A BET Protein Inhibitor Targeting Mononuclear Myeloid Cells Affects Specific Inflammatory Mediators and Pathways in Crohn's Disease.

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.

Carboxylesterase-1 Assisted Targeting of HDAC Inhibitors to Mononuclear Myeloid Cells in Inflammatory Bowel Disease.

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.

Bromodomain inhibitor I-BET151 suppresses immune responses during fungal-immune interaction.

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.

Aiming to Miss a Moving Target: Bromo and Extra Terminal Domain (BET) Selectivity in Constrained ATAD2 Inhibitors.

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.

BET bromodomain inhibitors show anti-papillomavirus activity in vitro and block CRPV wart growth in vivo.

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 E1ˆE4 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 W12 cells compared to non-infected C33A cells. The molecular mechanism for the cytotoxicity to W12 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.

A Chemical Probe for the ATAD2 Bromodomain.

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.

Structure-Based Optimization of Naphthyridones into Potent ATAD2 Bromodomain Inhibitors.

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.

Fragment-Based Discovery of Low-Micromolar ATAD2 Bromodomain Inhibitors.

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.

Differential mobilization of subsets of progenitor cells from the bone marrow.

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.

The role of the bone marrow in neutrophil clearance under homeostatic conditions in the mouse.

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.

The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation.

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1alpha-mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 x 10(6) and 5.4 x 10(6) neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 x 10(6) neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

Neutrophil mobilization and clearance in the bone marrow.

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.

Differential roles of the co-stimulatory molecules GITR and CTLA-4 in the immune response to Trichinella spiralis.

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.

Amelioration of influenza-induced pathology in mice by coinfection with Trichinella spiralis.

Illness due to respiratory virus infection is often induced by excessive infiltration of cells into pulmonary tissues, leading to airway occlusion. We show here that infection with Trichinella spiralis results in lower levels of tumor necrosis factor in bronchoalveolar lavage fluid and inhibits cellular recruitment into the airways of mice coinfected with influenza A virus. Infiltration of neutrophils and CD4+ and CD8+ lymphocytes was reduced, resulting in animals gaining weight more rapidly following the initial phase of infection. Influenza resulted in a generalized increase in vascular permeability in pulmonary tissues, and this was suppressed by parasite infection, although the effects were restricted to the early phase of trichinosis. Moreover, the number of cells producing interleukin-10 (IL-10), and the local levels of this cytokine, were reduced, suggesting that amelioration of pulmonary pathology by parasite infection occurs independently of IL-10 production.