Efficacy of therapeutically administered gepotidacin in a rabbit model of inhalational anthrax.

Bacillus anthracis is a Gram-positive Centers for Disease Control and Prevention category "A" biothreat pathogen. Without early treatment, inhalation of anthrax spores with progression to inhalational anthrax disease is associated with high fatality rates. Gepotidacin is a novel first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is being evaluated for use against biothreat and conventional pathogens. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode and has in vitro activity against a collection of B. anthracis isolates including antibacterial-resistant strains, with the MIC90 ranging from 0.5 to 1 µg/mL. In vivo activity of gepotidacin was also evaluated in the New Zealand White rabbit model of inhalational anthrax. The primary endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. The trigger for treatment was the presence of anthrax protective antigen in serum. New Zealand White rabbits were dosed intravenously for 5 days with saline or gepotidacin at 114 mg/kg/d to simulate a dosing regimen of 1,000 mg intravenous (i.v.) three times a day (TID) in humans. Gepotidacin provided a survival benefit compared to saline control, with 91% survival (P-value: 0.0001). All control animals succumbed to anthrax and were found to be blood- and organ culture-positive for B. anthracis. The novel mode of action, in vitro microbiology, preclinical safety, and animal model efficacy data, which were generated in line with Food and Drug Administration Animal Rule, support gepotidacin as a potential treatment for anthrax in an emergency biothreat situation.

Efficacy of Intravenously Administered Gepotidacin in Cynomolgus Macaques following a Francisella tularensis Inhalational Challenge.

Francisella tularensis (F. tularensis) is a Centers for Disease Control (CDC) category "A" Gram-negative biothreat pathogen. Inhalation of F. tularensis can cause pneumonia and respiratory failure and is associated with high mortality rates without early treatment. Gepotidacin is a novel, first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode, has activity against multidrug-resistant target pathogens, and has demonstrated in vitro activity against diverse collections of F. tularensis isolates (MIC90 of 0.5 to 1 µg/mL). Gepotidacin was evaluated in the cynomolgus macaque model of inhalational tularemia, using the SCHU S4 strain, with treatment initiated after exposure and sustained fever. Macaques were dosed via intravenous (i.v.) infusion with saline or gepotidacin at 72 mg/kg/day to support a human i.v. infusion dosing regimen of 1,000 mg three times daily. The primary study endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. Gepotidacin treatment resulted in 100% survival compared to 12.5% in the saline-treated control group (P < 0.0001) at Day 43 postinhalational challenge. All gepotidacin-treated animals were blood and organ culture negative for F. tularensis at the end of the study. In contrast, none of the saline control animals were blood and organ culture negative. Gepotoidacin's novel mechanism of action and the efficacy data reported here (aligned with the Food and Drug Administration Animal Rule) support gepotidacin as a potential treatment for pneumonic tularemia in an emergency biothreat situation.

In Vitro and In Vivo Activity of Omadacycline against Two Biothreat Pathogens, Bacillus anthracis and Yersinia pestis.

The in vitro activity and in vivo efficacy of omadacycline (OMC) were evaluated against the causative pathogens of anthrax and plague, Bacillus anthracis and Yersinia pestis, respectively. MICs of OMC were determined by broth microdilution according to CLSI guidelines for 30 isolates each of Y. pestis and B. anthracis The in vivo efficacy of omadacycline was studied at a range of dosages in both a postexposure prophylaxis (PEP) murine model of anthrax and plague as well as in a delayed treatment model of inhalational anthrax. Omadacycline was active in vitro against Y. pestis (MIC90 of 1 µg/ml) and B. anthracis (MIC90 of 0.06 µg/ml). Omadacycline was less active in vitro than ciprofloxacin (CIP) against Y. pestis (CIP MIC90 of 0.03 µg/ml) but was more potent in vitro against B. anthracis (CIP MIC90 of 0.12 µg/ml). In the mouse model of infection, the survival curves for all treatment cohorts differed significantly from the vehicle control (P = 0.004). The median survival for the vehicle-treated controls was 6 days postchallenge, while all antibiotic-treated mice survived the entire study. Omadacycline treatment with 5, 10, or 20 mg/kg of body weight twice daily for 14 days had significant efficacy over the vehicle control in the treatment of aerosolized B. anthracis Additionally, for postexposure prophylaxis treatment of mice infected with Y. pestis, the survival curves for omadacycline (40 mg/kg twice daily), ciprofloxacin, and doxycycline cohorts differed significantly from the vehicle control (P < 0.0001). Omadacycline is potent and demonstrates efficacy against both B. anthracis and Y. pestis The well-characterized oral and intravenous pharmacokinetics, safety, and tolerability warrant further assessment of the potential utility of omadacycline in combating these serious biothreat organisms.

In vitro antibiotic susceptibilities of Yersinia pestis determined by broth microdilution following CLSI methods.

In vitro susceptibilities to 45 antibiotics were determined for 30 genetically and geographically diverse strains of Yersinia pestis by the broth microdilution method at two temperatures, 28°C and 35°C, following Clinical and Laboratory Standards Institute (CLSI) methods. The Y. pestis strains demonstrated susceptibility to aminoglycosides, quinolones, tetracyclines, ß-lactams, cephalosporins, and carbapenems. Only a 1-well shift was observed for the majority of antibiotics between the two temperatures. Establishing and comparing antibiotic susceptibilities of a diverse but specific set of Y. pestis strains by standardized methods and establishing population ranges and MIC50 and MIC90 values provide reference information for assessing new antibiotic agents and also provide a baseline for use in monitoring any future emergence of resistance.

Origins of Yersinia pestis sensitivity to the arylomycin antibiotics and the inhibition of type I signal peptidase.

Yersinia pestis is the etiologic agent of the plague. Reports of Y. pestis strains that are resistant to each of the currently approved first-line and prophylactic treatments point to the urgent need to develop novel antibiotics with activity against the pathogen. We previously reported that Y. pestis strain KIM6+, unlike most Enterobacteriaceae, is susceptible to the arylomycins, a novel class of natural-product lipopeptide antibiotics that inhibit signal peptidase I (SPase). In this study, we show that the arylomycin activity is conserved against a broad range of Y. pestis strains and confirm that it results from the inhibition of SPase. We next investigated the origins of this unique arylomycin sensitivity and found that it does not result from an increased affinity of the Y. pestis SPase for the antibiotic and that alterations to each component of the Y. pestis lipopolysaccharide-O antigen, core, and lipid A-make at most only a small contribution. Instead, the origins of the sensitivity can be traced to an increased dependence on SPase activity that results from high levels of protein secretion under physiological conditions. These results highlight the potential of targeting protein secretion in cases where there is a heavy reliance on this process and also have implications for the development of the arylomycins as an antibiotic with activity against Y. pestis and potentially other Gram-negative pathogens.

Artificial Blood Development Implications for Military Medicine.

Massive hemorrhaging remains the most common cause of preventable battlefield deaths. Blood used for trauma care requires a robust donation network, capacity for long-term storage, and extensive and accurate testing. Bioengineering technologies could offer a remedy to these constraints in the form of blood substitutes-fluids that could be transfused into patients to provide oxygen, carry away waste, and aid in coagulation-that would be used in prolonged casualty care and in far-forward settings, overcoming the obstacles of distance and time. The different molecular properties of red blood cells (RBCs), blood substitutes, and platelet replacements contribute to their respective utilities, and each type is currently represented in ongoing clinical trials. Hemoglobin oxygen carriers (HBOCs) are the most advanced RBC replacements, many of which are currently being evaluated in clinical trials in the United States and other countries. Despite recent advancements, challenges remaining in the development of blood alternatives include stability, oxygen capacity, and compatibility. The continued research and investment in new technologies has the potential to significantly benefit the treatment of life-threatening emergency injuries, both on the battlefield and in the civilian sector. In this review, we discuss military blood-management practices and military-specific uses of individual blood components, as well as describe and analyze several artificial blood products that could be options for future battlefield use.

Gepotidacin is efficacious in a nonhuman primate model of pneumonic plague.

Gepotidacin is a first-in-class triazaacenaphthylene antibacterial agent that selectively inhibits bacterial DNA gyrase and topoisomerase IV through a unique binding mode and has the potential to treat a number of bacterial diseases. Development of this new agent to treat pneumonic plague caused by Yersinia pestis depends on the U.S. Food and Drug Administration Animal Rule testing pathway, as testing in humans is not feasible. Here, preclinical studies were conducted in the African green monkey (AGM) inhalational model of pneumonic plague to test the efficacy of gepotidacin. AGMs infected with Y. pestis were dosed intravenously with gepotidacin (48, 36, or 28 milligrams/kilogram per day) for 10 days to provide a plasma concentration that would support a rationale for a 1000 mg twice or thrice daily intravenous dose in humans or saline as a control. The primary end point was AGM survival with predefined euthanasia criteria. Secondary end points included survival duration and bacterial clearance. Gepotidacin showed activity in vitro against diverse Y. pestis isolates including antibiotic-resistant strains. All control animals in the inhalational plague studies succumbed to plague and were blood culture and organ culture positive for Y. pestis. Gepotidacin provided a 75 to 100% survival benefit with all dose regimens. All surviving animals were blood culture and organ culture negative for Y. pestis. Our randomized, controlled efficacy trials in the AGM pneumonic plague nonhuman primate model together with the in vitro Y. pestis susceptibility data support the use of gepotidacin as a treatment for pneumonic plague caused by Y. pestis.

Aspartoacylase is a regulated nuclear-cytoplasmic enzyme.

Mutations in the gene for aspartoacylase (ASPA), which catalyzes deacetylation of N-acetyl-L-aspartate in the central nervous system (CNS), result in Canavan Disease, a fatal dysmyelinating disease. Consistent with its role in supplying acetate for myelin lipid synthesis, ASPA is thought to be cytoplasmic. Here we describe the occurrence of ASPA within nuclei of rat brain and kidney, and in cultured rodent oligodendrocytes. Immunohistochemistry showed cytoplasmic and nuclear ASPA staining, the specificity of which was demonstrated by its absence from tissues of the Tremor rat, an ASPA-null mutant. Subcellular fractionation analysis revealed low enzyme activity against NAA in nuclear fractions from normal rats. Whereas two recent reports have indicated that ASPA exists as a dimer, size-exclusion chromatography of subcellular fractions showed ASPA is an active monomer in both subcellular fractions. Western blotting detected ASPA as a single 38 kD band. Because ASPA is small enough to passively diffuse into the nucleus, we constructed, expressed, and detected in COS-7 cells a green fluorescent protein-human ASPA (GFP-hASPA) fusion protein larger than the permissible size for the nuclear pore complex. GFP-hASPA was enzymatically active and showed mixed nuclear-cytoplasmic distribution. We conclude that ASPA is a regulated nuclear-cytoplasmic protein that may have distinct functional roles in the two cellular compartments.

Mutational analysis of aspartoacylase: implications for Canavan disease.

Mutations that result in near undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-l-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.

Canavan disease and the role of N-acetylaspartate in myelin synthesis.

Canavan disease (CD) is an autosomal-recessive neurodegenerative disorder caused by inactivation of the enzyme aspartoacylase (ASPA, EC 3.5.1.15) due to mutations. ASPA releases acetate by deacetylation of N-acetylaspartate (NAA), a highly abundant amino acid derivative in the central nervous system. CD results in spongiform degeneration of the brain and severe psychomotor retardation, and the affected children usually die by the age of 10. The pathogenesis of CD remains a matter of inquiry. Our hypothesis is that ASPA actively participates in myelin synthesis by providing NAA-derived acetate for acetyl CoA synthesis, which in turn is used for synthesis of the lipid portion of myelin. Consequently, CD results from defective myelin synthesis due to a deficiency in the supply of the NAA-derived acetate. The demonstration of the selective localization of ASPA in oligodendrocytes in the central nervous system (CNS) is consistent with the acetate deficiency hypothesis of CD. We have tested this hypothesis by determining acetate levels and studying myelin lipid synthesis in the ASPA gene knockout model of CD, and the results provided the first direct evidence in support of this hypothesis. Acetate supplementation therapy is proposed as a simple and inexpensive therapeutic approach to this fatal disease, and progress in our preclinical efforts toward this goal is presented.

Plantazolicin is an ultra-narrow spectrum antibiotic that targets the Bacillus anthracis membrane.

Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified natural product from Bacillus methylotrophicus FZB42 and Bacillus pumilus. Extensive tailoring to twelve of the fourteen amino acid residues in the mature natural product endows PZN with not only a rigid, polyheterocyclic structure, but also antibacterial activity. Here we report a remarkably discriminatory activity of PZN toward Bacillus anthracis, which rivals a previously-described gamma (γ) phage lysis assay in distinguishing B. anthracis from other members of the Bacillus cereus group. We evaluate the underlying cause of this selective activity by measuring the RNA expression profile of PZN-treated B. anthracis, which revealed significant upregulation of genes within the cell envelope stress response. PZN depolarizes the B. anthracis membrane like other cell envelope-acting compounds but uniquely localizes to distinct foci within the envelope. Selection and whole-genome sequencing of PZN-resistant mutants of B. anthracis implicate a relationship between the action of PZN and cardiolipin (CL) within the membrane. Exogenous CL increases the potency of PZN in wild type B. anthracis and promotes the incorporation of fluorescently tagged PZN in the cell envelope. We propose that PZN localizes to and exacerbates structurally compromised regions of the bacterial membrane, which ultimately results in cell lysis.

Membrane-Targeting DCAP Analogues with Broad-Spectrum Antibiotic Activity against Pathogenic Bacteria.

We performed a structure-activity relationship study of 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), which is an antibacterial agent that disrupts the membrane potential and permeability of bacteria. The stereochemistry of DCAP had no effect on the biological activity of DCAP. The aromaticity and electronegativity of the chlorine-substituted carbazole was required for activity, suggesting that its planar and dipolar characteristics orient DCAP in membranes. Increasing the hydrophobicity of the tail region of DCAP enhanced its antibiotic activity. Two DCAP analogues displayed promising antibacterial activity against the BSL-3 pathogens Bacillus anthracis and Francisella tularensis. Codosing DCAP analogues with ampicillin or kanamycin increased their potency. These studies demonstrate that DCAP and its analogues may be a promising scaffold for developing chemotherapeutic agents that bind to bacterial membranes and kill strains of slow-growing or dormant bacteria that cause persistent infections.

SH-SY5Y neuroblastoma cells: a model system for studying biosynthesis of NAAG.

N-Acetylaspartylglutamate (NAAG) is a neuropeptide that is thought to modulate neurotransmitter release through pre-synaptic mGluR3 receptors. Despite years of research into NAAG biochemistry, almost nothing is known about NAAG biosynthesis. To date, NAAG biosynthesis has only been demonstrated conclusively in explanted animal neural tissues, including frog retina, rat dorsal root ganglia and crayfish nerve cord, but not in human cells or tissues. We show here that a human neuroblastoma cell line, SH-SY5Y, provides a good model system for the study of NAAG biosynthesis. Radiolabled NAAG synthesis occurred using both L-[3H]glutamic acid and L-[3H]glutamine as precursors, with glutamine being the preferred substrate. Differentiation of SH-SY5Y cells with retinoic acid resulted in decreased radiolabel incorporation into NAAG, whereas differentiation with nerve growth factor did not affect radiolabel incorporation.

3-substituted indole inhibitors against Francisella tularensis FabI identified by structure-based virtual screening.

In this study, we describe novel inhibitors against Francisella tularensis SchuS4 FabI identified from structure-based in silico screening with integrated molecular dynamics simulations to account for induced fit of a flexible loop crucial for inhibitor binding. Two 3-substituted indoles, 54 and 57, preferentially bound the NAD(+) form of the enzyme and inhibited growth of F. tularensis SchuS4 at concentrations near that of their measured Ki. While 57 was species-specific, 54 showed a broader spectrum of growth inhibition against F. tularensis , Bacillus anthracis , and Staphylococcus aureus . Binding interaction analysis in conjunction with site-directed mutagenesis revealed key residues and elements that contribute to inhibitor binding and species specificity. Mutation of Arg-96, a poorly conserved residue opposite the loop, was unexpectedly found to enhance inhibitor binding in the R96G and R96M variants. This residue may affect the stability and closure of the flexible loop to enhance inhibitor (or substrate) binding.