Inhibition of Metalloprotease Botulinum Serotype A from a Pseudo-peptide Binding Mode to a Small Molecule That Is Active in Primary Neurons

Abstract

An efficient research strategy integrating empirically guided, structure-based modeling and chemoinformatics was used to discover potent small molecule inhibitors of the botulinum neurotoxin serotype A light chain. First, a modeled binding mode for inhibitor 2-mercapto-3-phenylpropionyl-RATKML (K i ‫؍‬ 330 nM) was generated, and required the use of a molecular dynamic conformer of the enzyme displaying the reorientation of surface loops bordering the substrate binding cleft. These flexible loops are conformationally variable in x-ray crystal structures, and the model predicted that they were pivotal for providing complementary binding surfaces and solvent shielding for the pseudo-peptide. The docked conformation of 2-mercapto-3-phenylpropionyl-RATKML was then used to refine our pharmacophore for botulinum serotype A light chain inhibition. Data base search queries derived from the pharmacophore were employed to mine small molecule (non-peptidic) inhibitors from the National Cancer Institute's Open Repository. Four of the inhibitors possess K i values ranging from 3.0 to 10.0 M.Of these, NSC 240898 is a promising lead for therapeutic development, as it readily enters neurons, exhibits no neuronal toxicity, and elicits dose-dependent protection of synaptosomalassociated protein (of 25 kDa) in a primary culture of embryonic chicken neurons. Isothermal titration calorimetry showed that the interaction between NSC 240898 and the botulinum A light chain is largely entropy-driven, and occurs with a 1:1 stoichiometry and a dissociation constant of 4.6 M. Botulinum neurotoxins (BoNTs)3 are the most potent of the biological toxins (1), and are listed as category A (highest priority) bioterror agents by the Centers for Disease Control and Prevention. They may be delivered by aerosol route (1, 2), and consequently represent a serious threat to both military personnel and civilians (3, 4). Moreover, BoNTs are now established biotherapeutics for a range of physical ailments and cosmetic treatments (2, 5-8), making their misuse and/or adverse side effects (9) more likely. Neither the currently available BoNT antitoxin nor antibodies can counter these toxins once they are inside neurons; currently, critical care mechanical ventilation is the only life-saving treatment option. However, the effects of internalized BoNTs can last for months (10), and mechanical ventilation would be impractical if even a limited number of individuals were clandestinely/accidentally intoxicated. Thus, there is an urgent need to identify and develop small molecule (non-peptidic) inhibitors (SMNPIs) that can serve as both prophylactics and post-exposure therapeutics.BoNTs are composed of a heavy chain and a light chain (LC) that are connected by a disulfide bridge (11). The heavy chain binds to neurons and transports the LC into the cytosol (12). The LC is a zinc metalloprotease. Each of the seven BoNT serotypes (A-G) cleaves a component of the SNARE (soluble NSFethylmaleimide-sensitive factor attachment protein receptor) proteins (13), which mediate ...

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

“…Compounds 1-4 were docked in the BoNT/A LC substrate binding cleft to determine if they would also engage in intermolecular contacts that were comparable to those predicted for other structurally diverse SMNPIs. [31][32][33] Similar to our previously docked models of ACQ inhibitors, 31,32 the 7-chloro substituents of 1-3 ( Figure 3A-C) and one of the ACQs of 4 ( Figure 4A and B) engage in favorable hydrophobic contacts with residues Phe 162, Phe 163, and Thr 219 of hydrophobic binding subsite 1 [31][32][33] (also described as the S1′ binding site 34 ). At the same time, the quinoline nitrogens of the inhibitors are positioned so that this heteroatom may interfere with the enzyme's catalytic engine.…”

“…31 In a subsequent molecular dynamics study, we demonstrated that conformationally flexible loops surrounding the BoNT/A LC substrate binding cleft may reorient to decrease the solvent accessibility of the cleft (as compared to respective energy refined structures), while simultaneously providing more hydropathically feasible binding contacts for SMNPIs. 32 The molecular dynamics studies were pivotal for identifying a binding mode 33 for the potent pseudo-peptide inhibitor 2-mercapto-3-phenylpropionyl-RATKML (mpp-RAT-KML, K i ) 330 nM 34 ). In the same study, we also proposed new pharmacophore components and constraints based on the docked model of mpp-RATKML, and, using this information, we identified more potent SMNPIs.…”

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

“…Indeed, one compound (NA-A1B2C10) showed the least protection of SNAP-25 cleavage in cellular assays at 25 M, whereas the other (2,4-dichlorocinnamic hydroxamic acid) was found to be cytotoxic in neuroblastoma cells at concentrations three orders of magnitude less than those tested in animals (i.e., 5 M). Numerous cellbased assays have been developed for assessing BoNT toxicity, including cultured murine neuroblastoma cells (Neuro-2a) (38), primary rodent fetal spinal cord cells (35,39,40), cultured chicken spinal motor neurons (16), and rat adrenal pheochromocytoma cells (PC12) (41). One of the major advantages of a secondary screen such as cell-based assays is a considerable reduction in the number of animals used, time expended, and cost incurred, especially when used for assessing large numbers of target molecules.…”

“…A complementary approach used by our laboratory and others has focused on metal chelators, which presumably bind the active site zinc cation, thereby rendering the BoNT LC inactive (12)(13)(14). Analogous to these approaches, others have used an in silico screen of a large compound library in an effort to identify BoNT/A-selective inhibitors and establish a pharmacophore for BoNT/A LC inhibition; again, the most effective compounds identified by using this approach operated by zinc chelation (15,16). However, it is important to note that this pharmacophore has been established from in vitro methods, with scant in vivo data for any reported compound.…”

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

“…BoNT antagonist development has been largely limited to studies of small sets of compounds or peptides chosen through rational design, computer-assisted, or other methods [54][55][56][57][58][59]. Although these studies have provided a considerable amount of information about BoNT catalytic mechanism and substrate requirements, few of the small molecule inhibitors that have emerged from these studies have sufficient potency to be effective BoNT therapeutics.…”