proteins in E. coli and have excellent hydrodynamic properties.

• These antibodies are also generally superior for enzyme neutralization as they bind better into “pockets” such as found in enzyme active sites.

Progress:

• We hyper-immunized two alpacas in New Zealand with A-LC and prepared a VHH phage display library.

• We obtained five unique A-LC binding positives screening at high stringency, three with particularly high apparent affinity.

VHHs as targeting domains

2.5e6 6.43216080040002e410e45e5

ELISA vs BoNT/A LCSDS-PAGE (Coomassie)

A6 E3 D4 G6 B8VHH ELISA on BoNT/A LC

0

0.5

1

1.5

2

2.5

3

3.5

300 60 12.5 2.5 0.5 0.1 0.02

nM VHH

Ab

sorb

ance

A6

E3

D4

G6

B8

Series1

Series2

Series3

Series4

Series5

A6

E3

D4

G6

B8

350 ng each

SDS-PAGE (Coomassie)

A6 E3 D4 G6 B8

350 ng each

VHH-B8 selected as having the highest affinity for BoNT/A LC

Elisa analysis of Anti-BoNT/A Lc VHH clones

GST-VHH-B8 (ug)

YF

P-S

NA

P25

-CF

P

clea

vag

e ac

tivi

ty (

%)

25

50

75

100

VHH-B8 was expressed as a GST fusion protein (GST-VHH-B8). Assays were conducted with 0.2 ug BoNT/A LC in 100 ul reaction volume (25 nM), 0.5 ug YFP-SNAP25-CFP substrate (80 nM), and increasing concentrations of GST-VHH (B8). Inhibition of BoNT/A LC activity by GST-VHH (B8) was near stoichiometric.

0.10 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.90

100% inhibition of 2.5 pM of BoNT/A LC by 0.4 ug, or ~10 pM, of VHH-B8

GST-VHH-B8 potently inhibits BoNT/A Lc

YFP Channel CFP Channel

+ YFP-VHH-RING

- CFP-BoNT/A LC

+ YFP-VHH-RING

+ CFP-BoNT/A LC

- YFP-VHH-RING

+ CFP-BoNT/A LC

Anti-A-LC VHH co-localizes with A-LC in cells

Anti-A-LC VHH localizes to cytosol in transfected Neuro2a

cells.

When co-expressed with BoNT/A LC, the VHH localizes with A-

LC at the plasma membrane.

Western Blot for Steady State level of CFP-BoNT/A LC with YFP-VHH-RING Designer ligases

-BoNT/A LC

Con

trol (

Y-SN

AP25

-C)

YFP-

B8-R

ING

YFP-

D4-

RIN

G

N2a cells Expressing Yes-SNAP25-Cer FRET Indicator

YesFP

CerFP FRETYesFP

CerFPSNAP25 (1-206)

FRET ratio changes from 1.3 to 0.60 over 24 hr treatment with 10 nM BoNT/A in media

Cer BoNT LC Cer BoNT LC

Y B8 onlyCer BoNT LC

Y B8 RingCer BoNT LC

Y B8 TrCP

VHH-B8 inhibits A-LC co-expressed in cells

Transfected BoNT/A LC activity in N2a cell lysates is inhibited when co-transfected with VHH-B8 constructions using YFP/SNAP25/CFP

FRET reduction assay

TrCP designer ligases themselves turnover rapidly

Anti XFP 1:5000

YFP/VHH-B8/TrCP

M - 4 hr o/n

MG135 treatment

Inhibition of proteasomes with MG135 stabilizes TrCP fusion

protein and leads to accumulation of poly-ubiquitinated forms

75

50

37

100

150

25

250

1: YFP-B8 +/A24+24

2: YFP-B8 +/control

3: Indicator only +/A24+24

4: YFP-VHH B8-Trcp +/A24+24

5: YFP-VHH B8-Trcp+/control

6:YFP-VHH B8-Trcp +/A24

7: Indicator only +/A24

8: Indicator only +/control

9: YFP-VHH B8-RING+/A24+24

10: YFP-VHH B8-RING +/control

11: No transfection +/A24+24

12: No transfection +/control

1 2 3 4 5 6 7 8 M 9 10 11 12

Jun. 11th-15th.2007

Anti XFP 1:5000

Anti SNAP 1:5000

1 2 3 4 5 6 7 8 M 9 10 11 12

YFP-B8

NC YFP-SNAP25-CFP

C YFP-SNAP25-CFP

YFP-VHH B8-RING

VHH based designer ligases prevent YFP-SNAP25-CFP cleavage in intoxicated M17 cells.

YFP-B8 YFP-VHH B8-Trcp

YFP-VHH B8-RING

Designer E3 ligases that target toxins for proteasome degradation

Preferred strategy for targeted destruction of BoNT: a smaller, modular “designer E3 ligase”

E3 ligase targeting domain, e.g. minimal TrCP (F-box)

LC bindingagent

VHH-LC targeting domain

Delivery vehicle to neuronal cytosol

E3-ligase

BoNT LC

Note that the targeting domain

can be interchanged to create botulism therapeutics for each serotype once an A-LC prototype has

been developed.

BoNT Lc

BoNT Hc-N

BoNT Hc-C

a. b.

c. d.

BoNT/A Heavy Chain can be used for trafficking cargo to hippocampal organotypic neurons

Conclusions:

1. BoNT/A and /E LC are plasma membrane localized.

2. BoNT/E is degraded much more rapidly than BoNT/A LC in cells.

3. BoNT/E is ubiquitinated and degraded by the proteasome rapidly.

4. Designer E3 ligases can be constructed to accelerate BoNT/A degradation.

5. VHH camelid antibodies have been generated against BoNT/A LC.

6. VHH-based designer E3 ligases are effective in degrading BoNT/A LC.

7. Delivery to intoxicated neurons of VHH-based designer E3 ligases may offer novel post-exposure therapies for BoNT intoxication.

Acknowledgements:Tufts Team:

Chuck Shoemaker PhD

Saul Tzipori DVM PhD

Chueh-Ling Kuo

Jong Beak Park PhD

Ira Herman PhD

University of Maryland:

Paul Fishman MD PhD

Yien Che Tsai PhD (now NCI)

Johns Hopkins:

Daniel Drachman MD

Michael Betenbaugh PhD

Synaptic Research:

George A Oyler MD PhD

James R Oyler

USAMRICD:

Michael Adler PhD

James Eric Keller PhD (now FDA)

Metabiologics:

Michael Goodnough PhD

University of Wisconsin:

Eric Johnson PhD

UMass Dartmouth:

Bal Ram Singh PhD

This work was supported by contract NO1-AI30050 from the National Institutes of Health (NIH) and the National Institute of Allergy and Infectious Diseases (NIAID) and Bioshield NIAID 1R01AI 67504-01 to George A. Oyler MD, PhD