The botulinum neurotoxins (BoNTs) represent a group of the most poisonous biological substances known (1- 3). Seven different serotypes of BoNTs (BoNT/A through BoNT/G) are secreted by the anaerobic spore- forming bacteria Clostridium botulinum, Clostridium butyricum and Clostridium baratii (4-7). Together with the tetanus neurotoxin (TeNT), which is produced by Clostridium tetani, the BoNTs comprise the clostridial neurotoxin (CNT) family (8). The lethal intravenous dose of BoNT serotype A (BoNT/A) in humans is 1-5ng/kg (9,10). If accidental exposure to BoNT occurs (e.g., from contaminated foodstuffs), loss of life or life-threatening paralysis can occur (11). Most importantly, the BoNTs have already been """"""""weaponized"""""""" in a highly toxic aerosol form, and the BoNTs consequently pose a significant threat to both civilian and military populations (9, 12). Once inhaled into the lung, BoNTs are taken up by the blood stream, target the peripheral cholinergic nerve endings, and cause death by interrupting autonomic nerve function. The zinc-dependent endopeptidase light chain (LC) portion of BoNTs impair neuronal exocytosis through proteolysis of essential SNARE (soluble NSF-ethylmaleimide-sensitive factor attachment protein receptor) components of neurotransmission. The overall goal or this project is to develop small molecule inhibitors from multiple scaffolds of the BoNT/A light chain (LC) metalloprotease activity to treat botulinum poisoning. Small molecule quinoline-based inhibitors of BoNT /A LC have been identified by screening the NCI chemical diversity set (13, 14). These validated hit compounds are suitable starting points for BoNT/A inhibitor drug discovery by using structure-based drug design (SBDD) to improve and optimize their potencies and """"""""drug-like"""""""" properties. In Phase I we will use proven techniques of medicinal and parallel synthetic chemistry, to produce """"""""drug-like"""""""" molecules. We will use molecular modeling approaches and X-ray crystallography to explore the structural features of enzyme bound inhibitor complexes, and use parallel synthesis to prepare focused libraries of compounds related to idealized inhibitors. In an iterative process we will probe these focused compound libraries for structural features that contribute to tighter binding and more potent inhibition of the metalloprotase by measuring the enzymatic and cellular activities, and specificity of the enzyme inhibitors. We will use the growing data set to develop refined pharmacophore models that will guide the development of the structure activity relationships (SARs). Also, to accelerate the drug development process, and dramatically reduce the number of animal experiments needed in Phase II of this project, we will routinely assess all of our target compounds for optimal in vitro ADME-T (Absorption, Distribution, Metabolism, and Elimination and cyto- Toxicity) properties. We will produce an optimized lead compound and a back-up compound from a different scaffold. In Phase II, we will further optimize these leads for in vivo efficacy, pharmacokinetic properties, toxicity (in two species) and safety pharmacology to develop them as pre-IND clinical candidates (Phase III). """"""""The botulinum neurotoxins are some of the most poisonous biological substances known. Loss of life or life-threatening paralysis can occur following exposure to these neurotoxins from contaminated foodstuffs or acts of bioterrorism. This proposal describes the preparation and development of novel drugs to treat botulinum poisoning."""""""" ? ? ?
