Development of multi-probe fluorescence-based assay for boNTA detection

Abstract

Botulinum neurotoxins (BoNTs) cause the lethal disease botulism through the inhibition of acetyl choline secretion by the cleavage of crucial SNARE proteins. Determination of critical residues in the protein sequence of BoNT serotype A was the primary step to identify novel fluorescence recognition agents for BoNTA. Computational and experimental studies were employed to identify paclitaxel as a new inhibitor (IC50 equal to 5.2 μM) for the proteolytic activity of BoNTA light chain (LC) using Fluorescence Resonance Energy Transfer (FRET) assay. A fluorescent derivative of paclitaxel (PAC-BDP) exhibited binding to complex BoNTA. A Primary Amines Database comprised of 1,153 compounds suitable for fluorescent labeling was computationally screened to select 6-aminofluorescein (6-AFLU) and aspartame (APM) as recognition agent candidates. Fluorescent labeled APM (APM-BDP) was synthesised and the purity of the compound was confirmed using liquid chromatography mass spectrometry (LC/MS) and nuclear magnetic resonance (NMR). 6-AFLU exhibited good binding affinity to BoNTA heavy chain (HC) with an EC50 of 546 ± 60nM, whereas APM-BDP displayed binding to BoNTA LC with an EC50 of 20.96 ± 10 nM, as determined by fluorescence polarization (FP) assay. APM was shown to compete with APM-BDP for the same binding site in BoNTA LC, but showed no binding to BoNTA HC in FP competition assay. Also, aminopterin (AMN) and 6-AFLU exhibited binding to the same site of BoNTA HC, whereas desmosine (DES) showed affinity to a different binding site in BoNTA HC. Additionally, PAC exhibited binding to BoNTA LC, however paclitaxel (PAC) did not compete with APM-BDP for the same binding region. PAC-BDP showed binding to both BoNTA LC and BoNTA HC and did not compete with APM-BDP for the same binding site in BoNTA LC. A library consisting of 1,624 commercially available radiolabeled ligands were screened computationally to select the ligands with binding affinity against BoNTA LC and HC. The binding of [3H] Aminopterin and [3H] desmosine was shown to be concentration-dependent with EC50of 703 ± 98 nM and 1.6 ± 0.3μM, respectively, against BoNTA HC using scintillation proximity assay (SPA). [3H]Solanesyl pyrophosphate (Solanesyl PP) exhibited high binding to both BoNTA LC and BoNTA HC. However its related compound, [3H]Solanesol, show no binding against BoNTA LC or BoNTA HC using SPA assay. The development of a fast, simple, reliable assay for BoNTA detection is essential since mouse lethality assay (MLA), the only trustable assay, is a costly, time consuming and complicated assay. In addition, detection of BoNTA in the initial steps of contamination is critical for successful treatment. This study demonstrated that FP can be used as a platform for BoNTA detection and that PAC-BDP, APM-BDP and 6-AFLU can be used simultaneously since they bind to different binding regions of BoNTA. The identified recognition agents can potentially be used in a multi-probe FP assay against the whole BoNTA complex.