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 ...

Botulinum toxin is an exceedingly potent inhibitor of neurotransmission across the neuromuscular junction, causing flaccid paralysis and death. The potential for misuse of this deadly poison as a bioweapon has added a greater urgency to the search for effective therapeutics. The development of sensitive and efficient cell-based assays for the evaluation of toxin antagonists is crucial to the rapid and successful identification of therapeutic compounds. The authors evaluated the sensitivity of primary cultures from 4 distinct regions of the embryonic chick nervous system to botulinum neurotoxin A (BoNT/A) cleavage of synaptosomal-associated protein of 25 kD (SNAP-25). Although differences in sensitivity were apparent, SNAP-25 cleavage was detectable in neuronal cells from each of the 4 regions within 3 h at BoNT/A concentrations of 1 nM or lower. Co-incubation of chick neurons with BoNT/A and toxin-neutralizing antibodies inhibited SNAP-25 cleavage, demonstrating the utility of these cultures for the assay of BoNT/A antagonists. (Journal of Biomolecular Screening 2007:370-377)

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Coordinated small‐molecule drug discovery research efforts for the treatment of botulism by the public sector, especially the U.S. Department of Defense (DoD) and Department of Health and Human Services (DHHS), began in the 1990s and represent a significant resource investment. Organization of an effective botulism therapeutic drug program, however, presents formidable technical and logistical challenges. Seven distinct BoNT serotypes are known, each representing a different target. Moreover, BoNT exerts its action inside peripheral cholinergic neurons, and some serotypes may persist functionally within nerve cells for weeks or months. Clinical botulism occurs infrequently, and the effectiveness of prolonged mechanical ventilation to treat poisoning further limits experimental drug testing. The efficacy of experimental compounds must be extrapolated from disparate cell‐ or tissue‐based or rodent models. Numerous compounds with moderate efficacy in experimental laboratory assays have been reported, but may not possess the necessary safety, efficacy, and pharmacokinetic profile to support therapeutic development. To mitigate these challenges, we propose product development tools to assist in management of the BoNT portfolio and to clearly define the desired therapeutic product. Establishing a target product profile (TPP) is proposed to guide public sector managers toward critical aspects of the desired therapeutic product. Additional product development tools to assist in shaping research portfolios and to inform decisions regarding lead candidates to pursue are also discussed. Product development tools that facilitate the characterization of the ideal therapeutic product, and assist in the maintenance of a robust portfolio, will ameliorate the inherent financial risk in drug development for treating BoNT intoxication. Drug Dev Res 70:303–326, 2009. Published 2009 Wiley‐Liss, Inc.

We tested the hypothesis that a decrease in the blood-to-tissue movement of albumin contributes to the recovery of plasma albumin and plasma volume after acute plasma protein depletion (plasmapheresis). Awake and unrestrained male Sprague-Dawley rats (220-320 g) fitted with jugular catheters were plasmapheresed, and plasma volume, plasma albumin, and total plasma protein content were measured at 1, 5, 24, and 48 h postplasmapheresis. Plasma volume recovered to baseline within 1 h (4.6 +/- 0.42 vs. 4.7 +/- 0.46 mL/100 g body weight (bw), remained at baseline from 5 h to 24 h but increased to 5.5 + 0.57 mL/100 g bw at 48 h (P < 0.05). Plasma albumin and total protein content recovered rapidly but remained below baseline levels at 1 h (10.05 +/- 0.98 vs. 12.33 +/- 1.29 and 19.75 +/- 1.75 vs. 24.73 +/- 2.56 mg/100 g bw, respectively). Plasma protein content retumed to baseline by 5 h of recovery. Tissue uptake of I125-labeled albumin decreased in the heart, skin, skeletal muscle, and small Intestines of plasmapheresed rats (P < 0.05). These data support the hypothesis that a reduction in albumin efflux from the vascular space contrlbutes to the recovery of plasma albumin and total protein content during plasma volume recovery and eventual expansion after plasmapheresis.