Presentation on theme: "UMass Dartmouth Botulinum Research Center Introduction Symposium Insights into the mechanism of BoNT/A neuronal persistence and avenues for novel therapies."— Presentation transcript:
1UMass 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
4Proposed 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:
5YFP-BoNT/A LC is trafficked through multiple vesicle compartments in neuronal cells
6GFP-BoNT/A LC is trafficked in a polarized fashion and accumulates in specific sites of neuronal cells
7YFP-BoNT/E LC is also trafficked to plasma membrane
8Differential compartmentalization alone cannot account for differences in persistence YFP-LCERFP-LCAMergedN18 neuroblastoma
9BoNT/A and /E LC stability in SH-SY5Y cells YFP-LCEYFP-LCACHX:1246812468anti-GFPanti-actin
15Enhanced proteasomal turnover of BoNT LC Therapeutic fusion protein Designer E3 ligases that target toxins forproteasome degradationEnhanced proteasomal turnover of BoNT LCUbUb“Designer E3 ligase”E3-ligaseLC bindingagentE3-ligaseLC bindingagentUbUb“fast”UbUbUbBoNT lcUbubiquitinationBoNT LCTherapeutic fusion proteinProteasomerecognitionUbUbBoNT lcAcceleratedBoNTdegradationProteasomeComplexUbDegraded BoNT lc
16Antidotes 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.
17Zn Zn Zn C H C C C C C C C H BIR1 BIR2 BIR3 SNAP-25/NC XIAP BIR1-3 domains recognizes and binds caspase substrate for ubiquitinationXIAP RING isCatalytic E3 domainSNAP-25/NCCCZnCCCSNAP25 replaces XIAP BIR1-3 domains and recognizes BoNT as substrate for ubiquitinationCZnCHBoNT/A and E noncleavable C-terminus of SNAP25
18Relative amount of 35S labelled YFP-LC Designer E3 ligase accelerates BoNT/A LC turnover in N18 cellsSNAP-25/NC-RING + proteasome inhibitor (MG132)cells aloneRelative amount of 35S labelled YFP-LCSNAP-25/NC ControlSNAP-25/NC-RING510152025Time (hours)SNAP-25/NC-RING “designer E3 ligase” substantially accelerates proteasome-mediated degradation of recombinant BoNT/A in transfected neurons
20Camelid VHH forms a compact well-folding single peptide structure
21VHHs 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.
22Elisa analysis of Anti-BoNT/A Lc VHH clones SDS-PAGE (Coomassie)350 ng eachSDS-PAGE (Coomassie)A6E3D4G6B8ELISA vs BoNT/A LCVHH ELISA on BoNT/A LCA6E3D4G6B83.532.5A6E3D4G6B8Series1Series2Series3Series4Series5Absorbance21.510.52.5e66.43216080040002e410e45e53006012.52.50.50.10.02nM VHHVHH-B8 selected as having the highest affinity for BoNT/A LC
23GST-VHH-B8 potently inhibits BoNT/A Lc 100YFP-SNAP25-CFP cleavage activity (%)100% inhibition of 2.5 pM of BoNT/A LC by 0.4 ug, or ~10 pM, of VHH-B87550250.10.20.30.22.214.171.124.80.9GST-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.
24Anti-A-LC VHH co-localizes with A-LC in cells YFP ChannelCFP ChannelAnti-A-LC VHH localizes to cytosol in transfected Neuro2a cells.+ YFP-VHH-RING- CFP-BoNT/A LCWhen 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
25Western Blot for Steady State level of CFP-BoNT/A LC with YFP-VHH-RING Designer ligases Control (Y-SNAP25-C)YFP-B8-RINGYFP-D4-RING-BoNT/A LC
26N2a cells Expressing Yes-SNAP25-Cer FRET Indicator YesFPCerFPFRETYesFPCerFPSNAP25 (1-206)FRET ratio changes from 1.3 to 0.60 over 24 hr treatment with 10 nM BoNT/A in media
27VHH-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 assayCer BoNT LCCer BoNT LC Y B8 onlyCer BoNT LC Y B8 RingCer BoNT LC Y B8 TrCP
28TrCP designer ligases themselves turnover rapidly MG135 treatmentM hr o/nInhibition of proteasomes with MG135 stabilizes TrCP fusion protein and leads to accumulation of poly-ubiquitinated formsYFP/VHH-B8/TrCPAnti XFP 1:5000
29VHH based designer ligases prevent YFP-SNAP25-CFP cleavage in intoxicated M17 cells. Anti XFP 1:50001: YFP-B8 +/A24+242: YFP-B8 +/control3: Indicator only +/A24+244: YFP-VHH B8-Trcp +/A24+245: YFP-VHH B8-Trcp+/control6:YFP-VHH B8-Trcp +/A247: Indicator only +/A248: Indicator only +/control9: YFP-VHH B8-RING+/A24+2410: YFP-VHH B8-RING +/control11: No transfection +/A24+2412: No transfection +/controlYFP-B8YFP-VHHB8-TrcpYFP-VHHB8-RING250150100NC YFP-SNAP25-CFP75YFP-VHH B8-RINGC YFP-SNAP25-CFP50YFP-B837M25Anti SNAP 1:5000MJun. 11th-15th.2007
30Designer E3 ligases that target toxins for proteasome degradation Preferred strategy for targeted destruction of BoNT: a smaller, modular “designer E3 ligase”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 targeting domain, e.g. minimal TrCP (F-box)E3-ligaseLC bindingagentVHH-LC targeting domainBoNT LCDelivery vehicle to neuronal cytosol
32BoNT/A Heavy Chain can be used for trafficking cargo to hippocampal organotypic neurons
33Conclusions: 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.
34Acknowledgements: Synaptic Research: George A Oyler MD PhD James R OylerUSAMRICD:Michael Adler PhDJames Eric Keller PhD (now FDA)Metabiologics:Michael Goodnough PhDUniversity of Wisconsin:Eric Johnson PhDUMass Dartmouth:Bal Ram Singh PhDAcknowledgements:Tufts Team:Chuck Shoemaker PhDSaul Tzipori DVM PhDChueh-Ling KuoJong Beak Park PhDIra Herman PhDUniversity of Maryland:Paul Fishman MD PhDYien Che Tsai PhD (now NCI)Johns Hopkins:Daniel Drachman MDMichael Betenbaugh PhD