HDAC7 is an immunometabolic switch triaging danger signals for engagement of antimicrobial versus inflammatory responses in macrophages.

The immune system must be able to respond to a myriad of different threats, each requiring a distinct type of response. Here, we demonstrate that the cytoplasmic lysine deacetylase HDAC7 in macrophages is a metabolic switch that triages danger signals to enable the most appropriate immune response. Lipopolysaccharide (LPS) and soluble signals indicating distal or far-away danger trigger HDAC7-dependent glycolysis and proinflammatory IL-1ß production. In contrast, HDAC7 initiates the pentose phosphate pathway (PPP) for NADPH and reactive oxygen species (ROS) production in response to the more proximal threat of nearby bacteria, as exemplified by studies on uropathogenic Escherichia coli (UPEC). HDAC7-mediated PPP engagement via 6-phosphogluconate dehydrogenase (6PGD) generates NADPH for antimicrobial ROS production, as well as D-ribulose-5-phosphate (RL5P) that both synergizes with ROS for UPEC killing and suppresses selective inflammatory responses. This dual functionality of the HDAC7-6PGD-RL5P axis prioritizes responses to proximal threats. Our findings thus reveal that the PPP metabolite RL5P has both antimicrobial and immunomodulatory activities and that engagement of enzymes in catabolic versus anabolic metabolic pathways triages responses to different types of danger for generation of inflammatory versus antimicrobial responses, respectively.

Oxazole-Benzenesulfonamide Derivatives Inhibit HIV-1 Reverse Transcriptase Interaction with Cellular eEF1A and Reduce Viral Replication.

HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

An HDAC6 Inhibitor Confers Protection and Selectively Inhibits B-Cell Infiltration in DSS-Induced Colitis in Mice.

Small molecule histone deacetylase (HDAC) inhibitors with anti-inflammatory activity may be candidates for targeting intestinal inflammatory pathways in inflammatory bowel disease (IBD). This study investigated whether treatment with a potent HDAC6 inhibitor, BML-281, could protect against colonic inflammation and prevent inflammatory cell infiltration into the colon to drive disease pathology in a mouse model of acute dextran sodium sulfate (DSS) colitis. Control and acute DSS-colitis mice were treated with BML-281 (1 mg/kg per day s.c. and 10 mg/kg per day s.c.) for 8 days. Changes in disease pathology, colonic structure, function, alterations in inflammatory milieu, together with colonic inflammatory cell flux, were assessed by weight loss and disease activity index in vivo and by flow cytometry, gene expression, and histology ex vivo. Anti-inflammatory responses of BML-281 on human polymorphonuclear leukocytes were assessed in vitro. Administration of BML-281 to DSS-treated mice attenuated colitis, weight loss, and disease pathology, including changes in colon structure and function, by eliciting broad-spectrum anti-inflammatory effects and preventing infiltration and activation of key immune cells in the lamina propria of the intestinal epithelium. Among different immune cells, BML-281 particularly suppressed the infiltration of CD19+ B-cells into the inflamed colonic lamina propria. This study supports the targeting of HDAC6 as an anti-inflammatory strategy for treating colon inflammation progressing to IBD. Some HDAC inhibitors are used in the clinic to treat cancer, and the results here for BML-281 highlight the potential for HDAC6 inhibitors to be used in a clinical setting for preventing and treating colonic inflammation and IBD in humans.

Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor.

Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (Kd) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pKi of 8.6 ± 0.2 and Ki of 2.5 nM) and C3aR ligands TR16 (pKi of 6.8 ± 0.1 and Ki of 138 nM), BR103 (pKi of 6.7 ± 0.1 and Ki of 185 nM), BR111 (pKi of 6.3 ± 0.2 and Ki of 544 nM) and SB290157 (pKi of 6.3 ± 0.1 and Ki of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.

Differential Anti-inflammatory Activity of HDAC Inhibitors in Human Macrophages and Rat Arthritis.

Vorinostat and other inhibitors of different histone deacetylase (HDAC) enzymes are currently being sought to modulate a variety of human conditions, including chronic inflammatory diseases. Some HDAC inhibitors are anti-inflammatory in rodent models of arthritis and colitis, usually at cytotoxic doses that may cause side effects. Here, we investigate the dose-dependent pro- and anti-inflammatory efficacy of two known inhibitors of multiple HDACs, vorinostat and BML281, in human macrophages and in a rat model of collagen-induced arthritis by monitoring effects on disease progression, histopathology, and immunohistochemistry. Both HDAC inhibitors differentially modulated lipopolysaccharide (LPS)-induced cytokine release from human macrophages, suppressing release of some inflammatory mediators (IL12p40, IL6) at low concentrations (<3 µM) but amplifying production of others (TNF, IL1ß) at higher concentration (>3 µΜ). This trend translated in vivo to rat arthritis, with anti-inflammatory activity inversely correlating with dose. Both compounds were efficacious only at a low dose (1 mg⋅kg(-1)⋅day(-1) s.c.), whereas a higher dose (5 mg⋅kg(-1)⋅day(-1) s.c.) showed no positive effects on reducing pathology, even showing signs of exacerbating disease. These striking effects suggest a smaller therapeutic window than previously reported for HDAC inhibition in experimental arthritis. The findings support new investigations into repurposing HDAC inhibitors for anti-inflammatory therapeutic applications. However, HDAC inhibitors should be reinvestigated at lower, rather than higher, doses for enhanced efficacy in chronic diseases that require long-term treatment, with careful management of efficacy and long-term safety.

Benzylamide antagonists of protease activated receptor 2 with anti-inflammatory activity.

Activation of protease activated receptor 2 (PAR2) has been implicated in inflammatory and metabolic disorders and its inhibition may yield novel therapeutics. Here, we report a series of PAR2 antagonists based on C-terminal capping of 5-isoxazolyl-L-cyclohexylalanine-L-isoleucine, with benzylamine analogues being effective new PAR2 antagonists. 5-Isoxazolyl-L-cyclohexylalanine-L-isoleucine-2-methoxybenzylamine (10) inhibited PAR2-, but not PAR1-, induced release of Ca(2+) (IC50 0.5 µM) in human colon cells, IL-6 and TNFα secretion (IC50 1-5 µM) from human kidney cells, and was anti-inflammatory in acute rat paw inflammation (ED50 5 mg/kg sc). These findings show that new benzylamide antagonists of PAR2 have anti-inflammatory activity.

Histone Deacetylase Inhibitors Promote Mitochondrial Reactive Oxygen Species Production and Bacterial Clearance by Human Macrophages.

Broad-spectrum histone deacetylase inhibitors (HDACi) are used clinically as anticancer agents, and more isoform-selective HDACi have been sought to modulate other conditions, including chronic inflammatory diseases. Mouse studies suggest that HDACi downregulate immune responses and may compromise host defense. However, their effects on human macrophage antimicrobial responses are largely unknown. Here, we show that overnight pretreatment of human macrophages with HDACi prior to challenge with Salmonella enterica serovar Typhimurium or Escherichia coli results in significantly reduced intramacrophage bacterial loads, which likely reflect the fact that this treatment regime impairs phagocytosis. In contrast, cotreatment of human macrophages with HDACi at the time of bacterial challenge did not impair phagocytosis; instead, HDACi cotreatment actually promoted clearance of intracellular S. Typhimurium and E. coli. Mechanistically, treatment of human macrophages with HDACi at the time of bacterial infection enhanced mitochondrial reactive oxygen species generation by these cells. The capacity of HDACi to promote the clearance of intracellular bacteria from human macrophages was abrogated when cells were pretreated with MitoTracker Red CMXRos, which perturbs mitochondrial function. The HDAC6-selective inhibitor tubastatin A promoted bacterial clearance from human macrophages, whereas the class I HDAC inhibitor MS-275, which inhibits HDAC1 to -3, had no effect on intracellular bacterial loads. These data are consistent with HDAC6 and/or related HDACs constraining mitochondrial reactive oxygen species production from human macrophages during bacterial challenge. Our findings suggest that, whereas long-term HDACi treatment regimes may potentially compromise host defense, selective HDAC inhibitors may have applications in treating acute bacterial infections.

Potent complement C3a receptor agonists derived from oxazole amino acids: Structure-activity relationships.

Potent ligands for the human complement C3a receptor (C3aR) were developed from the almost inactive tripeptide Leu-Ala-Arg corresponding to the three C-terminal residues of the endogenous peptide agonist C3a. The analogous Leu-Ser-Arg was modified by condensing the serine side chain with the leucine carbonyl with elimination of water to form leucine-oxazole-arginine. Subsequent elaboration with a variety of N-terminal amide capping groups produced agonists as potent as human C3a itself in stimulating Ca(2+) release from human macrophages. Structure-activity relationships are discussed.

Histone deacetylase 7 promotes Toll-like receptor 4-dependent proinflammatory gene expression in macrophages.

Broad-spectrum inhibitors of histone deacetylases (HDACs) constrain Toll-like receptor (TLR)-inducible production of key proinflammatory mediators. Here we investigated HDAC-dependent inflammatory responses in mouse macrophages. Of the classical Hdacs, Hdac7 was expressed at elevated levels in inflammatory macrophages (thioglycollate-elicited peritoneal macrophages) as compared with bone marrow-derived macrophages and the RAW264 cell line. Overexpression of a specific, alternatively spliced isoform of Hdac7 lacking the N-terminal 22 amino acids (Hdac7-u), but not the Refseq Hdac7 (Hdac7-s), promoted LPS-inducible expression of Hdac-dependent genes (Edn1, Il-12p40, and Il-6) in RAW264 cells. A novel class IIa-selective HDAC inhibitor reduced recombinant human HDAC7 enzyme activity as well as TLR-induced production of inflammatory mediators in thioglycollate-elicited peritoneal macrophages. Both LPS and Hdac7-u up-regulated the activity of the Edn1 promoter in an HDAC-dependent fashion in RAW264 cells. A hypoxia-inducible factor (HIF) 1 binding site in this promoter was required for HDAC-dependent TLR-inducible promoter activity and for Hdac7- and HIF-1α-mediated trans-activation. Coimmunoprecipitation assays showed that both Hdac7-u and Hdac7-s interacted with HIF-1α, whereas only Hdac7-s interacted with the transcriptional repressor CtBP1. Thus, Hdac7-u positively regulates HIF-1α-dependent TLR signaling in macrophages, whereas an interaction with CtBP1 likely prevents Hdac7-s from exerting this effect. Hdac7 may represent a potential inflammatory disease target.

Stereoelectronic effects dictate molecular conformation and biological function of heterocyclic amides.

Heterocycles adjacent to amides can have important influences on molecular conformation due to stereoelectronic effects exerted by the heteroatom. This was shown for imidazole- and thiazole-amides by comparing low energy conformations (ab initio MP2 and DFT calculations), charge distribution, dipole moments, and known crystal structures which support a general principle. Switching a heteroatom from nitrogen to sulfur altered the amide conformation, producing different three-dimensional electrostatic surfaces. Differences were attributed to different dipole and orbital alignments and spectacularly translated into opposing agonist vs antagonist functions in modulating a G-protein coupled receptor for inflammatory protein complement C3a on human macrophages. Influences of the heteroatom were confirmed by locking the amide conformation using fused bicyclic rings. These findings show that stereoelectronic effects of heterocycles modulate molecular conformation and can impart strikingly different biological properties.