Paclitaxel Is an Inhibitor and Its Boron Dipyrromethene Derivative Is a Fluorescent Recognition Agent for Botulinum Neurotoxin Subtype A

Abstract

We have successfully identified one new inhibitor and one new fluorescent recognition agent for the botulinum neurotoxin subtype A (BoNT/A) using the virtual screening protocol Protein Scanning with Virtual Ligand Screening (PSVLS). Hit selection used an in-house developed Holistic Binding scoring method. Selected hits were tested experimentally for inhibitory activity using Fluorescence Resonance Energy Transfer (FRET) assays against the light chain (catalytic domain) of BoNT/A. Ligand binding was determined against the light and heavy chain BoNT/A complex either through radiolabeled ligand binding assays (non-fluorescent ligands) or fluorescence intensity assays (fluorescent ligands). These experimental assays have confirmed one compound (paclitaxel) to inhibit BoNT/A’s proteolytic activity experimentally with an IC50 of 5.2 μM. A fluorescent derivative was also confirmed to bind to the toxin, and therefore is a suitable candidate for the rational design of new detection agents and for the development of fluorescence-based multi-probe detection assays.

Systematic discovery of molecular probes targeting multiple non-orthosteric and spatially distinct sites in the botulinum neurotoxin subtype A (BoNT/A)

“…PSVLS was used to screen a virtual library of 1,624 commercially-available radiolabeled ligands against BoNT/A in order to identify new inhibitors and molecular probes targeting the catalytic site of BoNT/A[19], a metalloprotease. The results of that screening for all 33 binding sites of BoNT/A were further analyzed in the present work with the intent of identifying non-orthosteric ligands for BoNT/A.…”

“…However, most of these studies focused on identification of novel recognition agents and inhibitors for the active site of BoNT/A. Our group has also targeted the active site of BoNT/A[19], successfully identifying a novel inhibitor and a fluorescent recognition agent. In that work, a library consisting of 1,624 compounds including commercially available radiolabeled ligands was computationally screened against BoNT/A using the Protein Scanning with Virtual Ligands Screening (PSVLS) method.…”

“…Here we report the analysis of the PSVLS results in Dadgar et al, 2013 [19] for all 33 binding regions explored by PSVLS, with the intent of identifying non-orthosteric ligands for BoNT/A. Non-orthostheric ligands may serve as subtype-specific molecular probes that take opportunity of unique binding pockets in BoNT/A compared to other subtypes or other homologous proteins (potential confounders in a detection assay).…”

“…We selected four ligands based on their PSVLS binding profiles. A previously identified inhibitor [19], paclitaxel, was used as positive control. D-fructose, a non-binder [19], was used as negative control.…”

“…A previously identified inhibitor [19], paclitaxel, was used as positive control. D-fructose, a non-binder [19], was used as negative control. Selected ligands were tested against BoNT/A HC and BoNT/A LC separately using scintillation proximity assay (SPA).…”