Current Topics in Medicinal Chemistry - Volume 14, Issue 18, 2014

Botulinum neurotoxins (BoNTs) are endopeptidases that target motor neurons and block acetylcholine neurotransmitter release. This action results in the muscle paralysis that defines the disease botulism. To date, there are no FDA-approved therapeutics to treat BoNT-mediated paralysis after intoxication of the motor neuron. Importantly, the rationale for pursuing treatments to counter these toxins is driven by their potential misuse. Current drug discovery efforts have mainly focused on small molecules, peptides, and peptidomimetics that can directly and competitively inhibit BoNT light chain proteolytic activity. Although this is a rational approach, direct inhibition of the Zn2+ metalloprotease activity has been elusive as demonstrated by the dearth of candidates undergoing clinical evaluation. Therefore, broadening the scope of viable targets beyond that of active site protease inhibitors represents an additional strategy that could move the field closer to the clinic. Here we review the rationale, and discuss the outcomes of earlier approaches and highlight potential new targets for BoNT inhibition. These include BoNT uptake and processing inhibitors, enzymatic inhibitors, and modulators of neuronal processes associated with toxin clearance, neurotransmitter potentiation, and other pathways geared towards neuronal recovery and repair.

Botulinum neurotoxins (BoNTs) are a class of bacterial neurotoxins that are the most potent toxic compounds reported to date. Exposure to relatively low concentrations of the toxin protein can result in major muscle paralysis, which may result in death in severe cases. In addition to their role in natural human disease, BoNTs are currently under close scrutiny because of their potential to be used as biowarfare agents. Clinical treatment options for botulism are currently limited, and finite stockpiles of antitoxin exist. In light of current bioterrorist threats, researchers have focused on identifying new molecules that can be applied to either sensitive toxin detection or improved clinical treatment. High-throughput screening (HTS) is a laboratory technique commonly employed to screen large libraries of diverse compounds based on specific compound binding capabilities or function. Here we review existing HTS platforms that have been applied to identify novel BoNT diagnostic or therapeutic agents. HTS platforms for screening antibodies, peptides, small molecules, and aptamers are described, as well as the screening results and current progress of the identified compounds.

Delivering therapeutic cargos to specific cell types in vivo poses many technical challenges. There is currently a plethora of drug leads and therapies against numerous diseases, ranging from small molecule compounds to nucleic acids to peptides to proteins with varying binding or enzymatic functions. Many of these candidate therapies have documented potential for mitigating or reversing disease symptoms, if only a means for gaining access to the intracellular target were available. Recent advances in our understanding of the biology of cellular uptake and transport processes and the mode of action of bacterial protein toxins have accelerated the development of toxin-based cargo-delivery vehicle platforms. This review provides an updated survey of the status of available platforms for targeted delivery of therapeutic cargos, outlining various strategies that have been used to deliver different types of cargo into cells. Particular emphasis is placed on the application of toxin-based approaches, examining critical issues that have hampered realization of post-intoxication antitoxins against botulism.

We describe here the state of the art of certain aspects concerning potential small molecule therapy directed toward botulism, by inhibition of the zinc-protease containing light chain (LC) of botulinum neurotoxin BoNT/A from the anaerobic bacillus Clostridium botulinum. Botulinum neurotoxins (BoNTs) are comprised of eight serologically-distinct proteins (A – H), several of which are further divided, such as BoNT/A which has five subtypes. The BoNTs are the most toxic substances known to mankind, causing a form of flaccid paralysis that can be rapid and is often lethal. BoNT/A is comprised of a ~100 kDa heavy chain (HC) attached via a single disulfide Cys-Cys bond to a ~50 kDa LC. The HC mediates transport to and uptake by presynaptic glutamatergic neurons, where the LC cleaves the protein SNAP-25 and thus prevents vesicular trafficking and release of acetylcholine. The Zn-endoprotease activity of the LC of BoNT/A is a target for the development of small molecule inhibitors of BoNT/A-mediated toxicity. A variety of BoNT/A LC inhibitors have been described to date and we focus here primarily on the Zn-binding 8-hydroxyquinoline structural type as well as some of the previously-described hydroxamic acids.

The anthrax toxin lethal factor (LF) and matrix metalloproteinase-3 (MMP-3, stromelysin-1) are popular zinc metalloenzyme drug targets, with LF primarily responsible for anthrax-related toxicity and host death, while MMP-3 is involved in cancer- and rheumatic disease-related tissue remodeling. A number of in silico screening techniques, most notably docking and scoring, have proven useful for identifying new potential drug scaffolds targeting LF and MMP-3, as well as for optimizing lead compounds and investigating mechanisms of action. However, virtual screening outcomes can vary significantly depending on the specific docking parameters chosen, and systematic statistical significance analyses are needed to prioritize key parameters for screening small molecules against these zinc systems. In the current work, we present a series of chi-square statistical analyses of virtual screening outcomes for cocrystallized LF and MMP-3 inhibitors docked into their respective targets, evaluated by predicted enzyme-inhibitor dissociation constant and root-meansquare deviation (RMSD) between predicted and experimental bound configurations, and we present a series of preferred parameters for use with these systems in the industry-standard Surflex-Dock screening program, for use by researchers utilizing in silico techniques to discover and optimize new scaffolds.

Activation of the innate immune system can enhance resistance to a variety of bacterial and viral infections. In situations where the etiological agent of disease is unknown, such as a bioterror attack, stimulation of innate immunity may be particularly useful as induced immune responses are often capable of providing protection against a broad range of pathogens. In particular, the threat of an intentional release of a highly virulent bacterial pathogen that is either intrinsically resistant to antibiotics, or has been weaponized via the introduction of antibiotic resistance, makes immunopotentiation an attractive complementary or alternative strategy to enhance resistance to bacterial biothreat agents. Francisella tularensis, Yersinia pestis, Bacillus anthracis, and Burkholderia mallei or pseudomallei can all be easily disseminated via the respiratory route and infections can result in high mortality rates. Therefore, there has been a marked increase in research on immunotherapeutics against these Tier 1 select agents over the last 10 years that will be covered in this review. In addition, immunopotentiation against non-Tier 1 select agents such as Brucella spp., and Coxiella burnetii has also been studied and will be reviewed here. In particular, we will focus on cellular targets, such as toll-like receptors (TLRs), carbohydrate receptors and cytokine receptors, which have been exploited by immunomodulatory regimens that confer broad-spectrum protection against virulent bacterial pathogens.