2412 Chem. Commun., 2012, 48, 2412–2414 This journal is c The Royal Society of Chemistry 2012 Cite this: Chem. Commun., 2012, 48, 2412–2414 Discovery of a fluorene class of compounds as inhibitors of botulinum neurotoxin serotype E by virtual screeningw Gyanendra Kumar,z Rakhi Agarwalz and Subramanyam Swaminathan* Received 17th November 2011, Accepted 6th January 2012 DOI: 10.1039/c2cc17158a Botulinum neurotoxins are one of the most poisonous biological substances known to humans and present a potential bioterrorism threat. There are no therapeutic interventions developed so far. Here, we report the first small molecule non-peptide inhibitor for botulinum neurotoxin serotype E discovered by structure-based virtual screening and propose a mechanism for its inhibitory activity. Botulinum neurotoxin serotype E (BoNT/E) is one of the seven serotypes (A to G) of neurotoxins secreted by the bacterium Clostridium botulinum. With a lethal dose in the range of 0.1–1 ng kg À1 for humans, these could be potentially used as bioweapons, and hence they are classified as category A bioterrorism agents by the Centers for Disease Control and Prevention. Generally, each of these neurotoxins is produced as a single, inactive polypeptide chain (B 150 kDa) and then proteolytically cleaved before release into active di-chains, a heavy (HC, B100 kDa) and a light (LC, B50 kDa) chain linked by a disulfide bond. The HC itself is comprised of two domains of B50 kDa each. The C-terminal domain (H C ) plays a role in binding to specific receptors on the cell membrane and internalization of the toxin into cholinergic neurons followed by the N-terminal domain (H N ) facilitated release of LC from endosomes into the cytosol. 1–3 The LC is a zinc-dependent protease that cleaves and inactivates SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) proteins. Cleavage of SNARE protein(s) by BoNT disrupts the release of acetylcholine from synaptic terminals leading to botulism. 4 The BoNT catalytic domains share B36% sequence identity across serotypes and show specificity toward their substrates. Serotypes A and E cleave SNAP-25 (SyNaptosomal- Associated Protein of 25 kDa), serotype C cleaves SNAP-25 and syntaxin, serotypes B, D, F and G cleave VAMP (Vesicle- Associated Membrane Protein), all of them cleave at unique peptide bonds. 5–9 This makes it difficult to target all the serotypes with a single drug candidate for botulism. Hence, efforts are being made to design inhibitors targeting individual serotypes. The three most common serotypes of botulinum are A, B and E. A recent study shows that BoNT/E blocks neurotrans- mission faster compared to all other serotypes. 10 Although BoNT serotypes A and E both cleave SNAP-25, yet they cause synaptic blockade by very different properties. While BoNT/A cleaved SNAP-25 maintains its association with plasma membrane syntaxin, cleavage by BoNT/E severely compromises SNAP-25 association with syntaxin. 11 While a number of small molecule non-peptide inhibitors (SMNPIs) have been reported for BoNT/A 12–16 and a few for BoNT/B, 17 none have been reported for BoNT/E yet. We had earlier determined the crystal structures of the BoNT/E catalytic domain in native form and in complex with its substrate-based peptide inhibitor RIME. 18,19 Here, we report the first SMNPIs of BoNT/E identified by docking-based virtual screening. We screened the Diversity Set of the NCI Compound Database, which is a representative set of 1990 compounds made out of 140 000 that represent the diversity of compounds in collection in terms of pharmacophore features and were available with the NCI in the amount of 1 g or more. The docking program AutoDock 3.05 was used for virtual screening on the Brookhaven Linux Cluster. 20 A flowchart of this research work starting from virtual screening and leading to the discovery of first hit compound is depicted in Scheme 1. Scheme 1 Flowchart for the discovery of a fluorene class of BoNT/E inhibitors. Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA. E-mail: swami@bnl.gov; Fax: +1 631 344 3407; Tel: +1 631 344 3187 w Electronic supplementary information (ESI) available: Table of compound structures and BoNT/E inhibition data. See DOI: 10.1039/ c2cc17158a z These authors contributed equally to this work. ChemComm Dynamic Article Links www.rsc.org/chemcomm COMMUNICATION Downloaded by BNL Research Library on 01 February 2012 Published on 10 January 2012 on http://pubs.rsc.org | doi:10.1039/C2CC17158A View Online / Journal Homepage / Table of Contents for this issue