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We describe the stereocontrolled synthesis of Resolvin D4 and 17(R)-Resolvin D4. These molecules possess protective activities in resolving inflammation.

Figure S1 shows the gating strategy for PMN apoptosis analysis, RT-PCR, immunofluorescence staining, and flow cytometry analyses of FPR2/ALX-transfected HEK cells, effect of NAP1051 on dTHP-1 cells with selective kinase inhibitors, and Western blot with normal HEK cells and FPR2/ALX-transfected HEK cells

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PDF file, 55K, Classical apoptosis pathways.

Colorectal cancer is the second most common cause of cancer-related death in the United States. Recent studies showed that interleukin-8 (IL-8) and its receptors (CXCR1 and CXCR2) are significantly upregulated in both the tumor and its microenvironment, and act as key regulators of proliferation, angiogenesis, and metastasis. Our previous study showed that IL-8 overexpression in colorectal cancer cells triggers the upregulation of the CXCR2-mediated proliferative pathway. The aim of this study was to investigate whether the CXCR2 antagonist, SCH-527123, inhibits colorectal cancer proliferation and if it can sensitize colorectal cancer cells to oxaliplatin both in vitro and in vivo. SCH-527123 showed concentration-dependent antiproliferative effects in HCT116, Caco2, and their respective IL-8–overexpressing variants colorectal cancer cell lines. Moreover, SCH-527123 was able to suppress CXCR2-mediated signal transduction as shown through decreased phosphorylation of the NF-κB/mitogen-activated protein kinase (MAPK)/AKT pathway. These findings corresponded with decreased cell migration and invasion, while increased apoptosis in colorectal cancer cell lines. In vivo results verified that SCH-527123 treatment decreased tumor growth and microvessel density when compared with vehicle-treated tumors. Importantly, these preclinical studies showed that the combination of SCH-527123 and oxaliplatin resulted in a greater decrease in cell proliferation, tumor growth, apoptosis, and angiogenesis that was superior to single-agent treatment. Taken together, these findings suggest that targeting CXCR2 may block tumor proliferation, migration, invasion, and angiogenesis. In addition, CXCR2 blockade may further sensitize colorectal cancer to oxaliplatin treatment. Mol Cancer Ther; 11(6); 1353–64. ©2012 AACR.

Table S1 lists flow cytometry antibodies and panel design. Table S2 lists primers for RT-PCR analyses. Table S3 includes antibodies for immunohistochemistry and immunofluorescent staining.

Perillyl alcohol (POH) is a monoterpene that has been used orally for the treatment of systemic cancer. However, when used orally significant gastrointestinal side effects and lack of overall efficacy were documented. Recently, in a phase II trial in Brazil for the treatment of temozolomide (TMZ)-resistant malignant gliomas, POH was well tolerated when administered intranasally. The present study explores the effects and mechanisms of POH on TMZ-sensitive and TMZ-resistant glioma cells. In vitro studies showed that POH was cytotoxic to TMZ-resistant as well as TMZ-sensitive glioma cells, and this effect was independent of O6-methylguanine-DNA methyltransferase expression. POH induced cytotoxicity, in part, through the endoplasmic reticulum (ER) stress pathway as shown by the increased expression of glucose-regulated protein-78 (GRP78), activating transcription factor 3, and C/EBP-homologous protein. In addition, POH impeded survival pathways, such as mTOR and Ras. As well, POH reduced the invasive capacity of sensitive and resistant glioma cells. POH alone and/or in combination with other ER stress–inducing cytotoxic drugs (i.e., 2, 5-dimethyl-celecoxib, nelfinavir) further induced apoptosis in TMZ-sensitive and TMZ-resistant glioma cells. To show whether intranasal delivery of POH was effective for the treatment of TMZ-resistant gliomas, animals bearing intracranial tumors were given POH intranasally. Animals treated through intranasal administration of POH exhibited a decrease in tumor growth and an increase in survival. Our data show that POH is an effective anti-glioma cytotoxic agent for TMZ-resistant gliomas when administered intranasally. Mol Cancer Ther; 11(11); 2462–72. ©2012 AACR.

PDF file, 38K, POH does not affect microvessel density.

PDF file, 5.9MB, Clonogenicity assay.

PDF file, 45K, MGMT protein expressions in the different cell lines used in this study.

PDF file, 770K, SCH-527123 treatment inhbits downstream activation of CXCR2 by decreasing phosphoryaltion of NF-kB and MAPK signaling cascades in CRC cell line models.

PDF file, 570K, ERCC1 mRNA expression in colon cancer cell lines.

The first stereocontrolled total synthesis of 4(S),5(S)-oxido-17(S)-hydroxy-6(E),8(E),10(Z),13(Z),15(E),19(Z)-docosahexaenoic acid (1) is reported.

Resolving tumor-associated inflammation in the tumor microenvironment (TME) may promote antitumor effects. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. Here, a biomimetic of LXA4, NAP1051, was shown to have LXA4-like in vitro properties and antitumor activity in colorectal cancer xenograft models. NAP1051 inhibited neutrophil chemotaxis toward fMLP and dose-dependently promoted dTHP-1 efferocytosis which was equipotent to aspirin-triggered lipoxin A4 (ATLA). In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT similar to formyl peptide receptor 2 (FPR2/ALX) agonists. In two mouse xenograft colorectal cancer models, NAP1051 significantly inhibited tumor growth when given orally at 4.8 to 5 mg/kg/day. Flow cytometric analyses showed that NAP1051 reduced splenic and intratumoral neutrophil and myeloid-derived suppressor cell populations, which correlated to the antitumor effect. In addition, NAP1051 reduced NETosis in the TME while stimulating T-cell recruitment. Overall, these results show that NAP1051 possesses key lipoxin-like properties and has antitumor activity against colorectal cancer via modulation of neutrophils and NETosis in the TME.

The total synthesis of Resolvin D4 and its 17(

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic collapse around the world. Effective treatments to mitigate this viral infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells (HCEC) in culture challenged with IFNγ as models of the eye surface to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 Spike (S) protein to angiotensin-converting enzyme 2 (ACE2). We found that the lipid mediators, elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i) decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ-stimulated HCEC. There was also a concomitant decrease in the binding of Spike RBD with the lipid treatments. Using RNA-seq analysis, we uncovered that the lipid mediators also attenuated the expression of pro-inflammatoy cytokines participating in hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to open therapeutic avenues to counteract virus attachment and entrance to the body.

OBJECTIVE: Acute ischemic stroke triggers complex neurovascular, neuroinflammatory, and synaptic alterations. This study explores whether blocking pro-inflammatory platelet-activating factor receptor (PAF-R) plus selected docosanoids after middle cerebral artery occlusion (MCAo) would lead to neurological recovery. The following small molecules were investigated: (a) LAU-0901, a PAF-R antagonist that blocks pro-inflammatory signaling; and (b) derivatives of docosahexaenoic acid (DHA), neuroprotectin D1 (NPD1), and aspirin-triggered NPD1 (AT-NPD1), which activates cell survival pathways and are exert potent anti-inflammatory activity in the brain. MATERIALS AND METHODS: Sprague-Dawley rats received 2 h MCAo and LAU-0901 (30 or 60 mg/kg, 2 h after stroke), NPD1, and AT-NPD1 (333 μg/kg), DHA (5 mg/kg), and their combination were administered intravenous at 3 h after stroke. Behavior testing and ex vivo magnetic resonance imaging were conducted on day 3 or 14 to assess lesion characteristics and lipidomic analysis on day 1. Series 1 (LAU-0901 + NPD1, 14d), Series 2 (LAU-0901 + AT-NPD1, 3d), and Series 3 (LAU-0901 + DHA, 1d). RESULTS: All combinatory groups improved behavior compared to NPD1, AT-NPD1, or DHA treatments alone. Total lesion volumes were reduced with LAU-0901 + NPD1 by 62% and LAU-0901 + AT-NPD1 by 90% treatments versus vehicle groups. LAU-0901 and LAU-0901 + DHA increased the production of vasoactive lipid mediators (prostaglandins: PGE2, PGF2-α, 6-keto-PGF1-α, and PGD2) as well an inflammatory regulating mediator hydroxyoctadecadienoic acid. In contrast, LAU-0901 and LAU-0901 + DHA decreased the production of 12-hydroxyeicosatetraenoic acid, a pro-inflammatory mediator. CONCLUSION: Combination therapy with LAU-0901 and selected docosanoids is more effective than the single therapy, affording synergistic neuroprotection, with restored pro-homeostatic lipid mediators and improved neurological recovery. Altogether, our findings support the combinatory therapy as the basis for future therapeutics for ischemic stroke.

Significance The resolution of the acute inflammatory response is an active, highly organized process essential for tissue regeneration, wound healing, and the return to homeostasis that involves specialized proresolving mediators (SPMs). The elucidation of mediators that play roles in these processes is of general interest since many widely occurring diseases display unresolved inflammation in their pathobiology. Here, we document the pivotal role of 4 S ,5 S -epoxy-resolvin as an intermediate in the biosynthesis of resolvin D3 and resolvin D4 and identified a unique cysteinyl-resolvin isomer that is biosynthesized in a cell type–specific manner. Results from cell- and model organism–based systems each defined the potent bioactivity of the cysteinyl-resolvin isomer.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures(1-4) and ultra-large libraries of virtual compounds(5,6). However, to keep pace with the explosive growth of virtual libraries, such as REadily AvaiLable for synthesis (REAL) combinatorial libraries(7), new approaches to compound screening are needed(8,9). Here we introduce a novel synthon-based approach, V-SYNTHES, to perform hierarchical structure-based screening of REAL library of more than 11 billion compounds. V-SYNTHES first identifies the best synthon-scaffold 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 allows rapid detection of the best-scoring compounds in the Giga-scale chemical space while performing docking of only a small fraction (

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 R R‐ or S R‐configuration. The two compounds were chemically synthesized. Using chiral phase HPLC, four peaks of RvD6si1‐4 from tears were resolved. The R R‐RvD6 standard eluted as a single peak with RvD61 while pure S R‐RvD6 eluted with RvD63. The addition of these pure mediators prompted a trigeminal ganglion transcriptome response in injured corneas and showed that R R‐RvD6 was the more potent, increasing cornea sensitivity and nerve regeneration. R R‐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.

Significance This study uncovers biosynthetic pathway insufficiencies of prohomeostatic/neuroprotective mediators neuroprotectin D1 and elovanoids in the retina during early pathogenesis in transgenic Alzheimer’s disease 5xFAD mouse. These changes correlate with photoreceptor cell functional impairments preceding their loss. Amyloid beta (Aβ) peptide accumulates in drusen in AMD. Thus, injecting oligomeric Aβ in wild-type mice behind the retina leads to photoreceptor cell degeneration and transcriptional disruptions including upregulation of a senescence program and of senescence-associated secretory phenotype (SASP). Similar changes take place in human retinal pigment epithelium cells in culture. Novel lipid mediators, the elovanoids, restore Aβ-peptide-induced gene expression changes and SASP secretome and, in turn, protect these cells. This study opens avenues of potential therapeutic exploration of elovanoids for AMD., 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 β-peptide (Aβ) 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 β-amyloid (OAβ) and observed that ELVs administration counteracted their effects, protecting these cells. In addition, ELVs prevented OAβ-induced changes in gene expression engaged in senescence, inflammation, autophagy, extracellular matrix remodeling, and AMD. Moreover, as OAβ targets the RPE, we used primary human RPE cell cultures and demonstrated that OAβ caused cell damage, while ELVs protected and restored gene expression as in mouse. Our data show OAβ 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 OAβ-induced expression of genes engaged in AMD, autophagy, and extracellular matrix remodeling. Overall, our data uncovered that ELVs downplay OAβ-senescence program induction and inflammatory transcriptional events and protect RPE cells and PRC, and therefore have potential as a possible therapeutic avenue for AMD.

ObjectiveWe 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 methodsMale 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.ResultsLAU 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.ConclusionsWe 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.

The pro-homeostatic lipid mediators elovanoids (ELVs) attenuate cell binding and entrance of the SARS-CoV-2 receptor-binding domain (RBD) as well as of the SARS-CoV-2 virus in human primary alveoli cells in culture. We uncovered that very-long-chain polyunsaturated fatty acid precursors (VLC-PUFA, n-3) activate ELV biosynthesis in lung cells. Both ELVs and their precursors reduce the binding to RBD. ELVs downregulate angiotensin-converting enzyme 2 (ACE2) and enhance the expression of a set of protective proteins hindering cell surface virus binding and upregulating defensive proteins against lung damage. In addition, ELVs and their precursors decreased the signal of spike (S) protein found in SARS-CoV-2 infected cells, suggesting that the lipids curb viral infection. These findings open avenues for potential preventive and disease-modifiable therapeutic approaches for COVID-19.

Structure-based virtual ligand screening is emerging as a key paradigm for early drug discovery owing to the availability of high-resolution target structures

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.

The pro-homeostatic lipid mediators elovanoids (ELVs) attenuate cell binding and entrance of the SARS-CoV-2 receptor-binding domain (RBD) in human primary alveoli cells in culture. We uncovered that very-long-chain polyunsaturated fatty acid precursors (VLC-PUFA,n-3) activate ELV biosynthesis in lung cells. Both ELVs and their precursors reduce the binding to RBD. ELVs downregulate angiotensin-converting enzyme 2 (ACE2) and enhance the expression of a set of protective proteins hindering cell surface virus binding and upregulating defensive proteins against lung damage. These findings open avenues for potential preventive and disease-modifiable therapeutic approaches for COVID-19.

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.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection that causes coronavirus disease 2019 (COVID-19) has resulted in a pandemic affecting the most vulnerable in society, triggering a public health crisis and economic tall around the world. Effective treatments to mitigate this virus infection are needed. Since the eye is a route of virus entrance, we use an in vivo rat model of corneal inflammation as well as human corneal epithelial cells in culture challenged with IFNγ to study this issue. We explore ways to block the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). Elovanoid (ELV)-N32 or Resolvin D6-isomer (RvD6i), among the lipid mediators studied, consistently decreased the expression of the ACE2 receptor, furin, and integrins in damaged corneas or IFNγ stimulated human corneal epithelial cells (HCEC). There was also a concomitant decrease in the binding of spike RBD with the lipid treatments. Concurrently, we uncovered that the lipid mediators also attenuated the expression of cytokines that participate in the cytokine storm, hyper-inflammation and senescence programming. Thus, the bioactivity of these lipid mediators will contribute to opening therapeutic avenues for COVID-19 by counteracting virus attachment and entrance to the eye and other cells and the ensuing disruptions of homeostasis.

Objectives: Ischemic stroke triggers a pattern of cellular and molecular disturbances that include lipid peroxidation, uncompensated oxidative stress, and neuronal injury. Recently, we have uncovered and characterized a novel neuroprotective signaling mechanism, which involves the activation of the biosynthesis of a family of lipid mediators in the brain made from omega-3 very-long-chain polyunsaturated fatty acids, that we named Elovanoids (ELVs). The present study evaluated ELVs, made of 32 and 34 C atoms in length (ELV-N32 and ELV-N34) and their potential mechanisms of action in cerebral ischemia. Methods: Male Sprague-Dawley rats were subjected to 2 h of middle cerebral artery occlusion (MCAo). Sodium salts (Na) or methyl esters (Me) ELVs were dissolved in artificial CSF and administered into right lateral ventricle at 3 h after onset of stroke. There were five groups: ELV-N32-Na, ELV-N32-Me, ELV-N34-Na, ELV-N34-Me (5μg/50μl), and CSF (50μl). Neurological function was evaluated on days 1, 3, and 7 after MCAo. Ex vivo MRI and immunohistochemistry were conducted on day 7. Results: All ELV treatments greatly improved neurologic scores in a sustained fashion up to the 7-day survival period. Ischemic core and penumbra volumes (computed from T2WI) were significantly reduced by all ELV treatments, and total lesion volumes were significantly reduced by ELV-N32-Na, ELV-N32-Me, ELV-N34-Na, and ELV-N34-Me compared to CSF-treated group (by 60%, 56%, 99%, and 91%, respectively). ELV-treated rats showed less infarction with an increased number of NeuN- and GFAP-positive cells as well as SMI-71-positive vessels in the cortex and less IgG staining in the cortex. ELV-mediated protection was extensive in the frontal-parietal cortex (tissue was salvaged by 57-96%), subcortex (73-75%), and total infarct volume, correction for brain swelling was dramatically reduced in all ELV-treated groups by 55-91%. Conclusion: We have shown that the administration of ELVs provides high-grade neurobehavioral recovery, decreases ischemic core and penumbra volumes, as well as attenuates cellular damage, blood vessel integrity, and BBB disruption.

Purpose: Resolving tumor-associated inflammation in the tumor microenvironment (TME) can restore the immune response against malignant tissue. Lipoxin A4 (LXA4) is a short-lived endogenous bioactive lipid with potent anti-inflammatory and pro-resolving properties. We developed a LXA4 biomimetic, NAP1051, with increased stability and longer half-life, and demonstrated its properties using in vitro assays and in vivo xenograft tumor models and explored the underlying mechanism of action (MoA). Methods: Differentiated HL-60 and THP-1 induced by DMSO and PMA, respectively, were used as models. dHL-60 was used to determine the effect of NAP1051 on fMLP-induced neutrophil chemotaxis. NAP1051-induced dTHP-1 efferocytosis of apoptotic dHL-60 was measured using fluorescent microscopy. Concentration escalation and time of NAP1051 exposure were used to probe the molecular mechanism using Western Blot and RT-PCR. The efficacy of oral NAP1051 was evaluated in CT26 (CRC) xenograft model established in immunocompetent Balb/c mice with a dosage escalation design. Results: NAP1051 inhibited neutrophil chemotaxis towards fMLP by > 40% at 1, 10 and 100 nM (p < 0.05). NAP1051 dose-dependently promoted dTHP-1 efferocytosis (p < 0.05) and was equipotent to ATLA4. In dTHP-1 cells, NAP1051 induced strong phosphorylation on ERK1/2 and AKT from 10 nM to 1 µM. When compared to ATLA4 and W-peptide, NAP1051 caused strong phosphorylation at both S473 and T308 of AKT. The NAP1051-induced p-ERK1/2 and p-AKT were in parallel and in a dose-dependent and time-dependent way. MoA studies revealed that such p-ERK1/2 was mediated by MEK1/2. However, NAP1051 led to p-AKT at both S473 and T308 despite PI3K inhibition, indicating a PI3K-independent pathway was involved. In the mouse xenograft CRC model, we demonstrated that NAP1051 significantly inhibit tumor growth when given p.o. at 5 mg/kg/day. Flow cytometry on splenic cells showed that NAP1051 reduced splenic neutrophil and MDSC populations, which correlated to the tumor sizes. IHC analyses revealed that NAP1051 decreased the intratumoral Ly6G+ neutrophils most significantly at 5 mg/kg/day (p Conclusions: NAP1051 has LXA4-like activity including inhibiting fMLP-mediated neutrophil chemotaxis and promoting macrophage efferocytosis of apoptotic cells. The molecular MoA of NAP1051 involves ERK1/2 and AKT signaling pathways which are interrelated closely. A PI3K-independent pathway may be activated and resulted in AKT activation. NAP1051 inhibited tumor growth significantly at 5 mg/kg/day in a CRC model with significant changes in splenic and intratumoral immune cell populations. With these properties, NAP1051 may be an effective component in the treatment of CRCs by modulating the inflammatory immune cells in the TME. Citation Format: Tiange Dong, Priyal Dave, Brandon Ebright, Kabir Ahluwalia, Eugene Zhou, Isaac Asante, Malika Salimova, Hua Pei, Tracey Lin, Andrew Mead, Zeyang Li, Nicos Petasis, Stan Louie. Mechanistic investigation of NAP1051, a lipoxin A4 biomimetic, in treating colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1436.

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): 4 S ,11 R ,17 S -trihydroxydocosa-5 Z ,7 E ,9 E ,13 Z ,15 E ,19 Z -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: 4 S ,11 R ,17 R -trihydroxydocosa-5 Z ,7 E ,9 E ,13 Z ,15 E ,19 Z -hexaenoic acid, a 17 R -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.

Background: Cancer survival is dependent on the increase of protein synthesis for cell migration, proliferation and metastasis. Cancer adaptive mechanisms include upregulation of endoplasmic reticulum (ER) activities to accommodate increases in misfolded proteins. Sarcoendoplasmic reticulum calcium ATPase (SERCA) are important regulators of calcium found in the endoplasmic reticulum, where inhibition can lead to protein misfolding. Since breast cancer cells express higher level of SERCA2, its overexpression maybe a target for drug development. Method: Using an in vitro screening method to guide our development, we have developed a pharmacophore and made structural changes to enhance anti-cancer activity while reducing of COX-2 inhibition. In this effort, we have identified C1, a coxib analogue with improved antitumor activities with minimal COX-2 inhibitory properties. We have further tested C1 in nu/nu athymic mice xenograft with human breast cancer cell line (MDA MB 231). Tumor volume and animal weights were measured three times a week for 28 days. At the end of the study, tumor volume was correlated with molecular markers using RT-PCR and IHC staining. Results: Our screening identified C1 as a promising anti-cancer compound in vitro and affirmed in xenograft triple negative breast cancer (TNBC) animal model. C1 has EC50 of 85% tumor reduction when compared to vehicle after 28 days of treatment. The safety of C1 was assessed for 48 days at 100 mg/kg/day, where no detectable biochemical, hematologic or histological toxicities were detected. RT-PCR analysis of residual tumor correlated with cell death indicated through the activation of CHOP and DR5 marker. Finally, C1 treatment also decreased angiogenesis markers VEGF-α and IL-8, corresponding to reduction in microvessel formation. Conclusion: C1 is a bioavailable coxib analogue with minimal COX-2 inhibition while possessing broad spectrum antitumor activity, including TNBC. Treatment with C1 leads to dosage dependent antitumor properties as a consequence of specific SERCA2 inhibition. In dosage escalation studies up to 100 mg/kg/day, no detectable biochemical and histological toxicities were detected. With these properties, C1 may be an effective component in the treatment of TNBCs, where effective therapy(ies) is much needed. Citation Format: Tracey J. Lin1, Julie Yoo2, Priyal Dave, Andrew Mead, Kabir Ahluwalia, Tiange Dong, Eugene Zhou, Hua Pei, Isaac Asante, Nicos Petasis, Stan Louie Citation Format: Tracey Lin, Julie Yoo, Priyal Dave, Andrew Mead, Kabir Ahluwalia, Tiange Dong, Eugene Zhou, Hua Pei, Isaac Asante, Nicos Petasis, Stan Louie. Oral administration of novel compound targeting triple negative breast cancer (TNBC) through the inhibition of SERCA2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4172.

A one-step, three-component condensation of allenyl boronic acids or allenyl pinacolboronates with amines and aldehydes affords α-allenyl or α-propargyl α-amino acids and anti-β-amino alcohols. This process gives the allenyl or propargyl product depending on the amine and boron components. Secondary amines generate exclusively α-allenyl α-amino acids, while primary aliphatic amines lead to α-propargyl α-amino acids. Secondary aliphatic amines react with chiral α-hydroxy aldehydes and allenyl boron derivatives to form stereoselectively allenyl anti-β-amino alcohol products.

Docosahexaenoic acid (DHA, 22:6 n-3) is abundant in the retina and is enzymatically converted into pro-homeostatic docosanoids. The DHA- or eicosapentaenoic acid (EPA)-derived 26 carbon fatty acid is a substrate of elongase ELOVL4, which is expressed in photoreceptor cells and generates very long chain (≥C28) polyunsaturated fatty acids including n-3 (VLC-PUFAs,n-3). While ELOVL4 mutations are linked to vision loss and neuronal dysfunctions, the roles of VLC-PUFAs remain unknown. Here we report a novel class of lipid mediators biosynthesized in human retinal pigment epithelial (RPE) cells that are oxygenated derivatives of VLC-PUFAs,n-3; we termed these mediators elovanoids (ELV). ELVs have structures reminiscent of docosanoids but with different physicochemical properties and alternatively-regulated biosynthetic pathways. The structures, stereochemistry, and bioactivity of ELVs were determined using synthetic materials produced by stereo-controlled chemical synthesis. ELVs enhance expression of pro-survival proteins in cells undergoing uncompensated oxidative stress. Our findings unveil a novel autocrine/paracrine pro-homeostatic RPE cell signaling that aims to sustain photoreceptor cell integrity and reveal potential therapeutic targets for retinal degenerations.

We report the discovery of a novel family of prohomeostatic and neuroprotective lipid mediators called elovanoids., We report the characterization of a novel class of lipid mediators termed elovanoids (ELVs) (ELV-N32 and ELV-N34), which are dihydroxylated derivatives of 32:6n3 and 34:6n3, respectively. The precursors of ELVs are made by elongation of a 22:6n3 fatty acid and catalyzed by ELOVL4 (elongation of very-long-chain fatty acids–4). The structure and stereochemistry of ELVs were established using synthetic compounds produced by stereocontrolled total synthesis. We report that ELV-mediated protection is induced in neuronal cultures undergoing either oxygen/glucose deprivation or N-methyl-d-aspartate receptor–mediated excitotoxicity, as well as in experimental ischemic stroke. The methyl ester or sodium salt of ELV-N32 and ELV-N34 resulted in reduced infarct volumes, promoted cell survival, and diminished neurovascular unit disruption when administered 1 hour following 2 hours of ischemia by middle cerebral artery occlusion. Together, our data reveal a novel prohomeostatic and neuroprotective lipid-signaling mechanism aiming to sustain neural cell integrity.

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/EBPβ 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.

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). Prior 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 GABA ARs 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 GABAA receptor 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 first total synthesis of stereochemically pure resolvin D3 and aspirin-triggered resolvin D3 is reported. These enzymatic metabolites of docosahexaenoic acid (DHA) have potent anti-inflammatory and pro-resolving actions. The convergent synthetic strategy is based on enantiomerically pure starting materials, and it is highly stereocontrolled.

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

SummaryResolvins are a family of n-3 lipid mediators initially identified in resolving inflammatory exudates that temper inflammatory responses to promote catabasis. Here, temporal metabololipidomics with self-limited resolving exudates revealed that resolvin (Rv) D3 has a distinct time frame from other lipid mediators, appearing late in the resolution phase. Using synthetic materials prepared by stereocontrolled total organic synthesis and metabololipidomics, we established complete stereochemistry of RvD3 and its aspirin-triggered 17R-epimer (AT-RvD3). Both synthetic resolvins potently regulated neutrophils and mediators, reducing murine peritonitis and dermal inflammation. RvD3 and AT-RvD3 displayed leukocyte-directed actions, e.g., blocking human neutrophil transmigration and enhancing macrophage phagocytosis and efferocytosis. These results position RvD3 uniquely within the inflammation-resolution time frame to vantage and contribute to the beneficial actions of aspirin and essential n-3 fatty acids.

Resolvins of the D-series are specialized pro-resolving lipid mediators that regulate cellular response by orchestrating resolution networks involved in host responses to injury and infection. Here, endogenous resolvin D4 was identified in human tissues and found to persist late into the resolution phase of acute murine Staphylococcus aureus infections. Completion of the first total synthesis of resolvin D4 established the absolute stereochemical configuration of RvD4 confirmed by matching with endogenous RvD4 from resolving exudates in dorsal pouch S. aureus infections. In vivo, RvD4 (ng/mouse) reduced neutrophilic infiltration (~40%) and enhanced uptake of apoptotic PMN (51%) by human dermal fibroblasts at concentrations as low as 0.1 nM. These results establish the complete stereochemistry of RvD4 as 4S,5R,17S-trihydroxydocosa-6E,8E,10Z,13Z,15E,19Z-hexaenoic acid and its novel pro-resolving actions in S. aureus infections as well as its potent ability to stimulate clearance of apoptotic cells by skin fibroblasts.

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.

Lymphedema is mainly caused by lymphatic obstruction and manifested as tissue swelling, often in the arms and legs. Lymphedema is one of the most common post-surgical complications in breast cancer patients and presents a painful and disfiguring chronic illness that has few treatment options. Here, we evaluated the therapeutic potential of interleukin (IL)-8 in lymphatic regeneration independent of its pro-inflammatory activity. We found that IL-8 promoted proliferation, tube formation, and migration of lymphatic endothelial cells (LECs) without activating the VEGF signaling. Additionally, IL-8 suppressed the major cell cycle inhibitor CDKN1C/p57(KIP2) by downregulating its positive regulator PROX1, which is known as the master regulator of LEC-differentiation. Animal-based studies such as matrigel plug and cornea micropocket assays demonstrated potent efficacy of IL-8 in activating lymphangiogenesis in vivo. Moreover, we have generated a novel transgenic mouse model (K14-hIL8) that expresses human IL-8 in the skin and then crossed with lymphatic-specific fluorescent (Prox1-GFP) mouse. The resulting double transgenic mice showed that a stable expression of IL-8 could promote embryonic lymphangiogenesis. Moreover, an immunodeficient IL-8-expressing mouse line that was established by crossing K14-hIL8 mice with athymic nude mice displayed an enhanced tumor-associated lymphangiogenesis. Finally, when experimental lymphedema was introduced, K14-hIL8 mice showed an improved amelioration of lymphedema with an increased lymphatic regeneration. Together, we report that IL-8 can activate lymphangiogenesis in vitro and in vivo with a therapeutic efficacy in post-surgical lymphedema.