Lisa M. Eubanks

Technological advancements for the detection of and protection against biological and chemical warfare agents

There is a growing need for technological advancements to combat agents of chemical and biological warfare, particularly in the context of the deliberate use of a chemical and/or biological warfare agent by a terrorist organization. In this tutorial review, we describe methods that have been developed both for the specific detection of biological and chemical warfare agents in a field setting, as well as potential therapeutic approaches for treating exposure to these toxic species. In particular, nerve agents are described as a typical chemical warfare agent, and the two potent biothreat agents, anthrax and botulinum neurotoxin, are used as illustrative examples of potent weapons for which countermeasures are urgently needed.

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

Among the agents classified as “Category A” by the U.S. Centers for Disease Control and Prevention, botulinum neurotoxin (BoNT) is the most toxic protein known, with microgram quantities of the protein causing severe morbidity and mortality by oral or i.v. routes. Given that this toxin easily could be used in a potential bioterrorist attack, countermeasures urgently are needed to counteract the pathophysiology of BoNT. At a molecular level, BoNT exerts its paralytic effects through intracellular cleavage of vesicle docking proteins and subsequent organism-wide autonomic dysfunction. In an effort to identify small molecules that would disrupt the interaction between the light-chain metalloprotease of BoNT serotype A and its cognate substrate, a multifaceted screening effort was undertaken. Through the combination of in vitro screening against an optimized variant of the light chain involving kinetic analysis, cellular protection assays, and in vivo mouse toxicity assays, molecules that prevent BoNT/A-induced intracellular substrate cleavage and extend the time to death of animals challenged with lethal toxin doses were identified. Significantly, the two most efficacious compounds in vivo showed less effective activity in cellular assays intended to mimic BoNT exposure; indeed, one of these compounds was cytotoxic at concentrations three orders of magnitude below its effective dose in animals. These two lead compounds have surprisingly simple molecular structures and are readily amenable to optimization efforts for improvements in their biological activity. The findings validate the use of high-throughput screening protocols to define previously unrecognized chemical scaffolds for the development of therapeutic agents to treat BoNT exposure.

Bimodal modulation of the botulinum neurotoxin protein-conducting channel

Clostridium botulinum neurotoxin (BoNT) is the causative agent of botulism, a neuroparalytic disease. We describe here a semisynthetic strategy to identify inhibitors based on toosendanin, a traditional Chinese medicine reported to protect from BoNT intoxication. Using a single molecule assay of BoNT serotypes A and E light chain (LC) translocation through the heavy chain (HC) channel in neurons, we discovered that toosendanin and its tetrahydrofuran analog selectively arrest the LC translocation step of intoxication with subnanomolar potency, and increase the unoccluded HC channel propensity to open with micromolar efficacy. The inhibitory profile on LC translocation is accurately recapitulated in 2 different BoNT intoxication assays, namely the mouse protection and the primary rat spinal cord cell assays. Toosendanin has an unprecedented dual mode of action on the protein-conducting channel acting as a cargo-dependent inhibitor of translocation and as cargo-free channel activator. These results imply that the bimodal modulation by toosendanin depends on the dynamic interactions between channel and cargo, highlighting their tight interplay during the progression of LC transit across endosomes.

Repositioning of an existing drug for the neglected tropical disease Onchocerciasis

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm’s metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel’s effects, a retro-fragment-based study was used to define structural elements critical for closantel’s chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness.

Flagellin as carrier and adjuvant in cocaine vaccine development.

Cocaine abuse is problematic, directly and indirectly impacting the lives of millions, and yet existing therapies are inadequate and usually ineffective. A cocaine vaccine would be a promising alternative therapeutic option, but efficacy is hampered by variable production of anticocaine antibodies. Thus, new tactics and strategies for boosting cocaine vaccine immunogenicity must be explored. Flagellin is a bacterial protein that stimulates the innate immune response via binding to extracellular Toll-like receptor 5 (TLR5) and also via interaction with intracellular NOD-like receptor C4 (NLRC4), leading to production of pro-inflammatory cytokines. Reasoning that flagellin could serve as both carrier and adjuvant, we modified recombinant flagellin protein to display a cocaine hapten termed GNE. The resulting conjugates exhibited dose-dependent stimulation of anti-GNE antibody production. Moreover, when adjuvanted with alum, but not with liposomal MPLA, GNE-FliC was found to be better than our benchmark GNE-KLH. This work represents a new avenue for exploration in the use of hapten-flagellin conjugates to elicit antihapten immune responses.

Vitamin B2-mediated cellular photoinhibition of botulinum neurotoxin A

Botulinum neurotoxin (BoNT) is the most toxic species known to humans and has been identified as a potential bioterrorist threat. Unfortunately, the only existing countermeasures for BoNT intoxication involve vaccinations that are only effective prior to entry of the toxin into neuronal cells. Herein, we disclose the ability of the micronutrient riboflavin (vitamin B2) to photooxidatively inactivate BoNT in cell‐based assays without the need for toxin and riboflavin pre‐exposure. In total, this study suggests that botulism neurotoxicity may be blunted with photodynamic therapy technology.

A cross-over inhibitor of the botulinum neurotoxin light chain B: a natural product implicating an exosite mechanism of action

Clostridium botulinum produces the most lethal toxins known to man, as such they are high risk terrorist threats, and alarmingly there is no approved therapeutic. We report the first cross-over small molecule inhibitor of these neurotoxins and propose a mechanism by which it may impart its inhibitory activity.

Investigations of Enantiopure Nicotine Haptens Using an Adjuvanting Carrier in Anti-Nicotine Vaccine Development

Despite efforts to produce suitable smoking cessation aids, addiction to nicotine continues to carry a substantive risk of recidivism. An attractive alternative to current therapies is the pharmacokinetic strategy of antinicotine vaccination. A major hurdle in the development of the strategy has been to elicit a sufficiently high antibody concentration to curb nicotine distribution to the brain. Herein, we detail investigations into a new hapten design, which was able to elicit an antibody response of significantly higher specificity for nicotine. We also explore the use of a mutant flagellin carrier protein with adjuvanting properties. These studies underlie the feasibility of improvement in antinicotine vaccine formulations to move toward clinical efficacy.

Biologically templated organic polymers with nanoscale order.

Methods for the construction of ordered nanoscale arrays have been implicated in fields ranging from separation technologies to microelectronics. Yet, despite the plethora of nanoscale structures assembled in nature that use a templating strategy, chemists have been unable to replicate this success. A technology is reported for templated organic polymers composed of filamentous bacteriophage-polyacrylamide biomacromolecules that self-assemble into highly ordered helical bundles displaying hexagonal close packing. The results align with a previously reported mathematical prediction for the close packing of flexible tubes. This biopolymeric assembly can be viewed as a magnification of the inherent microscopic chirality and helicity present in individual phage particles at the macroscale level.

Identification of α2 Macroglobulin as a Major Serum Ghrelin Esterase

Obesity / excessive body weight leads to morbidity known collectively as metabolic syndrome and afflicts perhaps 1 billion people worldwide.[1] Reduction in caloric input continues to be the most effective means of treatment with approaches ranging from simple dieting to the extreme of bariatric surgery.[2] Regulating the desire to eat through pharmaceutical intervention is a lofty goal with a troubled history mostly because of an inability to separate regulation of satiety from the body’s fundamental reward system. The end product, depending on mechanism, is that potential therapeutic agents become drugs of abuse or lead to depression.[2b]