Et højt indhold Imaging Assay til identifikation af botulinumneurotoksin inhibitorer
Summary
Botulinum neurotoxin is one of the most potent toxins among Category-A biothreat agents, yet a post-exposure therapeutic is not available. The high content imaging approach is a powerful methodology for identifying novel inhibitors as it enables multiparameter screening using biologically relevant motor neurons, the primary target of this toxin.
Abstract
Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system.
Introduction
Bakterien Clostridium botulinum producerer botulinum neurotoksin, et af de mest potente biologiske toksiner, man kender 1. Der er 7 forskellige BoNT serotyper (BoNT / AG). BoNT / A-Gall fremkalde lammelse ved den neuromuskulære junction grund SNARE kompleks proteolyse 2,3. SNARE proteolyse forhindrer neurotransmitter vesikel-membran fusion og derfor blokerer neurotransmitter exocytose 4. Den specifikke SNARE målet afhænger af den særlige BoNT serotype involveret i forgiftning proces. BoNT / A og BoNT / E spalter SNAP-25 hvorimod BoNT / C spalter både SNAP-25 og syntaxin 5. Den resterende serotyper spalter synaptobrevin (også kaldet vesikelassocieret membranprotein (VAMP). BoNT / A blev valgt til assay udvikling, da den er ansvarlig for en stor del af naturligt forekommende botulisme, og har den længste virkningstid 6. Udvikling af lille molekyle lægemidler mod BoNT / A er et vigtigt mål forvores drug discovery program og har benyttet traditionelle målrettede metoder til at identificere aktive websted proteolytiske inhibitorer 7, 8-10. Imidlertid vil etablering af aktivt sted inhibitorer med bredspektret aktivitet mod flere serotyper og post-eksponering effekt sandsynligvis være udfordrende.
Vi har derfor gennemført en innovativ, fænotypisk drug discovery tilgang, der bruger BoNT SNAP-25 spaltning som en funktionel endepunkt for at identificere små molekyler, der kan blokere BoNT-medieret motor neuron beruselse. SNAP-25 er nødvendig for neurotransmitterfrigivelse, som nedbrydning af SNAP-25 er prædiktive for lammelse og letalitet in vivo. For eksempel kan celle-baserede screening føre til opdagelsen af nye modulatorer af cellulære faktorer, der er ansvarlige for toksin inaktivering eller inhibering af toksin veje inde i målcellerne. En vigtig faktor i fænotypisk assay udvikling er det udvalg af fysiologisk relevante biologiske modeller. We og andre har beskrevet mus embryonale stamceller (ES) celleafledte motoriske neuroner, der rekapitulere immunologisk karakter af primære motoriske neuroner, herunder afgivelse af SNAP-25 11- 13. Vigtigere, disse cellulære systemer er meget følsomme over BoNT / A forgiftning og viser dosis-afhængig spaltning af SNAP-25 som reaktion på stigende koncentrationer af toksin 11,12. De differentierede motoriske neuroner er også produceret i mængder, der er tilstrækkelige til high throughput plade baseret analyse og tilladt udformningen af en vifte af cellulære assays.
Den fænotypiske analyse er en immunfluorescens fremgangsmåde, der anvender to forskellige antistoffer til at kvantificere spaltning af endogent udtrykt fuldlængde SNAP-25 i BoNT / A forgiftning af muse motor neuron kultur. En carboxylterminale BoNT / A-sensitive spaltning (BACS) antistof, der genkender kun fuld længde SNAP-25 tillader vurdering af BoNT / A-medieret proteolyse af SNAP-25ekspression i muse motorneuroner 10. Et skematisk diagram af HCI assay er vist i figur 1.
Protocol
Representative Results
Discussion
Den høje styrke af botulinumneurotoksiner og den relative lethed af deres bevæbning har resulteret i deres klassificering som kategori A (højeste prioritet) biothreat midler ved det amerikanske Centers for Disease Control og Forebyggelse. Desværre er der ingen FDA godkendt terapi til bekæmpelse af BoNT forgiftning efter toksinet er blevet internaliseret af de motoriske neuroner. Enhver druggable mekanisme, der fremmer neuronal nyttiggørelse fra BoNT forgiftning kan føre mod udviklingen af en potentiel terap…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Funding was provided by the Joint Science and Technology Office – Chemical Biological Defense (JSTO-CBD) Defense Threat Reduction Agency (DTRA) under sponsor project number CCAR# CB3675 and National Institutes of Health (1 R21 AI101387-01 and 5 U01AI082051-05).
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Botulinum neurotoxin A | Metabiologics | NA | No catalog number |
| Microtitre plates | Greiner | 655946 | Poly-D-Lysine 96-well plates |
| BACs antibody | Lampire Biological | NA | |
| Microchem | National | 0255 | |
| Methanol | Thermo Scientific | A412-20 | |
| Formaldehyde | Thermo Scientific | 28908 | |
| Horse serum | Invitrogen | 16050 | |
| PE JANUS MDT Mini Automated Workstation | Perkin Elmer | AJMDT01 | |
| Opera | Perkin Elmer | OP-QEHS-01 | |
| Triton X 100 | Sigma-Aldrich | 9002-93-1 | |
| BIII tubulin antibody | R&D Systems | BAM1195 | |
| Tween 20 | Sigma | P1379-1L | |
| Hoechst 33342dye | Invitrogen | 3570 | |
| Antimouse IgG | Invitrogen | A21236 | |
| Anti rabbit IgG | Invitrogen | A10042 | |
| Columbus Image analysis software | Perkin Elmer | Ver 2.4 | |
| Spotfire | Perkin Elmer | Ver 5.5 | |
| Clorox bleach | Fisher Scientific | 18-861-284 | |
| PlateStack |
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