Conformational Sampling of the Botulinum Neurotoxin Serotype a Light Chain: Implications for Inhibitor Binding

Botulinum neurotoxins (BoNTs) are the most potent of the known biological toxins, and consequently are listed as category A biowarfare agents. Currently, the only treatments against BoNTs include preventative antitoxins and long-term supportive care. Consequently, there is an urgent need for therapeutics to counter these enzymes––post exposure. In a previous study, we identified a number of small, nonpeptidic lead inhibitors of BoNT serotype A light chain (BoNT/A LC) metalloprotease activity, and we identified a common pharmacophore for these molecules. In this study, we have focused on how the dynamic movement of amino acid residues in and surrounding the substrate binding cleft of the BoNT/A LC might affect inhibitor binding modes. The X-ray crystal structures of two BoNT/A LCs (PDB refcodes = 3BTA and 1E1H) were examined. Results from these analyses indicate that the core structural features of the examined BoNT/A LCs, including a-helices and b-sheets, remained relatively unchanged during 1ns dynamics trajectories. However, conformational flexibility was observed in surface loops bordering the substrate binding clefts in both examined structures. Our analyses indicate that these loops may possess the ability to decrease the solvent accessibility of the substrate binding cleft, while at the same time creating new residue contacts for the inhibitors. Loop movements and conformational/ positional analyses of residues within the substrate binding cleft are discussed with respect to BoNT/A LC inhibitor binding and our common pharmacophore for inhibition. The results from these studies may aid in the future identification/development of more potent small molecule inhibitors that take advantage of new binding contacts in the BoNT/A LC

Novel Small Molecule Inhibitors of Botulinum Neurotoxin a Metalloprotease Activity

Botulinum neurotoxins (BoNTs) are among the most lethal biological substances to have been weaponized and are listed as biodefense category A agents. Currently, no small molecule (non-peptidic) therapeutics exist to counter this threat; hence, identifying and developing compounds that inhibit BoNTs is a high priority. In the present study, a high-throughput assay was used to identify small molecules that inhibit the metalloprotease activity of BoNT serotype A light chain (BoNT/A LC). All inhibitors were further verified using a HPLC-based assay. Conformational analyses of these compounds, in conjunction with molecular docking studies, were used to predict structural features that contribute to inhibitor binding and potency. Based on these results, a common pharmacophore for BoNT/A LC inhibitors is proposed. This is the first study to report small molecules (non-peptidics) that inhibit BoNT/A LC metalloprotease activity in the low lM range

A Refined Pharmacophore Identifies Potent 4-Amino-7-chloroquinoline-Based Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease

We previously identified structurally diverse small molecule (non-peptidic) inhibitors (SMNPIs) of the botulinum neurotoxin serotype A (BoNT/A) light chain (LC). Of these, several (including antimalarial drugs) contained a 4-amino-7-chloroquinoline (ACQ) substructure and a separate positive ionizable amine component. The same antimalarials have also been found to interfere with BoNT/A translocation into neurons, via pH elevation of the toxin-mediated endosome. Thus, this structural class of small molecules may serve as dual-function BoNT/A inhibitors. In this study, we used a refined pharmacophore for BoNT/A LC inhibition to identify four new, potent inhibitors of this structural class (IC50’s ranged from 3.2 to 17 íM). Molecular docking indicated that the binding modes for the new SMNPIs are consistent with those of other inhibitors that we have identified, further supporting our structure-based pharmacophore. Finally, structural motifs of the new SMNPIs, as well as two structure-based derivatives, were examined for activity, providing valuable information about pharmacophore component contributions to inhibition

A threonine turnstile defines a dynamic amphiphilic binding motif in the AAA ATPase p97 allosteric binding site

The turnstile motion of two neighboring threonines sets up a dynamic side chain interplay that can accommodate both polar and apolar ligands in a small molecule allosteric protein binding site. A computational model based on SAR data and both X-ray and cryo-EM structures of the AAA ATPase p97 was used to analyze the effects of paired threonines at the inhibitor site. Specifically, the Thr side chain hydroxyl groups form a hydrogen bonding network that readily accommodates small, highly polar ligand substituents. Conversely, diametric rotation of the χ1 torsion by 150-180° orients the side chain β-methyl groups into the binding cleft, creating a hydrophobic pocket that can accommodate small, apolar substituents. This motif was found to be critical for rationalizing the affinities of a structurally focused set of inhibitors of p97 covering a > 2000-fold variation in potencies, with a preference for either small-highly polar or small-apolar groups. The threonine turnstile motif was further validated by a PDB search that identified analogous binding modes in ligand interactions in PKB, as well as by an analysis of NMR structures demonstrating additional gear-like interactions between adjacent Thr pairs. Combined, these data suggest that the threonine turnstile motif may be a general feature of interest in protein binding pockets