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.

C5aR and C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling.

Mammalian survival depends on metabolizing nutrients, storing energy, and combating infection. Complement activation in blood triggers energy-depleting immune responses to fight infections. Here we identify surprising energy-conserving roles for complement proteins C5a and C3a and their receptors, C5aR and C3aR, roles that are contraindicated in complement biology. Rats fed a high-carbohydrate high-fat diet developed obesity, visceral adiposity, adipose inflammation, glucose/insulin intolerance, and cardiovascular dysfunction that correlated with increased plasma C3a, adipose C5aR, and C3aR. These in vivo changes were dramatically attenuated by receptor-selective antagonists of either C5aR (5 mg/kg/d p.o.) or C3aR (30 mg/kg/d p.o.), which both reduced proinflammatory adipokines and altered expression of inflammatory genes in adipose tissue. In vitro C5a and C3a (100 nM) exhibited novel insulin-like effects on 3T3-L1 adipocytes, promoting energy conservation by increasing glucose and fatty acid uptake while inhibiting cAMP signaling and lipolysis, and induced PGE(2) release from macrophages, effects all blocked by each respective antagonist (10 µM). These studies reveal important new links between complement signaling and metabolism, highlight new complement functions on adipocytes and in adipose tissue, demonstrate how aberrant immune responses may exacerbate obesity and metabolic dysfunction, and show that targeting C3aR or C5aR with antagonists is a new strategy for treating metabolic dysfunction.

Ras-Related Protein Rab5a Regulates Complement C5a Receptor Trafficking, Chemotaxis, and Chemokine Secretion in Human Macrophages.

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits ß-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with ß-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or ß-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-ß-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.

The histone deacetylase Hdac7 supports LPS-inducible glycolysis and Il-1ß production in murine macrophages via distinct mechanisms.

TLRs reprogram macrophage metabolism, enhancing glycolysis and promoting flux through the tricarboxylic acid cycle to enable histone acetylation and inflammatory gene expression. The histone deacetylase (HDAC) family of lysine deacetylases regulates both TLR-inducible glycolysis and inflammatory responses. Here, we show that the TLR4 agonist LPS, as well as agonists of other TLRs, rapidly increase enzymatic activity of the class IIa HDAC family (HDAC4, 5, 7, 9) in both primary human and murine macrophages. This response was abrogated in murine macrophages deficient in histone deacetylase 7 (Hdac7), highlighting a selective role for this specific lysine deacetylase during immediate macrophage activation. With the exception of the TLR3 agonist polyI:C, TLR-inducible activation of Hdac7 enzymatic activity required the MyD88 adaptor protein. The rapid glycolysis response, as assessed by extracellular acidification rate, was attenuated in Hdac7-deficient mouse macrophages responding to submaximal LPS concentrations. Surprisingly however, reconstitution of these cells with either wild-type or an enzyme-dead mutant of Hdac7 enhanced LPS-inducible glycolysis, whereas only the former promoted production of the inflammatory mediators Il-1ß and Ccl2. Thus, Hdac7 enzymatic activity is required for TLR-inducible production of specific inflammatory mediators, whereas it acts in an enzyme-independent fashion to reprogram metabolism in macrophages responding to submaximal LPS concentrations. Hdac7 is thus a bifurcation point for regulated metabolism and inflammatory responses in macrophages. Taken together with existing literature, our findings support a model in which submaximal and maximal activation of macrophages via TLR4 instruct glycolysis through distinct mechanisms, leading to divergent biological responses.

Temporal perturbation of histone deacetylase activity reveals a requirement for HDAC1-3 in mesendoderm cell differentiation.

Histone deacetylases (HDACs) are a class of enzymes that control chromatin state and influence cell fate. We evaluated the chromatin accessibility and transcriptome dynamics of zinc-containing HDACs during cell differentiation in vitro coupled with chemical perturbation to identify the role of HDACs in mesendoderm cell fate specification. Single-cell RNA sequencing analyses of HDAC expression during human pluripotent stem cell (hPSC) differentiation in vitro and mouse gastrulation in vivo reveal a unique association of HDAC1 and -3 with mesendoderm gene programs during exit from pluripotency. Functional perturbation with small molecules reveals that inhibition of HDAC1 and -3, but not HDAC2, induces mesoderm while impeding endoderm and early cardiac progenitor specification. These data identify unique biological functions of the structurally homologous enzymes HDAC1-3 in influencing hPSC differentiation from pluripotency toward mesendodermal and cardiac progenitor populations.

QSAR Classification Models for Prediction of Hydroxamate Histone Deacetylase Inhibitor Activity against Malaria Parasites.

Malaria, caused by Plasmodium parasites, results in >400,000 deaths annually. There is no effective vaccine, and new drugs with novel modes of action are needed because of increasing parasite resistance to current antimalarials. Histone deacetylases (HDACs) are epigenetic regulatory enzymes that catalyze post-translational protein deacetylation and are promising malaria drug targets. Here, we describe quantitative structure-activity relationship models to predict the antiplasmodial activity of hydroxamate-based HDAC inhibitors. The models incorporate P. falciparum in vitro activity data for 385 compounds containing a hydroxamic acid and were subject to internal and external validation. When used to screen 22 new hydroxamate-based HDAC inhibitors for antiplasmodial activity, model A7 (external accuracy 91%) identified three hits that were subsequently verified as having potent in vitro activity against P. falciparum parasites (IC50 = 6, 71, and 84 nM), with 8 to 51-fold selectivity for P. falciparum versus human cells.

Inhibition of inflammation and fibrosis by a complement C5a receptor antagonist in DOCA-salt hypertensive rats.

The anaphylatoxin C5a generated by activation of the innate immunity complement system is a potent inflammatory peptide mediator through the G-protein-coupled receptor C5aR (CD88) present in immune-inflammatory cells, including monocytes, macrophages, neutrophils, T cells, and mast cells. Inflammatory cells infiltrate and initiate the development of fibrosis in the chronically hypertensive heart. In this study, we have investigated whether treatment with a selective C5aR antagonist prevents cardiovascular remodeling in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Control and DOCA-salt rats were treated with PMX53 (AcF-[OPdChaWR], 1 mg·kg·d oral gavage) for 32 days; structural and functional changes in cardiovascular system were determined. DOCA-salt hypertension increased leukocyte extravasation into ventricular tissue, increasing collagen deposition and ventricular stiffness; PMX53 treatment attenuated these changes, thereby improving cardiac function. Further, treatment with PMX53 suppressed an increased expression of C5aR in the left ventricle from DOCA-salt rats, consistent with the reduced infiltration of inflammatory cells. Vascular endothelial dysfunction in thoracic aortic rings was attenuated by PMX53 treatment, but systolic blood pressure was unchanged in DOCA-salt rats. In the heart, PMX53 treatment attenuated inflammatory cell infiltration, fibrosis, and ventricular stiffness, indicating that C5aR is critically involved in ventricular remodeling by regulating inflammatory responses in the hypertensive heart.

Histone deacetylase inhibitor AR-42 and achiral analogues kill malaria parasites in vitro and in mice.

Malaria is caused by infection with Plasmodium parasites and results in significant health and economic impacts. Malaria eradication is hampered by parasite resistance to current drugs and the lack of a widely effective vaccine. Compounds that target epigenetic regulatory proteins, such as histone deacetylases (HDACs), may lead to new therapeutic agents with a different mechanism of action, thereby avoiding resistance mechanisms to current antimalarial drugs. The anticancer HDAC inhibitor AR-42, as its racemate (rac-AR-42), and 36 analogues were investigated for in vitro activity against P. falciparum. Rac-AR-42 and selected compounds were assessed for cytotoxicity against human cells, histone hyperacetylation, human HDAC1 inhibition and oral activity in a murine malaria model. Rac-AR-42 was tested for ex vivo asexual and in vitro exoerythrocytic stage activity against P. berghei murine malaria parasites. Rac-AR-42 and 13 achiral analogues were potent inhibitors of asexual intraerythrocytic stage P. falciparum 3D7 growth in vitro (IC50 5-50 nM), with four of these compounds having >50-fold selectivity for P. falciparum versus human cells (selectivity index 56-118). Rac-AR-42 induced in situ hyperacetylation of P. falciparum histone H4, consistent with PfHDAC(s) inhibition. Furthermore, rac-AR-42 potently inhibited P. berghei infected erythrocyte growth ex vivo (IC50 40 nM) and P. berghei exoerythrocytic forms in hepatocytes (IC50 1 nM). Oral administration of rac-AR-42 and two achiral analogues inhibited P. berghei growth in mice, with rac-AR-42 (50 mg/kg/day single dose for four days) curing all infections. These findings demonstrate curative properties for HDAC inhibitors in the oral treatment of experimental mouse malaria.

HDAC7 Inhibition by Phenacetyl and Phenylbenzoyl Hydroxamates.

The zinc-containing histone deacetylase enzyme HDAC7 is emerging as an important regulator of immunometabolism and cancer. Here, we exploit a cavity in HDAC7, filled by Tyr303 in HDAC1, to derive new inhibitors. Phenacetyl hydroxamates and 2-phenylbenzoyl hydroxamates bind to Zn2+ and are 50-2700-fold more selective inhibitors of HDAC7 than HDAC1. Phenylbenzoyl hydroxamates are 30-70-fold more potent HDAC7 inhibitors than phenacetyl hydroxamates, which is attributed to the benzoyl aromatic group interacting with Phe679 and Phe738. Phthalimide capping groups, including a saccharin analogue, decrease rotational freedom and provide hydrogen bond acceptor carbonyl/sulfonamide oxygens that increase inhibitor potency, liver microsome stability, solubility, and cell activity. Despite being the most potent HDAC7 inhibitors to date, they are not selective among class IIa enzymes. These strategies may help to produce tools for interrogating HDAC7 biology related to its catalytic site.

De novo macrocyclic peptides for inhibiting, stabilizing, and probing the function of the retromer endosomal trafficking complex.

The retromer complex (Vps35-Vps26-Vps29) is essential for endosomal membrane trafficking and signaling. Mutation of the retromer subunit Vps35 causes late-onset Parkinson's disease, while viral and bacterial pathogens can hijack the complex during cellular infection. To modulate and probe its function, we have created a novel series of macrocyclic peptides that bind retromer with high affinity and specificity. Crystal structures show that most of the cyclic peptides bind to Vps29 via a Pro-Leu­containing sequence, structurally mimicking known interactors such as TBC1D5 and blocking their interaction with retromer in vitro and in cells. By contrast, macrocyclic peptide RT-L4 binds retromer at the Vps35-Vps26 interface and is a more effective molecular chaperone than reported small molecules, suggesting a new therapeutic avenue for targeting retromer. Last, tagged peptides can be used to probe the cellular localization of retromer and its functional interactions in cells, providing novel tools for studying retromer function.

Antimalarial histone deacetylase inhibitors containing cinnamate or NSAID components.

Malaria is the most lethal parasite-mediated tropical infectious disease, killing 1-2 million people each year. An emerging drug target is the enzyme Plasmodium falciparum histone deacetylase 1 (PfHDAC1). We report 26 compounds designed to bind the zinc and exterior surface around the entrance to the active site of PfHDAC1, 16 displaying potent in vitro antimalarial activity (IC(50)<100 nM) against P. falciparum. Selected compounds were shown to cause hyperacetylation of P. falciparum histones and be >10-fold more cytotoxic towards P. falciparum than a normal human cell type (NFF). Twenty-two inhibitors feature cinnamic acid derivatives or non-steroidal anti-inflammatory drugs (NSAIDs) as HDAC-binding components. A homology model of PfHDAC1 enzyme gives new insights to interactions likely made by some of these inhibitors. Results support PfHDAC1 as a promising new antimalarial drug target.