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.

Our group has recently demonstrated that virtual screening is a useful technique for the identification of target-specific molecular probes. In this paper, we discuss some of our proof-of-concept results involving two biologically relevant target proteins, and report the development of a computational script to generate large databases of fluorescence-labelled compounds for computer-assisted molecular design. The virtual screening of a small library of 1,153 fluorescently-labelled compounds against two targets, and the experimental testing of selected hits reveal that this approach is efficient at identifying molecular probes, and that the screening of a labelled library is preferred over the screening of base compounds followed by conjugation of confirmed hits. The automated script for library generation explores the known reactivity of commercially available dyes, such as NHS-esters, to create large virtual databases of fluorescence-tagged small molecules that can be easily synthesized in a laboratory. A database of 14,862 compounds, each tagged with the ATTO680 fluorophore was generated with the automated script reported here. This library is available for downloading and it is suitable for virtual ligand screening aiming at the identification of target-specific fluorescent molecular probes.

The development of molecular probes targeting proteins has traditionally relied on labeling compounds already known to bind to the protein of interest. These known ligands bind to orthosteric or allosteric sites in their target protein as a way to control their activity. Binding pockets other than known orthosteric or allosteric sites may exist that are large enough to accommodate a ligand without significantly disrupting protein activity. Such sites may provide opportunities to discriminate between subtypes or other closely related proteins, since they are under less evolutionary pressure to be conserved. The Protein Scanning with Virtual Ligand Screening (PSVLS) approach was previously used to identify a novel inhibitor and a fluorescent probe against the catalytic site of the botulinum neurotoxin subtype A (BoNT/A). PSVLS screens compound databases against multiple sites within a target protein, and the results for all the sites probed against BoNT/A, not only the catalytic site, are available online. Here, we analyze the PSVLS data for multiple sites in order to identify molecular probes with affinity for binding pockets other than the catalytic site of BoNT/A. BoNT/A is a large protein with a light (LC) and a heavy (HC) chain that can be assayed separately. We used scintillation proximity assay (SPA) to test experimentally 5 probe candidates predicted computationally to have affinity for different non-orthosteric binding regions within the HC and LC, and one compound predicted not to have affinity for either domain. The binding profiles obtained experimentally confirmed the targeting of multiple and spatially distinct pockets within BoNT/A. Moreover, inhibition assay results indicate that some of these probes do not significantly interfere with the catalytic activity of BoNT/A.