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1.
Bioorg Med Chem Lett ; 24(21): 4931-8, 2014 Nov 01.
Article in English | MEDLINE | ID: mdl-25288184

ABSTRACT

The Toll-like receptors (TLRs) are critical components of the innate immune system that regulate immune recognition in part through NF-κB activation. A human cell-based high throughput screen (HTS) revealed substituted 4-aminoquinazolines to be small molecular weight activators of NF-κB. The most potent hit compound predominantly stimulated through the human TLR4/MD2 complex, and had less activity with the mouse TLR4/MD2. There was no activity with other TLRs and the TLR4 activation was MD-2 dependent and CD14 independent. Synthetic modifications of the quinazoline scaffold at the 2 and 4 positions revealed trends in structure-activity relationships with respect to TLR dependent production of the NF-κB associated cytokine IL-8 in human peripheral blood mononuclear cells, as well as IL-6 in mouse antigen presenting cells. Furthermore, the hit compound in this series also activated the interferon signaling pathway resulting in type I interferon production. Substitution at the O-phenyl moiety with groups such as bromine, chlorine and methyl resulted in enhanced immunological activity. Computational studies indicated that the 4-aminoquinazoline compounds bind primarily to human MD-2 in the TLR4/MD-2 complex. These small molecules, which preferentially stimulate human rather than mouse innate immune cells, may be useful as adjuvants or immunotherapeutic agents.


Subject(s)
Interleukin-6/metabolism , Interleukin-8/metabolism , Leukocytes, Mononuclear/metabolism , NF-kappa B/metabolism , Quinazolines/chemistry , Toll-Like Receptor 4/metabolism , Toll-Like Receptors/metabolism , Animals , High-Throughput Screening Assays , Humans , Immunity, Innate , Leukocytes, Mononuclear/cytology , Ligands , Mice , Models, Molecular , Molecular Structure , Quinazolines/metabolism , Signal Transduction , Structure-Activity Relationship
2.
J Innate Immun ; 6(3): 315-24, 2014.
Article in English | MEDLINE | ID: mdl-24192551

ABSTRACT

Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.


Subject(s)
Adenine/analogs & derivatives , Anthrax/drug therapy , Bacillus anthracis/immunology , Communicable Diseases/drug therapy , Dendritic Cells/drug effects , Encephalitis Virus, Venezuelan Equine/immunology , Encephalomyelitis, Venezuelan Equine/drug therapy , Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/drug therapy , Lung/drug effects , Orthomyxoviridae Infections/drug therapy , Phosphatidic Acids/adverse effects , Phospholipids/administration & dosage , Purines/administration & dosage , Toll-Like Receptor 7/agonists , Adenine/administration & dosage , Adenine/adverse effects , Adenine/chemical synthesis , Administration, Intranasal , Animals , Anthrax/immunology , Bronchoalveolar Lavage Fluid/immunology , Communicable Diseases/immunology , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/immunology , Disease Models, Animal , Encephalomyelitis, Venezuelan Equine/immunology , Female , Humans , Immunity, Innate , Influenza, Human/immunology , Injections, Spinal , Ligands , Lung/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Orthomyxoviridae Infections/immunology , Phosphatidic Acids/administration & dosage , Phosphatidic Acids/chemical synthesis , Phospholipids/adverse effects , Phospholipids/chemical synthesis , Purines/adverse effects , Purines/chemical synthesis
3.
J Med Chem ; 56(11): 4206-23, 2013 Jun 13.
Article in English | MEDLINE | ID: mdl-23656327

ABSTRACT

A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators.


Subject(s)
Indoles/chemical synthesis , Pyrimidines/chemical synthesis , Toll-Like Receptor 4/agonists , Animals , Cells, Cultured , Chemokine CXCL10/metabolism , High-Throughput Screening Assays , Humans , Immunity, Innate/drug effects , Indoles/chemistry , Indoles/pharmacology , Interleukin-6/metabolism , Interleukin-8/metabolism , Ligands , Lipopolysaccharides/pharmacology , Lymphocyte Antigen 96/agonists , Lymphocyte Antigen 96/metabolism , Mice , Mice, Inbred C57BL , Models, Molecular , NF-kappa B/metabolism , Pyrimidines/chemistry , Pyrimidines/pharmacology , Structure-Activity Relationship , Toll-Like Receptor 4/metabolism
4.
PLoS One ; 7(9): e45860, 2012.
Article in English | MEDLINE | ID: mdl-23029281

ABSTRACT

The Toll-like receptors (TLR) have been advocated as attractive therapeutic targets because TLR signaling plays dual roles in initiating adaptive immune responses and perpetuating inflammation. Paradoxically, repeated stimulation of bone marrow mononuclear cells with a synthetic TLR7 ligand 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (called 1V136) leads to subsequent TLR hyporesponsiveness. Further studies on the mechanism of action of this pharmacologic agent demonstrated that the TLR7 ligand treatment depressed dendritic cell activation, but did not directly affect T cell function. To verify this mechanism, we utilized experimental allergic encephalitis (EAE) as an in vivo T cell dependent autoimmune model. Drug treated SJL/J mice immunized with proteolipid protein (PLP)(139-151) peptide had attenuated disease severity, reduced accumulation of mononuclear cells in the central nervous system (CNS), and limited demyelination, without any apparent systemic toxicity. Splenic T cells from treated mice produced less cytokines upon antigenic rechallenge. In the spinal cords of 1V136-treated EAE mice, the expression of chemoattractants was also reduced, suggesting innate immune cell hyposensitization in the CNS. Indeed, systemic 1V136 did penetrate the CNS. These experiments indicated that repeated doses of a TLR7 ligand may desensitize dendritic cells in lymphoid organs, leading to diminished T cell responses. This treatment strategy might be a new modality to treat T cell mediated autoimmune diseases.


Subject(s)
Adenine/analogs & derivatives , Encephalomyelitis, Autoimmune, Experimental/immunology , Immunity, Innate/drug effects , Immunosuppressive Agents/pharmacology , Membrane Glycoproteins/agonists , Toll-Like Receptor 7/agonists , Adenine/pharmacology , Adenine/therapeutic use , Animals , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , Cells, Cultured , Chemokines/metabolism , Cytokines/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Female , Immunosuppressive Agents/therapeutic use , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Microglia/drug effects , Microglia/immunology , Microglia/physiology , Neutrophil Infiltration/drug effects , Spinal Cord/drug effects , Spinal Cord/immunology , Spinal Cord/pathology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Toll-Like Receptor 7/metabolism
5.
Bioconjug Chem ; 22(3): 445-54, 2011 Mar 16.
Article in English | MEDLINE | ID: mdl-21338093

ABSTRACT

Toll-like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid, or poly(ethylene glycol) (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6-10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1ß, and type 1 interferon, as well as for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.


Subject(s)
Polyethylene Glycols/chemical synthesis , Polyethylene Glycols/metabolism , Toll-Like Receptor 7/metabolism , Animals , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , Cell Line , Cell Proliferation/drug effects , Cytokines/metabolism , Endosomes/metabolism , Female , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Ligands , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Polyethylene Glycols/pharmacokinetics , Polyethylene Glycols/pharmacology , Protein Transport
6.
Melanoma Res ; 21(1): 66-75, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21030882

ABSTRACT

There remains a compelling need for the development of treatments for unresectable melanoma. Agents that stimulate the innate immune response could provide advantages for cell-based therapies. However, there are conflicting reports concerning whether toll-like receptor (TLR) signaling controls tumor growth. The objective of this study was to evaluate the effect of intralesional administration of a TLR7 agonist in melanoma therapy. B16cOVA melanoma was implanted to TLR7 mice to evaluate the roles of stromal TLR7 on melanoma growth. To capitalize on the potential deleterious effects of TLR7 stimulation on the tumor growth, we injected melanoma tumor nodules with a newly developed and potent TLR7 agonist. B16 melanoma nodules expanded more rapidly in TLR7-deficient and MyD88 mice compared with TLR9 and wild type mice. Repeated injections with low doses of unconjugated TLR7 agonist were more effective at attenuating nodule size than a single high dose injection. To improve the efficacy we conjugated the agonist to phospholipid or phospholipids-polyethylene glycol, which retained TLR7 specificity. The phospholipid conjugate was indeed more effective in reducing lesion size. Furthermore, intralesional administration of the phospholipid TLR7 agonist conjugate enhanced the antimelanoma effects of systemic treatment with interleukin (IL)-2 and prolonged the survival of mice compared with IL-2 alone. Our study showed that: (1) TLR7/MyD88 signaling in the stroma is involved in melanoma growth; and (2) intralesional administration of a TLR7 agonist reduces the growth of melanoma nodules and enhances the antimelanoma effects of IL-2.


Subject(s)
Adenine/analogs & derivatives , Antineoplastic Agents/pharmacology , Interleukin-2/metabolism , Melanoma/metabolism , Membrane Glycoproteins/agonists , Membrane Glycoproteins/metabolism , Phosphatidic Acids/pharmacology , Phospholipids/pharmacology , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/metabolism , Adenine/pharmacology , Animals , Cell Line, Tumor , Cytokines/metabolism , Drug Synergism , Female , Flow Cytometry , Immune System , Interleukin-2/pharmacology , Melanoma/drug therapy , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Differentiation Factor 88/genetics , Neoplasm Transplantation , Toll-Like Receptor 9/genetics
7.
Proc Natl Acad Sci U S A ; 106(8): 2764-9, 2009 Feb 24.
Article in English | MEDLINE | ID: mdl-19188597

ABSTRACT

Activation of Toll-like receptors (TLR) contributes to the initiation and maintenance of chronic inflammation in autoimmune diseases, yet repeated exposure to a TLR agonist can induce hyporesponsiveness to subsequent TLR stimulation. Here, we used a synthetic TLR7 agonist, 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (SM360320, 1V136) to study TLR7 induced attenuation of inflammatory responses and its application to autoimmune diseases. Repeated low dose administration of this TLR7 agonist induced hyporesponsiveness or tolerance to TLR2, -7, and -9 activators and limited the course of neural inflammation in an experimental allergic encephalomyelitis model. The hyporesponsiveness did not depend on T or B lymphocytes, but did require bone marrow derived cells. In addition, TLR7 tolerance reduced inflammation in a passive antibody mediated arthritis model. TLR7 tolerance did not cause global immunosuppression, because susceptibility to Listeria monocytogenes infection was not altered. The mechanism of TLR7 tolerance involved the up-regulation of 2 inhibitors of TLR signaling: Interleukin 1 Receptor Associated Kinase (IRAK) M, and Src homology 2 domain-containing inositol polyphosphate phosphatase (SHIP)-1. These findings suggest that induction of TLR7 tolerance might be a new therapeutic approach to subdue inflammation in autoimmune diseases.


Subject(s)
Autoimmune Diseases/prevention & control , Immune Tolerance , Toll-Like Receptor 7/immunology , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Encephalomyelitis, Autoimmune, Experimental/immunology , Mice , Mice, Inbred C57BL , Toll-Like Receptor 7/agonists
8.
Am J Physiol Regul Integr Comp Physiol ; 295(1): R123-32, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18480244

ABSTRACT

Systemic viral infections produce a highly regulated set of responses in sickness behavior, such as fever, anorexia, and adipsia. Toll-like receptor (TLR)7, activated by viral RNA during infection, potently stimulates the innate and adaptive immune responses that aid in viral clearance. However, the physiological consequences of TLR7 activation have not been thoroughly studied. In these experiments, we used a potent synthetic TLR7 ligand, 9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine (SM360320; 1V136), to investigate the consequences of TLR7 activation in genetically defined strains of mice. Administration of the drug by the nasal, intragastric, or intraperitoneal routes caused transient hypophagia, hypodypsia, and hypothermia. Analyses of mutant mouse strains indicated that these effects were dependent on the expression of TLR7, its adaptor protein MyD88, and TNF-alpha, and independent of IL-1beta, IL-6 and cyclo-oxygenase-1 (COX1). Partial roles were also implied for mast cells and COX2. Although plasma TNF-alpha levels were significantly higher after systemic drug delivery, the behavioral effects were maximal when the agent was administered to the mucosa. Tissue and mucosal mast cells are known to express high levels of TLR7 and to rapidly release TNF-alpha upon TLR7 ligation. Mice deficient in tissue mast cells, W/W(v), had significantly less anorexia after TLR7 activation, and this response was restored with mast cell reconstitution. Our results thus suggest that tissue mast cells may play a role in the anorexia induced by mucosal activation of TLR7.


Subject(s)
Anorexia/etiology , Hypothermia/etiology , Mast Cells/metabolism , Membrane Glycoproteins/metabolism , Toll-Like Receptor 7/metabolism , Adenine/administration & dosage , Adenine/analogs & derivatives , Adenine/pharmacology , Administration, Intranasal , Animals , Appetite Depressants/administration & dosage , Appetite Depressants/pharmacology , Cytokines/genetics , Cytokines/metabolism , Drug Administration Routes , Female , Gene Deletion , Gene Expression Regulation/drug effects , Ligands , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mucous Membrane , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Random Allocation , Time Factors , Toll-Like Receptor 7/genetics
9.
Bioorg Med Chem Lett ; 16(17): 4559-63, 2006 Sep 01.
Article in English | MEDLINE | ID: mdl-16784848

ABSTRACT

Several 9-benzyl adenine derivatives bearing various substituted amines at the 8-position have been prepared and evaluated for interferon induction in peripheral blood mononuclear cells (PBMC) from healthy human donors. The 8-bromoadenine derivative 5 was used as a versatile intermediate for all substitutions. The most active 8-substituted amino compound was found to be the 8-morpholinoethylamino derivative 19 which had an EC(50) in the submicromolar range.


Subject(s)
Adenine/analogs & derivatives , Toll-Like Receptor 7/agonists , Toll-Like Receptor 7/immunology , Adenine/chemical synthesis , Adenine/chemistry , Adenine/pharmacology , Amination , Humans , Leukocytes/drug effects , Molecular Structure , Structure-Activity Relationship
10.
J Med Chem ; 48(20): 6409-22, 2005 Oct 06.
Article in English | MEDLINE | ID: mdl-16190767

ABSTRACT

The chemoprotection of cells from apoptosis induced by toxins or ionizing radiation could be important for biodefense and in the treatment of acute injuries. We describe a series of small heterocycles, including fused benzothiazoles, benzimidazoles, and related compounds, that abrogate thymocyte apoptosis induced by dexamethasone and gamma-irradiation. To optimize the protective activity of the previously reported pifithrin-alpha (PFT-alpha, 1), various derivatives and analogues of this and the corresponding ring-closed imidazobenzothiazole (IBT, 39) were synthesized. The aromatic analogues of 39 were more protective than 39, while the aromatic analogues of 1 were not active. Compound 19 containing a pyrrolidinyl substituent on the phenyl ring provided potent antiapoptotic activity (EC50 of 1.31 microM compared to 4.16 microM for 1). Modification of aromatic 39 with a pyrrolidinyl para substituent (compound 60) enhanced the activity, lowering the EC50 to 0.35 microM. Also, 60 provided significant protection against gamma-irradiation-induced apoptosis, as expected. Compounds 19 and 60 may be promising for potential clinical development.


Subject(s)
Apoptosis/drug effects , Imidazoles/chemical synthesis , Lymphocytes/drug effects , Thiazoles/chemical synthesis , Toluene/analogs & derivatives , Animals , Benzimidazoles/chemical synthesis , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Benzothiazoles , Dexamethasone/pharmacology , Gamma Rays , Imidazoles/chemistry , Imidazoles/pharmacology , In Vitro Techniques , Lymphocytes/cytology , Lymphocytes/radiation effects , Mice , Mice, Inbred C57BL , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistry , Pyrrolidines/pharmacology , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Thymus Gland/cytology , Toluene/chemical synthesis , Toluene/chemistry , Toluene/pharmacology , Tumor Suppressor Protein p53/physiology
11.
Bioorg Med Chem Lett ; 15(7): 1785-8, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15780606

ABSTRACT

The synthesis of several quinolinium salts and related compounds and their ability to inhibit glucocorticoid-induced apoptosis in murine thymocytes are described. Interestingly, 1-[2-methoxyimino-2-(4-pyrrolidin-1-yl-phenyl)ethyl]quinolinium bromide (11) showed a potent protective effect with an EC(50) of 0.013 microM, which was at least 300-fold more potent than the reference compound pifithrin-alpha.


Subject(s)
Antineoplastic Agents/chemical synthesis , Apoptosis/drug effects , Lymphocytes/drug effects , Quinolinium Compounds/chemical synthesis , Salts/chemistry , Toluene/analogs & derivatives , Antineoplastic Agents/pharmacology , Benzothiazoles , Glucocorticoids/pharmacology , Humans , Lymphocytes/cytology , Quinolinium Compounds/pharmacology , Structure-Activity Relationship , Thiazoles/pharmacology , Toluene/pharmacology , Tumor Cells, Cultured
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