The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. By using in situ lead identification chemistry we have uncovered the first class of inhibitors that display nanomolar potency. From a 15 µM lead compound, structure activity relationship studies were performed granting the most potent BoNT/A inhibitor reported to date that displays an inhibition constant of 300 nM.Botulinum neurotoxin (BoNT), an agent responsible for the deadly food poisoning disease botulism and a dreaded biological weapon, is one of the most toxic proteins currently known (~100 billion times more toxic than cyanide). 1 Clostridium botulinum is classified into seven strains (A-G) each of which can cause flaccid muscle paralysis and subsequent death by blocking the release of a neurotransmitter, acetylcholine, at neuromuscular junctions. 2 Structurally, BoNT consists of three functional domains; catalytic, translocation, and binding; BoNT toxicity results from the catalytic activity of its light chain, a Zn(II) endopeptidase.The catalytic domain of BoNT is a compact globule consisting of a mixture of α-helices, β-sheets and strands with a gorge-like zinc containing metalloprotease active site (15-20Å deep depending on serotype). 3 The metalloprotease activity is responsible for BoNT's neurotoxicity through the hydrolytic cleavage of one of three SNARE (soluble NSF-attachment protein receptor) proteins that are involved in neuronal synaptic vesicle function. Moreover, the hydrolytic cleavage sites of these SNARE proteins (SNAP-25, VAMP, Sb-1) differ across the BoNT serotypes; however, any degradation of these SNARE proteins disables the exocytosis of acetylcholine, resulting in paralysis and potentially death. 2 Current therapy for BoNT intoxication involves "passive immunization" with equine antitoxin. 4 Unfortunately, treatment must start shortly after intoxication, and several safety concerns exist 2 over the use of antitoxins in the general population. 5 Therefore, inhibition of the catalytic light chain protease with a small molecule inhibitor may provide an attractive approach to counter the effects of botulism poisoning.BoNT serotype A (BoNT/A) is the most toxic form of BoNT's and is considered the most threatening for biological attacks due to a prolonged half-life in vivo and ease of its production.kdjanda@scripps.edu. While there are reports of success treating BoNT/A toxicity with multiple monoclonal antibodies 6 as antitoxins this is of limited therapeutic utility since the antibodies must be administered prior to, or shortly after, toxin exposure (<12 hrs). NIH Public AccessPresently, there are only modest small molecule, non-peptidic, protease inhibitors for BoNT/ A with IC 50 values in the range of > 20 µM. 7 We established a high-throughput screen for the identification of inhibitors of BoNT/A LC protease. 8 Using this screen we have analyzed a library of hydroxamate-based compounds generated using in situ chemistry to reveal the le...

BackgroundThe prevalence of obesity has increased dramatically worldwide. The obesity epidemic begs for novel concepts and therapeutic targets that cohesively address “food-abuse” disorders. We demonstrate a molecular link between impairment of a central kinase (Akt) involved in insulin signaling induced by exposure to a high-fat (HF) diet and dysregulation of higher order circuitry involved in feeding. Dopamine (DA) rich brain structures, such as striatum, provide motivation stimuli for feeding. In these central circuitries, DA dysfunction is posited to contribute to obesity pathogenesis. We identified a mechanistic link between metabolic dysregulation and the maladaptive behaviors that potentiate weight gain. Insulin, a hormone in the periphery, also acts centrally to regulate both homeostatic and reward-based HF feeding. It regulates DA homeostasis, in part, by controlling a key element in DA clearance, the DA transporter (DAT). Upon HF feeding, nigro-striatal neurons rapidly develop insulin signaling deficiencies, causing increased HF calorie intake.Methodology/Principal FindingsWe show that consumption of fat-rich food impairs striatal activation of the insulin-activated signaling kinase, Akt. HF-induced Akt impairment, in turn, reduces DAT cell surface expression and function, thereby decreasing DA homeostasis and amphetamine (AMPH)-induced DA efflux. In addition, HF-mediated dysregulation of Akt signaling impairs DA-related behaviors such as (AMPH)-induced locomotion and increased caloric intake. We restored nigro-striatal Akt phosphorylation using recombinant viral vector expression technology. We observed a rescue of DAT expression in HF fed rats, which was associated with a return of locomotor responses to AMPH and normalization of HF diet-induced hyperphagia.Conclusions/SignificanceAcquired disruption of brain insulin action may confer risk for and/or underlie “food-abuse” disorders and the recalcitrance of obesity. This molecular model, thus, explains how even short-term exposure to “the fast food lifestyle” creates a cycle of disordered eating that cements pathological changes in DA signaling leading to weight gain and obesity.

A novel animal model highlights the link between Akt dysfunction, reduced cortical dopamine function, norepinephrine transporters, and schizophrenia-like behaviors.

Botulinum neurotoxins (BoNTs), etiological agents of the deadly food poisoning disease botulism, are the most toxic proteins currently known. Although only a few hundred cases of botulism are reported in the United States annually, there is growing interest in BoNTs attributable to their potential use as biological warfare agents. Neurotoxicity results from cleavage of the soluble NSF-attachment protein receptor complex proteins of the presynaptic vesicles by the BoNT light chain subunit, a Zn endopeptidase. Few effective inhibitors of BoNT/A LC (light chain) activity are known, and the discovery process is hampered by the lack of an efficient high-throughput assay for screening compound libraries. To alleviate this bottleneck, we have synthesized the peptide SNAPtide and have developed a robust assay for the high-throughput evaluation of BoNT/A LC inhibitors. Key aspects for the development of this optimized assay include the addition of a series of detergents, cosolvents, and salts, including 0.01% w/v Tween 20 to increase BoNT/A LC catalysis, stability, and ease of small molecule screening. To evaluate the effectiveness of the assay, a series of hydroxamate-based small molecules were synthesized and examined with BoNT/A LC. The methodology described is superior to other assays reported to date for the high-throughput identification of BoNT/A inhibitors.