means of treating botulinum neurotoxin persistence
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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
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