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Proposed Mechanisms of Persistence

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, PhD Friday August 24, 2007. Proposed Mechanisms of Persistence.

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Proposed Mechanisms of Persistence

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  1. UMass Dartmouth Botulinum Research Center Introduction SymposiumInsights into the mechanism of BoNT/A neuronal persistence and avenues for novel therapiesGeorge A. Oyler MD, PhDFriday August 24, 2007

  2. 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:

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

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

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

  6. Differential compartmentalization alone cannot account for differences in persistence YFP-LCE RFP-LCA Merged N18 neuroblastoma

  7. BoNT/A and /E LC stability in SH-SY5Y cells YFP-LCE YFP-LCA CHX: 0 1 2 4 6 8 0 1 2 4 6 8 anti-GFP anti-actin

  8. Ubiquitin proteasome system

  9. YFP YFP-LCE YFP-LCA YFP YFP-LCE YFP-LCA + HA-Ub 250 100 75 50 25 IP: anti-GFP IP: anti-GFP IB: anti-GFP IB: anti-HA

  10. a-YFP a-Ubi Ubin YFP-BoNT LC YFP-LC YFP-LC/A YFP-LC/A YFP-LC/E YFP-LC/E YFP-BoNT/E LC is ubiquitinated more extensively than YFP-BoNT/A LC in N18 cells

  11. Cellular E3 ligase Ubiquitin E3-ligase Ubiquitin E3-ligase Ub Ub ubiquitination Target binding domain Target binding domain Ub Ub BoNT lc Proteasome recognition slow BoNT degradation Designer E3 ligases that target toxins for proteasome degradation Natural proteasomal turnover of BoNT LC Ub Ub Ub Ub Ub Ub “slow” Ub Ub BoNT lc BoNT LC Ub Proteasome Complex Ub Degraded BoNT lc

  12. E3-ligase E3-ligase Ub Ub ubiquitination Ub Ub BoNT lc LC binding agent LC binding agent Proteasome recognition Designer E3 ligases that target toxins for proteasome degradation Enhanced proteasomal turnover of BoNT LC Ub Ub “Designer E3 ligase” Ub “fast” Ub Ub Ub Ub Ub BoNT lc BoNT LC Therapeutic fusion protein Ub Accelerated BoNT degradation Proteasome Complex Ub Degraded BoNT lc

  13. 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: • Camelid antibodies as more effective LC targeting domains. • Optimal E3-ligase domain (e.g. F-box proteins). • Neuronal delivery vehicle.

  14. BIR1 C C Zn XIAP BIR1-3 domains recognizes and binds caspase substrate for ubiquitination C C C C Zn C BIR2 BIR3 H XIAP RING is Catalytic E3 domain SNAP-25/NC C C Zn C C C SNAP25 replaces XIAP BIR1-3 domains and recognizes BoNT as substrate for ubiquitination C Zn C H BoNT/A and E noncleavable C-terminus of SNAP25

  15. Designer E3 ligase accelerates BoNT/A LC turnover in N18 cells SNAP-25/NC-RING + proteasome inhibitor (MG132) cells alone Relative amount of 35S labelled YFP-LC SNAP-25/NC Control SNAP-25/NC-RING 0 5 10 15 20 25 Time (hours) SNAP-25/NC-RING “designer E3 ligase” substantially accelerates proteasome-mediated degradation of recombinant BoNT/A in transfected neurons

  16. Camelid VHH forms a compact well-folding single peptide structure

  17. VHHs as targeting domains • 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.

  18. A6 A6 A6 E3 D4 G6 B8 E3 E3 D4 D4 350 ng each G6 G6 SDS-PAGE (Coomassie) 350 ng each B8 B8 Series1 Series2 Series3 2.5e6 5e5 10e4 2e4 4000 800 160 32 6.4 Series4 Series5 Elisa analysis of Anti-BoNT/A Lc VHH clones SDS-PAGE (Coomassie) ELISA vs BoNT/A LC VHH ELISA on BoNT/A LC A6 E3 D4 G6 B8 3.5 3 2.5 Absorbance 2 1.5 1 0.5 0 300 60 12.5 2.5 0.5 0.1 0.02 nM VHH VHH-B8 selected as having the highest affinity for BoNT/A LC

  19. GST-VHH-B8 potently inhibits BoNT/A Lc 100 YFP-SNAP25-CFP cleavage activity (%) 100% inhibition of 2.5 pM of BoNT/A LC by 0.4 ug, or ~10 pM, of VHH-B8 75 50 25 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 GST-VHH-B8 (ug) 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.

  20. Anti-A-LC VHH co-localizes with A-LC in cells YFP Channel CFP Channel Anti-A-LC VHH localizes to cytosol in transfected Neuro2a cells. + YFP-VHH-RING - CFP-BoNT/A LC When co-expressed with BoNT/A LC, the VHH localizes with A-LC at the plasma membrane. + YFP-VHH-RING + CFP-BoNT/A LC - YFP-VHH-RING + CFP-BoNT/A LC

  21. Western Blot for Steady State level of CFP-BoNT/A LC with YFP-VHH-RING Designer ligases Control (Y-SNAP25-C) YFP-D4-RING YFP-B8-RING -BoNT/A LC

  22. N2a cells Expressing Yes-SNAP25-Cer FRET Indicator YesFP CerFP FRET YesFP CerFP SNAP25 (1-206) FRET ratio changes from 1.3 to0.60 over24 hr treatment with10 nM BoNT/Ain media

  23. 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 VHH-B8 inhibits A-LC co-expressed in cells Cer BoNT LC Cer BoNT LC Y B8 only Cer BoNT LC Y B8 Ring Cer BoNT LC Y B8 TrCP

  24. Inhibition of proteasomes with MG135 stabilizes TrCP fusion protein and leads to accumulation of poly-ubiquitinated forms TrCP designer ligases themselves turnover rapidly MG135 treatment M - 4 hr o/n YFP/VHH-B8/TrCP Anti XFP 1:5000

  25. VHH based designer ligases prevent YFP-SNAP25-CFP cleavage in intoxicated M17 cells. Anti XFP 1:5000 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 YFP-B8 YFP-VHH B8-Trcp YFP-VHH B8-RING 250 150 100 NC YFP-SNAP25-CFP 75 YFP-VHH B8-RING C YFP-SNAP25-CFP 50 YFP-B8 37 1 2 3 4 5 6 7 8 M 9 10 11 12 25 Anti SNAP 1:5000 1 2 3 4 5 6 7 8 M 9 10 11 12 Jun. 11th-15th.2007

  26. Note that the targeting domain can be interchanged to create botulism therapeutics for each serotype once an A-LC prototype has been developed. E3-ligase E3 ligase targeting domain, e.g. minimal TrCP (F-box) LC binding agent VHH-LC targeting domain Delivery vehicle to neuronal cytosol Designer E3 ligases that target toxins for proteasome degradation Preferred strategy for targeted destruction of BoNT: a smaller, modular “designer E3 ligase” BoNT LC

  27. BoNT Lc BoNT Hc-N BoNT Hc-C

  28. a. b. c. d. BoNT/A Heavy Chain can be used for trafficking cargo to hippocampal organotypic neurons

  29. 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.

  30. 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 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

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