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.