Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Ross, Justin A.; Gilmore, M.; Williams, Dudley; Aoki, K. Roger; Steward, Lance E.; Jameson, David M.

doi:10.1016/j.ab.2011.01.045

Cited by 12 publications

(4 citation statements)

References 39 publications

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“…Light Chain assays (ELISA [26]–[28], Endopep-MS [29], FRET [30], [31], HPLC-UPLC [32], and DARET [33], [34]) only measure activity of the catalytic domain and cannot detect non-functionality in other BoNT domains (i.e., translocation or binding domains). Previous cell-based assays to screen BoNT inhibitors relied on cells with low toxin sensitivity such as SH-SY5Y [35] or Neuro-2a cells [36], [37].…”

Section: Introductionmentioning

confidence: 99%

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

Fernández‐Salas

Wang²,

Molina³

et al. 2012

PLoS ONE

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112

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Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

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Section: Introductionmentioning

confidence: 99%

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

Fernández‐Salas

Wang²,

Molina³

et al. 2012

PLoS ONE

Self Cite

112

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“…To screen for substrate specificity, depolarization after resonance energy transfer (DARET) 25 , 26 was used to measure LC/A cleavage of SNAP23 and SNAP25. Here, > 70 residues of each substrate were sandwiched between GFP and BFP (shown schematically in Fig.…”

Section: Resultsmentioning

confidence: 99%

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

Dyer

Isoda

Salcedo

et al. 2022

Sci Rep

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The botulinum neurotoxin serotype A (BoNT/A) cuts a single peptide bond in SNAP25, an activity used to treat a wide range of diseases. Reengineering the substrate specificity of BoNT/A’s protease domain (LC/A) could expand its therapeutic applications; however, LC/A’s extended substrate recognition (≈ 60 residues) challenges conventional approaches. We report a directed evolution method for retargeting LC/A and retaining its exquisite specificity. The resultant eight-mutation LC/A (omLC/A) has improved cleavage specificity and catalytic efficiency (1300- and 120-fold, respectively) for SNAP23 versus SNAP25 compared to a previously reported LC/A variant. Importantly, the BoNT/A holotoxin equipped with omLC/A retains its ability to form full-length holotoxin, infiltrate neurons, and cleave SNAP23. The identification of substrate control loops outside BoNT/A’s active site could guide the design of improved BoNT proteases and inhibitors.

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“…Förster resonance energy transfer (FRET), a direct and sensitive technology, has been widely used for bioassays, biosensing, bioimaging, and photodynamic therapy [15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline

Zhang

Fang

et al. 2015

Analytical Biochemistry

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Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay

Cited by 12 publications

References 39 publications

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

Botulinum Neurotoxin Serotype a Specific Cell-Based Potency Assay to Replace the Mouse Bioassay

Reengineering the specificity of the highly selective Clostridium botulinum protease via directed evolution

Upconversion luminescence resonance energy transfer-based aptasensor for the sensitive detection of oxytetracycline

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