Tobin J. Dickerson

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.

Characterization of differences between blood sample matrices in untargeted metabolomics.

Large-scale proteomic and metabolomic technologies are increasingly gaining attention for their use in the diagnosis of human disease. In order to ensure the statistical power of relevant markers, such analyses must incorporate a large number of representative samples. While in a best-case scenario these samples are collected through a study design that is specifically tailored for the desired analysis, often studies must rely upon the analysis of large numbers of previously banked samples that may or may not have complete and accurate documentation of their associated collection and storage methods. In this study, several human blood matrices were analyzed and compared for the quality of metabolomic output. The sample types that were tested include plasma prepared with a variety of anticoagulants and serum collected by venipuncture and capillary blood collection protocols. Analysis with liquid chromatography-mass spectrometry (LC-MS) revealed only subtle differences between the various plasma preparation methods. Differences between the serum and plasma samples appear to be largely peptide/protein-based and are consistent with the biological distinction of the two matrices. Interestingly, the small molecule lysophosphatidylinositol was found to be in higher abundance in plasma, as a possible consequence of the effect of the intrinsic clotting cascade on adjacent metabolic pathways. Comparison of the small-molecule profiles of the capillary- and venipuncture-collected samples revealed 23 statistically significant compound differences between these sample types. Most of these features can be attributed to surfactants and detergents used to pretreat the skin in order to maintain the sterility of sample collection. However, several have identical mass and molecular formulas as endogenous human metabolites and could be erroneously attributed to actual metabolic perturbations. Understanding the extent of these matrix effects is important for control of systematic bias and ensuring the quality of metabolomic analysis.

Mephedrone (4-methylmethcathinone) supports intravenous self-administration in Sprague-Dawley and Wistar rats.

Recreational use of the drug 4‐methylmethcathinone (mephedrone; 4‐MMC) became increasingly popular in the United Kingdom in recent years, spurred in part by the fact that it was not criminalized until April 2010. Although several fatalities have been associated with consumption of 4‐MMC and cautions for recreational users about its addictive potential have appeared on Internet forums, very little information about abuse liability for this drug is available. This study was conducted to determine if 4‐MMC serves as a reinforcer in a traditional intravenous self‐administration model. Groups of male Wistar and Sprague‐Dawley rats were prepared with intravenous catheters and trained to self‐administer 4‐MMC in 1‐hour sessions. Per‐infusion doses of 0.5 and 1.0 mg/kg were consistently self‐administered, resulting in greater than 80% discrimination for the drug‐paired lever and mean intakes of about 2–3 mg/kg/hour. Dose‐substitution studies after acquisition demonstrated that the number of responses and/or the total amount of drug self‐administered varied as a function of dose. In addition, radiotelemetry devices were used to show that self‐administered 4‐MMC was capable of increasing locomotor activity (Wistar) and decreasing body temperature (Sprague‐Dawley). Pharmacokinetic studies found that the T1/2 of 4‐MMC was about 1 hour in vivo in rat plasma and 90 minutes using in vitro liver microsomal assays. This study provides evidence of stimulant‐typical abuse liability for 4‐MMC in the traditional pre‐clinical self‐administration model.

Effect of Ambient Temperature on the Thermoregulatory and Locomotor Stimulant Effects of 4-Methylmethcathinone in Wistar and Sprague-Dawley Rats

The drug 4-methylmethcathinone (4-MMC; aka, mephedrone, MMCAT, “plant food”, “bath salts”) is a recent addition to the list of popular recreational psychomotor-stimulant compounds. Relatively little information about this drug is available in the scientific literature, but popular media reports have driven recent drug control actions in the UK and several US States. Online user reports of subjective similarity to 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”) prompted the current investigation of the thermoregulatory and locomotor effects of 4-MMC. Male Wistar and Sprague-Dawley rats were monitored after subcutaneous administration of 4-MMC (1–10 mg/kg ) using an implantable radiotelemetry system under conditions of low (23°C) and high (27°C) ambient temperature. A reliable reduction of body temperature was produced by 4-MMC in Wistar rats at 23°C or 27°C with only minimal effect in Sprague-Dawley rats. Increased locomotor activity was observed after 4-MMC administration in both strains with significantly more activity produced in the Sprague-Dawley strain. The 10 mg/kg s.c. dose evoked greater increase in extracellular serotonin, compared with dopamine, in the nucleus accumbens. Follow-up studies confirmed that the degree of locomotor stimulation produced by 10 mg/kg 4-MMC was nearly identical to that produced by 1 mg/kg d-methamphetamine in each strain. Furthermore, hypothermia produced by the serotonin 1A/7 receptor agonist 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) was similar in each strain. These results show that the cathinone analog 4-MMC exhibits thermoregulatory and locomotor properties that are distinct from those established for methamphetamine or MDMA in prior work, despite recent evidence of neuropharmacological similarity with MDMA.

Contrasting effects of d-methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxypyrovalerone, and 4-methylmethcathinone on wheel activity in rats.

BACKGROUND Reports from U.S., U.K. and European drug policy entities, and ongoing media accounts, show increasing recreational use of 4-methylmethcathinone (4-MMC, mephedrone) and 3,4-methylenedioxypyrovalerone (MDPV). Severe sympathomimetic symptoms, hallucinations, psychoses, and even deaths have been reported, yet little scientific information is available on the effects of these compounds in laboratory models. Available studies on the neurochemistry of these drugs show that 4-MMC and MDPV enhance DA neurotransmission, while 4-MMC additionally enhances 5-HT neurotransmission--a pattern much like that reported for methamphetamine versus 3,4-methylenedioxymethamphetamine (MDMA). As is the case for designer amphetamines, these neurochemical distinctions may predict differential potential for repetitive versus episodic abuse and distinct lasting toxicities. METHODS This study determined relative locomotor stimulant effects of 4-MMC (1-10 mg/kg, s.c.) and MDPV (0.5-5.6 mg/kg, s.c.), in comparison with d-methamphetamine (MA; 0.5-5.6 mg/kg, s.c.) and MDMA (1-7.5 mg/kg, s.c.) on a measure of locomotor activity--voluntary wheel running--in male Wistar rats (N=8). RESULTS Compared to counts of wheel rotations after saline, a biphasic change in the pattern of counts was observed after injections of MA and MDPV, with relatively higher counts following lower doses and lower counts following the highest dose. However, monophasic, dose-dependent reductions in counts were observed in response to injections of MDMA and 4-MMC. CONCLUSION Thus, voluntary wheel running yielded the same categorical distinctions for these drugs as did prior experiments testing the effects of these drugs on monoaminergic neurotransmission. These data indicate that MDPV produces prototypical locomotor stimulant effects whereas 4-MMC is more similar to the entactogen MDMA.

A critical evaluation of a nicotine vaccine within a self-administration behavioral model.

(S)-Nicotine is a psychostimulant legal drug responsible for causing addiction to tobacco smoking. Tobacco smoking has been irrevocably linked to a number of serious diseases and at present is considered the leading cause of preventable death in the United States. Despite well-documented adverse medical consequences, nicotine addiction has historically been one of the hardest to break. Current therapies have offered limited success and show high rates of relapse, emphasizing the need to engineer alternative therapies to aid nicotine cessation. The current study presents a protein-based immunopharmacotherapy approach for the treatment of nicotine addiction. Immunopharmacotherapy aims to use highly specific antibodies to blunt passage of drug into the brain thus minimizing reinforcing effects on the reward pathways of the central nervous system. Generation of a successful vaccine heavily relies on appropriate optimization of hapten design, immunogenic carrier and adjuvant. Modification of a classical nicotine hapten in conjugation with three distinct carrier proteins allowed for priming of a nicotine vaccine able to elicit significant amounts of nicotine-specific antibodies. Increased self-administration with use of a high drug dose (0.03 mg/kg/infusion; approximately 2 cigarettes in human) was observed in the vaccinated versus control animals suggesting a compensatory pattern and possibly reduced passage of nicotine to the brain. These results support the hypothesis that proper optimization of vaccine formulations could lead to successful nicotine vaccines for human use.

Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

Sensitive and rapid detection of botulinum neurotoxins (BoNTs), the most poisonous substances known to date, is essential for studies of medical applications of BoNTs and detection of poisoned food, as well as for response to potential bioterrorist threats. Currently, the most common method of BoNT detection is the mouse bioassay. While this assay is sensitive, it is slow, quite expensive, has limited throughput and requires sacrificing animals. Herein, we discuss and compare recently developed alternative in vitro detection methods and assess their ability to supplement or replace the mouse bioassay in the analysis of complex matrix samples.

Metabolomics-based discovery of diagnostic biomarkers for onchocerciasis.

Background Development of robust, sensitive, and reproducible diagnostic tests for understanding the epidemiology of neglected tropical diseases is an integral aspect of the success of worldwide control and elimination programs. In the treatment of onchocerciasis, clinical diagnostics that can function in an elimination scenario are non-existent and desperately needed. Due to its sensitivity and quantitative reproducibility, liquid chromatography-mass spectrometry (LC-MS) based metabolomics is a powerful approach to this problem. Methodology/Principal Findings Analysis of an African sample set comprised of 73 serum and plasma samples revealed a set of 14 biomarkers that showed excellent discrimination between Onchocerca volvulus–positive and negative individuals by multivariate statistical analysis. Application of this biomarker set to an additional sample set from onchocerciasis endemic areas where long-term ivermectin treatment has been successful revealed that the biomarker set may also distinguish individuals with worms of compromised viability from those with active infection. Machine learning extended the utility of the biomarker set from a complex multivariate analysis to a binary format applicable for adaptation to a field-based diagnostic, validating the use of complex data mining tools applied to infectious disease biomarker discovery and diagnostic development. Conclusions/Significance An LC-MS metabolomics-based diagnostic has the potential to monitor the progression of onchocerciasis in both endemic and non-endemic geographic areas, as well as provide an essential tool to multinational programs in the ongoing fight against this neglected tropical disease. Ultimately this technology can be expanded for the diagnosis of other filarial and/or neglected tropical diseases.