WO2016191695A2 - Traitements d'infections intracellulaires strictes - Google Patents

Traitements d'infections intracellulaires strictes Download PDF

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WO2016191695A2
WO2016191695A2 PCT/US2016/034686 US2016034686W WO2016191695A2 WO 2016191695 A2 WO2016191695 A2 WO 2016191695A2 US 2016034686 W US2016034686 W US 2016034686W WO 2016191695 A2 WO2016191695 A2 WO 2016191695A2
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inhibitor
wnt
notch
chaffeensis
cells
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WO2016191695A3 (fr
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Jere W. Mcbride
Tian Lou
Taslima T. LINA
Paigs S. DUNPHY
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Research Developmetn Foundation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
    • A61K31/10—Sulfides; Sulfoxides; Sulfones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates generally to the field of molecular biology and medicine. More particularly, it concerns methods of treating bacterial infections. 2. Description of Related Art
  • Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the emerging life-threatening human zoonosis, human monocytotropic ehrlichiosis (HME) (Paddock and Childs, 2003).
  • HME human monocytotropic ehrlichiosis
  • E. chaffeensis selectively infects mononuclear phagocytes and resides in early-endosome-like membrane-bound vacuoles (Paddock and Childs, 2003). The mechanisms by which E.
  • TRP tandem repeat proteins
  • chaffeensis TRPs play a role in manipulating these important cellular processes to facilitate infection (Luo et al., 2011; Luo and McBride, 2012; Wakeel et al., 2011; Wakeel et al., 2009; Wakeel et al., 2010).
  • Recent findings of ehrlichial effector TRPs highlight the importance and complexity of interactions between bacterial effectors and host processes; however, the molecular mechanisms by which Ehrlichia modulates host cells are still not well understood (Dunphy et al., 2013; Luo et al., 2011; Luo and McBride, 2012; Wakeel et al., 2009).
  • Phagocytosis is a specific form of endocytosis and a major mechanism used to remove pathogens in the immune system. Usually the pathogen becomes trapped in a phagosome which then fuses with a lysosome to form a phagolysosome, but E. chaffeensis enters the monocyte through lipid raft-caveolae-mediated endocytosis and then it can reside within a cytoplasmic vacuole that resembles an early endosome and does not fuse with lysosomes, protecting it from killing (Lin and Rikihisa, 2003). However, the underlying molecular mechanism remains unclear.
  • E. chaffeensis TRPs were initially identified as major immunoreactive proteins that elicit strong antibody responses during infection (Luo and McBride, 2012; Doyle et al., 2006; Luo et al., 2009), and antibodies directed at continuous species-specific epitopes in tandem repeat regions are protective against infection (Kuriakose et al., 2012). Subsequently, understanding of the functional role of TRPs as effectors in ehrlichial pathobiology has significantly advanced through studies that have defined specific TRP-host protein and DNA interactions (Dunphy et al., 2013).
  • the present invention is based, in part, on the discovery that Ehrlichia bacteria utilize canonical and noncanonical host Wnt signaling pathways to stimulate phagocytosis and promote intracellular survival, and inhibition of Wnt signaling may be used to treat or inhibit bacterial infection by obligately intracellular bacteria such as Ehrlichia canis or Ehrlichia chaffeensis. Additionally, it has also been observed as described herein that Notch signaling can promote ehrlichial survival, and a Notch inhibitor may be used to treat infection by an obligately intracellular bacteria such as Ehrlichia canis or Ehrlichia chaffeensis.
  • RNAi or small molecule inhibitors may be used to inhibit canonical and/or non-canonical Wnt signaling to decrease bacterial load or treat ehrlichiosis.
  • a Wnt inhibitor may be used to treat (e.g., inhibit phagocytosis and replication) of an obligately intracellular bacteria, an obligately intracellular protozoa, or an obligately intracellular fungi in a host.
  • chaffeensis infection canonical and noncanonical Wnt signaling pathways were observed to be significantly stimulated at the very early stage (1-3 h) of infection as determined by ⁇ -catenin and NFATC1 nuclear translocation, but repressed at the later stage (72 h) of infection.
  • expression levels of approximately 46% of Wnt component and signaling target genes were observed to be modulated during E. chaffeensis infection, indicating early activation and late repression of the pathway.
  • Knockdown of critical components of Wnt signaling pathways by RNAi including Wnt5a ligand, receptor and co- receptor, regulator, other signaling molecules, transcription factor and target, resulted in significant reductions in ehrlichial load.
  • small molecule inhibitors of the canonical and noncanonical (Ca 2+ and PCP) Wnt signaling pathways significantly decreased ehrlichial infection in vitro, including casein kinase I ⁇ /GSK3 ⁇ activator pyrvinium pamoate, calmodulin kinase II inhibitor KN93 and inhibitor of Wnt secretion, IWP-2, which dramatically reduced bacterial and TRP coated latex bead internalization.
  • Latex beads coated with E. chaffeensis TRP120 and TRP32 were observed to be phagocytized through Wnt5a-directed mechanisms, demonstrating a key role of these effectors in Wnt5a-mediated phagocytosis.
  • E. chaffeensis exploits canonical and noncanonical Wnt pathways to induce phagocytosis and promote intracellular survival.
  • TRP secreted tandem repeat protein
  • Notch signaling pathway components chaffeensis or recombinant TRP120, resulting in upregulation of Notch signaling pathway components and target genes notch1, adam17, hes and hey.
  • Significant differences in canonical Notch signaling gene expression levels (>40%) were observed during early and late stages of infection, indicating activation of the Notch pathway.
  • Notch pathway activation was linked specifically to the TRP120 effector, which was observed to directly interact with the Notch metalloprotease ADAM17.
  • pharmacological inhibitors and siRNAs against ⁇ -secretase enzyme Notch transcription factor complex, Notch1 and ADAM17, Notch signaling was demonstrated o be required for ehrlichial survival.
  • An aspect of the present invention relates to a method of treating a bacterial infection in a mammalian subject, comprising administering a therapeutically effective amount of a Wnt inhibitor to said subject, wherein said bacterial infection comprises an obligately intracellular bacteria.
  • the obligately intracellular bacteria may be Ehrlichia, Chlamydia, Rickettsia, Coxiella, Orientia, or Mycobacteria.
  • the obligately intracellular bacteria is Ehrlichia chaffeensis.
  • the obligately intracellular bacteria is Ehrlichia canis.
  • the subject may be a human.
  • the subject may be a dog.
  • the Wnt inhibitor is a small interfering RNA (RNAi), an antibody, or a small molecule Wnt inhibitor.
  • RNAi small interfering RNA
  • the Wnt inhibitor may be administered orally, intravenously, or parenterally.
  • the Wnt inhibitor may be an inhibitor of a canonical Wnt pathway.
  • the Wnt inhibitor selectively inhibits PI3K, CKII, CK1 ⁇ , or ⁇ -catenin/TCF/LEF, or the Wnt inhibitor selectively activates casein kinase I ⁇ /GSK3 ⁇ . In some embodiments, the Wnt inhibitor selectively activates casein kinase I ⁇ /GSK3 ⁇ . In some embodiments, the Wnt inhibitor is pyrvinium pamoate, TBCA, SB202190, LY294002, or FH535. In some embodiments, the Wnt inhibitor is pyrvinium pamoate. The Wnt inhibitor may be an inhibitor of a non-canonical Wnt pathway.
  • the Wnt inhibitor is an inhibitor of a Wnt/Ca 2+ pathway.
  • the Wnt inhibitor may selectively inhibit calmodulin kinase II (CaMKII) or IKK.
  • the Wnt inhibitor selectively inhibits calmodulin kinase II.
  • the Wnt inhibitor is KN93.
  • the Wnt inhibitor may be an inhibitor of a Wnt/PCP pathway.
  • the Wnt inhibitor selectively inhibits PI3K, Akt, or IKK.
  • the Wnt inhibitor selectively inhibits Akt.
  • the Wnt inhibitor is pyrvinium pamoate, LY294002, or BAY 11-7082.
  • the Wnt inhibitor is pyrvinium pamoate.
  • the Wnt inhibitor may selectively inhibit Wnt secretion.
  • the Wnt inhibitor selectively inhibits PORCN.
  • the Wnt inhibitor is IWP-2.
  • the Wnt inhibitor is comprised in a pharmaceutical preparation.
  • the pharmaceutical preparation may be formulated, e.g., for oral, intravenous, topical, or parenteral administration.
  • Another aspect of the present invention relates to a method of treating a bacterial infection in a mammalian subject, comprising administering a therapeutically effective amount of a Notch inhibitor to said subject, wherein said bacterial infection comprises an obligately intracellular bacteria.
  • the obligately intracellular bacteria may be Ehrlichia, Chlamydia, Rickettsia, Coxiella, Orientia, or Mycobacteria.
  • the obligately intracellular bacteria is Ehrlichia chaffeensis.
  • the obligately intracellular bacteria is Ehrlichia canis.
  • the subject may be a human or a dog.
  • the Notch inhibitor may be a small interfering RNA (RNAi), an antibody, or a small molecule Notch inhibitor.
  • the Notch inhibitor is administered orally, intravenously, or parenterally.
  • the Notch inhibitor selectively inhibits ⁇ -secretase, Notch transcription factor complex, Notch1, ADAM17, or ADAM10. In some embodiments, the Notch inhibitor selectively inhibits ⁇ -secretase. In some embodiments, the Notch inhibitor is DAPT, BMS-906024, MK0752, PF-03084014, MRK0003, or RO4929097. In some embodiments, the Notch inhibitor is an antibody, an antibody fragment, or a Notch blocking peptide.
  • the Notch inhibitor may be a blocking peptide (e.g., MAM peptide agonist SAHM1), an antibody fragment or a neutralizing antibody (e.g., OMP-59R5 (anti- Notch 2/3 mAb), NRR1 anti-Notch1 mAB, NRR2 anti-Notch2 mAb, NRR3 anti-Notch3 mAb, OMP-21M18 anti-DLL4 mAb, a DLL1-Fc Fc anti-delta like 1 chimeric mAb, a JAG1 Fc or jagged 1 Fc chimeric mAb, or A5622A anti-nicastrin mAb), a monoclonal antibody (mAb), or a Notch decoy (e.g., a soluble Notch1, Dll1, jagged1).
  • a blocking peptide e.g., MAM peptide agonist SAHM1
  • an antibody fragment or a neutralizing antibody e.g., OMP-59R5 (
  • the Notch inhibitor may be comprised in a pharmaceutical preparation.
  • the pharmaceutical preparation is formulated for oral, intravenous, topical, or parenteral administration.
  • the bacterial infection is not Salmonella typhimurium, Mycobacterium bovis (M. bovis) BSG, Bacillus anthracis, or Clostridium difficile.
  • both the Wnt inhibitor and the Notch inhibitor are administered to the subject to treat the bacterial infection.
  • FIG. 1 Expression levels of seven host genes at different time points postinfection of E. chaffeensis by Wnt signaling pathway PCR array. Fold regulation > 1 indicates an upregulation, and ⁇ 1 indicates a downregulation, compared to that in uninfected cells.
  • FIG 2 Expression levels of six target genes of Wnt signaling at different time points postinfection of E. chaffeensis by Wnt signaling target PCR array.
  • FIGS. 3A-B Knockdown of Wnt signaling pathway component or target influences ehrlichial infection of macrophages. THP ⁇ 1 cells were transfected with specific or control siRNA and then infected with E. chaffeensis.
  • FIG. 3A Bacterial numbers were determined by qPCR at 1 day and 2 days p.i. All experiments were repeated three times, and the values are means ⁇ standard deviations (*, P ⁇ 0.05).
  • FIG. 3B Western blottings confirmed the reduction of FZD9, DVL2, GSK3 ⁇ and JUN proteins at 2 days p.i.
  • FIGS.4A-B Wnt signaling pathway inhibitors reduced ehrlichial infection of host cells.
  • FIG.4A Percentages of infected cells were determined by Diff ⁇ Quik staining and counting of 100 cells at 3 days p.i. An infected cell culture without the inhibitor served as a positive control, and an uninfected culture served as a negative control. Results are from three independent experiments, and the values are means ⁇ standard deviations (*, P ⁇ 0.05).
  • FIG. 4B Bright ⁇ field images (magnification, ⁇ 40) of Diff ⁇ Quik ⁇ stained samples collected at 3 days p.i.
  • FIGS. 5A-B E. chaffeensis upregulatesWnt signaling.
  • FIG. 5A ⁇ catenin translocates to nucleus in THP ⁇ 1 cells at 3 h postinfection of E. chaffeensis.
  • FIG. 5A ⁇ catenin translocates to nucleus in THP ⁇ 1 cells at 3 h postinfection of E. chaffeensis.
  • FIGS. 6A-C Wnt pathway inhibitor pyrvinium abolished the stimulation of phagocytosis by the ehrlichial tandem repeat protein TRP120 in macrophages.
  • FIG.6A and FIG. 6B Compared with control TRP120N-coated beads, TRP120 ⁇ coated beads were phagocytosed by the THP ⁇ 1 cell dramatically.
  • FIG. 7 Illustration of canonical and noncanonical Wnt signaling pathways, potent inhibitors and involved TRPinteracting proteins. Wnt pathways regulate cellular processes including gene transcription, phagocytosis, and cytoskeletal reorganization. Small molecule inhibitors disrupt components of Wnt pathways. Ehrlichial TRPs interact with proteins that regulate Wnt signaling.
  • FIGS.8A-C E. chaffeensis activates Notch signaling pathway in THP-1 cells. (FIG.
  • FIG.8B Expression levels of Notch signaling components in THP-1 cells were analyzed using real time RT-PCR in uninfected and E. chaffeensis infected THP-1 cells, 2 h (open bar) and 72 h p.i. (closed bar).
  • FIGS. 9A-C Expression array analysis of Notch signaling pathway genes during E. chaffeensis infection.
  • FIGS. 9A Heat map showing relative expression levels of Notch signaling pathway components and its downstream target genes at 12, 24, 48 and 72 h p.i. Each individual well in the heat map represents individual gene, and the scale bar shows differential expression from the mean gene expression level of uninfected cells. The levels of induction/repression are shown.
  • FIG. 9B Scatter plot showing the Notch PCR array data. Genes displaying upregulation, no significant change of expression, and downregulation are shown.
  • FIG. 9C List of genes with their fold-change which showed differential expression (up and down-regulation) at 24 h p.i. Notch signaling pathway target genes, Notch pathway components and other genes involved in signaling pathway that cross talks with the Notch signaling pathway are grouped as shown. [0027] FIGS.
  • 10A-D Inhibition of Notch pathway decreases E. chaffeensis load.
  • THP-1 cells were treated with pharmacological inhibitors against ⁇ -secretase enzyme (DAPT) and RBPj ⁇ transcription complex (SAHM1). Cells were infected with E. chaffeensis after 1 h post treatment.
  • Bacterial loads were determined at 24 and 48 h p.i. either by (FIG. 10A) calculating the percentage of infected cells by counting 100 Diff-Quik-stained cells or (FIG. 10B) using qPCR measurement of dsb copy number.
  • FIG.10C THP-1 cells were transfected with specific or control siRNA to knockdown Notch1/ADAM17/RBPj ⁇ and then infected with E. chaffeensis (1 day post transfection).
  • FIG. 11 ADAM17 and Notch1 interact with TRP120 expressing E. chaffeensis.
  • chaffeensis-infected or uninfected THP-1 cells (48 h) were fixed, permeabilized and probed with (A) anti-TRP120 (green), anti-ADAM17 (red) (B) anti-TRP120 (green), anti- Notch1 (red). ADAM17 and Notch1 protein co-localization with TRP120 was observed.
  • C HeLa cells were transfected with GFP-TRP120 WT or GFP-control plasmids and probed with anti-ADAM17 (red) antibody (24 h post transfection). Direct ADAM17 and TRP120 interaction through co-localization was observed. Cells were visualized by immunofluoresence microscopy (40x; Bars, 10 ⁇ m). DAPI shows DNA, blue.
  • FIGS. 12A-E Activation of Notch signaling pathway by E. chaffeensis TRP120.
  • FIG. 12A THP-1 cells were treated with TRP120 coated beads, fixed, permeabilized, and probed with anti-NICD (red) and DNA (DAPI, blue) then visualized by immunofluoresence microscopy (40x). NICD nuclear translocation after 15 min stimulation with TRP120-coated beads was observed, (Bars, 10 ⁇ m).
  • FIG. 12B Expression levels of notch1, hes1 and hes5 in THP-1 cells were analyzed using real time RT-PCR.
  • RNA was isolated from cells stimulated with TRP120 or thioredoxin coated beads (2 h). mRNA level was normalized to GAPDH and compared with the level of control cells (student’s t test: *p ⁇ 0.05, ** p ⁇ 0.01, n 3). Notch pathway PCR array was performed to analyze gene expression level in THP-1 cells stimulated with TRP120 coated beads compared to thioredoxin control (24 h).
  • FIG. 12C Heat map showing the expression level of Notch signaling genes after TRP120 stimulation. For each gene, the scale bar shows differential expression from the mean gene expression level of thioredoxin stimulated cells. The level of induction (red) or repression (green) are shown. (FIG.
  • FIGS. 13A-D Notch signaling regulates ERK1/2 and p38 MAPK signaling during ehrlichial infection.
  • THP-1 cells were infected with E. chaffeensis (MOI 100, 1 h post treatment) in the presence or absence of Notch inhibitor SAHM1 (10 ⁇ M). Medium or LPS (100 ng/ml) was added at the indicated time and cells were incubated for 30 min.
  • FIGS. 14A-D E. chaffeensis mediated downregulation of PU.1 depends on Notch signaling pathway. THP-1 cells were transfected with either control siRNA or RBPj ⁇ siRNA, incubated for 24 h and then infected with E.
  • FIGS. 15A-D Notch signaling pathway plays critical role in inhibition of TLR2/4 expression during E. chaffeensis infection.
  • THP-1 cells were treated with either vehicle (DMSO), or with Notch inhibitor DAPT and SAHM1 for 1 h then infected with E. chaffeensis.
  • Expression of TLR2 and 4 was measured using RT-PCR (24 h p.i.) and Western blot (48 h p.i.) after 1 h stimulation with LPS (100 ng/ml).
  • FIG. 16A-D TRP120 mediated downregulation of PU.1, TLR2 and TLR4 expression.
  • THP-1 cells were treated with thioredoxin (control) or TRP120 coated latex beads or TRP120 in suspension (1 ⁇ g/ml) for 24 h and stimulated with LPS for 1 h. IFA analysis was done to measure (FIG.16A) PU.1 (FIG.16B) TLR2 and (FIG.16C) TLR4 expression.
  • FIG. 17 Proposed model for E.
  • chaffeensis TRP120 mediated activation of canonical Notch signaling pathway and inhibition of TLR2/4 expression.
  • E. chaffeensis TRP120 effector interaction with ADAM17 activates the metalloprotease resulting in cleavage of the substrate Notch1 and subsequent cleavage by ⁇ -secretase causes (2) nuclear translocation of NICD, the transcriptionally active form which binds with RBPj ⁇ and MAML proteins.
  • This tri-protein complex activates transcription of Notch target genes which causes inhibition of ERK1/2 and p38 MAPK pathway (4).
  • the downstream transcription factor PU.1 expression is repressed, which causes further inhibition of monocyte TLR2/4 expression.
  • Inhibition of TLR2/4 expression causes both inhibition of E. chaffeensis recognition and TLR mediated proinflammatory cytokine production needed for the activation of monocytes and clearance of ehrlichiae.
  • the present invention provides, in some aspects, methods for treating an infection by an obligately intracellular pathogen, such as an obligately intracellular bacteria, in a subject by administering a pharmaceutically effective amount of a Wnt inhibitor or a Notch inhibitor to the subject.
  • an obligately intracellular pathogen such as an obligately intracellular bacteria
  • a Wnt inhibitor or a Notch inhibitor to the subject.
  • inhibition of Wnt signaling in cells by modulating canonical or non-canonical Wnt signaling at a variety of stages in the Wnt signaling pathway can decrease infection of cells by obligately intracellular bacteria such as Ehrlichia (e.g., E. chaffeensis or E. canis).
  • the bacteria is tuberculosis or chlamydia.
  • the present invention provides methods for treating an obligately intracellular bacteria by administering a pharmacologically effective dose of a Notch inhibitor to the subject.
  • the method may comprise administering both a Wnt inhibitor and a Notch inhibitor to treat an obligately intracellular bacteria such as, e.g., E. chaffeensis or E. canis.
  • E. chaffeensis e.g., E. chaffeensis or E. canis.
  • the inventors further analyzed the underlying survival mechanism and showed that E. chaffeensis and TRP120-mediated activation of the Notch pathway causes inhibition of ERK1/2 and p38 MAPK signaling pathways and expression of transcription factor PU.1, which represses TLR2/4 expression.
  • this investigation is the first to demonstrate pathogen exploitation of Notch signaling to modulate PRR expression and to promote intracellular survival. I. Wnt Signaling and Wnt Inhibitors
  • Wnt signaling was first identified for its role in carcinogenesis, but has since been recognized for its central role in embryonic development, differentiation, cell proliferation, cell motility, cell polarity, and adult tissue homeostasis (Klaus and Birchmeier, 2008; Nusse et al., 1984). The importance of Wnt signaling has been demonstrated by mutations that lead to a variety of diseases, including breast and prostate cancer, glioblastoma, type II diabetes, and others ( Komiya and Habas, 2008; Logan and Nusse, 2004). Wnt signaling pathways are highly evolutionarily conserved (Nusse and Varmus, 2012; Nusse and Varmus, 1992).
  • At least three Wnt signaling pathways have been characterized: a canonical Wnt/ ⁇ - catenin pathway and two noncanonical ⁇ -catenin-independent pathways, the Wnt/PCP (planar cell polarity) pathway, and the Wnt/Ca 2+ pathway. All three Wnt signaling pathways are activated by the binding of a Wnt ligand to a transmembrane receptor Frizzled (FZD), which activates the protein Dishevelled (DVL) inside the cell (Habas and Dawid, 2005; Rao and Kuhl, 2010).
  • FZD transmembrane receptor Frizzled
  • Dishevelled recruits the protein complex containing axis inhibitor (Axin), adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase-3 (GSK-3), leading to the inhibition of phosphorylation of ⁇ -catenin by these kinases.
  • Axin axis inhibitor
  • APC adenomatous polyposis coli
  • CK1 casein kinase 1
  • GSK-3 glycogen synthase kinase-3
  • Unphosphorylated ⁇ -catenin accumulates and subsequently translocates to the nucleus, where it associates with TCF/LEF (lymphoid-enhancing factor) family transcription factors to induce the expression of Wnt target genes (Rao and Kuhl, 2010; MacDonald et al., 2009; Staal and clevers, 2000).
  • the noncanonical Wnt/PCP pathway involves the Rho and Rac GTPases, Rho kinase (ROCK), MAP kinases and c-Jun N-terminal kinase (JNK), and regulates cell motility and tissue polarity ( Komiya and Habas, 2008; Gordon and Nusse, 2006).
  • the noncanonical Wnt/Ca 2+ pathway involves stimulation of heterotrimeric G-proteins, which further activates phospholipase C (PLC), leading to increased intracellular Ca 2+ release and activation of calcium/calmodulin-dependent protein kinase II (CaMKII), calcineurin and protein kinase C (PKC) ( Komiya and Habas, 2008).
  • PLC phospholipase C
  • CaMKII calcium/calmodulin-dependent protein kinase II
  • PKC protein kinase C
  • NFAT nuclear factor of activated T-cells
  • CREB cAMP response element-binding protein
  • chaffeensis binding and entry appear to involve one or more glycosylphosphatidylinositol (GPI)-anchored proteins associated with caveolae at the cell surface, inducing receptor-mediated endocytosis that triggers Wnt signaling-like events including transglutamination, tyrosine phosphorylation, phospholipase C ⁇ 2 (PLC- ⁇ 2) activation, inositol-(1,4,5)-trisphosphate (IP 3 ) production and intracellular calcium release (Lin and Rikihisa, 2003; Lin et al., 2002). Moreover, multiple studies have shown the importance of E.
  • GPI glycosylphosphatidylinositol
  • FIG.7 A simplified diagram showing the canonical and non-canonical Wnt pathways is shown in FIG.7.
  • RNAi small interfering RNA
  • antibodies antibodies
  • small molecule Wnt inhibitors may be used to inhibit Wnt signaling.
  • the Wnt inhibitor does not selectively inhibit Wnt5a. It is anticipated that additional Wnt inhibitors that may be subsequently discovered may be used in embodiments of the present invention.
  • Wnt inhibtors may be used to inhibit canonical Wnt signaling to treat infection by an obligately intracellular bacteria such as an Ehrlichia.
  • the Wnt inhibitor may selectively inhibit phosphoinositide 3-kinase (PI3K), Casein kinase 2 (CKII), Casein kinase 1 epsilon (CK1 ⁇ ), ⁇ -catenin/TCF/LEF, or Protein kinase B (AKT), or the Wnt inhibitor may selectively activate casein kinase I ⁇ /GSK3 ⁇ .
  • PI3K phosphoinositide 3-kinase
  • CKII Casein kinase 2
  • CK1 ⁇ Casein kinase 1 epsilon
  • ⁇ -catenin/TCF/LEF ⁇ -catenin/TCF/LEF
  • PKA Protein kinase B
  • pyrvinium pamoate (2-[(E)-2-(2,5-Dimethyl-1- phenylpyrrol-3-yl)ethenyl]-N,N,1-trimethylquinolin-1-ium-6-amine), TBCA ( (E)-3-(2,3,4,5- Tetrabromophenyl)acrylic acid), SB202190 (4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4- pyridyl)1H-imidazole, FHPI), LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4- one), or FH535 (N-(2-Methyl-4-nitro)-2,4-dichlorosulfonamide) may be used to inhibit canonical Wnt signaling.
  • about 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x of the IC50 amount of the Wnt inhibitor may be administered to treat infection by an obligately intracellular bacteria such as an Ehrlichia.
  • an obligately intracellular bacteria such as an Ehrlichia.
  • a Wnt inhibitor may be used to selectively inhibit non- canonical Wnt signaling to treat infection by an obligately intracellular bacteria such as an Ehrlichia.
  • an Wnt inhibitor may be used to selectively inhibit the Wnt/Ca 2+ pathway.
  • the Wnt inhibitor selectively inhibits calmodulin kinase II (CaMKII), I ⁇ B kinase (IKK), or Protein kinase B (AKT).
  • the Wnt inhibitor is KN93 (2-[N-(2-hydroxyethyl)]-N-(4- methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine)).
  • an Wnt inhibitor may be used to selectively inhibit the Wnt/PCP pathway.
  • the Wnt inhibitor selectively inhibits Phosphoinositide 3-kinase (PI3K), Protein kinase B (Akt), or I ⁇ B kinase (IKK).
  • pyrvinium pamoate (2-[(E)-2-(2,5-Dimethyl-1-phenylpyrrol-3-yl)ethenyl]- N,N,1-trimethylquinolin-1-ium-6-amine), LY294002 (2-(4-Morpholinyl)-8-phenyl-4H-1- benzopyran-4-one), or BAY 11-7082 ( (E)3-[(4-Methylphenyl)sulfonyl]-2-propenenitrile) is used to inhibit Wnt signaling.
  • a Wnt inhibitor may be used to selectively inhibit Wnt secretion.
  • a Wnt inhibitor may inhibit Wnt secretion by selectively inhibiting X- chromosomal porcupine homolog (PORCN).
  • the Wnt inhibitor is IWP- 2 (N-(6-Methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2- d]pyrimidin-2-yl)thio]-acetamide).
  • Notch signaling pathway is evolutionarily conserved in eukaryotes and plays important roles in cell proliferation, differentiation, and apoptosis, thereby influencing cell fate (Fortini 2012; Artavanis-Tsakonas et al., 1999; Hoyne 2003, Radtke et al., 2013).
  • Three proteolytic cleavage steps are essential for the production of fully functional Notch receptor signaling. The first occurs at site 1 (S1) by furin in the trans-golgi (Sasamura et al., 2003; Shi and Stanley 2003), resulting in translocation of the heterodimer to the cell surface.
  • the canonical Notch pathway is activated when the extracellular domain of Notch receptor (NECD) binds to the ligand (DLL1, 3, 4 and Jagged 1, 2) expressed on the membrane of neighboring cells.
  • NECD Notch receptor
  • DLL1, 3, 4 and Jagged 1, 2 expressed on the membrane of neighboring cells.
  • S2 site 2
  • S3 cleavage ⁇ -secretase enzyme
  • NICD translocates to the nucleus where it forms a tri- protein complex with DNA-binding transcription factor RBPj ⁇ (CSL) and transcriptional coactivator Mastermind (MAM), activating Notch target gene transcription (Barrick and Kopan 2006; Kovall 2007).
  • CSL DNA-binding transcription factor
  • MAM transcriptional coactivator Mastermind
  • TRP120 interacts with the ADAM17 metalloprotease (Luo et al., 2011), and also acts as a nucleomodulin, binding target genes associated with the Notch signaling pathway, including notch1 (Zhu et al., 2011).
  • notch1 Zhu et al., 2011
  • Notch inhibitors may be used to treat an obligately intracellular pathogen such as, e.g., Ehrlichia, Chlamydia trachomatis, or Mycobacterium tuberculosis.
  • the Notch inhibitor selectively inhibits ⁇ -secretase, Notch transcription factor complex, Notch1, ADAM17, or ADAM10.
  • the Notch inhibitor is a ⁇ -secretase inhibitor, such as:
  • a blocking peptide e.g., MAM peptide agonist SAHM1
  • an antibody fragment or a neutralizing antibody e.g., OMP-59R5 (anti-Notch 2/3 mAb), NRR1 anti-Notch1 mAB, NRR2 anti-Notch2 mAb, NRR3 anti-Notch3 mAb, OMP-21M18 anti-DLL4 mAb, DLL1-Fc and JAG1 Fc anti-delta like 1 and jagged 1 Fc chimeric mAbs, A5622A anti-nicastrin mAb), or a Notch decoy (e.g., a soluble Notch1, Dll1, jagged1).
  • Additional Notch inhibitors are also described in Espinoza and Miele (2013) that may be used in various aspects of the present invention, e.g., to treat an infection by an obligately intracellular bacteria.
  • Obligately intracellular pathogens are capable of growing and reproducing inside the cells of a host.
  • Obligately intracellular pathogens include bacteria, protozoa, and fungi. Generally, obligately intracellular pathogens cannot reproduce outside their host cell, and the reproduction is primarily or entirely reliant on intracellular host resources.
  • Obligately intracellular bacteria include Ehrlichia bacteria, such as Ehrlichia chaffeensis and Ehrlichia canis.
  • Ehrlichiosis is a bacterial illness resulting from infection of a human or dog host by Ehrlichia chaffeensis or Ehrlichia canis, respectively. Ehrlichiosis is transmitted by ticks, and typically results in flu-like symptoms.
  • Ehrlichia chaffeensis is an obligately intracellular bacterium responsible for the life-threatening tick transmitted zoonosis, human monocytotropic ehrlichiosis. E. chaffeensis invades and survives in mononuclear phagocytes by modulating cell processes and evading host defenses, but the mechanisms are not fully defined. Recently it has been observed that E.
  • chaffeensis tandem repeat proteins are type 1 secreted effectors involved in functionally diverse interactions with host targets, including components of the evolutionarily conserved canonical and non-canonical Wnt signaling pathways.
  • Other obligately intracellular bacteria that may be treated with a Wnt inhibitor in various embodiments include, e.g., Chlamydia, Rickettsia, Coxiella, certain Mycobacterium such as Mycobacterium leprae, Mycobacterium tuberculosis.
  • a Wnt inhibitor may be used to treat an obligately intracellular protozoa.
  • Obligately intracellular protozoa include, e.g., Apicomplexans (e.g., Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum), Trypanosomatids (e.g., Leishmania spp., Trypanosoma cruzi).
  • Apicomplexans e.g., Plasmodium spp., Toxoplasma gondii, Cryptosporidium parvum
  • Trypanosomatids e.g., Leishmania spp., Trypanosoma cruzi
  • a Wnt inhibitor may be used to treat an obligately intracellular fungi.
  • Obligately intracellular fungi include, e.g., Pneumocystis jirovecii, and Histoplasma capsulatum.
  • compositions or pharmaceutical preparations of the present invention comprise an effective amount of one or more compounds of the present invention, e.g., a Wnt inhibitor, or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that contains at least one compound or Wnt inhibitor or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21 st Ed.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference).
  • preservatives e.g., antibacterial agents, antifungal agents
  • isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like
  • the compound, Wnt inhibitor, or Notch inhibitor of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18 th Ed.
  • the compound, Wnt inhibitor, or Notch inhibitor of the present invention may be formulated into a composition in a free base, neutral or salt form.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as formulated for parenteral administrations such as injectable solutions, or aerosols for delivery to the lungs, or formulated for alimentary administrations such as drug release capsules and the like.
  • the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semi-solid (e.g., pastes) or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • the composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • various antibacterial and antifungal agents including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification,
  • the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, e.g., denaturation in the stomach.
  • stabilizers for use in an the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc.
  • the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include a compound or Wnt inhibitor of the present invention, one or more lipids, and an aqueous solvent.
  • lipid will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term“lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • neutral fats phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • lipids are also encompassed by the compositions and methods of the present invention.
  • One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle.
  • the compound or Wnt inhibitor of the present invention may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • compositions of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. [0066] In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound.
  • the an active compound may comprise between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound.
  • Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • about 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x of the IC 50 amount of the Wnt inhibitor or Notch inhibitor may be administered to treat infection by an obligately intracellular bacteria such as an Ehrlichia.
  • a Wnt inhibitor or Notch inhibitor can be formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • these compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each specifically incorporated herein by reference in its entirety).
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as, for
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present invention may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one containing sodium borate, glycerin and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically- effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • Additional formulations which are suitable for other modes of alimentary administration include suppositories.
  • Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • a compound, Wnt inhibitor, or Notch inhibitor of the present invention may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered for example, but not limited to intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils.
  • a coating such as lecithin
  • surfactants for example
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035- 1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • the active compound, Wnt inhibitor, or Notch inhibitor may be formulated for administration via various miscellaneous routes, for example, topical or transdermal administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
  • Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder. Ointments include all oleaginous, adsorption, emulsion and water-solubly based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin.
  • Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also comprise the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
  • the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos.5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Likewise, the delivery of drugs using intranasal microparticle resins (Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat.
  • aerosol refers to a colloidal system of finely divided solid of liquid particles dispersed in a liquefied or pressurized gas propellant.
  • the typical aerosols that may be used can include a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • Administration of the aerosol will vary according to subject’s age, weight and the severity and response of the symptoms. V. Examples
  • InhibitorSelect Wnt signaling pathway inhibitor panel was purchased from Calbiochem/EMD (Billerica, MA).
  • This panel contains 15 potent and selective inhibitors for the study of the Wnt signaling pathways: casein kinase I inhibitor D4476, casein kinase II inhibitor III TBCA, ⁇ -catenin/TCF inhibitor FH535, GSK-3 inhibitor IX BIO, protein kinase G I ⁇ inhibitor DT3, protein kinase A inhibitor H89 dihydrochloride, JNK inhibitor II SP600125, protein kinase inhibitor K252a Nocardiopsis sp., CaM kinase II inhibitor KN93, PI3 kinase inhibitor LY294002, TAK1 inhibitor (5Z)-7- oxozeaenol Curvularia sp., Src family kinase inhibitor PP2, mTOR inhibitor rapamycin, casein kinase I ⁇ / ⁇ and p38 MAP kinase inhibitor SB202190, and MEK1/2 inhibitor U0126.
  • Wnt signaling inhibitors included casein kinase I ⁇ /GSK3 activator (Akt/PKB inhibitor) pyrvinium pamoate salt hydrate and inhibitor of Wnt production II IWP-2 (Sigma).
  • Akt/PKB inhibitor casein kinase I ⁇ /GSK3 activator
  • IWP-2 Wnt production II IWP-2
  • MCD methyl- ⁇ –cyclodextrin
  • OGP n-octyl- ⁇ –D-glucopyranoside
  • BAY11-7082 were from Sigma.
  • Rabbit or mouse anti-TRP32 antibodies have been described previously.
  • mice anti-human ⁇ -tubulin were mouse anti-human ⁇ -tubulin, NFATC1 (Santa Cruz Biotechnology, Santa Cruz, CA) and ⁇ -catenin (Pierce, Rockford, IL) and rabbit anti-human FZD9 (Pierce), JUN (Santa Cruz), DVL2 and GSK3 ⁇ (Cell signaling, Beverly, MA).
  • siRNAs of human DKK3, DVL2, FZD9, JUN, NFATC1 (NFAT2), NFATC3 (NFAT4), PP2B-A ⁇ (Calcineurin PPP3CA), PP2B-A ⁇ (Calcineurin PPP3CB), TCF4, WNT10A, WNT6, and control-A were purchased from Santa Cruz.
  • PCR array The RT2 Profiler PCR arrays (version 4.0; SABiosciences, Valencia, CA) were used, including human Wnt signaling pathway plus PCR array and human Wnt signaling targets PCR array (see SABiosciences website for gene list and functional gene grouping).
  • the human Wnt signaling pathway plus PCR array profiles the expression of 84 genes related to Wnt-mediated signal transduction, including Wnt signaling ligands, receptors and regulators as well as downstream signaling molecules and target proteins for all three Wnt pathways, and uses experimentally derived signature biomarker genes along with classification algorithms to generate the pathway activity score and determines whether Wnt pathway activity is activated or repressed in experimental samples.
  • the human Wnt signaling targets PCR array profiles the expression of 84 key genes responsive to Wnt signal transduction, including Wnt signaling pathway transcription factors and highly relevant target genes identified by multiple studies, and can be used to analyze activation or inhibition of Wnt signaling. PCR arrays were performed according to the PCR array handbook from the manufacturer.
  • RNA FlashGel RNA FlashGel
  • Real-time PCR was performed using RT 2 Profiler PCR array in combination with RT 2 SYBR Green mastermix (Qiagen) on a Mastercycler EP Realplex 2 S (Eppendorf, Germany). Cycling conditions were as follows: 95°C for 10 min and 40 cycles of 95°C for 15 s, 60°C for 1 min.
  • the real-time cycler software RealPlex 1.5 (Eppendorf) was used for PCR and data collection. The baseline was set automatically, the threshold was defined manually, and then the threshold cycle (CT) for each well was calculated by RealPlex.
  • the threshold was set in the proper location and at the same level for all PCR arrays in the same analysis so that the values of the positive PCR control (PPC) assays on all arrays were between 18 C T and 22 C T .
  • the C T values for all wells were exported for analysis using Web- based PCR array data analysis software (version 3.5; SABiosciences). PCR array quality checks were performed by the software before data analysis, including PCR array reproducibility, reverse transcription efficiency control, genomic DNA contamination control and positive PCR control.
  • RNA interference THP-1 cells (1 ⁇ 10 5 /well on a 96-well plate) were transfected with 5 pmol human siRNA using Lipofectamine 2000 (Invitrogen).
  • a control-A siRNA consisting of a scrambled sequence was used as a negative control, and an Alexa Fluor 488-labeled negative siRNA was used as a control to monitor transfection efficiency.
  • MOI multiplicity of infection
  • the cells were infected by cell-free E. chaffeensis at a multiplicity of infection (MOI) of ⁇ 50. Then, the cells were collected at 1 day and 2 days p.i., and subjected to quantitative PCR (qPCR) and Western blot. [0085] Quantification of E. chaffeensis by qPCR.
  • Treated THP-1 cells were pelleted, washed by PBS, lysed in SideStep lysis and stabilization buffer (Agilent, Santa Clara, CA) for 30 min at room temp, and analyzed for bacterial load using realtime qPCR.
  • Amplification of the integral ehrlichial gene dsb was performed using Brilliant II SYBR Green mastermix (Agilent), 200 nM forward primer (5'-gctgctccaccaataaatgtatccct-3') and 200 nM reverse primer (5'-gtttcattagccaagaattccgacact-3').
  • the qPCR thermal cycling protocol (denaturation at 95°C 10 min, then 40 cycles of 95°C 30 s, 58°C 1 min, 72°C 1 min) was performed on the Mastercycler EP Realplex 2 S (Eppendorf).
  • a standard plasmid pBAD-dsb was constructed by cloning the ehrlichial dsb gene using the TOPO TA cloning kit (Invitrogen).
  • chaffeensis dsb copy number in the cells was determined against the standard curve or the fold change of dsb copy number relative to the control was normalized to qPCR-detected levels of the host genomic glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene.
  • gapdh host genomic glyceraldehyde-3-phosphate dehydrogenase
  • Western immunoblot The THP-1 cell lysates were prepared using CytoBuster protein extraction reagent (Novagen/EMD, Gibbstown, NJ), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose.
  • THP-1 cells were plated in FBS-free medium and treated with different concentrations of inhibitor or DMSO control, and then infected with cell-free E. chaffeensis at a multiplicity of infection (MOI) of ⁇ 50. Percentages of infected cells were monitored daily over 3 days by Diff-Quik staining and counting of 100 cells.
  • MOI multiplicity of infection
  • THP-1 cells were collected, and the indirect immunofluorescent antibody assay was performed as previously described, except that anti- ⁇ -catenin or NFATC1 antibody (1:100) and anti- TRP32 antibody (1:10,000) were used.
  • TRP E. chaffeensis tandem repeat proteins
  • FluoSpheres® sulfate microspheres (1.0 ⁇ m, yellow- green fluorescent; Invitrogen) (10 ⁇ l, ⁇ 3.6 ⁇ 10 8 beads) were washed by 40 mM 2-(N- morpholino)ethanesulfonic acid (MES) buffer, pH 6.1, and then incubated with 15 ⁇ g of TRP protein in 500 ⁇ l MES buffer at room temperature for 1 h with mixing at 20 rpm. The coated beads were collected by centrifugation, washed twice in MES buffer and resuspended in RPMI medium, then gently sonicated to disperse the beads. Protein coating of the beads were confirmed by dot blot assay.
  • MES 2-(N- morpholino)ethanesulfonic acid
  • TRP-coated or control protein-coated beads were added to THP- 1 cells at a multiplicity of approximately 50 beads per cell and incubated for 2 h at 37°C with 5% CO2. Unbound beads were removed by washing and low-speed centrifugation for three times, and then cells were collected by cytospin onto the slide and fixed with 3% paraformaldehyde to observe internalized beads. For estimating inhibition of phagocytosis, designated inhibitors were added 2 h before addition of coated beads.
  • FIG.1 shows the expression levels of seven important host genes at different time points p.i. by Wnt signaling pathway PCR array.
  • Wnt signaling target proteins include development and differentiation, calcium binding and signaling, adhesion, migration, cell cycle, proteolysis, signal transduction, and transcription factors, which are associated with major biological processes of host cells.
  • FIG. 2 shows the expression levels of six host target genes at different time points p.i. by Wnt signaling target PCR array, including cyclin D1 (CCND1), fibroblast growth factor 9 (FGF9), fibronectin 1 (FN1), MET proto-oncogene (MET), matrix metallopeptidase 2 (MMP2), and secreted frizzled-related protein 2 (SFRP2).
  • CCND1 cyclin D1
  • FGF9 fibroblast growth factor 9
  • FN1 fibronectin 1
  • MET MET proto-oncogene
  • MMP2 matrix metallopeptidase 2
  • SFRP2 secreted frizzled-related protein 2
  • Wnt signaling pathway components influences ehrlichial infection of macrophages.
  • the role of host Wnt signaling pathways in ehrlichial infection was further confirmed using RNA interference.
  • 16 siRNAs were used, including those of some important components of Wnt signaling pathways, such as Wnt ligand, receptor and co-receptor, regulator, transcription factor and target (FIG. 3A).
  • the decrease of most target proteins (12 proteins at 1 day p.i. and 11 proteins at 2 days p.i.) influenced E.
  • 3B shows examples that protein expression of four target genes FZD9, DVL2, GSK3 ⁇ and JUN was reduced in specific siRNA- transfected cells, respectively, compared with the unrelated control siRNA-transfected cells.
  • Small molecule inhibitors of Wnt signaling pathways repress E. chaffeensis infection of host cells. To confirm the role of host Wnt signaling pathways in E.
  • chaffeensis infection a large panel of 17 Wnt signaling pathway inhibitors was examined on ehrlichial infection of host cells, and five inhibitors were found to have significant impact on ehrlichial infection but without apparent toxicity to host cells, including casein kinase I ⁇ /GSK3 activator (Akt/PKB inhibitor) pyrvinium pamoate, CaM kinase II inhibitor KN93, inhibitor of Wnt production II IWP-2, casein kinase II inhibitor III TBCA, and casein kinase I ⁇ / ⁇ inhibitor SB202190 (FIG.4A and FIG.4B). Pyrvinium, KN93 and IWP-2 were highly potent inhibitors of ehrlichial infection, and could block the infection almost completely.
  • Akt/PKB inhibitor casein kinase I ⁇ /GSK3 activator
  • Two inhibitors TBCA and SB202190 reduced ehrlichial infection significantly.
  • two other inhibitors ⁇ -catenin/TCF inhibitor FH535 and PI3 kinase inhibitor LY294002 could influence ehrlichial infection but showed some toxicity to host cells.
  • three most potent inhibitors pyrvinium, KN93 and IWP-2 target the canonical Wnt pathway, the noncanonical Wnt/Ca 2+ pathway and Wnt production, respectively, indicating that the importance of both canonical and noncanonical Wnt signaling pathways in ehrlichial infection.
  • MICs of pyrvinium, KN93 and IWP-2 for ehrlichial infection were determined to be 20 nM, 4 ⁇ M, and 0.3 ⁇ M, respectively, by using two-fold serial dilutions.
  • Host ⁇ -catenin and NFATC1 proteins translocate to the nucleus after E. chaffeensis infection.
  • ⁇ -catenin and NFATC1 are important nuclear factors involved in Wnt/ ⁇ -catenin and Wnt/Ca 2+ pathways, respectively; thus, the localization of these two proteins after infection was examined using immunofluorescence microscopy. Remarkable redistribution of ⁇ -catenin and NFATC1 proteins in E.
  • ⁇ -catenin was diffusely distributed mainly in the cytoplasm and associated with cell membrane, but in E. chaffeensis-infected cells at 3 h p.i., ⁇ -catenin accumulated and was punctately distributed mainly in the nucleus (FIG.5A).
  • NFATC1 was diffusely distributed mainly in the cytoplasm of uninfected THP-1 cells, but translocated to the nucleus as early as 1 h p.i. of E.
  • E. chaffeensis TRP120 interacts with host targets involved in Wnt signaling that influence infection. Since E. chaffeensis TRPs have been identified as bacterial effector proteins and interact with multiple host proteins involved in Wnt signaling, the role of these host proteins in ehrlichial infection was confirmed using RNA interference.
  • TRP120-interacting host proteins Similar to the previous result of TRP32-interacting protein DAZAP2, knockdown of five TRP120-interacting host proteins, including ARID1B, KDM6B, IRF2BP2, PPP3R1 and VPS29, influenced ehrlichial infection of macrophages significantly (Table 2).
  • the bacterial load in all specific siRNA-transfected cells decreased at both 1 day and 2 days p.i. (fold regulation ⁇ -2), except that the bacterial load in ARID1B siRNA-transfected cells increased at 1 day p.i.
  • ARID1B is an AT-rich DNA interacting domain-containing protein and a component of the SWI/SNF chromatin remodeling complex and has been reported to interact with ⁇ -catenin to suppress Wnt signaling.
  • E. chaffeensis tandem repeat proteins stimulate phagocytosis of macrophages and Wnt pathway inhibitors abolished the stimulation. Recently Wnt signaling pathway has been demonstrated to stimulate phagocytosis but not bacterial killing. Ehrlichial tandem repeat proteins were observed to stimulate the internalization of latex beads by macrophages.
  • FIG. 6A and FIG. 6B show that TRP120-coated latex beads were phagocytosed by the THP-1 cell dramatically compared with the control protein TRP120 N- terminal region only (TRP120N)-coated latex beads.
  • FIG. 6C shows that TRP120 could not promote the internalization of latex beads by the THP-1 cell after treatment with CKI ⁇ /GSK3 ⁇ activator (Akt/protein kinase B inhibitor) pyrvinium (30 nM).
  • CKI ⁇ /GSK3 ⁇ activator Akt/protein kinase B inhibitor
  • LY94002 Some other Wnt pathway inhibitors, including CaMKII inhibitor KN93 and PI3K inhibitor LY94002, also blocked TRP-stimulated phagocytosis.
  • IWP-2 did not inhibit the phagocytosis mediated by TRP.
  • TRP-mediated phagocytosis by the lipid raft disrupting agents MCD and OGP and the I ⁇ B kinase inhibitor BAY11-7082.
  • chaffeensis replication occurs in a 72 h life cycle.
  • the developmental cycle starts with the dense-cored cells (DC), which attaches to and enters into the host cell (Zhang et al., 2007) .
  • the DC dense-cored cells
  • RC reticulate cell
  • the mature DCs are released and start a new cycle of infection.
  • the general entry time of DC into the host cell is about 1 h to 3 h (Popov et al., 2000; Zhang et al., 2007), but without synchronization of bacteria and cells, the internalization may take longer. So these time point results indicates that host Wnt signaling pathways may be utilized by Ehrlichia for its entry, transformation, and exit. Ehrlichia may activate host Wnt signaling for the entry and transformation, and suppress the Wnt signaling for the exit. Comparison of pathway activity scores and P values of 3h and 72 h suggests that regulation of Wnt signaling at 3 h p.i. is more significant than that at 72 h.
  • the pathway activity score determined by PCR array was calculated by a data analysis software and represents an overall activity of the pathway, which may reflect a balance of regulations of multiple components. Different components may be regulated by Ehrlichia at different time points in a different way.
  • the pathway activities determined by PCR array are based on canonical Wnt signaling pathway due to its good documentation, but Wnt signaling pathway PCR array does include many components from other two noncanonical Wnt pathways.
  • Genes with significant changes of expression after infection include ligand, receptor, inhibitor, signaling molecule, transcription factor and target of Wnt signaling pathways and are involved in both canonical and noncanonical Wnt pathways, e.g., ligands WNT6 and WNT7b are also included in the Wnt/Ca 2+ pathway (Schmidt et al., 2007; Zhang et al., 2007), and receptor FZD5 and target VANGL2 (Vang-like 2) are involved in the regulation of planar cell polarity (Croce et al., 2006; Shafer et al., 2011) (Table S1).
  • Wnt signaling pathways include Wnt ligand, receptor and co-receptor, signaling molecule, transcription factor and target as well as TRP120-interacting proteins.
  • Wnt signaling pathway inhibitors reduce ehrlichial infection significantly, three of which, pyrvinium pamoate, KN93 and IWP- 2 were highly potent.
  • KN93 selectively binds to the CaM binding site of CaM kinase II and prevents the association of CaM with CaM kinase II (Sumi et al., 1991); IWP-2 inhibits the cellular Wnt processing and secretion via selective blockage of Porcn-mediated Wnt palmitoylation (Chen et al., 2009).
  • FIG. 7 illustrates canonical and noncanonical Wnt signaling pathways, potent inhibitors and involved TRP-interacting proteins.
  • Phagocytosis is a specific form of endocytosis and a major mechanism used to remove pathogens in the immune system. Usually the pathogen becomes trapped in a phagosome which then fuses with a lysosome to form a phagolysosome, but E. chaffeensis enters the monocyte through lipid raft-caveolae-mediated endocytosis and then it can reside within a cytoplasmic vacuole that resembles an early endosome and does not fuse with lysosomes, protecting it from killing. However, the underlying molecular mechanism remains unclear.
  • PI3 kinase and I ⁇ B kinase as well as lipid rafts play important roles in TRP-induced phagocytosis and infection.
  • Activation of PI3 kinase and I ⁇ B kinase might stimulate the assembly of scavenger receptors through lipid raft and supports cytoskeletal rearrangements for enhanced internalization (Lafont et al., 2005; Seveau et al., 2007; Yin and Janmey, 2003).
  • Akt/protein kinase B and CaM kinase II are also involved.
  • TRP-induced phagocytosis might occur through lipid raft as a noncanonical mode of Wnt signaling, similar to Wnt5a-induced phagocytosis.
  • Wnt production inhibitor IWP-2 could not inhibit the phagocytosis mediated by TRP, suggesting that Wnt ligand secretion/recycling may be involved in the ehrlichial infection but not in TRP-induced phagocytosis, or the phagocytosis is either Wnt ligand-independent or IWP-2-resistant Wnt ligand-dependent, such as WNT3a, since IWP-2 was found to have a much more significant effect on Wnt5a secretion than on Wnt3a secretion (Maiti et al., 2012).
  • Example 3 It is envisioned that one or more of the above experiments may be performed for other obligately intracellular bacteria such as, e.g., Chlamydia, Rickettsia, Orientia, and Mycobacteria, and it is envisioned that similar effects including, e.g., inhibition of phagocytosis and/or reduction of bacterial load, may be observed regarding these and other obligately intracellular bacteria.
  • obligately intracellular bacteria such as, e.g., Chlamydia, Rickettsia, Orientia, and Mycobacteria
  • E. chaffeensis Human monocytic leukemia cells (THP-1) were propagated in RPMI medium 1640 with L-glutamine and 25 mM HEPES buffer (Invitrogen), supplemented with 1 mM sodium pyruvate (Sigma, St. Louis, MO), 2.5 g/L D-(+)-glucose (Sigma), and 10% fetal bovine serum at 37 ⁇ C in a 5% CO2 atmosphere.
  • E. chaffeensis (Arkansas strain) was cultivated in THP-1 cells as previously described (8).
  • antibodies that were used in this study include anti- Hes1 (ab71559) (Abcam, Cambridge, MA), anti-Notch1 (C-20), anti-cleaved Notch1 (m1711), anti- ⁇ tubulin (B7), anti-TLR2 (TL2.1), anti-TLR4 (15), anti-PU.1 (A-7) (Santa Cruz Biotechnology, NY), anti-ADAM17 (TACE, D22H4), anti-Notch1 (D1E11), anti-RBPSUH (D10A4) (Cell signaling Technology, Inc) and anti-GAPDH (clone 6C5, EMD Millipore, CA).
  • ⁇ -secretase inhibitor IX named DAPT or N-[N-(3, 5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t- Butyl Ester (Calbiochem, Canada), and Notch transcription factor inhibitor SAHM1 (Calbiochem, Canada).
  • siRNAs and transfection were used: ⁇ -secretase inhibitor IX named DAPT or N-[N-(3, 5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t- Butyl Ester (Calbiochem, Canada), and Notch transcription factor inhibitor SAHM1 (Calbiochem, Canada).
  • THP-1 cells (1 ⁇ 10 5 /well on a 96-well plate) were transfected with siRNA for TACE (ADAM17), RBP-j ⁇ or Notch1 (Santa Cruz Biotechnology, NY) using the lipofectamine 2000 reagent (Life Technologies, CA) according to the manufacturer’s instructions with a cocktail of 5 picomole siRNA or a negative control siRNA (Santa Cruz Biotechnology, NY).
  • RT-PCR Total RNA from E. chaffeensis infected, TRP 120 or thioredoxin stimulated, and control THP-1 cells, was isolated using an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions.
  • cDNA was synthesized from 1 ⁇ g of total RNA using a qScript cDNA SuperMix kit (Quanta Biosciences). Gene expression level of target host genes were quantitated by qPCR using brilliant II SYBR green qPCR master mix (Agilent Technologies) with gene-specific primers and a thermal cycling protocol consisting of an initial denaturation step of 95°C for 10 min and 40 cycles of 95°C for 30 s, 58°C for 1:00 m, and 72°C for 30 s. Gene expression values were calculated on the basis of the 2 - ⁇ CT method and normalized with GAPDH.
  • chaffeensis serum (1:100), goat anti-NICD (1:50), mouse anti-ADAM17 (1:50), goat anti- Notch1 (1:50), rabbit anti-Hes1 (1:50), mouse anti-TLR2 (1:50, mouse anti-TLR4 (1:50) and mouse anti-PU.1 (1:50) for 1 h, washed and incubated with Alexa Fluor 488 IgG (H+L) and Alexa Fluor 568 IgG (H+L) secondary antibodies (1:100, Molecular probes) for 30 min. Slides were mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) after washing.
  • HeLa cells transfected with the GFP-TRP120 plasmids or GFP-control plasmids were fixed in chamber slides, permeabilized and stained with anti-ADAM17 using the same protocol. Images were obtained using Olympus BX61 epifluorescence microscope and analyzed using Slidebook software (version 5.0; Intelligent Imaging Innovations, Denver, CO). [00112] Pharmacological inhibitor treatment and determination of bacterial load. THP-1 cells were treated with DAPT, SAHM1 or DMSO and incubated for at least 1 h and then infected with cell free E. chaffeensis at a multiplicity of infection (MOI) of 50.
  • MOI multiplicity of infection
  • chaffeensis dsb copy number was determined using a standard curve and was normalized to qPCR-detected levels of the host genomic gapdh gene. To confirm host cell death did not account for decreased ehrlichial inclusions, differences in cell viability were assessed at day 1, 2 and 3 p.i. using trypan blue staining.
  • Western immunoblot THP-1 cells infected with E. chaffeensis in the presence of DMSO or Notch inhibitor SAHM1, and uninfected cells were harvested after 1 and 2 days p.i. and LPS stimulation (100 ng/ml for 1 h). Cell lysates were prepared as previously described (Wakeel et al., 2011).
  • mice anti-ADAM17 rabbit anti- Hes1, mouse anti- ⁇ -tubulin, mouse anti-PU.1, mouse anti-TLR4, mouse anti-TLR2 and mouse anti-GAPDH were used.
  • horseradish peroxidase-labeled goat anti-rabbit, or mouse IgG (heavy and light chains) conjugate was used.
  • E. chaffeensis TRP120 protein (thioredoxin-fused) was expressed and purified as described previously (Doyle et al., 2011; Luo et al., 2008; Luo et al., 2009). Purified proteins were desalted (Zeba Spin desalting column, Thermo Scientific) to change the buffer to 40 mM MES [2-(N-morpholino) ethanesulfonic acid].
  • Recombinant purified TRP120 or thioredoxin were coated on FluoroSpheres sulfate microsphere beads (1.0 ⁇ m, yellow-green fluorescent; Invitrogen) using the following protocol. Briefly, 10 ⁇ l ( ⁇ 3.6 ⁇ 10 8 beads) of beads were washed two times with 10 volumes of 40 mM MES buffer (5000g for 5 min), re-suspended in 10 ⁇ g of TRP120 desalted protein in 500 ⁇ l MES buffer and incubated at RT for 2 h in a rotor. After incubation beads were washed twice with 500 ⁇ l MES buffer (10,000g for 8 min) and re-suspended in RPMI media.
  • TRP120 or thioredoxin-coated beads were used to treat THP-1 cells for different time points, and the cells were incubated at 37oC with 5% CO 2 . After incubation unbound beads were washed by centrifuging at least 4 times at 400g.
  • Bio-Plex The level of total and phosphorylated ERK1/2 and p38MAPK proteins in THP-1 cells infected with E. chaffeensis in the presence and absence of Notch inhibitor, SAHM1 (10 ⁇ M) and with or without LPS stimulation (100 ng/ml) were measured using Luminex array (Millipore, Beillerica, MA) according to manufacturer’s instructions.
  • chaffeensis TRP120 interacts with many different cellular targets at different times during infection, including interaction with ADAM17 and binding to the promoter region of notch1 (Zhu et al., 2011; Luo et al., 2011). Since E. chaffeensis interacts with a component of the Notch signaling receptor complex the inventors sought to investigate whether E. chaffeensis might somehow alter the function of this pathway. Activation of Notch receptor following interaction with its ligand and proteolytic cleavage by the ADAM17 and ⁇ -secretase enzyme involves nuclear translocation of NICD (Brou et al., 2000; Wolfe and Kopan 2004).
  • NICD translocation to the nucleus was observed within 2 h of E. chaffeensis infection (FIG. 8A). Since nuclear translocation of NICD results in activation of specific Notch target genes (Barrick and Kopan 2006; Kovall 2007), next the expression of different Notch signaling components and target genes were examined in E. chaffeensis infected cells.
  • Notch target genes are the families of basic helix-loop-helix proteins, hairy and enhancer of split (Hes) and hairy and enhancer of split with YRPW motif (Hey) (Fisher and Gessler 2007). These DNA binding proteins function as transcriptional repressors and are the primary effectors of Notch signaling.
  • RT-PCR data showed notch1, hes1, hes5 and hey2 mRNA expression was significantly increased as early as 2 h p.i., reaching a maximum at 72 h p.i. (FIG. 8B). Consistent with RT-PCR data, increased expression of Hes1 and ADAM17 protein was also observed by Western immunoblot after 2 days p.i.
  • Heat maps were constructed depicting the differential expression of the Notch signaling pathway genes in the E. chaffeensis infected and uninfected cells (FIG.9A). The intensities of the red and green in the heat map represents the level of induction and repression, respectively.
  • PCR array data identified activation of canonical Notch signaling pathway by E. chaffeensis at 12 h, 24 h, 48 h and 72 h p.i.
  • the expression patterns of genes that were consistently upregulated throughout all different time points included Notch target genes: hes1, hey2, NF ⁇ B1, NF ⁇ B2, nr4a2, pax5, fosl1, chuk and ccne1; Notch pathway component genes, e.g.
  • notch1 receptor
  • dll4 ligand
  • maml2 transcription complex protein
  • the transcription factor rbpj ⁇ and E3 ubiquitin ligase dtx1 which play important roles in Notch pathway activation and regulation were upregulated at 48 and 72 h p.i. Only a small percentage of genes were downregulated during the infection, including genes for the Notch pathway components dll1 and mmp7 (FIG.9A).
  • FIG.9B the scatter plot shows the comparison of the normalized expression of all genes in the Notch PCR array between infected and uninfected cells.
  • the central line indicates unchanged gene expression (2-fold regulation cut-off), the red dots represent the genes which were upregulated, the green dots represent genes that were downregulated, and the black dots represent genes with no significant difference in expression level.
  • the gene expression pattern was similar throughout the different time points, maximum changes in Notch gene expression occurred at 24 h p.i.
  • 38 genes showed significant differential expression (p ⁇ 0.05), including 28 (33.33 %) that were up-regulated and 10 (11.90 %) were down-regulated (FIG. 9C).
  • Notch signaling is required not only for the cell growth and proliferation, but also plays important role in determining the fate of mature immune cells (Hoyne 2003; Radtke et al., 2013). Since Notch pathway regulates both innate and adaptive immune responses, and this pathway is activated during E. chaffeensis infection, the role of this pathway in ehrlichial survival was examined. To that end, cells were treated with Notch signaling transcription complex inhibitor SAHM1 and ⁇ -secretase inhibitor DAPT. SAHM1 is a cell permeable small peptide that targets critical protein-protein interaction in Notch transcription complex and prevents their assembly (Moellering et al., 2009).
  • Gamma-secretase inhibitor DAPT is a dipeptide and targets the C terminal fragment of presenilin that is a component of ⁇ -secretase protein (Morohasi et al., 2006 ).
  • THP-1 cells were treated with different concentrations of SAHM1 (1 ⁇ M, 5 ⁇ M and 10 ⁇ M) and DAPT (0.5 ⁇ g, 1 ⁇ g and 5 ⁇ g/ml) and cells were infected with cell free E. chaffeensis (at MOI 50).
  • a dose dependent effect of both of inhibitors on bacterial load was observed as percent of ehrlichiae infected cells was determined using Diff- Quik staining.
  • siRNAs were used to knock-down expression of the receptor Notch1, metalloprotease ADAM17 and transcription factor RBPj ⁇ in THP-1 cells, then infected them with E. chaffeensis. These components were selected since they play critical role in canonical Notch signal transduction. Bacterial load was measured using real time qPCR by amplification of the integral ehrlichial gene dsb. Consistent with Notch pathway inhibitor experiments, significant reduction of bacterial load was also found in cells in which Notch component genes were knocked down (FIG.10C). Protein expression of Notch1, ADAM17 and RBPj ⁇ was reduced in siRNA-transfected cells compared with the control siRNA-transfected cells (FIG. 10D).
  • E. chaffeensis TRP120 protein interacts with Notch receptor complex.
  • E. chaffeensis activates canonical Notch signaling, which is requisite for survival.
  • the mechanism of Ehrlichia-induced Notch activation remains undefined.
  • E. chaffeensis TRP effectors are the major immunoprotective proteins, and contains species specific epitopes (Gooz 2010).
  • TRP120 functions as an adhesin, facilitating ehrlichial entry and is a nucleomodulin (Popov et al., 2000).
  • the Y2H data showed TRP120 interacts with ADAM17 and binds to the promoter region of notch1 (Zhu et al., 2011; Luo et al., 2011).
  • cells were stained with anti-ADAM17 and anti-TRP120 antibody.
  • FIG. 11A Immunofluorescence microscopy showed diffused cytoplasmic localization of ADAM17 in uninfected THP-1 cells; however consistent with previous Y2H data, co-localization of ADAM17 with morulae expressing TRP120 was observed (FIG. 11A). Since Notch1 and ADAM17 are components of the receptor complex, colocalization of Notch1 with E. chaffeensis morulae was also examined. In contrast to the diffused cytoplasmic localization of Notch1 in uninfected cells colocalization of Notch1 with TRP120 expressing morulae was observed. (FIG. 11B). This data was further validated by transfecting HeLa cells with GFP- tagged TRP120 or GFP-control plasmids.
  • TRP120 activates canonical Notch signaling pathway.
  • TRP120 coated FluoroSphere sulfate microspheres was used to stimulate the THP-1 cells. Notch activation involves proteolytic release of NICD from Notch by the furin, ADAM17 and ⁇ -secretase enzymes and translocation of NICD to the nucleus. Thus, the expression of NICD was used to monitor activation of Notch pathway.
  • NICD basal level expression was confirmed in the cytoplasm of untreated cells. However, within 5 min of stimulation condensed expression of NICD was observed near the nucleus which then translocated to nucleus within 15 min of stimulation (Fig. 12A). Since, thioredoxin tag was used as the TRP120 fusion protein; cells were treated with thioredoxin as control. Nuclear translocation of NICD was not observed in untreated cells, or in cells treated with thioredoxin.
  • FIG. 12C shows the graphical representation of gene expression pattern of all the 84 genes involved in Notch signaling, in cells stimulated with TRP120, and normalized to control cells treated with thioredoxin. Examination of individual gene expression identified 33 (39.3%) genes were differentially expressed (p ⁇ 0.05).
  • the scatter plot in Fig 5D shows differential expression of Notch pathway genes during TRP120 stimulation compared to the thioredoxin control, where the up and downregulation of genes are represented as red and green dots (2-fold cutoff), respectively.
  • FIG. 12E shows the list of genes and the fold change during the TRP120 stimulation.
  • TRP120 stimulated cells showed increased expression of 16 Notch pathway components (19%) including receptors (notch1, notch3); ligands (dll 1, 3, 4), transcription factor complex proteins (maml2, rbpj ⁇ ); ⁇ -secretase protein (psen1) and the TRP120 interacting enzyme adam17.
  • Significant induction of 12 target genes (14%) including hes1, hes5, hey2, NF ⁇ B 1, NF ⁇ B 2, il2ra and lor, and downregulation of 4 genes (5%) (lmo2, id1, fosl1 and lrp5) was also observed.
  • chaffeensis represses ERK1/2 and p38 MAPK pathway through Notch signaling.
  • Previous studies reported down-regulation of PU.1 and TLR2/4 expression during E. chaffeensis infection, and demonstrated that host cells become progressively less responsive to LPS mediated stimulation.
  • the underlying mechanism involved inhibition of ERK1/2 and p38 MAPK pathway (Lin and Rikihisa 2004). Since recent studies linked Notch signaling pathway with inhibition of TLR triggered inflammation and inhibition of ERK1/2 (Zhang et al., 2012), the inventors sought to determine the role of E. chaffeensis mediated activation of Notch signaling in inhibition of ERK1/2 and p38 MAPK pathways.
  • Control siRNA and RBPj ⁇ siRNA transfected cells were infected with E. chaffeensis one day post transfection, and stimulated with LPS (100 ng/ml) for 1 h after 1 d.p.i.
  • LPS 100 ng/ml
  • Using immunofluorescent microscopy high levels and predominant localization of PU.1 in the nucleus of uninfected and control siRNA treated THP-1 cells was observed, but there was a reduction of expression in E. chaffeensis infected cells.
  • the inventors observed reconstitution of PU.1 expression level in the nucleus of THP-1 cells which were treated with RBPj ⁇ siRNA to inhibit Notch signaling before infection (FIG.14A).
  • THP-1 cells were treated with either 1 ⁇ g/ml of TRP120 or thioredoxin (control) in soluble form, or TRP120 coated on latex beads and incubated at 37oC for 24 h. TRP120 treated and control cells were then stimulated with LPS (100 ng/ml) for 1 h and expression of PU.1, TLR2 and TLR4 was determined using immunofluorescence microscopy.
  • FIG.16A shows, strong PU.1 expression in the nucleus of thioredoxin treated cells in response to LPS stimulation.
  • LPS treatment reduction in the expression level of PU.1 in response to TRP120 stimulation (both bead bound and in suspension) was observed.
  • PU.1 transcription factor regulates TLR2/4 expression the effect of TRP120 in TLR expression was also determined using the same method.
  • Thioredoxin treated cells showed strong TLR2 expression in both cytoplasm and nucleus of the cells in response to LPS; however cells stimulated with TRP120 showed reduced expression of TLR2 (FIG. 16B). Similar results were found when TLR4 expression was measured in response to TRP120 stimulation (FIG. 16C).
  • TRP120 E. chaffeensis T1S effector protein TRP120 can manipulate Notch signaling to regulate immune recognition through inhibition of TLR expression to promote survival.
  • TRP120 is found on the surface of the infectious DC form, but is also secreted into the host cell where it interacts with a variety of host cell targets and DNA (Zhu et al., 2011; Luo et al., 2011; Luo and McBride 2012). TRPs can also be released from the E. chaffeensis infected cell during infection (Luo et al., 2008).
  • TRP120 bound to a substrate or in soluble form can activate Notch signaling pathway and modulate PU.1 and TLR2/4 expression.
  • Kuriakose et al. Molecular basis of antibody mediated immunity against Ehrlichia chaffeensis involves species-specific linear epitopes in tandem repeat proteins. Microbes Infect 14: 1054-1063, 2012.
  • variable-length PCR target protein of Ehrlichia chaffeensis contains major
  • C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]- S-phenylglycine t-butyl ester).
  • DAPT dipeptidic gamma-secretase-specific inhibitor
  • Vangl2 promotes Wnt/planar cell polarity-like signaling by antagonizing Dvl1- mediated feedback inhibition in growth cone guidance.
  • Shi and Stanley Protein O-fucosyltransferase 1 is an essential component of Notch signaling pathways. Proc Natl Acad Sci U S A 100:5234-5239, 2003.

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Abstract

L'invention concerne des compositions et des procédés utilisés pour le traitement d'infections bactériennes, par exemple d'infections par bactéries intracellulaires strictes. Selon certains modes de réalisation, un inhibiteur de Wnt peut être administré à un sujet mammifère pour traiter l'ehrlichiose
PCT/US2016/034686 2015-05-28 2016-05-27 Traitements d'infections intracellulaires strictes WO2016191695A2 (fr)

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WO2020168290A1 (fr) * 2019-02-14 2020-08-20 University Of Kentucky Research Foundation N-aryl-benzènesulfonamides destinés à être utilisés dans le traitement de cancers, de maladies bactériennes, de maladies métaboliques et d'une lésion cérébrale traumatique
US10933061B2 (en) 2017-12-21 2021-03-02 Shepherd Therapeutics, Inc. Pyrvinium pamoate therapies and methods of use
CN112933066A (zh) * 2021-02-26 2021-06-11 河南省农业科学院 抑制剂kn-93抑制猪繁殖与呼吸综合征病毒体外感染的方法及应用

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US20130323284A1 (en) * 2012-06-05 2013-12-05 Jazzya Investments USE OF mTOR INHIBITORS TO TREAT BACTERIAL INFECTION

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10933061B2 (en) 2017-12-21 2021-03-02 Shepherd Therapeutics, Inc. Pyrvinium pamoate therapies and methods of use
WO2020168290A1 (fr) * 2019-02-14 2020-08-20 University Of Kentucky Research Foundation N-aryl-benzènesulfonamides destinés à être utilisés dans le traitement de cancers, de maladies bactériennes, de maladies métaboliques et d'une lésion cérébrale traumatique
CN112933066A (zh) * 2021-02-26 2021-06-11 河南省农业科学院 抑制剂kn-93抑制猪繁殖与呼吸综合征病毒体外感染的方法及应用
CN112933066B (zh) * 2021-02-26 2023-05-02 河南省农业科学院 抑制剂kn-93抑制猪繁殖与呼吸综合征病毒体外感染的方法及应用

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