George A. Oyler

President and Founder

Synaptic Research

Ubiquitin ligase therapeutics to treat BoNTpersistence

The BoN/A, B, and C Lc protease activity inside neurons is long due to evading

degradation pathways. Our goal is to harness the neurons own machinery to

degrade the BoNT Lc inside neurons using the ubiquitin proteasome system.

Current Opinion in Pharmacology 2005, 5:274–279

Ubiquitin proteasome system

90-100 DUBs in

human genome

E1 = 2

E2 = 40

E3 = 600

schematic of conventional Ab

Antibody (Ab) structure

Camelid single domain VHH or Nanobody

schematic of camelid“heavy-chain only” Ab

VHH or nanobody

nanobody

Fv domain

Alpacas were immunized with both fragments of BoNT and toxoid to develop several nanobody phage display libraries

Designer E3 Ubiquitin ligase based on F-box and VHH to form a complete SCF Ubiquitin ligase complex

F-box VHH

F-box

YFP B8 TrCPSBP

WD40 repeats

YFP B8SBPD1

D2

D3

D4

YFP B8SBP

YFP B8SBP

YFP B8SBP

D6 B8SBP

F

F

F

F

F

D7 B8SBP F

D7* B8*SBP F*

TrCP current

D5 B8SBP F

F

Examples of VHH-Fbox fusion designer E3 ligases

Reduction of mammalian expressed BoNT Lc by

corresponding VHH based designer E3 ligase

P< 0.001 P= 0.001

0

0.1

0.2

0.3

0.4

0.5

0.6

YFP-D5-B8 YFP-D5-B10

CF

P-A

Lc

sig

nal

(O

D 4

50)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

YFP-D5-B8 YFP-D5-B10

CF

P-B

Lc

sig

nal

(O

D 4

50)

ALc-NTF BLc-NTF ALc-NTF BLc-NTF

CFP-BLcCFP-ALc

Reduction of mammalian expressed BoNT Lc in cell lines

stably expressing the BoNT Lc Designer Ligases (NTF)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

16 40 64

Rela

tive

CF

P-A

Lc

sig

na

l

hours post transfection

Reduction of CFP-ALc

M17LentiD5B8 M17LentiD5B10ALc NTF line BLc NTF line

Toxin protein based delivery systems

• BoNT A, C, or D based atoxic holotoxin.

• BoNT Hc delivery systems (Brenda Wilson).

• Tcd retargeted delivery system.

• Botulinum exotoxin C2 based system

Viral based delivery systems

• Retargeted Adenoviral system

• Pseudotyped Lentiviral system

BoNT Lc

BoNT Hc-N

BoNT Hc-C

Atoxic BoNT holotoxin proteins delivery vehicles for

designer ligases (expressed in algae for oral delivery)

Cargo attached to atoxic LC

Jank, T. et al. Glycobiology 2007 17:15R-22R

Structure and intoxication pathway of toxin A and B from C. difficult (Tcd)

C2I-N C2I-C

C2II

Cleavage activation site

Glycosylation receptor binding site

C2I-N

C2II

Cleavage activation site

BoNT/C Hc targeting neurons

Designer Ligase Cargo

Conclusions from Designer E3 Ligase

Research and Development

• BoNT Lc persistence can be reduced by harnessing the cell’s own UPS.

• SR and collaborators (Shoemaker group) have developed an effective VHH based E3 ligase to BoNT.

• Protein toxin-based and viral delivery systems are being developed.

Acknowledgements

Tufts:

Charles Shoemaker

Greice Krautz-Peterson

Chuehling Kuo

Claudia Abeijon

Hanping Feng

Synaptic Research:

Barry Gertz

Archana Kotiya

Kevin McIntosh

Kyle Krady

Julian Rosenberg

NCI:

YienCheTsai

Allan Weissman

USAMRIID:

Len Smith

Bob Webb

Ashraf Ahmed

USAMRICD:

Patrick McNutt

Michael Alder

Funding: NIH NIAID, DTRA, SR Development funds