Mark S. Hixon

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.

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.

Botulinum Neurotoxin A Protease: Discovery of Natural Product Exosite Inhibitors

A new mechanistic class of BoNT/A zinc metalloprotease inhibitors, from Echinacea, exemplified by the natural product d-chicoric acid (I1) is disclosed. A detailed evaluation of chicoric acids mechanism of inhibition reveals that the inhibitor binds to an exosite, displays noncompetitive partial inhibition, and is synergistic with a competitive active site inhibitor when used in combination. Other components found in Echinacea, I3 and I4, were also inhibitors of the protease.

Catalytic antibody degradation of ghrelin increases whole-body metabolic rate and reduces refeeding in fasting mice

Obesity is a chronic, costly, and globally prevalent condition, with excess caloric intake a suspected etiologic factor. Nonsurgical treatments are modestly efficacious, and weight loss maintenance is hampered by anti-famine homeostatic mechanisms. Ghrelin, a gastric hormone linked to meal initiation, energy expenditure, and fuel partitioning, is hypothesized to facilitate weight gain and impede weight loss. Unique among known animal peptides, the serine-3 residue of ghrelin is posttranslationally acylated with an n-octanoic acid, a modification important for the peptides active blood-brain transport and growth hormone secretagogue receptor-1 agonist activity. Pharmacological degradation of ghrelin would be hypothesized to reduce ghrelins biological effects. To study endogenous ghrelins role in appetite and energy expenditure, we generated antibodies that hydrolyze the octanoyl moiety of ghrelin to form des-acyl ghrelin. The most proficient antibody catalyst, GHR-11E11, was found to display a second-order rate constant of 18 M−1·s−1 for the hydrolysis of ghrelin to des-acyl ghrelin. I.v. administration of GHR-11E11 (50 mg/kg) maintained a greater metabolic rate in fasting C57BL/6J mice as compared with mice receiving a control antibody and suppressed 6-h refeeding after 24 h of food deprivation. Indirect respiratory measures of metabolism after refeeding and relative fuel substrate utilization were unaffected. The results support the hypothesis that acylated ghrelin stimulates appetite and curbs energy expenditure during deficient energy intake, whereas des-acyl ghrelin does not potently share these functions. Catalytic anti-ghrelin antibodies might thereby adjunctively aid consolidation of caloric restriction-induced weight loss and might also be therapeutically relevant to Prader–Willi syndrome, characterized after infancy by hyperghrelinemia, hyperphagia, and obesity.

Biochemical Characterization of TAK-593, a Novel VEGFR/PDGFR Inhibitor with a Two-Step Slow Binding Mechanism

Inhibition of tumor angiogenesis leads to a lack of oxygen and nutrients in the tumor and therefore has become a standards of care for many solid tumor therapies. Dual inhibition of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) protein kinase activities is a popular strategy for targeting tumor angiogenesis. We discovered that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, potently inhibits tyrosine kinases from the VEGFR and PDGFR families. TAK-593 was highly selective for these families, with an IC(50) >1 μM when tested against more than 200 protein and lipid kinases. TAK-593 displayed competitive inhibition versus ATP. In addition, TAK-593 inhibited VEGFR2 and PDGFRβ in a time-dependent manner, classifying it as a type II kinase inhibitor. Analysis of enzyme-inhibitor preincubation experiments revealed that the binding of TAK-593 to VEGFR2 and PDGFRβ occurs via a two-step slow binding mechanism. Dissociation of TAK-593 from VEGFR2 was extremely slow (t(1/2) >17 h), and the affinity of TAK-593 at equilibrium (K(i)*) was less than 25 pM. Ligand displacement analysis with a fluorescent tracer confirmed the slow dissociation of TAK-593. The dissociation rate constants were in good agreement between the activity and ligand displacement data, and both analyses supported slow dissociation of TAK-593. The long residence time of TAK-593 may achieve an extended pharmacodynamic effect on VEGFR2 and PDGFRβ kinases in vivo that differs substantially from its observed pharmacokinetic profile.

Toward the discovery of potent inhibitors of botulinum neurotoxin A: development of a robust LC MS based assay operational from low to subnanomolar enzyme concentrations.

The development of a sensitive, yet reliable assay for the analysis of botulinum neurotoxin A (BoNT/A) inhibitors is described; using this assay a new protease inhibitor was characterized and found to be one of the most potent inhibitors reported to date.

Chirality Holds the Key for Potent Inhibition of the Botulinum Neurotoxin Serotype A Protease

Botulinum neurotoxin serotype A (BoNT/A) is the most toxic protein known to man and also a bioterrorism agent. As defined by our previous research targeting the etiological agent responsible for BoNT/A intoxication, a protease, we now report on the asymmetric synthesis of four new BoNT/A inhibitors; the most potent of this series is roughly 2-fold more active than the best small molecule inhibitor currently known.

Onchocerca volvulus-neurotransmitter tyramine is a biomarker for river blindness

Onchocerciasis, also known as “river blindness”, is a neglected tropical disease infecting millions of people mainly in Africa and the Middle East but also in South America and Central America. Disease infectivity initiates from the filarial parasitic nematode Onchocerca volvulus, which is transmitted by the blackfly vector Simulium sp. carrying infectious third-stage larvae. Ivermectin has controlled transmission of microfilariae, with an African Program elimination target date of 2025. However, there is currently no point-of-care diagnostic that can distinguish the burden of infection—including active and/or past infection—and enable the elimination program to be effectively monitored. Here, we describe how liquid chromatography-MS–based urine metabolome analysis can be exploited for the identification of a unique biomarker, N-acetyltyramine-O,β-glucuronide (NATOG), a neurotransmitter-derived secretion metabolite from O. volvulus. The regulation of this tyramine neurotransmitter was found to be linked to patient disease infection, including the controversial antibiotic doxycycline treatment that has been shown to both sterilize and kill adult female worms. Further clues to its regulation have been elucidated through biosynthetic pathway determination within the nematode and its human host. Our results demonstrate that NATOG tracks O. volvulus metabolism in both worms and humans, and thus can be considered a host-specific biomarker for onchocerciasis progression. Liquid chromatography-MS–based urine metabolome analysis discovery of NATOG not only has broad implications for a noninvasive host-specific onchocerciasis diagnostic but provides a basis for the metabolome mining of other neglected tropical diseases for the discovery of distinct biomarkers and monitoring of disease progression.

Probing Active Cocaine Vaccination Performance through Catalytic and Noncatalytic Hapten Design

Presently, there are no FDA-approved medications to treat cocaine addiction. Active vaccination has emerged as one approach to intervene through the rapid sequestering of the circulating drug, thus terminating both psychoactive effects and drug toxicity. Herein, we report our efforts examining two complementary, but mechanistically distinct active vaccines, i.e., noncatalytic and catalytic, for cocaine treatment. A cocaine-like hapten GNE and a cocaine transition-state analogue GNT were used to generate the active vaccines, respectively. GNE-KLH (keyhole limpet hemocyannin) was found to elicit persistent high-titer, cocaine-specific antibodies and blunt cocaine-induced locomotor behaviors. Catalytic antibodies induced by GNT-KLH were also shown to produce potent titers and suppress locomotor response in mice; however, upon repeated cocaine challenges, the vaccines protecting effects waned. In depth kinetic analysis suggested that loss of catalytic activity was due to antibody modification by cocaine. The work provides new insights for the development of active vaccines for the treatment of cocaine abuse.

Biochemical characterization of a novel type-II VEGFR2 kinase inhibitor: comparison of binding to non-phosphorylated and phosphorylated VEGFR2.

A pyrrolo[3,2-d]pyrimidine-based type-II vascular endothelial growth factor receptor 2 (VEGFR2) kinase inhibitor, compound 20d, displayed time-dependent inhibition of the non-phosphorylated catalytic domain of VEGFR2. In contrast, 20d did not show time-dependent inhibition of the phosphorylated enzyme. Dissociation of 20d from non-phosphorylated VEGFR2 was slow and the half-life of the complex was longer than 4h. In contrast, dissociation of 20d from the phosphorylated enzyme was very fast (half-life <5min). A fluorescent tracer based displacement assay and surface plasmon resonance (SPR) analysis confirmed the slow dissociation of 20d from only non-phosphorylated VEGFR2. Thus, activity based and binding kinetic analyses both supported slow dissociation of 20d from only non-phosphorylated VEGFR2. Additionally SPR analysis revealed that association rates were rapid and nearly identical for these two phosphorylation forms of VEGFR2. From these results, the preferential effect of 20d on non-phosphorylated VEGFR2 is dominated by its slow dissociation from the enzyme and this characteristically long residence time may increase its potency in vivo. The present findings may assist in the design of novel type-II kinase inhibitors.