ABSTRACT
Latent reservoirs of HIV-1-infected cells are refractory to antiretroviral therapies (ART) and remain the major barrier to curing HIV-1. Because latently infected cells are long-lived, immunologically invisible, and may undergo homeostatic proliferation, a "shock and kill" approach has been proposed to eradicate this reservoir by combining ART with inducers of viral transcription. However, all attempts to alter the HIV-1 reservoir in vivo have failed to date. Using humanized mice, we show that broadly neutralizing antibodies (bNAbs) can interfere with establishment of a silent reservoir by Fc-FcR-mediated mechanisms. In established infection, bNAbs or bNAbs plus single inducers are ineffective in preventing viral rebound. However, bNAbs plus a combination of inducers that act by independent mechanisms synergize to decrease the reservoir as measured by viral rebound. Thus, combinations of inducers and bNAbs constitute a therapeutic strategy that impacts the establishment and maintenance of the HIV-1 reservoir in humanized mice.
Subject(s)
Antibodies, Neutralizing/administration & dosage , HIV Infections/immunology , HIV-1/drug effects , Transcription, Genetic/drug effects , Virus Latency/drug effects , Animals , Anti-HIV Agents/therapeutic use , Antibodies, Neutralizing/immunology , CD4-Positive T-Lymphocytes/immunology , CTLA-4 Antigen/administration & dosage , HIV Infections/virology , HIV-1/genetics , HIV-1/physiology , Heterocyclic Compounds, 4 or More Rings/administration & dosage , Humans , Hydroxamic Acids/administration & dosage , Immunoglobulin Fc Fragments/immunology , Mice , Receptors, Fc/immunology , VorinostatABSTRACT
AIMS: To improve the tolerability and therapeutic application of histone deacetylase inhibitors (HDACi), by application of an esterase-sensitive motif (ESM), to target pharmacological activity directly to mononuclear myeloid cells expressing the processing enzyme carboxylesterase-1 (CES1). METHODS: This first-in-human study comprised single and multiple ascending dose cohorts to determine safety and tolerability. Pharmacodynamic parameters included acetylation, cytokine inhibition and intracellular concentrations of processed acid metabolite in isolated monocytes. Mechanistic work was conducted in vitro and in a CES1/Es1elo mouse strain. RESULTS: ESM-HDAC391 showed transient systemic exposure (plasma half-life of 21-30 min) but selective retention of processed acid for at least 12 hours, resulting in robust targeted mechanistic engagement (increased acetylation in monocytes plus inhibition of ex vivo stimulated cytokine production). ESM-HDAC391 was well tolerated and clinical toxicities common to non-targeted HDACi were not observed. ESM-HDAC391 treatment was accompanied by the novel finding of a dose-dependent monocyte depletion that was transient and reversible and which plateaued at 0.06 × 109 monocytes/L after repeat dosing with 20 or 40 mg. Characterisation of monocyte depletion in transgenic mice (CES1/Es1elo ) suggested that colony stimulating factor 1 receptor loss on circulating cells contributed to ESM-HDAC-mediated depletion. Further mechanistic investigations using human monocytes in vitro demonstrated HDACi-mediated change in myeloid fate through modulation of colony stimulating factor 1 receptor and downstream effects on cell differentiation. CONCLUSION: These findings demonstrate selective targeting of monocytes in humans using the ESM approach and identify monocytopaenia as a novel outcome of ESM-HDACi treatment, with implications for potential benefit of these molecules in myeloid-driven diseases.
Subject(s)
Esterases , Histone Deacetylase Inhibitors , Humans , Animals , Mice , Histone Deacetylase Inhibitors/pharmacology , Macrophage Colony-Stimulating Factor , CytokinesABSTRACT
Changes in the epigenetic landscape of immune cells are a crucial component of gene activation during the induction of inflammatory responses, therefore it has been hypothesized that epigenetic modulation could be employed to restore homeostasis in inflammatory scenarios. Fungal pathogens cause a large burden of morbidity and even mortality due to the hyperinflammatory processes that induce mucosal, allergic or systemic infections. Bromodomain and extraterminal domain (BET) proteins are considered as one as the most tantalizing pharmacological targets for the modulation of inflammatory responses at the epigenetic level. Nothing is known of the role of BET inhibitors on the inflammation induced by fungal pathogens. In the present study, we assessed the in vitro efficacy of the small molecular histone mimic BET inhibitor I-BET151 to modulate innate immune responses during fungal-immune interaction with the clinically relevant fungal pathogens Candida albicans and Aspergillus fumigatus. Our results prove that BET inhibitors (I-BETs) represent an important modulator of inflammation induced by fungal pathogens: both direct production of proinflammatory cytokines and the induction of trained immunity were inhibited by I-BET151. These modulatory effects are likely to have important potential implications in clinically relevant situations.
Subject(s)
Gene Expression Regulation/drug effects , Heterocyclic Compounds, 4 or More Rings/pharmacology , Host-Pathogen Interactions/drug effects , Immunologic Factors/pharmacology , Monocytes/drug effects , Neutrophils/drug effects , Aspergillus fumigatus/immunology , Aspergillus fumigatus/pathogenicity , Candida albicans/immunology , Candida albicans/pathogenicity , Endocytosis/drug effects , Endocytosis/genetics , Endocytosis/immunology , Gene Expression Regulation/immunology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Humans , Interferon-gamma/genetics , Interferon-gamma/immunology , Interleukin-10/genetics , Interleukin-10/immunology , Interleukin-17/genetics , Interleukin-17/immunology , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/antagonists & inhibitors , Interleukin-6/genetics , Interleukin-6/immunology , Interleukins/genetics , Interleukins/immunology , Monocytes/immunology , Monocytes/microbiology , Neutrophils/immunology , Neutrophils/microbiology , Primary Cell Culture , Signal Transduction , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Interleukin-22ABSTRACT
Pulmonary hypertension is a co-morbidity, which strongly participates in morbi-mortality in patients with chronic obstructive pulmonary disease (COPD). Recent findings showed that bromodomain-containing proteins, in charge of reading histone acetylation, could be involved in pulmonary arterial hypertension. Our aim was to study the effect of I-BET151, an inhibitor of bromodomain and extra-terminal domain (BET), on the right ventricle hypertrophy and pulmonary hypertension, induced by a combination of chronic hypoxia and pulmonary inflammation, as the two main stimuli encountered in COPD. Adult Wistar male rats, exposed to chronic hypoxia plus pulmonary inflammation (CHPI), showed a significant right ventricle hypertrophy (+57%, p < 0.001), an increase in systolic pressure (+46%, p < 0.001) and in contraction speed (+36%, p < 0.001), when compared to control animals. I-BET151 treated animals (CHPI-iB) showed restored hemodynamic parameters to levels similar to control animals, despite chronic hypoxia plus exposure to pulmonary inflammation. They displayed lower right ventricle hypertrophy and hematocrit compared to the CHPI group (respectively -16%, p < 0.001; and -9%, p < 0.05). Our descriptive study shows a valuable effect of the inhibition of bromodomain and extra-terminal domain proteins on hemodynamic parameters, despite the presence of chronic hypoxia and pulmonary inflammation. This suggests that such inhibition could be of potential interest for COPD patients with pulmonary hypertension. Further studies are needed to unravel the underlying mechanisms involved and the net benefits of inhibiting adaptations to chronic hypoxia.
Subject(s)
Heterocyclic Compounds, 4 or More Rings/therapeutic use , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/etiology , Hypertrophy, Right Ventricular/drug therapy , Hypertrophy, Right Ventricular/etiology , Hypoxia/complications , Pneumonia/complications , Transcription Factors/antagonists & inhibitors , Animals , Blood Pressure/drug effects , Hypertension, Pulmonary/pathology , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/pathology , Hypertrophy, Right Ventricular/physiopathology , Hypoxia/pathology , Hypoxia/physiopathology , Male , Pneumonia/pathology , Pneumonia/physiopathology , Pulmonary Disease, Chronic Obstructive/complications , Pulmonary Disease, Chronic Obstructive/pathology , Pulmonary Disease, Chronic Obstructive/physiopathology , Rats, WistarABSTRACT
ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of that target class. Starting from a potent lead, permeability and selectivity were improved through a dual approach: 1) using CF2 as a sulfone bio-isostere to exploit the unique properties of fluorine, and 2) using 1,3-interactions to control the conformation of a piperidine ring. This resulted in the first reported low-nanomolar, selective and cell permeable chemical probe for ATAD2.
ABSTRACT
BACKGROUND: Neutrophils are a critical part of the innate immune system. Their ability to migrate into infected or injured tissues precedes their role in microbial killing and clearance. We have previously shown that Rab27a can promote neutrophil migration by facilitating uropod release through protease secretion from primary granule exocytosis at the cell rear. Rab27b has been implicated in primary granule exocytosis but its role in neutrophil migration has not been investigated. RESULTS: Here we found Rab27b to be expressed in bone marrow derived neutrophils and Rab27b knockout (Rab27b KO) along with Rab27a/b double knockout (Rab27DKO) neutrophils exhibited impaired transwell migration in vitro in response to chemokines MIP-2 and LTB4. Interestingly, no additional defect in migration was observed in Rab27DKO neutrophils compared with Rab27b KO neutrophils. In vivo, Rab27DKO mice displayed severe impairment in neutrophil recruitment to the lungs in a MIP-2 dependent model but not in an LPS dependent model. CONCLUSIONS: These data taken together implicate Rab27b in the regulation of neutrophil chemotaxis, likely through the regulation of primary granule exocytosis.
Subject(s)
Chemotaxis, Leukocyte , Lung/immunology , Neutrophils/cytology , rab GTP-Binding Proteins/immunology , Animals , Cell Movement , Chemokine CXCL2/immunology , Gene Expression , Gene Knockdown Techniques , Inflammation/genetics , Inflammation/immunology , Lipopolysaccharides/immunology , Lung/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Neutrophils/immunology , Neutrophils/metabolism , Receptors, Interleukin-8B/analysis , rab GTP-Binding Proteins/genetics , rab27 GTP-Binding ProteinsABSTRACT
BACKGROUND: Myeloid cells are critical determinants of the sustained inflammation in Crohn's Disease (CD). Targeting such cells may be an effective therapeutic approach for refractory CD patients. Bromodomain and extra-terminal domain protein inhibitors (iBET) are potent anti-inflammatory agents; however, they also possess wide-ranging toxicities. In the current study, we make use of a BET inhibitor containing an esterase sensitive motif (ESM-iBET), which is cleaved by carboxylesterase-1 (CES1), a highly expressed esterase in mononuclear myeloid cells. METHODS: We profiled CES1 protein expression in the intestinal biopsies, peripheral blood, and CD fistula tract (fCD) cells of CD patients using mass cytometry. The anti-inflammatory effect of ESM-iBET or its control (iBET) were evaluated in healthy donor CD14+ monocytes and fCD cells, using cytometric beads assay or RNA-sequencing. RESULTS: CES1 was specifically expressed in monocyte, macrophage, and dendritic cell populations in the intestinal tissue, peripheral blood, and fCD cells of CD patients. ESM-iBET inhibited IL1ß, IL6, and TNFα secretion from healthy donor CD14+ monocytes and fCD immune cells, with 10- to 26-fold more potency over iBET in isolated CD14+ monocytes. Transcriptomic analysis revealed that ESM-iBET inhibited multiple inflammatory pathways, including TNF, JAK-STAT, NF-kB, NOD2, and AKT signaling, with superior potency over iBET. CONCLUSIONS: We demonstrate specific CES1 expression in mononuclear myeloid cell subsets in peripheral blood and inflamed tissues of CD patients. We report that low dose ESM-iBET accumulates in CES1-expressing cells and exerts robust anti-inflammatory effects, which could be beneficial in refractory CD patients.
Subject(s)
Anti-Inflammatory Agents , Crohn Disease , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Carboxylic Ester Hydrolases , Crohn Disease/drug therapy , Crohn Disease/metabolism , Humans , Inflammation Mediators , Interleukin-6 , Myeloid Cells/metabolism , NF-kappa B , Proto-Oncogene Proteins c-akt , RNA , Tumor Necrosis Factor-alphaABSTRACT
Latency reversal strategies for HIV cure using inhibitor of apoptosis protein (IAP) antagonists (IAPi) induce unprecedented levels of latent reservoir expression without immunotoxicity during suppressive antiretroviral therapy (ART). However, full targeting of the reservoir may require combinatorial approaches. A Jurkat latency model screen for IAPi combination partners demonstrated synergistic latency reversal with bromodomain (BD) and extraterminal domain protein inhibitors (BETi). Mechanistic investigations using CRISPR-CAS9 and single-cell RNA-Seq informed comprehensive ex vivo evaluations of IAPi plus pan-BET, bD-selective BET, or selective BET isoform targeting in CD4+ T cells from ART-suppressed donors. IAPi+BETi treatment resulted in striking induction of cell-associated HIV gag RNA, but lesser induction of fully elongated and tat-rev RNA compared with T cell activation-positive controls. IAPi+BETi resulted in HIV protein induction in bulk cultures of CD4+ T cells using an ultrasensitive p24 assay, but did not result in enhanced viral outgrowth frequency using a standard quantitative viral outgrowth assay. This study defines HIV transcriptional elongation and splicing as important barriers to latent HIV protein expression following latency reversal, delineates the roles of BET proteins and their BDs in HIV latency, and provides a rationale for exploration of IAPi+BETi in animal models of HIV latency.
Subject(s)
HIV Infections , HIV-1 , Animals , CD4-Positive T-Lymphocytes , HIV Infections/drug therapy , HIV Infections/genetics , HIV-1/physiology , Human Immunodeficiency Virus Proteins , NF-kappa B/genetics , NF-kappa B/metabolism , Nuclear Proteins/metabolism , RNA , Transcription Factors/metabolism , Virus Activation , Virus LatencyABSTRACT
BACKGROUND AND AIMS: Histone deacetylase inhibitors [HDACi] exert potent anti-inflammatory effects. Because of the ubiquitous expression of HDACs, clinical utility of HDACi is limited by off-target effects. Esterase-sensitive motif [ESM] technology aims to deliver ESM-conjugated compounds to human mononuclear myeloid cells, based on their expression of carboxylesterase 1 [CES1]. This study aims to investigate utility of an ESM-tagged HDACi in inflammatory bowel disease [IBD]. METHODS: CES1 expression was assessed in human blood, in vitro differentiated macrophage and dendritic cells, and Crohn's disease [CD] colon mucosa, by mass cytometry, quantitative polymerase chain reaction [PCR], and immunofluorescence staining, respectively. ESM-HDAC528 intracellular retention was evaluated by mass spectrometry. Clinical efficacy of ESM-HDAC528 was tested in dextran sulphate sodium [DSS]-induced colitis and T cell transfer colitis models using transgenic mice expressing human CES1 under the CD68 promoter. RESULTS: CES1 mRNA was highly expressed in human blood CD14+ monocytes, in vitro differentiated and lipopolysaccharide [LPS]-stimulated macrophages, and dendritic cells. Specific hydrolysis and intracellular retention of ESM-HDAC528 in CES1+ cells was demonstrated. ESM-HDAC528 inhibited LPS-stimulated IL-6 and TNF-α production 1000 times more potently than its control, HDAC800, in CES1high monocytes. In healthy donor peripheral blood, CES1 expression was significantly higher in CD14++CD16- monocytes compared with CD14+CD16++ monocytes. In CD-inflamed colon, a higher number of mucosal CD68+ macrophages expressed CES1 compared with non-inflamed mucosa. In vivo, ESM-HDAC528 reduced monocyte differentiation in the colon and significantly improved colitis in a T cell transfer model, while having limited potential in ameliorating DSS-induced colitis. CONCLUSIONS: We demonstrate that monocytes and inflammatory macrophages specifically express CES1, and can be preferentially targeted by ESM-HDAC528 to achieve therapeutic benefit in IBD.
Subject(s)
Carboxylic Ester Hydrolases , Colitis , Crohn Disease , Histone Deacetylase Inhibitors , Inflammatory Bowel Diseases , Animals , Carboxylic Ester Hydrolases/metabolism , Colitis/chemically induced , Colitis/drug therapy , Colitis/metabolism , Crohn Disease/drug therapy , Crohn Disease/metabolism , Histone Deacetylase Inhibitors/pharmacology , Humans , Inflammatory Bowel Diseases/metabolism , Intestinal Mucosa/metabolism , Lipopolysaccharides , Mice , Monocytes , Myeloid CellsABSTRACT
In humans, 10(11) neutrophils are released from the bone marrow per day, and these cells have a half-life in the blood of only approximately 6.5 h. Although it is generally believed that neutrophils are cleared from the circulation via the liver and spleen, in this study using (111)In-labeled senescent neutrophils, we show that in mice, 32% of neutrophils are cleared from the circulation via the bone marrow. We have previously shown that senescent neutrophils home to the bone marrow in a CXCR4-dependent manner, and we show here that pretreatment of neutrophils with pertussis toxin significantly inhibits neutrophil clearance via the bone marrow (75%), consistent with a role for chemokines in this process. By labeling senescent neutrophils with inert fluorescent microspheres, we have tracked their fate and shown that in vivo, they are ultimately phagocytosed by bone marrow stromal macrophages. Finally, we show that under noninflammatory conditions, circulating levels of neutrophils are regulated by granulocyte-colony stimulating factor (G-CSF), but not interleukin-17. Interestingly, we report that the uptake of apoptotic neutrophils by bone marrow macrophages stimulates their production of G-CSF in vitro. Taken together, these data provide evidence that the bone marrow represents a major site of neutrophil clearance in mice.
Subject(s)
Bone Marrow/physiology , Chemotaxis, Leukocyte/physiology , Homeostasis/physiology , Animals , Apoptosis/physiology , Female , Granulocyte Colony-Stimulating Factor/biosynthesis , Interleukin-17/physiology , Macrophages/physiology , Mice , Mice, Inbred BALB C , Neutrophils/physiologyABSTRACT
Monocytes and macrophages are key drivers in the pathogenesis of inflammatory diseases. Epigenetic targets have been shown to control the transcriptional profile and phenotype of these cells. Since histone deacetylase protein inhibitors demonstrate profound anti-inflammatory activity, we wanted to test whether HDAC inhibition within monocytes and macrophages could be applied to suppress inflammation in vivo. ESM technology conjugates an esterase-sensitive motif (ESM) onto small molecules to allow targeting of cells that express carboxylesterase 1 (CES1), such as mononuclear myeloid cells. This study utilized an ESM-HDAC inhibitor to target monocytes and macrophages in mice in both an acute response model and an atherosclerosis model. We demonstrate that the molecule blocks the maturation of peritoneal macrophages and inhibits pro-inflammatory cytokine production in both models but to a lesser extent in the atherosclerosis model. Despite regulating the inflammatory response, ESM-HDAC528 did not significantly affect plaque size or phenotype, although histological classification of the plaques demonstrated a significant shift to a less severe phenotype. We hereby show that HDAC inhibition in myeloid cells impairs the maturation and activation of peritoneal macrophages but shows limited efficacy in a model of atherosclerosis.
ABSTRACT
The bromodomain of ATAD2 has proved to be one of the least-tractable proteins within this target class. Here, we describe the discovery of a new class of inhibitors by high-throughput screening and show how the difficulties encountered in establishing a screening triage capable of finding progressible hits were overcome by data-driven optimization. Despite the prevalence of nonspecific hits and an exceptionally low progressible hit rate (0.001%), our optimized hit qualification strategy employing orthogonal biophysical methods enabled us to identify a single active series. The compounds have a novel ATAD2 binding mode with noncanonical features including the displacement of all conserved water molecules within the active site and a halogen-bonding interaction. In addition to reporting this new series and preliminary structure-activity relationship, we demonstrate the value of diversity screening to complement the knowledge-based approach used in our previous ATAD2 work. We also exemplify tactics that can increase the chance of success when seeking new chemical starting points for novel and less-tractable targets.
Subject(s)
ATPases Associated with Diverse Cellular Activities/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Drug Design , Drug Discovery/methods , High-Throughput Screening Assays/methods , Protein Domains , Small Molecule Libraries/pharmacology , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/metabolism , Biophysical Phenomena , Catalytic Domain , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Models, Molecular , Molecular Structure , Protein Binding/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolismABSTRACT
The bone marrow is the site of neutrophil production, a process that is regulated by the cytokine granulocyte colony-stimulating factor (G-CSF). Mature neutrophils are continually released into the circulation, with an estimated 10(11) neutrophils exiting the bone marrow daily under basal conditions. These leucocytes have a short half-life in the blood of approximately 6.5 hr, and are subsequently destroyed in the spleen, liver and indeed the bone marrow itself. Additionally, mature neutrophils are retained in the bone marrow by the stromal cell-derived factor (SDF-1alpha)/chemokine (C-X-C motif) receptor 4 (CXCR4) chemokine axis and form the bone marrow reserve. Following infection or inflammatory insult, neutrophil release from the bone marrow reserve is substantially elevated and this process is mediated by the co-ordinated actions of cytokines and chemokines. In this review we discuss the factors and molecular mechanisms regulating the neutrophil mobilization and consider the mechanisms and functional significance of neutrophil clearance via the bone marrow.
Subject(s)
Bone Marrow/immunology , Chemotaxis, Leukocyte/immunology , Neutrophils/immunology , Chemokines/immunology , Humans , Inflammation/immunology , Integrins/immunology , Macrophages/immunologyABSTRACT
ATAD2 is a cancer-associated protein whose bromodomain has been described as among the least druggable of its class. In our recent disclosure of the first chemical probe against this bromodomain, GSK8814 (6), we described the use of a conformationally constrained methoxy piperidine to gain selectivity over the BET bromodomains. Here we describe an orthogonal conformational restriction strategy of the piperidine ring to give potent and selective tropane inhibitors and show structural insights into why this was more challenging than expected. Greater understanding of why different rational approaches succeeded or failed should help in the future design of selectivity in the bromodomain family.
Subject(s)
ATPases Associated with Diverse Cellular Activities/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Drug Discovery , Nuclear Proteins/antagonists & inhibitors , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Transcription Factors/antagonists & inhibitors , Cell Cycle Proteins , Humans , Models, Molecular , Molecular Structure , Protein Conformation , Protein Domains , Structure-Activity RelationshipABSTRACT
The DNA papillomaviruses infect squamous epithelium and can cause persistent, benign and sometimes malignant hyperproliferative lesions. Effective antiviral drugs to treat human papillomavirus (HPV) infection are lacking and here we investigate the anti-papillomavirus activity of novel epigenetic targeting drugs, BET bromodomain inhibitors. Bromodomain and Extra-Terminal domain (BET) proteins are host proteins which regulate gene transcription, they bind acetylated lysine residues in histones and non-histone proteins via bromodomains, functioning as scaffold proteins in the formation of transcriptional complexes at gene regulatory regions. The BET protein BRD4 has been shown to be involved in the papillomavirus life cycle, as a co-factor for viral E2 and also mediating viral partitioning in some virus types. We set out to study the activity of small molecule BET bromodomain inhibitors in models of papillomavirus infection. Several BET inhibitors reduced HPV11 E1ËE4 mRNA expression in vitro and topical therapeutic administration of an exemplar compound I-BET762, abrogated CRPV cutaneous wart growth in rabbits, demonstrating translation of anti-viral effects to efficacy in vivo. Additionally I-BET762 markedly reduced viability of HPV16 infected W12â¯cells compared to non-infected C33A cells. The molecular mechanism for the cytotoxicity to W12â¯cells is unknown but may be through blocking viral-dependent cell-survival factors. We conclude that these effects, across multiple papillomavirus types and in vivo, highlight the potential to target BET bromodomains to treat HPV infection.
Subject(s)
Benzodiazepines/therapeutic use , Heterocyclic Compounds, 4 or More Rings/therapeutic use , Nuclear Proteins/antagonists & inhibitors , Papillomaviridae/drug effects , Transcription Factors/antagonists & inhibitors , Warts/drug therapy , Acetylation , Animals , Cell Line, Tumor , Cell Survival , Epigenesis, Genetic , Lysine , Male , Papillomaviridae/genetics , Protein Domains , Rabbits , Warts/virologyABSTRACT
Some of the Chronic Obstructive Pulmonary Disease (COPD) patients engaged in exercise-based muscle rehabilitation programs are unresponsive. To unravel the respective role of chronic hypoxia and pulmonary inflammation on soleus muscle hypertrophic capacities, we challenged male Wistar rats to repeated lipopolysaccharide instillations, associated or not with a chronic hypoxia exposure. Muscle hypertrophy was initiated by bilateral ablation of soleus agonists 1 week before sacrifice. To understand the role played by the histone acetylation, we also treated our animals with an inhibitor of bromodomains and extra terminal proteins (I-BET) during the week after surgery. Pulmonary inflammation totally inhibited this hypertrophy response under both normoxic and hypoxic conditions (26% lower than control surgery, p < 0.05), consistent with the S6K1 and myogenin measurements. Changes in histone acetylation and class IIa histone deacetylases expression, following pulmonary inflammation, suggested a putative role for histone acetylation signaling in the altered hypertrophy response. The I-BET drug restored the hypertrophy response suggesting that the non-response of muscle to a hypertrophic stimulus could be modulated by epigenetic mechanisms, including histone-acetylation dependant pathways. Drugs targeting such epigenetic mechanisms may open therapeutic perspectives for COPD patients with systemic inflammation who are unresponsive to rehabilitation.
Subject(s)
Heterocyclic Compounds, 4 or More Rings/pharmacology , Hypertrophy/complications , Hypoxia/complications , Muscle, Skeletal/pathology , Muscular Diseases/complications , Pneumonia/complications , Pulmonary Disease, Chronic Obstructive/complications , Acetylation/drug effects , Animals , Histones/metabolism , Humans , Hypertrophy/drug therapy , Hypertrophy/metabolism , Hypertrophy/pathology , Hypoxia/drug therapy , Hypoxia/metabolism , Hypoxia/pathology , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Diseases/drug therapy , Muscular Diseases/metabolism , Muscular Diseases/pathology , Pneumonia/drug therapy , Pneumonia/metabolism , Pneumonia/pathology , Protein Domains , Pulmonary Disease, Chronic Obstructive/drug therapy , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Rats, WistarABSTRACT
We investigated the roles of the regulatory molecules glucocorticoid-induced TNF receptor family-related protein (GITR) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) in murine infection with the nematode parasite Trichinella spiralis. Expression of GITR and CTLA-4 was rapidly upregulated on cells in the mesenteric lymph nodes and spleen, with approximately 80% of CD4+ lymphocytes expressing GITR by day 7 post-infection, coinciding with release and dissemination of newborn larvae. As the infection progressed to the chronic muscle phase, expression of GITR returned to normal, whereas CTLA-4 was sustained as late as day 60. Mice treated with anti-GITR antibodies rapidly developed higher titres of parasite-specific IgG1, IgG2a, IgG2b and IgM than controls. This was accompanied by elevated background lymphocyte proliferation, but parasite establishment in the intestine or the muscle was unaffected. In contrast, treatment with anti-CTLA-4 antibody resulted in elevated serum IgE, enhanced production of interleukin-4 and interleukin-10, and lower numbers of parasites recovered from skeletal muscle. These results reveal different temporal and regulatory roles for CTLA-4 and GITR in immune responses to helminth infection.
Subject(s)
Antigens, CD/immunology , Antigens, Differentiation/immunology , Receptors, Nerve Growth Factor/immunology , Receptors, Tumor Necrosis Factor/immunology , Trichinella spiralis/immunology , Trichinellosis/immunology , Animals , Antibodies, Helminth/blood , Antigens, CD/biosynthesis , Antigens, Differentiation/biosynthesis , CD4-Positive T-Lymphocytes/immunology , CTLA-4 Antigen , Cell Proliferation , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Flow Cytometry , Glucocorticoid-Induced TNFR-Related Protein , Immunoglobulin E/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Interleukin-10 , Interleukin-4/analysis , Intestines/parasitology , Lymph Nodes/immunology , Lymphocyte Subsets/immunology , Mice , Muscle, Skeletal/parasitology , Receptors, Nerve Growth Factor/biosynthesis , Receptors, Tumor Necrosis Factor/biosynthesis , Spleen/immunology , Up-RegulationABSTRACT
ATAD2 is a bromodomain-containing protein whose overexpression is linked to poor outcomes in a number of different cancer types. To date, no potent and selective inhibitors of the bromodomain have been reported. This article describes the structure-based optimization of a series of naphthyridones from micromolar leads with no selectivity over the BET bromodomains to inhibitors with sub-100 nM ATAD2 potency and 100-fold BET selectivity.
Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Naphthyridines/chemistry , Naphthyridines/pharmacology , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/chemistry , DNA-Binding Proteins/chemistry , Models, Molecular , Molecular StructureABSTRACT
Overexpression of ATAD2 (ATPase family, AAA domain containing 2) has been linked to disease severity and progression in a wide range of cancers, and is implicated in the regulation of several drivers of cancer growth. Little is known of the dependence of these effects upon the ATAD2 bromodomain, which has been categorized as among the least tractable of its class. The absence of any potent, selective inhibitors limits clear understanding of the therapeutic potential of the bromodomain. Here, we describe the discovery of a hit from a fragment-based targeted array. Optimization of this produced the first known micromolar inhibitors of the ATAD2 bromodomain.
Subject(s)
Adenosine Triphosphatases/antagonists & inhibitors , Adenosine Triphosphatases/chemistry , Drug Discovery , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Adenosine Triphosphatases/metabolism , Amino Acid Sequence , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Humans , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Quinolones/chemistry , Quinolones/pharmacologyABSTRACT
G-CSF stimulates mobilization of hematopoietic progenitor cells (HPCs) from bone marrow by disrupting the CXCR4/SDF-1alpha retention axis. We show here that distinct factors and mechanisms regulate the mobilization of endothelial (EPCs) and stromal progenitor cells (SPCs). Pretreatment of mice with VEGF did not disrupt the CXCR4/SDF-1alpha chemokine axis but stimulated entry of HPCs into the cell cycle via VEGFR1, reducing their migratory capacity in vitro and suppressing their mobilization in vivo. In contrast, VEGF pretreatment enhanced EPC mobilization via VEGFR2 in response to CXCR4 antagonism. Furthermore, SPC mobilization was detected when the CXCR4 antagonist was administered to mice pretreated with VEGF, but not G-CSF. Thus, differential mobilization of progenitor cell subsets is dependent upon the cytokine milieu that regulates cell retention and proliferation. These findings may inform studies investigating mechanisms that regulate progenitor cell recruitment in disease and can be exploited to provide efficacious stem cell therapy for tissue regeneration.