Modification of N-hydroxycytidine yields a novel lead compound exhibiting activity against the Venezuelan equine encephalitis virus.

Nucleoside and nucleobase analogs capable of interfering with nucleic acid synthesis have played essential roles in fighting infectious diseases. However, many of these agents are associated with important and potentially lethal off-target intracellular effects that limit their use. Based on the previous discovery of base-modified 2'-deoxyuridines, which showed high anticancer activity while exhibiting lower toxicity toward rapidly dividing normal human cells compared to antimetabolite chemotherapeutics, we hypothesized that a similar modification of the N4-hydroxycytidine (NHC) molecule would provide novel antiviral compounds with diminished side effects. This presumption is due to the substantial structural difference with natural cytidine leading to less recognizability by host cell enzymes. Among the 42 antimetabolite species that have been synthesized and screened against VEEV, one hit compound was identified. The structural features of the modifying moiety were similar to those of the anticancer lead 2'-deoxyuridine derivative reported previously, providing an opportunity to pursue further structure-activity relationship (SAR) studies directed to lead improvement, and obtain insight into the mechanism of action, which can lead to identifying drug candidates against a broad spectrum of RNA viral infections.

In Vitro Antibacterial Activity and In Vivo Efficacy of Sulbactam-Durlobactam against Pathogenic Burkholderia Species.

The Gram-negative bacterial genus Burkholderia includes several hard-to-treat human pathogens: two biothreat species, Burkholderia mallei (causing glanders) and B. pseudomallei (causing melioidosis), and the B. cepacia complex (BCC) and B. gladioli, which cause chronic lung infections in persons with cystic fibrosis. All Burkholderia spp. possess an Ambler class A Pen ß-lactamase, which confers resistance to ß-lactams. The ß-lactam-ß-lactamase inhibitor combination sulbactam-durlobactam (SUL-DUR) is in clinical development for the treatment of Acinetobacter infections. In this study, we evaluated SUL-DUR for in vitro and in vivo activity against Burkholderia clinical isolates. We measured MICs of SUL-DUR against BCC and B. gladioli (n = 150), B. mallei (n = 30), and B. pseudomallei (n = 28), studied the kinetics of inhibition of the PenA1 ß-lactamase from B. multivorans and the PenI ß-lactamase from B. pseudomallei by durlobactam, tested for blaPenA1 induction by SUL-DUR, and evaluated in vivo efficacy in a mouse model of melioidosis. SUL-DUR inhibited growth of 87.3% of the BCC and B. gladioli strains and 100% of the B. mallei and B. pseudomallei strains at 4/4 µg/ml. Durlobactam potently inhibited PenA1 and PenI with second-order rate constant for inactivation (k2/K) values of 3.9 × 106 M-1 s-1 and 2.6 × 103 M-1 s-1 and apparent Ki (Kiapp) of 15 nM and 241 nM, respectively, by forming highly stable covalent complexes. Neither sulbactam, durlobactam, nor SUL-DUR increased production of PenA1. SUL-DUR demonstrated activity in vivo in a murine melioidosis model. Taken together, these data suggest that SUL-DUR may be useful as a treatment for Burkholderia infections.

A direct spectropolarimetric assay of arabinose 5-phosphate isomerase.

Arabinose 5-phosphate isomerase (API) catalyzes the reversible isomerization of Ribulose 5-phosphate (Ru5P) to Arabinose 5-Phosphate (Ar5P) for the production of 3-deoxy-2-octulosonic acid 8-phosphate (KDO), a component of bacterial lipopolysaccharide (LPS) of gram-negative bacteria. API is an attractive target for therapeutic development against gram-negative bacterial pathogens. The current assay method of API activity utilizes a general reaction for keto sugar determination in a secondary, 3-h color development reaction with 25 N sulfuric acid which poses hazard to both personnel and instrumentation. We therefore aimed to develop a more user friendly assay of the enzyme. Since Ru5P absorbs in the UV region and contains at least 2 chiral centers, it can be expected to display circular dichroism (CD). A wavelength scan revealed indeed Ru5P displays a pronounced negative ellipticity of 30,560 mDeg M-1cm-1 at 279 nm in Tris buffer pH 9.1 but Ar5P does not have any CD. API enzymatic reactions were monitored directly and continuously in real time by following the disappearance of CD from the Ru5P substrate, or by the appearance of CD from Ar5P substrate. The CD signal at this wavelength was not affected by absorption of the enzyme protein or of small molecules, or turbidity of the solution. Common additives in protein and enzyme reaction mixtures such as detergents, metals, and 5% dimethylsulfoxide did not interfere with the CD signal. Assay reactions of 1-3 min consistently yielded reproducible results. Introduction of accessories in a spectropolarimeter will easily adapt this assay to high throughput format for screening thousands of small molecules as inhibitor candidates of API.

Combinations of early generation antibiotics and antimicrobial peptides are effective against a broad spectrum of bacterial biothreat agents.

The misuse of infectious disease pathogens as agents of deliberate attack on civilians and military personnel is a serious national security concern, which is exacerbated by the emergence of natural or genetically engineered multidrug resistant strains. In this study, the therapeutic potential of combinations of an antibiotic and a broad-spectrum antimicrobial peptide (AMP) was evaluated against five bacterial biothreats, the etiologic agents of glanders (Burkholderia mallei), melioidosis (Burkholderia pseudomallei), plague (Yersinia pestis), tularemia (Francisella tularensis), and anthrax (Bacillus anthracis). The therapeutics included licensed early generation antibiotics which are now rarely used. Three antibiotics and one 24- amino acid AMP were selected based on MIC assay data. Combinations of the AMP and tigecycline, minocycline, or novobiocin were screened for synergistic activity by checkerboard MIC assay. The combinations each enhanced the susceptibility of several strains. The tetracycline-peptide combinations increased the sensitivities of Y. pestis, F. tularensis, B. anthracis and B. pseudomallei, and the novobiocin-AMP combination augmented the sensitivity of all five. In time-kill assays, down-selected combinations of the peptide and minocycline or tigecycline enhanced killing of B. anthracis, Y. pestis, F. tularensis, and Burkholderia mallei but not B. pseudomallei. The novobiocin-AMP pair significantly reduced viability of all strains except B. mallei, which was very sensitive to the antibiotic alone. The results suggested that antibiotic-AMP combinations are useful tools for combating diverse pathogens. Future studies employing cell culture and animal models will utilize virulent strains of the agents to investigate the in vivo availability, host cytotoxicity, and protective efficacy of these therapeutics.

Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430.

Filoviruses are emerging pathogens and causative agents of viral haemorrhagic fever. Case fatality rates of filovirus disease outbreaks are among the highest reported for any human pathogen, exceeding 90% (ref. 1). Licensed therapeutic or vaccine products are not available to treat filovirus diseases. Candidate therapeutics previously shown to be efficacious in non-human primate disease models are based on virus-specific designs and have limited broad-spectrum antiviral potential. Here we show that BCX4430, a novel synthetic adenosine analogue, inhibits infection of distinct filoviruses in human cells. Biochemical, reporter-based and primer-extension assays indicate that BCX4430 inhibits viral RNA polymerase function, acting as a non-obligate RNA chain terminator. Post-exposure intramuscular administration of BCX4430 protects against Ebola virus and Marburg virus disease in rodent models. Most importantly, BCX4430 completely protects cynomolgus macaques from Marburg virus infection when administered as late as 48 hours after infection. In addition, BCX4430 exhibits broad-spectrum antiviral activity against numerous viruses, including bunyaviruses, arenaviruses, paramyxoviruses, coronaviruses and flaviviruses. This is the first report, to our knowledge, of non-human primate protection from filovirus disease by a synthetic drug-like small molecule. We provide additional pharmacological characterizations supporting the potential development of BCX4430 as a countermeasure against human filovirus diseases and other viral diseases representing major public health threats.

Characterization of the plasma proteome of nonhuman primates during Ebola virus disease or melioidosis: a host response comparison.

BACKGROUND: In-depth examination of the plasma proteomic response to infection with a wide variety of pathogens can assist in the development of new diagnostic paradigms, while providing insight into the interdependent pathogenic processes which encompass a host's immunological and physiological responses. Ebola virus (EBOV) causes a highly lethal infection termed Ebola virus disease (EVD) in primates and humans. The Gram negative non-spore forming bacillus Burkholderia pseudomallei (Bp) causes melioidosis in primates and humans, characterized by severe pneumonia with high mortality. We sought to examine the host response to infection with these two bio-threat pathogens using established animal models to provide information on the feasibility of pre-symptomatic diagnosis, since the induction of host molecular signaling networks can occur before clinical presentation and pathogen detection. METHODS: Herein we report the quantitative proteomic analysis of plasma collected at various times of disease progression from 10 EBOV-infected and 5 Bp-infected nonhuman primates (NHP). Our strategy employed high resolution LC-MS/MS and a peptide-tagging approach for relative protein quantitation. In each infection type, for all proteins with > 1.3 fold abundance change at any post-infection time point, a direct comparison was made with levels obtained from plasma collected daily from 5 naïve rhesus macaques, to determine the fold changes that were significant, and establish the natural variability of abundance for endogenous plasma proteins. RESULTS: A total of 41 plasma proteins displayed significant alterations in abundance during EBOV infection, and 28 proteins had altered levels during Bp infection, when compared to naïve NHPs. Many major acute phase proteins quantitated displayed similar fold-changes between the two infection types but exhibited different temporal dynamics. Proteins related to the clotting cascade, immune signaling and complement system exhibited significant differential abundance during infection with EBOV or Bp, indicating a specificity of the response. CONCLUSIONS: These results advance our understanding of the global plasma proteomic response to EBOV and Bp infection in relevant primate models for human disease and provide insight into potential innate immune response differences between viral and bacterial infections.

The Biophysical Interaction of the Danger-Associated Molecular Pattern (DAMP) Calreticulin with the Pattern-Associated Molecular Pattern (PAMP) Lipopolysaccharide.

The endoplasmic reticulum (ER) chaperone protein, calreticulin (CRT), is essential for proper glycoprotein folding and maintaining cellular calcium homeostasis. During ER stress, CRT is overexpressed as part of the unfolded protein response (UPR). In addition, CRT can be released as a damage-associated molecular pattern (DAMP) molecule that may interact with pathogen-associated molecular patterns (PAMPs) during the innate immune response. One such PAMP is lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall. In this report, we show that recombinant and native human placental CRT strongly interacts with LPS in solution, solid phase, and the surface of gram-negative and gram-positive bacteria. Furthermore, LPS induces oilgomerization of CRT with a disappearance of the monomeric form. The application of recombinant CRT (rCRT) to size exclusion and anion exchange chromatography shows an atypical heterogeneous elution profile, indicating that LPS affects the conformation and ionic charge of CRT. Interestingly, LPS bound to CRT is detected in sera of bronchiectasis patients with chronic bacterial infections. By ELISA, rCRT dose-dependently bound to solid phase LPS via the N- and C-domain globular head region of CRT and the C-domain alone. The specific interaction of CRT with LPS may be important in PAMP innate immunity.

Bacterial genome engineering and synthetic biology: combating pathogens.

BACKGROUND: The emergence and prevalence of multidrug resistant (MDR) pathogenic bacteria poses a serious threat to human and animal health globally. Nosocomial infections and common ailments such as pneumonia, wound, urinary tract, and bloodstream infections are becoming more challenging to treat due to the rapid spread of MDR pathogenic bacteria. According to recent reports by the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC), there is an unprecedented increase in the occurrence of MDR infections worldwide. The rise in these infections has generated an economic strain worldwide, prompting the WHO to endorse a global action plan to improve awareness and understanding of antimicrobial resistance. This health crisis necessitates an immediate action to target the underlying mechanisms of drug resistance in bacteria. RESEARCH: The advent of new bacterial genome engineering and synthetic biology (SB) tools is providing promising diagnostic and treatment plans to monitor and treat widespread recalcitrant bacterial infections. Key advances in genetic engineering approaches can successfully aid in targeting and editing pathogenic bacterial genomes for understanding and mitigating drug resistance mechanisms. In this review, we discuss the application of specific genome engineering and SB methods such as recombineering, clustered regularly interspaced short palindromic repeats (CRISPR), and bacterial cell-cell signaling mechanisms for pathogen targeting. The utility of these tools in developing antibacterial strategies such as novel antibiotic production, phage therapy, diagnostics and vaccine production to name a few, are also highlighted. CONCLUSIONS: The prevalent use of antibiotics and the spread of MDR bacteria raise the prospect of a post-antibiotic era, which underscores the need for developing novel therapeutics to target MDR pathogens. The development of enabling SB technologies offers promising solutions to deliver safe and effective antibacterial therapies.

Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging.

BACKGROUND: Tissue samples should be fixed and permanently stabilized as soon as possible ex-vivo to avoid variations in proteomic content. Tissues collected from studies involving infectious microorganisms, must face the additional challenge of pathogen inactivation before downstream proteomic analysis can be safely performed. Heat fixation using the Denator Stabilizor System (Gothenburg, Sweden) utilizes conductive heating, under a mild vacuum, to rapidly eliminate enzymatic degradation in tissue samples. Although many studies have reported on the ability of this method to stop proteolytic degradation and other sample changes immediately and permanently, pathogen inactivation has not been studied. RESULTS: We examined the ability of the heat fixation workflow to inactivate bacterial and viral pathogens and the suitability of this tissue for Matrix Assisted Laser Desorption Ionization mass spectrometry imaging (MALDI-MSI). Mice were infected with viral or bacterial pathogens representing two strains of Venezuelan Equine Encephalitis virus (VEEV) and two strains of Burkholderia. Additionally, a tissue mimetic model was employed using Escherichia, Klebsiella and Acinetobacter isolates. Infected tissue samples harvested from each animal or mimetic model were sectioned in half. One half was heat fixed and the other remained untreated. Lysates from each sample were checked for organism viability by performing plaque (infectivity) assays or plating on nutrient agar for colony forming unit (CFU) calculation. Untreated infected control tissue demonstrated the presence of each viable pathogen by positive plaque or colony formation, whereas heat fixation resulted in complete inactivation of both the viral and bacterial pathogens. MALDI-MSI images produced from heat fixed tissue were reflective of molecular distributions within brain, spleen and lung tissue structures. CONCLUSIONS: We conclude that heat fixation inactivates viral and bacterial pathogens and is compatible with proteomic analysis by MALDI-MSI. This treatment will enable the use of infected tissue from studies performed in bio-safety level 3 laboratories with VEEV and Burkholderia to be safely used for proteomic, small molecule drug detection, and imaging mass spectrometry analysis.

Characterization of the murine macrophage response to infection with virulent and avirulent Burkholderia species.

BACKGROUND: Burkholderia pseudomallei (Bp) and Burkholderia mallei (Bm) are Gram-negative facultative intracellular pathogens, which are the causative agents of melioidosis and glanders, respectively. Depending on the route of exposure, aerosol or transcutaneous, infection by Bp or Bm can result in an extensive range of disease - from acute to chronic, relapsing illness to fatal septicemia. Both diseases are associated with difficult diagnosis and high fatality rates. About ninety five percent of patients succumb to untreated septicemic infections and the fatality rate is 50 % even when standard antibiotic treatments are administered. RESULTS: The goal of this study is to profile murine macrophage-mediated phenotypic and molecular responses that are characteristic to a collection of Bp, Bm, Burkholderia thailandensis (Bt) and Burkholderia oklahomensis (Bo) strains obtained from humans, animals, environment and geographically diverse locations. Burkholderia spp. (N = 21) were able to invade and replicate in macrophages, albeit to varying degrees. All Bp (N = 9) and four Bm strains were able to induce actin polymerization on the bacterial surface following infection. Several Bp and Bm strains showed reduced ability to induce multinucleated giant cell (MNGC) formation, while Bo and Bp 776 were unable to induce this phenotype. Measurement of host cytokine responses revealed a statistically significant Bm mediated IL-6 and IL-10 production compared to Bp strains. Hierarchical clustering of transcriptional data from 84 mouse cytokines, chemokines and their corresponding receptors identified 29 host genes as indicators of differential responses between the Burkholderia spp. Further validation confirmed Bm mediated Il-1b, Il-10, Tnfrsf1b and Il-36a mRNA expressions were significantly higher when compared to Bp and Bt. CONCLUSIONS: These results characterize the phenotypic and immunological differences in the host innate response to pathogenic and avirulent Burkholderia strains and provide insight into the phenotypic alterations and molecular targets underlying host-Burkholderia interactions.

High-throughput screen using a single-cell tyrosine phosphatase assay reveals biologically active inhibitors of tyrosine phosphatase CD45.

Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.

A high-content imaging assay for the quantification of the Burkholderia pseudomallei induced multinucleated giant cell (MNGC) phenotype in murine macrophages.

BACKGROUND: Burkholderia pseudomallei (Bp), a Gram-negative, motile, facultative intracellular bacterium is the causative agent of melioidosis in humans and animals. The Bp genome encodes a repertoire of virulence factors, including the cluster 3 type III secretion system (T3SS-3), the cluster 1 type VI secretion system (T6SS-1), and the intracellular motility protein BimA, that enable the pathogen to invade both phagocytic and non-phagocytic cells. A unique hallmark of Bp infection both in vitro and in vivo is its ability to induce cell-to-cell fusion of macrophages to form multinucleated giant cells (MNGCs), which to date are semi-quantitatively reported following visual inspection. RESULTS: In this study we report the development of an automated high-content image acquisition and analysis assay to quantitate the Bp induced MNGC phenotype. Validation of the assay was performed using T6SS-1 (∆hcp1) and T3SS-3 (∆bsaZ) mutants of Bp that have been previously reported to exhibit defects in their ability to induce MNGCs. Finally, screening of a focused small molecule library identified several Histone Deacetylase (HDAC) inhibitors that inhibited Bp-induced MNGC formation of macrophages. CONCLUSIONS: We have successfully developed an automated HCI assay to quantitate MNGCs induced by Bp in macrophages. This assay was then used to characterize the phenotype of the Bp mutants for their ability to induce MNGC formation and identify small molecules that interfere with this process. Successful application of chemical genetics and functional reverse genetics siRNA approaches in the MNGC assay will help gain a better understanding of the molecular targets and cellular mechanisms responsible for the MNGC phenotype induced by Bp, by other bacteria such as Mycobacterium tuberculosis, or by exogenously added cytokines.

Synthesis and antibacterial evaluation of new, unsymmetrical triaryl bisamidine compounds.

Herein we describe the synthesis and antibacterial evaluation of a new, unsymmetrical triaryl bisamidine compound series, [Am]-[indole]-[linker]-[HetAr/Ar]-[Am], in which [Am] is an amidine or amino group, [linker] is a benzene, thiophene or pyridine ring, and [HetAr/Ar] is a benzimidazole, imidazopyridine, benzofuran, benzothiophene, pyrimidine or benzene ring. When the [HetAr/Ar] unit is a 5,6-bicyclic heterocycle, it is oriented such that the 5-membered ring portion is connected to the [linker] unit and the 6-membered ring portion is connected to the [Am] unit. Among the 34 compounds in this series, compounds with benzofuran as the [HetAr/Ar] unit showed the highest potencies. Introduction of a fluorine atom or a methyl group to the triaryl core led to the more potent analogs. Bisamidines are more active toward bacteria while the monoamidines are more active toward mammalian cells (as indicated by low CC50 values). Importantly, we identified compound P12a (MBX 1887) with a relatively narrow spectrum against bacteria and a very high CC50 value. Compound P12a has been scaled up and is currently undergoing further evaluations for therapeutic applications.

Orally available nucleoside analog UMM-766 provides protection in a murine model of orthopox disease.

Although smallpox has been eradicated, other orthopoxviruses continue to be a public health concern as exemplified by the ongoing Mpox (formerly monkeypox) global outbreak. While medical countermeasures (MCMs) previously approved by the Food and Drug Administration for the treatment of smallpox have been adopted for Mpox, previously described vulnerabilities coupled with the questionable benefit of at least one of the therapeutics during the 2022 Mpox outbreak reinforce the need for identifying and developing other MCMs against orthopoxviruses. Here, we screened a panel of Merck proprietary small molecules and identified a novel nucleoside inhibitor with potent broad-spectrum antiviral activity against multiple orthopoxviruses. Efficacy testing of a 7-day dosing regimen of the orally administered nucleoside in a murine model of severe orthopoxvirus infection yielded a dose-dependent increase in survival. Treated animals had greatly reduced lesions in the lung and nasal cavity, particularly in the 10 µg/mL dosing group. Viral levels were also markedly lower in the UMM-766-treated animals. This work demonstrates that this nucleoside analog has anti-orthopoxvirus efficacy and can protect against severe disease in a murine orthopox model.IMPORTANCEThe recent monkeypox virus pandemic demonstrates that members of the orthopoxvirus, which also includes variola virus, which causes smallpox, remain a public health issue. While currently FDA-approved treatment options exist, risks that resistant strains of orthopoxviruses may arise are a great concern. Thus, continued exploration of anti-poxvirus treatments is warranted. Here, we developed a template for a high-throughput screening assay to identify anti-poxvirus small-molecule drugs. By screening available drug libraries, we identified a compound that inhibited orthopoxvirus replication in cell culture. We then showed that this drug can protect animals against severe disease. Our findings here support the use of existing drug libraries to identify orthopoxvirus-targeting drugs that may serve as human-safe products to thwart future outbreaks.

Characterization of the Burkholderia thailandensis SOS response by using whole-transcriptome shotgun sequencing.

The bacterial SOS response is a well-characterized regulatory network encoded by most prokaryotic bacterial species and is involved in DNA repair. In addition to nucleic acid repair, the SOS response is involved in pathogenicity, stress-induced mutagenesis, and the emergence and dissemination of antibiotic resistance. Using high-throughput sequencing technology (SOLiD RNA-Seq), we analyzed the Burkholderia thailandensis global SOS response to the fluoroquinolone antibiotic, ciprofloxacin (CIP), and the DNA-damaging chemical, mitomycin C (MMC). We demonstrate that a B. thailandensis recA mutant (RU0643) is ∼4-fold more sensitive to CIP in contrast to the parental strain B. thailandensis DW503. Our RNA-Seq results show that CIP and MMC treatment (P < 0.01) resulted in the differential expression of 344 genes in B. thailandensis and 210 genes in RU0643. Several genes associated with the SOS response were induced and include lexA, uvrA, dnaE, dinB, recX, and recA. At the genome-wide level, we found an overall decrease in gene expression, especially for genes involved in amino acid and carbohydrate transport and metabolism, following both CIP and MMC exposure. Interestingly, we observed the upregulation of several genes involved in bacterial motility and enhanced transcription of a B. thailandensis genomic island encoding a Siphoviridae bacteriophage designated E264. Using B. thailandensis plaque assays and PCR with B. mallei ATCC 23344 as the host, we demonstrate that CIP and MMC exposure in B. thailandensis DW503 induces the transcription and translation of viable bacteriophage in a RecA-dependent manner. This is the first report of the SOS response in Burkholderia spp. to DNA-damaging agents. We have identified both common and unique adaptive responses of B. thailandensis to chemical stress and DNA damage.

Potent and broad-spectrum antibacterial activity of indole-based bisamidine antibiotics: synthesis and SAR of novel analogs of MBX 1066 and MBX 1090.

The prevalence of drug-resistant bacteria in the clinic has propelled a concerted effort to find new classes of antibiotics that will circumvent current modes of resistance. We have previously described a set of bisamidine antibiotics that contains a core composed of two indoles and a central linker. The first compounds of the series, MBX 1066 and MBX 1090, have potent antibacterial properties against a wide range of Gram-positive and Gram-negative bacteria. We have conducted a systematic exploration of the amidine functionalities, the central linker, and substituents at the indole 3-position to determine the factors involved in potent antibacterial activity. Some of the newly synthesized compounds have even more potent and broad-spectrum activity than MBX 1066 and MBX 1090.

Anthrax toxin-induced rupture of artificial lipid bilayer membranes.

We demonstrate experimentally that anthrax toxin complexes rupture artificial lipid bilayer membranes when isolated from the blood of infected animals. When the solution pH is temporally acidified to mimic that process in endosomes, recombinant anthrax toxin forms an irreversibly bound complex, which also destabilizes membranes. The results suggest an alternative mechanism for the translocation of anthrax toxin into the cytoplasm.

Identification of a small-molecule entry inhibitor for filoviruses.

Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available. Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations (IC(50)s) of 10 µM and 12 µM, respectively. Time-of-addition and binding studies suggested that compound 7 binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schrödinger SiteMap calculations, using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine compounds that are suitable for optimization as potential inhibitors of filoviral infection.

A Novel Toll-Like Receptor 2 Agonist Protects Mice in a Prophylactic Treatment Model Against Challenge With Bacillus anthracis.

Current therapies for anthrax include the use of antibiotics (i.e., doxycycline, and ciprofloxacin), an anthrax vaccine (BioThrax) and Bacillus anthracis-specific, monoclonal antibody (mAb) (i.e., Raxibacumab and obiltoxaximab). In this study, we investigated the activity of immunomodulators, which potentiate inflammatory responses through innate immune receptors. The rationale for the use of innate immune receptor agonists as adjunctive immunomodulators for infectious diseases is based on the concept that augmentation of host defense should promote the antimicrobial mechanism of the host. Our aim was to explore the anti-B. anthracis effector function of Toll-like receptor (TLR) agonists using a mouse model. Amongst the six TLR ligands tested, Pam3CSK4 (TLR1/2 ligand) was the best at protecting mice from lethal challenge of B. anthracis. We then evaluated the activity of a novel TLR2 ligand, DA-98-WW07. DA-98-WW07 demonstrated enhanced protection in B. anthracis infected mice. The surviving mice that received DA-98-WW07 when re-challenged with B. anthracis 20 days post the first infection showed increased survival rate. Moreover, ciprofloxacin, when treated in adjunct with a suboptimal concentration of DA-98-WW07 demonstrated augmented activity in protecting mice from B. anthracis infection. Taken together, we report the prophylactic treatment potential of DA-98-WW07 for anthrax and the utility of immunomodulators in combination with an antibiotic to treat infections caused by the B. anthracis bacterium.

Screening of a Focused Ubiquitin-Proteasome Pathway Inhibitor Library Identifies Small Molecules as Novel Modulators of Botulinum Neurotoxin Type A Toxicity.

Botulinum neurotoxins (BoNTs) are known as the most potent bacterial toxins, which can cause potentially deadly disease botulism. BoNT Serotype A (BoNT/A) is the most studied serotype as it is responsible for most human botulism cases, and its formulations are extensively utilized in clinics for therapeutic and cosmetic applications. BoNT/A has the longest-lasting effect in neurons compared to other serotypes, and there has been high interest in understanding how BoNT/A manages to escape protein degradation machinery in neurons for months. Recent work demonstrated that an E3 ligase, HECTD2, leads to efficient ubiquitination of the BoNT/A Light Chain (A/LC); however, the dominant activity of a deubiquitinase (DUB), VCIP135, inhibits the degradation of the enzymatic component. Another DUB, USP9X, was also identified as a potential indirect contributor to A/LC degradation. In this study, we screened a focused ubiquitin-proteasome pathway inhibitor library, including VCIP135 and USP9X inhibitors, and identified ten potential lead compounds affecting BoNT/A mediated SNAP-25 cleavage in neurons in pre-intoxication conditions. We then tested the dose-dependent effects of the compounds and their potential toxic effects in cells. A subset of the lead compounds demonstrated efficacy on the stability and ubiquitination of A/LC in cells. Three of the compounds, WP1130 (degrasyn), PR-619, and Celastrol, further demonstrated efficacy against BoNT/A holotoxin in an in vitro post-intoxication model. Excitingly, PR-619 and WP1130 are known inhibitors of VCIP135 and USP9X, respectively. Modulation of BoNT turnover in cells by small molecules can potentially lead to the development of effective countermeasures against botulism.