Synthon-based ligand discovery in virtual libraries of over 11 billion compounds.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures1-4 and ultra-large libraries of virtual compounds5,6. However, to keep pace with the rapid growth of virtual libraries, such as readily available for synthesis (REAL) combinatorial libraries7, new approaches to compound screening are needed8,9. Here we introduce a modular synthon-based approach-V-SYNTHES-to perform hierarchical structure-based screening of a REAL Space library of more than 11 billion compounds. V-SYNTHES first identifies the best scaffold-synthon combinations as seeds suitable for further growth, and then iteratively elaborates these seeds to select complete molecules with the best docking scores. This hierarchical combinatorial approach enables the rapid detection of the best-scoring compounds in the gigascale chemical space while performing docking of only a small fraction (<0.1%) of the library compounds. Chemical synthesis and experimental testing of novel cannabinoid antagonists predicted by V-SYNTHES demonstrated a 33% hit rate, including 14 submicromolar ligands, substantially improving over a standard virtual screening of the Enamine REAL diversity subset, which required approximately 100 times more computational resources. Synthesis of selected analogues of the best hits further improved potencies and affinities (best inhibitory constant (Ki) = 0.9 nM) and CB2/CB1 selectivity (50-200-fold). V-SYNTHES was also tested on a kinase target, ROCK1, further supporting its use for lead discovery. The approach is easily scalable for the rapid growth of combinatorial libraries and potentially adaptable to any docking algorithm.

Human leukocytes selectively convert 4S,5S-epoxy-resolvin to resolvin D3, resolvin D4, and a cys-resolvin isomer.

Human phagocytes have key functions in the resolution of inflammation. Here, we assessed the role of the proposed 4S,5S-epoxy-resolvin intermediate in the biosynthesis of both resolvin D3 and resolvin D4. We found that human neutrophils converted this synthetic intermediate to resolvin D3 and resolvin D4. M2 macrophages transformed this labile epoxide intermediate to resolvin D4 and a previously unknown cysteinyl-resolvin isomer without appreciable amounts of resolvin D3. M2 macrophages play critical roles in the resolution of inflammation and in wound healing. Human M2 macrophages also converted leukotriene A4 to lipoxins. The cysteinyl-resolvin isomer significantly accelerated tissue regeneration of surgically injured planaria. In a model of human granuloma formation, the cysteinyl-resolvin isomer significantly inhibited granuloma development by human peripheral blood leukocytes. Together, these results provide evidence for a human cell type-specific role of 4S,5S-epoxy-resolvin in the biosynthesis of resolvin D3 by neutrophils, resolvin D4 by both M2 macrophages and neutrophils, and a unique cysteinyl-resolvin isomer produced by M2 macrophages that carries potent biological activities in granuloma formation and tissue regeneration.

Stereocontrolled total synthesis of Resolvin D4 and 17(R)-Resolvin D4.

The total synthesis of Resolvin D4 and its 17(R)-hydroxy-epimer is reported. These lipid-based natural products are biosynthesized from docosahexaenoic acid (DHA, C22:6) during the body's rapid cellular and chemical response to injurious stimuli and are part of a large class of bioactive molecules that resolve inflammation. Our convergent synthesis employed a chiral pool strategy starting from glycidol derivatives and D-erythrose to introduce stereogenic centers. A copper(I)-mediated cross coupling between propargyl bromide and terminal acetylenic precursors yielded core structures of late-stage key intermediates. A simultaneous Lindlar reduction of the skipped diynyl moiety followed by silyl group cleavage securely completed the synthesis. The synthetic availability of these molecules helped further elucidate their stereoselective biofunctions.

PCTR1 Enhances Repair and Bacterial Clearance in Skin Wounds.

Tissue injury elicits an inflammatory response that facilitates host defense. Resolution of inflammation promotes the transition to tissue repair and is governed, in part, by specialized pro-resolving mediators (SPM). The complete structures of a novel series of cysteinyl-SPM (cys-SPM) were recently elucidated, and proved to stimulate tissue regeneration in planaria and resolve acute inflammation in mice. Their functions in mammalian tissue repair are of interest. Here, nine structurally distinct cys-SPM were screened and PCTR1 uniquely enhanced human keratinocyte migration with efficacy similar to epidermal growth factor. In skin wounds of mice, PCTR1 accelerated closure. Wound infection increased PCTR1 that coincided with decreased bacterial burden. Addition of PCTR1 reduced wound bacteria levels and decreased inflammatory monocytes/macrophages, which was coupled with increased expression of genes involved in host defense and tissue repair. These results suggest that PCTR1 is a novel regulator of host defense and tissue repair, which could inform new approaches for therapeutic management of delayed tissue repair and infection.

Elucidating the structure and functions of Resolvin D6 isomers on nerve regeneration with a distinctive trigeminal transcriptome.

Innervation sustains cornea integrity. Pigment epithelium-derived factor (PEDF) plus docosahexaenoic acid (DHA) regenerated damaged nerves by stimulating the synthesis of a new stereoisomer of Resolvin D6 (RvD6si). Here, we resolved the structure of this lipid isolated from mouse tears after injured corneas were treated with PEDF + DHA. RvD6si synthesis was inhibited by fluvoxamine, a cytochrome P450 inhibitor, but not by 15- or 5-LOX inhibitors, suggesting that the 4- and 17-hydroxy of DHA have an RR- or SR-configuration. The two compounds were chemically synthesized. Using chiral phase HPLC, four peaks of RvD6si1-4 from tears were resolved. The RR-RvD6 standard eluted as a single peak with RvD61 while pure SR-RvD6 eluted with RvD63 . The addition of these pure mediators prompted a trigeminal ganglion transcriptome response in injured corneas and showed that RR-RvD6 was the more potent, increasing cornea sensitivity and nerve regeneration. RR-RvD6 stimulates Rictor and hepatocyte growth factor (hgf) genes specifically as upstream regulators and a gene network involved in axon growth and suppression of neuropathic pain, indicating a novel function of this lipid mediator to maintain cornea integrity and homeostasis after injury.

Elovanoids counteract oligomeric ß-amyloid-induced gene expression and protect photoreceptors.

The onset of neurodegenerative diseases activates inflammation that leads to progressive neuronal cell death and impairments in cognition (Alzheimer's disease) and sight (age-related macular degeneration [AMD]). How neuroinflammation can be counteracted is not known. In AMD, amyloid ß-peptide (Aß) accumulates in subretinal drusen. In the 5xFAD retina, we found early functional deficiencies (ERG) without photoreceptor cell (PRC) death and identified early insufficiency in biosynthetic pathways of prohomeostatic/neuroprotective mediators neuroprotectin D1 (NPD1) and elovanoids (ELVs). To mimic an inflammatory milieu in wild-type mouse, we triggered retinal pigment epithelium (RPE) damage/PRC death by subretinally injected oligomeric ß-amyloid (OAß) and observed that ELVs administration counteracted their effects, protecting these cells. In addition, ELVs prevented OAß-induced changes in gene expression engaged in senescence, inflammation, autophagy, extracellular matrix remodeling, and AMD. Moreover, as OAß targets the RPE, we used primary human RPE cell cultures and demonstrated that OAß caused cell damage, while ELVs protected and restored gene expression as in mouse. Our data show OAß activates senescence as reflected by enhanced expression of p16INK4a, MMP1, p53, p21, p27, and Il-6, and of senescence-associated phenotype secretome, followed by RPE and PRC demise, and that ELVs 32 and 34 blunt these events and elicit protection. In addition, ELVs counteracted OAß-induced expression of genes engaged in AMD, autophagy, and extracellular matrix remodeling. Overall, our data uncovered that ELVs downplay OAß-senescence program induction and inflammatory transcriptional events and protect RPE cells and PRC, and therefore have potential as a possible therapeutic avenue for AMD.

Synergistic Neuroprotection by a PAF Antagonist Plus a Docosanoid in Experimental Ischemic Stroke: Dose-Response and Therapeutic Window.

OBJECTIVE: We tested the hypothesis that blocking pro-inflammatory platelet-activating factor receptor (PAFR) with LAU-0901 (LAU) plus administering a selected docosanoid, aspirin-triggered neuroprotectin D1 (AT-NPD1), which activates cell-survival pathways after middle cerebral artery occlusion (MCAo), would lead to neurological recovery. Dose-response and therapeutic window were investigated. MATERIALS AND METHODS: Male SD rats were subjected to 2 hours of MCAo. Behavior testing (days 1-7) and ex vivo MRI on day 7 were conducted. In dose-response, rats were treated with LAU (45 and 60 mg/kg; IP), AT-NPD1 (111, 222, 333 µg/kg; IV), LAU+AT-NPD1 (LAU at 3 hours and AT-NPD1 at 3.15 hours) or vehicle. In the therapeutic window, vehicle, LAU (60 mg/kg), AT-NPD1 (222 µg/kg), and LAU+AT-NPD1 were administered at 3, 4, 5, and 6 hours after onset of MCAo. RESULTS: LAU and AT-NPD1 treatments alone improved behavior by 40-42% and 20-30%, respectively, and LAU+AT-NPD1 by 40% compared to the vehicle group. T2-weighted imaging (T2WI) volumes were reduced with all doses of LAU and AT-NPD1 by 73-90% and 67-83% and LAU+AT-NPD1 by 94% compared to vehicle. In the therapeutic window, LAU+AT-NPD1, when administered at 3, 4, 5, and 6 hours, improved behavior by 50, 56, 33, and 26% and reduced T2WI volumes by 93, 90, 82, and 84% compared to vehicle. CONCLUSIONS: We have shown here for the first time that LAU plus AT-NPD1 treatment affords high-grade neuroprotection in MCAo, equaling or exceeding that afforded by LAU or AT-NPD1 alone at considerably moderate doses. It has a broad therapeutic window extending to 6 hours after stroke onset.

Cyanine Nanocage Activated by Near-IR Light for the Targeted Delivery of Cyclosporine A to Traumatic Brain Injury Sites.

More than 2.8 million annually in the United States are afflicted with some form of traumatic brain injury (TBI), where 75% of victims have a mild form of TBI (MTBI). TBI risk is higher for individuals engaging in physical activities or involved in accidents. Although MTBI may not be initially life-threatening, a large number of these victims can develop cognitive and physical dysfunctions. These late clinical sequelae have been attributed to the development of secondary injuries that can occur minutes to days after the initial impact. To minimize brain damage from TBI, it is critical to diagnose and treat patients within the first or "golden" hour after TBI. Although it would be very helpful to quickly determine the TBI locations in the brain and direct the treatment selectively to the affected sites, this remains a challenge. Herein, we disclose our novel strategy to target cyclosporine A (CsA) into TBI sites, without the need to locate the exact location of the TBI lesion. Our approach is based on TBI treatment with a cyanine dye nanocage attached to CsA, a known therapeutic agent for TBI that is associated with unacceptable toxicities. In its caged form, CsA remains inactive, while after near-IR light photoactivation, the resulting fragmentation of the cyanine nanocage leads to the selective release of CsA at the TBI sites.

Maresin conjugates in tissue regeneration biosynthesis enzymes in human macrophages.

Macrophages are central in coordinating immune responses, tissue repair, and regeneration, with different subtypes being associated with inflammation-initiating and proresolving actions. We recently identified a family of macrophage-derived proresolving and tissue regenerative molecules coined maresin conjugates in tissue regeneration (MCTR). Herein, using lipid mediator profiling we identified MCTR in human serum, lymph nodes, and plasma and investigated MCTR biosynthetic pathways in human macrophages. With human recombinant enzymes, primary cells, and enantiomerically pure compounds we found that the synthetic maresin epoxide intermediate 13S,14S-eMaR (13S,14S-epoxy- 4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid) was converted to MCTR1 (13R-glutathionyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) by LTC4S and GSTM4. Incubation of human macrophages with LTC4S inhibitors blocked LTC4 and increased resolvins and lipoxins. The conversion of MCTR1 to MCTR2 (13R-cysteinylglycinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid) was catalyzed by γ-glutamyl transferase (GGT) in human macrophages. Biosynthesis of MCTR3 was mediated by dipeptidases that cleaved the cysteinyl-glycinyl bond of MCTR2 to give 13R-cysteinyl, 14S-hydroxy-4Z,7Z,9E,11E,13R,14S,16Z,19Z-docosahexaenoic acid. Of note, both GSTM4 and GGT enzymes displayed higher affinity to 13S,14S-eMaR and MCTR1 compared with their classic substrates in the cysteinyl leukotriene metabolome. Together these results establish the MCTR biosynthetic pathway and provide mechanisms in tissue repair and regeneration.

Resolvin D3 and Aspirin-Triggered Resolvin D3 Are Protective for Injured Epithelia.

Acute lung injury is a life-threatening condition caused by disruption of the alveolar-capillary barrier leading to edema, influx of inflammatory leukocytes, and impaired gas exchange. Specialized proresolving mediators biosynthesized from essential fatty acids, such as docosahexaenoic acid, have tissue protective effects in acute inflammation. Herein, we found that the docosahexaenoic acid-derived mediator resolvin D3 (RvD3): 4S,11R,17S-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid was present in uninjured lungs, and increased significantly 24 to 72 hours after hydrochloric acid-initiated injury. Because of its delayed enzymatic degradation, we used aspirin-triggered (AT)-RvD3: 4S,11R,17R-trihydroxydocosa-5Z,7E,9E,13Z,15E,19Z-hexaenoic acid, a 17R-epimer of RvD3, for in vivo experiments. Histopathological correlates of acid injury (alveolar wall thickening, edema, and leukocyte infiltration) were reduced in mice receiving AT-RvD3 1 hour after injury. AT-RvD3-treated mice had significantly reduced edema, as demonstrated by lower wet/dry weight ratios, increased epithelial sodium channel γ expression, and more lymphatic vessel endothelial hyaluronan receptor 1-positive vascular endothelial growth factor receptor 3-positive lymphatic vessels. Evidence for counterregulation of NF-κB by RvD3 and AT-RvD3 was seen in vitro and by AT-RvD3 in vivo. Increases in lung epithelial cell proliferation and bronchoalveolar lavage fluid levels of keratinocyte growth factor were observed with AT-RvD3, which also promoted cutaneous re-epithelialization. Together, these data demonstrate protective actions of RvD3 and AT-RvD3 for injured mucosa that accelerated restoration of epithelial barrier and function.

Cutting edge: maresin-1 engages regulatory T cells to limit type 2 innate lymphoid cell activation and promote resolution of lung inflammation.

Asthma is a chronic inflammatory disease that fails to resolve. Recently, a key role for type 2 innate lymphoid cells (ILC2s) was linked to asthma pathogenesis; however, mechanisms for ILC2 regulation remain to be determined. In this study, metabololipidomics of murine lungs identified temporal changes in endogenous maresin 1 (MaR1) during self-limited allergic inflammation. Exogenous MaR1 reduced lung inflammation and ILC2 expression of IL-5 and IL-13 and increased amphiregulin. MaR1 augmented de novo generation of regulatory T cells (Tregs), which interacted with ILC2s to markedly suppress cytokine production in a TGF-ß-dependent manner. Ab-mediated depletion of Tregs interrupted MaR1 control of ILC2 expression of IL-13 in vivo. Together, the findings uncover Tregs as potent regulators of ILC2 activation; MaR1 targets Tregs and ILC2s to restrain allergic lung inflammation, suggesting MaR1 as the basis for a new proresolving therapeutic approach to asthma and other chronic inflammatory diseases.

Maresin 1 biosynthesis during platelet-neutrophil interactions is organ-protective.

Unregulated acute inflammation can lead to collateral tissue injury in vital organs, such as the lung during the acute respiratory distress syndrome. In response to tissue injury, circulating platelet-neutrophil aggregates form to augment neutrophil tissue entry. These early cellular events in acute inflammation are pivotal to timely resolution by mechanisms that remain to be elucidated. Here, we identified a previously undescribed biosynthetic route during human platelet-neutrophil interactions for the proresolving mediator maresin 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid). Docosahexaenoic acid was converted by platelet 12-lipoxygenase to 13S,14S-epoxy-maresin, which was further transformed by neutrophils to MaR1. In a murine model of acute respiratory distress syndrome, lipid mediator metabololipidomics uncovered MaR1 generation in vivo in a temporally regulated manner. Early MaR1 production was dependent on platelet-neutrophil interactions, and intravascular MaR1 was organ-protective, leading to decreased lung neutrophils, edema, tissue hypoxia, and prophlogistic mediators. Together, these findings identify a transcellular route for intravascular maresin 1 biosynthesis via platelet-neutrophil interactions that regulates the extent of lung inflammation.

Protective actions of aspirin-triggered (17R) resolvin D1 and its analogue, 17R-hydroxy-19-para-fluorophenoxy-resolvin D1 methyl ester, in C5a-dependent IgG immune complex-induced inflammation and lung injury.

Increasing evidence suggests that the novel anti-inflammatory and proresolving mediators such as the resolvins play an important role during inflammation. However, the functions of these lipid mediators in immune complex-induced lung injury remain unknown. In this study, we determined the role of aspirin-triggered resolvin D1 (AT-RvD1) and its metabolically stable analog, 17R-hydroxy-19-para-fluorophenoxy-resolvin D1 methyl ester (p-RvD1), in IgG immune complex-induced inflammatory responses in myeloid cells and injury in the lung. We show that lung vascular permeability in the AT-RvD1- or p-RvD1-treated mice was significantly reduced when compared with values in mice receiving control vesicle during the injury. Furthermore, i.v. administration of either AT-RvD1 or p-RvD1 caused significant decreases in the bronchoalveolar lavage fluid contents of neutrophils, inflammatory cytokines, and chemokines. Of interest, AT-RvD1 or p-RvD1 significantly reduced bronchoalveolar lavage fluid complement C5a level. By EMSA, we demonstrate that IgG immune complex-induced activation of NF-κB and C/EBPß transcription factors in the lung was significantly inhibited by AT-RvD1 and p-RvD1. Moreover, AT-RvD1 dramatically mitigates IgG immune complex-induced NF-κB and C/EBP activity in alveolar macrophages. Also, secretion of TNF-α, IL-6, keratinocyte cell-derived chemokine, and MIP-1α from IgG immune complex-stimulated alveolar macrophages or neutrophils was significantly decreased by AT-RvD1. These results suggest a new approach to the blocking of immune complex-induced inflammation.

Molecular characterization of the boron adducts of the proteasome inhibitor bortezomib with epigallocatechin-3-gallate and related polyphenols.

The green tea polyphenol epigallocatechin-3-gallate (EGCG) was reported to effectively antagonize the ability of Bortezomib (BZM) to induce apoptosis in cancer cells. This interaction was attributed to the formation of a covalent adduct between a phenolic moiety of EGCG with the boronic acid group of Bortezomib. However, the structural details of this boron adduct and the molecular factors that contribute to its formation and its ability to inhibit Bortezomib's activity remain unclear. This paper describes the use of NMR spectroscopy and cell assays to characterize the structures and properties of the boron adducts of EGCG and related polyphenols. The observed boron adducts included both boronate and borate derivatives, and their structural characteristics were correlated with cell-based evaluation of the ability of EGCG and other phenols to antagonize the anticancer activity of Bortezomib. The enhanced stability of the BZM/EGCG adduct was attributed to electronic and steric reasons, and a newly identified intramolecular interaction of the boron atom of BZM with the adjacent amide bond. The reported approach provides a useful method for determining the potential ability of polyphenols to form undesired adducts with boron-based drugs and interfere with their actions.

Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis.

A growing body of evidence indicates that resolution of acute inflammation is an active process. Resolvins are a new family of lipid mediators enzymatically generated within resolution networks that possess unique and specific functions to orchestrate catabasis, the phase in which disease declines. Resolvin D2 (RvD2) was originally identified in resolving exudates, yet its individual contribution in resolution remained to be elucidated. Here, we establish RvD2's potent stereoselective actions in reducing excessive neutrophil trafficking to inflammatory loci. RvD2 decreased leukocyte-endothelial interactions in vivo by endothelial-dependent nitric oxide production, and by direct modulation of leukocyte adhesion receptor expression. In mice with microbial sepsis initiated by caecal ligation and puncture, RvD2 sharply decreased both local and systemic bacterial burden, excessive cytokine production and neutrophil recruitment, while increasing peritoneal mononuclear cells and macrophage phagocytosis. These multi-level pro-resolving actions of RvD2 translate to increased survival from sepsis induced by caecal ligation and puncture and surgery. Together, these results identify RvD2 as a potent endogenous regulator of excessive inflammatory responses that acts via multiple cellular targets to stimulate resolution and preserve immune vigilance.

Discovery of an orally active small-molecule irreversible inhibitor of protein disulfide isomerase for ovarian cancer treatment.

Protein disulfide isomerase (PDI), an endoplasmic reticulum chaperone protein, catalyzes disulfide bond breakage, formation, and rearrangement. The effect of PDI inhibition on ovarian cancer progression is not yet clear, and there is a need for potent, selective, and safe small-molecule inhibitors of PDI. Here, we report a class of propynoic acid carbamoyl methyl amides (PACMAs) that are active against a panel of human ovarian cancer cell lines. Using fluorescent derivatives, 2D gel electrophoresis, and MS, we established that PACMA 31, one of the most active analogs, acts as an irreversible small-molecule inhibitor of PDI, forming a covalent bond with the active site cysteines of PDI. We also showed that PDI activity is essential for the survival and proliferation of human ovarian cancer cells. In vivo, PACMA 31 showed tumor targeting ability and significantly suppressed ovarian tumor growth without causing toxicity to normal tissues. These irreversible small-molecule PDI inhibitors represent an important approach for the development of targeted anticancer agents for ovarian cancer therapy, and they can also serve as useful probes for investigating the biology of PDI-implicated pathways.

Avermectins differentially affect ethanol intake and receptor function: implications for developing new therapeutics for alcohol use disorders.

Our laboratory is investigating ivermectin (IVM) and other members of the avermectin family as new pharmaco-therapeutics to prevent and/or treat alcohol use disorders (AUDs). Earlier work found that IVM significantly reduced ethanol intake in mice and that this effect likely reflects IVM's ability to modulate ligand-gated ion channels. We hypothesized that structural modifications that enhance IVM's effects on key receptors and/or increase its brain concentration should improve its anti-alcohol efficacy. We tested this hypothesis by comparing the abilities of IVM and two other avermectins, abamectin (ABM) and selamectin (SEL), to reduce ethanol intake in mice, to alter modulation of GABAARs and P2X4Rs expressed in Xenopus oocytes and to increase their ability to penetrate the brain. IVM and ABM significantly reduced ethanol intake and antagonized the inhibitory effects of ethanol on P2X4R function. In contrast, SEL did not affect either measure, despite achieving higher brain concentrations than IVM and ABM. All three potentiated GABAAR function. These findings suggest that chemical structure and effects on receptor function play key roles in the ability of avermectins to reduce ethanol intake and that these factors are more important than brain penetration alone. The direct relationship between the effect of these avermectins on P2X4R function and ethanol intake suggest that the ability to antagonize ethanol-mediated inhibition of P2X4R function may be a good predictor of the potential of an avermectin to reduce ethanol intake and support the use of avermectins as a platform for developing novel drugs to prevent and/or treat AUDs.

The novel 13S,14S-epoxy-maresin is converted by human macrophages to maresin 1 (MaR1), inhibits leukotriene A4 hydrolase (LTA4H), and shifts macrophage phenotype.

Maresins are produced by macrophages from docosahexaenoic acid (DHA) and exert potent proresolving and tissue homeostatic actions. Maresin 1 (MaR1; 7R,14S-dihydroxy-docosa-4Z,8E,10E,12Z,16Z,19Z-hexaenoic acid) is the first identified maresin. Here, we investigate formation, stereochemistry, and precursor role of 13,14-epoxy-docosahexaenoic acid, an intermediate in MaR1 biosynthesis. The 14-lipoxygenation of DHA by human macrophage 12-lipoxygenase (hm12-LOX) gave 14-hydro(peroxy)-docosahexaenoic acid (14-HpDHA), as well as several dihydroxy-docosahexaenoic acids, implicating an epoxide intermediate formation by this enzyme. Using a stereo-controlled synthesis, enantiomerically pure 13S,14S-epoxy-docosa-4Z,7Z,9E,11E,16Z,19Z-hexaenoic acid (13S,14S-epoxy-DHA) was prepared, and its stereochemistry was confirmed by NMR spectroscopy. When this 13S,14S-epoxide was incubated with human macrophages, it was converted to MaR1. The synthetic 13S,14S-epoxide inhibited leukotriene B4 (LTB4) formation by human leukotriene A4 hydrolase (LTA4H) ∼40% (P<0.05) to a similar extent as LTA4 (∼50%, P<0.05) but was not converted to MaR1 by this enzyme. 13S,14S-epoxy-DHA also reduced (∼60%; P<0.05) arachidonic acid conversion by hm12-LOX and promoted conversion of M1 macrophages to M2 phenotype, which produced more MaR1 from the epoxide than M1. Together, these findings establish the biosynthesis of the 13S,14S-epoxide, its absolute stereochemistry, its precursor role in MaR1 biosynthesis, and its own intrinsic bioactivity. Given its actions and role in MaR1 biosynthesis, this epoxide is now termed 13,14-epoxy-maresin (13,14-eMaR) and exhibits new mechanisms in resolution of inflammation in its ability to inhibit proinflammatory mediator production by LTA4 hydrolase and to block arachidonate conversion by human 12-LOX rather than merely terminating phagocyte involvement.

Inhibition of the function of class IIa HDACs by blocking their interaction with MEF2.

Enzymes that modify the epigenetic status of cells provide attractive targets for therapy in various diseases. The therapeutic development of epigenetic modulators, however, has been largely limited to direct targeting of catalytic active site conserved across multiple members of an enzyme family, which complicates mechanistic studies and drug development. Class IIa histone deacetylases (HDACs) are a group of epigenetic enzymes that depends on interaction with Myocyte Enhancer Factor-2 (MEF2) for their recruitment to specific genomic loci. Targeting this interaction presents an alternative approach to inhibiting this class of HDACs. We have used structural and functional approaches to identify and characterize a group of small molecules that indirectly target class IIa HDACs by blocking their interaction with MEF2 on DNA.Weused X-ray crystallography and (19)F NMRto show that these compounds directly bind to MEF2. We have also shown that the small molecules blocked the recruitment of class IIa HDACs to MEF2-targeted genes to enhance the expression of those targets. These compounds can be used as tools to study MEF2 and class IIa HDACs in vivo and as leads for drug development.

Ataxin-1 poly(Q)-induced proteotoxic stress and apoptosis are attenuated in neural cells by docosahexaenoic acid-derived neuroprotectin D1.

Neurodegenerative diseases share two common features: enhanced oxidative stress and cellular inability to scavenge structurally damaged abnormal proteins. Pathogenesis of polyglutamine (poly(Q)) diseases involves increased protein misfolding, along with ubiquitin and chaperon protein-containing nuclear aggregates. In spinocerebellar ataxia, the brain and retina undergo degeneration. Neuroprotectin D1 (NPD1) is made on-demand in the nervous system and retinal pigment epithelial (RPE) cells in response to oxidative stress, which activates prosurvival signaling via regulation of gene expression and other processes. We hypothesized that protein misfolding-induced proteotoxic stress triggers NPD1 synthesis. We used ARPE-19 cells as a cellular model to assess stress due to ataxin-1 82Q protein expression and determine whether NPD1 prevents apoptosis. Ectopic ataxin-1 expression induced RPE cell apoptosis, which was abrogated by 100 nm docosahexaenoic acid, 10 ng/ml pigment epithelium-derived factor, or NPD1. Similarly, NPD1 was protective in neurons and primary human RPE cells. Furthermore, when ataxin-1 82Q was expressed in 15-lipoxygenase-1-deficient cells, apoptosis was greatly enhanced, and only NPD1 (50 nm) rescued cells from death. NPD1 reduced misfolded ataxin-1-induced accumulation of proapoptotic Bax in the cytoplasm, suggesting that NPD1 acts by preventing proapoptotic signaling pathways from occurring. Finally, NPD1 signaling interfered with ataxin-1/capicua repression of gene expression and decreased phosphorylated ataxin-1 in an Akt-independent manner, suggesting that NPD1 signaling modulates formation or stabilization of ataxin-1 complexes. These data suggest that 1) NPD1 synthesis is an early response induced by proteotoxic stress due to abnormally folded ataxin-1, and 2) NPD1 promotes cell survival through modulating stabilization of ataxin-1 functional complexes and pro-/antiapoptotic and inflammatory pathways.