Trimebutine prevents corneal inflammation in a rat alkali burn model.

Alkaline burns to the cornea lead to loss of corneal transparency, which is essential for normal vision. We used a rat corneal alkaline burn model to investigate the effect of ophthalmic trimebutine solution on healing wounds caused by alkaline burns. Trimebutine, an inhibitor of the high-mobility group box 1-receptor for advanced glycation end products, when topically applied to the burned cornea, suppressed macrophage infiltration in the early phase and neutrophil infiltration in the late phase at the wound site. It also inhibited neovascularization and myofibroblast development in the late phase. Furthermore, trimebutine effectively inhibited interleukin-1ß expression in the injured cornea. It reduced scar formation by decreasing the expression of type III collagen. These findings suggest that trimebutine may represent a novel therapeutic strategy for corneal wounds, not only through its anti-inflammatory effects but also by preventing neovascularization.

Mononuclear binding and catalytic activity of europium(III) and gadolinium(III) at the active site of the model metalloenzyme phosphotriesterase.

Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Šresolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.

Enantioselective Catalysis by the Umpolung of Conjugate Acceptors Involving N-Heterocyclic Carbene or Organophosphine 1,4-Addition.

ConspectusConjugate acceptors are one of the most common electrophilic functional groups in organic synthesis. While useful in a diverse range of transformations, their applications are largely dominated by the reactions from which their name is derived (i.e., as an acceptor of nucleophiles in the conjugate position). In 2014, we commenced studies focused on their ability to undergo polarity inversion through the conjugate addition of Lewis base catalysts. The first step in this process provides an enolate, from which the well-developed Rauhut-Currier (RC) and Morita-Baylis-Hillman (MBH) reactions can occur; however, tautomerization to provide a species in which the ß-carbon of the conjugate acceptor can now act as a donor is also possible. When we commenced studies on this topic, reaction designs with this type of species, particularly when accessed using N-heterocyclic carbenes (NHCs), had been reported on only a handful of occasions. Despite a lack of development, conceptually it was felt that reactions taking advantage of polarity switching by Lewis base conjugate addition have a number of desirable features. Perhaps the most significant is the potential to reimagine a ubiquitous functional group as an entirely new synthon, namely, a donor to electrophiles from the conjugate position.Our work has focused on catalysis with both simple conjugate acceptors and also those embedded within more complicated substrates; the latter has allowed a series of cycloisomerizations and annulation reactions to be achieved. In most cases, the reactions have been possible using enantioenriched chiral NHCs or organophosphines as the Lewis base catalysts thereby delivering enantioselective approaches to novel cyclic molecules. While related chemistry can be accessed with either family of catalyst, in all cases reactions have been designed to take advantage of one or the other. In addition, a fine balance exists between reactions that exploit the initially formed enolate and those that involve the polarity-inverted ß-anion. In our studies, this balance is addressed through substrate design, although catalyst control may also be possible. We consider the chemistry discussed in this Account to be in its infancy. Significant challenges remain to be addressed before our broad aim of discovering a universal approach to the polarity inversion of all conjugate acceptors can be achieved. These challenges broadly relate to chemoselectivity with substrates bearing multiple electrophilic functionalities, reliance upon the use of conjugate acceptors, and catalyst efficiency. To address these challenges, advances in catalyst design and catalyst cooperativity are likely required.

One carbon-four new bonds.

Stable carbenes deliver a carbon atom to simple amides, producing a range of cyclic compounds.

Disulfiram Ophthalmic Solution Inhibited Macrophage Infiltration by Suppressing Macrophage Pseudopodia Formation in a Rat Corneal Alkali Burn Model.

FROUNT is an intracellular protein that promotes pseudopodia formation by binding to the chemokine receptors CCR2 and CCR5 on macrophages. Recently, disulfiram (DSF), a drug treatment for alcoholism, was found to have FROUNT inhibitory activity. In this study, we investigated the effect of DSF eye drops in a rat corneal alkali burn model. After alkali burn, 0.5% DSF eye drops (DSF group) and vehicle eye drops (Vehicle group) were administered twice daily. Immunohistochemical observations and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses were performed at 6 h and 1, 4, and 7 days after alkali burn. Results showed a significant decrease in macrophage accumulation in the cornea in the DSF group, but no difference in neutrophils. RT-PCR showed decreased expression of macrophage-associated cytokines in the DSF group. Corneal scarring and neovascularization were also suppressed in the DSF group. Low-vacuum scanning electron microscopy imaging showed that macrophage length was significantly shorter in the DSF group, reflecting the reduced extension of pseudopodia. These results suggest that DSF inhibited macrophage infiltration by suppressing macrophage pseudopodia formation.

Prophylactic Instillation of Hydrogen-Rich Water Decreases Corneal Inflammation and Promotes Wound Healing by Activating Antioxidant Activity in a Rat Alkali Burn Model.

Many studies have demonstrated the therapeutic effects of hydrogen in pathological conditions such as inflammation; however, little is known about its prophylactic effects. The purpose of this study is to investigate the prophylactic effects of hydrogen-rich water instillation in a rat corneal alkali burn model. Hydrogen-rich water (hydrogen group) or physiological saline (vehicle group) was instilled continuously to the normal rat cornea for 5 min. At 6 h after instillation, the cornea was exposed to alkali. The area of corneal epithelial defect (CED) was measured every 6 h until 24 h after alkali exposure. In addition, at 6 and 24 h after injury, histological and immunohistochemical observations were made and real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to investigate superoxide dismutase enzyme (SOD)1, SOD2, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) mRNA expression. CED at 12 h and the number of inflammatory infiltrating cells at 6 h after injury were significantly smaller in the hydrogen group than the vehicle group. Furthermore, SOD1 expression was significantly higher in the hydrogen group than the vehicle group at both 6 and 24 h, and the number of PGC-1α-positive cells was significantly larger in the hydrogen group than the vehicle group at 6 h after injury. In this model, prophylactic instillation of hydrogen-rich water suppressed alkali burn-induced inflammation, likely by upregulating expression of antioxidants such as SOD1 and PGC-1α. Hydrogen has not only therapeutic potential but also prophylactic effects that may suppress corneal scarring following injury and promote wound healing.

Effects of Selective Peroxisome Proliferator Activated Receptor Agonists on Corneal Epithelial Wound Healing.

The effects of each subtype-selective peroxisome proliferator activated receptor (PPAR) agonist (α, ß/δ, γ) on corneal epithelial wound healing were investigated using a rat corneal alkali burn model. After the alkali burn, each PPAR agonist or vehicle ophthalmic solution was instilled topically onto the rat's cornea. Corneal epithelial healing processes were evaluated by fluorescein staining. Pathological analyses and real-time reverse transcription polymerase chain reactions were performed to evaluate Ki67 (proliferative maker) expression and inflammatory findings. The area of the corneal epithelial defect at 12 h and 24 h after the alkali burn was significantly smaller in each PPAR group than in the vehicle group. Ki67 mRNA expression was increased in the PPARß/δ group, whereas mRNA expressions of inflammatory cytokines were suppressed in all of the PPAR agonist groups. Nuclear factor kappa B (NF-κB) was the most suppressed in the PPARγ group. The accelerated corneal epithelial healing effects of each PPAR ligand were thought to be related to the promotion of proliferative capacity and inhibition of inflammation.

Photoenzymatic Reductions Enabled by Direct Excitation of Flavin-Dependent "Ene"-Reductases.

Non-natural photoenzymatic reactions reported to date have depended on the excitation of electron donor-acceptor complexes formed between substrates and cofactors within protein active sites to facilitate electron transfer. While this mechanism has unlocked new reactivity, it limits the types of substrates that can be involved in this area of catalysis. Here we demonstrate that direct excitation of flavin hydroquinone within "ene"-reductase active sites enables new substrates to participate in photoenzymatic reactions. We found that by using photoexcitation these enzymes gain the ability to reduce acrylamides through a single electron transfer mechanism.

Evaluation of the Utility of Capsular Stabilization Devices in a Zonular Fiber Defect Model with the Slit Side View System.

Capsular stabilization devices were evaluated in a zonular fiber defect model using the slit side view (SSV) system to confirm their utility for capsular stabilization during phacoemulsification. A zonular fiber defect model was made by cutting Zinn's zonule under observation with a slit lamp microscope in a porcine eye. Phacoemulsification was performed, and the movement of the lens capsule and the depth of the anterior chamber were observed using the SSV in three groups: control group: no surgical instruments used, CE group: a capsule expander was inserted, and CTR group: a capsular tension ring was inserted. In the control group, the equator of the lens was unstable and was easily suctioned to the port of the ultrasound handpiece. The lens capsule was stable in both in the CE and CTR groups. In the CTR group, the equator responsible for the zonular rupture also returned and closed true to its original position. The utility of the capsular stabilization devices in this zonular fiber defect model was confirmed with the SSV system.

Combination of Peroxisome Proliferator-Activated Receptor (PPAR) Alpha and Gamma Agonists Prevents Corneal Inflammation and Neovascularization in a Rat Alkali Burn Model.

Peroxisome proliferator-activated receptor alpha (PPARα) and gamma (PPARγ) agonists have anti-inflammatory and anti-neovascularization effects, but few reports have tested the combination of PPARα and PPARγ agonists. In this study, we investigated the therapeutic effects of ophthalmic solutions of agonists of PPARα, PPARγ, and the combination in a rat corneal alkali burn model. After alkali injury, an ophthalmic solution of 0.05% fenofibrate (PPARα group), 0.1% pioglitazone (PPARγ group), 0.05% fenofibrate + 0.1% pioglitazone (PPARα+γ group), or vehicle (vehicle group) was topically instilled onto the rat's cornea twice a day. After instillation, upregulation was seen of PPAR mRNA corresponding to each agonist group. Administration of agonists for PPARα, PPARγ, and PPARα+γ suppressed inflammatory cells, neovascularization, and fibrotic changes. In addition, the PPARγ agonist upregulated M2 macrophages, which contributed to wound healing, whereas the PPARα agonist suppressed immature blood vessels in the early phase. Administration of PPARα+γ agonists showed therapeutic effects in corneal wound healing, combining the characteristics of both PPARα and PPARγ agonists. The results indicate that the combination of PPARα and γ agonists may be a new therapeutic strategy.

Peroxisome Proliferator-Activated Receptor Beta/Delta Agonist Suppresses Inflammation and Promotes Neovascularization.

The effects of peroxisome proliferator-activated receptor (PPAR)ß/δ ophthalmic solution were investigated in a rat corneal alkali burn model. After alkali injury, GW501516 (PPARß/δ agonist) or vehicle ophthalmic solution was topically instilled onto the rat's cornea twice a day until day 7. Pathological findings were evaluated, and real-time reverse transcription polymerase chain reaction was performed. GW501516 strongly suppressed infiltration of neutrophils and pan-macrophages, and reduced the mRNA expression of interleukin-6, interleukin-1ß, tumor necrosis factor alpha, and nuclear factor-kappa B. On the other hand, GW501516 promoted infiltration of M2 macrophages, infiltration of vascular endothelial cells associated with neovascularization in the wounded area, and expression of vascular endothelial growth factor A mRNA. However, 7-day administration of GW501516 did not promote neovascularization in uninjured normal corneas. Thus, the PPARß/δ ligand suppressed inflammation and promoted neovascularization in the corneal wound healing process. These results will help to elucidate the role of PPARß/δ in the field of ophthalmology.

Immune Checkpoints Contribute Corneal Immune Privilege: Implications for Dry Eye Associated with Checkpoint Inhibitors.

The eye is provided with immune protection against pathogens in a manner that greatly reduces the threat of inflammation-induced vision loss. Immune-mediated inflammation and allograft rejection are greatly reduced in the eye, a phenomenon called 'immune privilege'. Corneal tissue has inherent immune privilege properties with underlying three mechanisms: (1) anatomical, cellular, and molecular barriers in the cornea; (2) an immunosuppressive microenvironment; and (3) tolerance related to regulatory T cells and anterior chamber-associated immune deviation. This review describes the molecular mechanisms of the immunosuppressive microenvironment and regulatory T cells in the cornea that have been elucidated from animal models of ocular inflammation, especially those involving corneal transplantation, it also provides an update on immune checkpoint molecules in corneal and systemic immune regulation, and its relevance for dry eye associated with checkpoint inhibitor therapy.

Photoenzymatic Hydrogenation of Heteroaromatic Olefins Using 'Ene'-Reductases with Photoredox Catalysts.

Flavin-dependent 'ene'-reductases (EREDs) are highly selective catalysts for the asymmetric reduction of activated alkenes. This function is, however, limited to enones, enoates, and nitroalkenes using the native hydride transfer mechanism. Here we demonstrate that EREDs can reduce vinyl pyridines when irradiated with visible light in the presence of a photoredox catalyst. Experimental evidence suggests the reaction proceeds via a radical mechanism where the vinyl pyridine is reduced to the corresponding neutral benzylic radical in solution. DFT calculations reveal this radical to be "dynamically stable", suggesting it is sufficiently long-lived to diffuse into the enzyme active site for stereoselective hydrogen atom transfer. This reduction mechanism is distinct from the native one, highlighting the opportunity to expand the synthetic capabilities of existing enzyme platforms by exploiting new mechanistic models.

Development of Benzenesulfonamide Derivatives as Potent Glutathione Transferase Omega-1 Inhibitors.

Glutathione transferase omega-1 (GSTO1-1) is an enzyme whose function supports the activation of interleukin (IL)-1ß and IL-18 that are implicated in a variety of inflammatory disease states for which small-molecule inhibitors are sought. The potent reactivity of the active-site cysteine has resulted in reported inhibitors that act by covalent labeling. In this study, structure-activity relationship (SAR) elaboration of the reported GSTO1-1 inhibitor C1-27 was undertaken. Compounds were evaluated for inhibitory activity toward purified recombinant GSTO1-1 and for indicators of target engagement in cell-based assays. As covalent inhibitors, the kinact/KI values of selected compounds were determined, as well as in vivo pharmacokinetics analysis. Cocrystal structures of key novel compounds in complex with GSTO1-1 were also solved. This study represents the first application of a biochemical assay for GSTO1-1 to determine kinact/KI values for tested inhibitors and the most extensive set of cell-based data for a GSTO1-1 inhibitor SAR series reported to date. Our research culminated in the discovery of 25, which we propose as the preferred biochemical tool to interrogate cellular responses to GSTO1-1 inhibition.

N-Heterocyclic Carbene Catalyzed (5+1) Annulations Exploiting a Vinyl Dianion Synthon Strategy.

Direct polarity inversion of conjugate acceptors provides a valuable entry to homoenolates. N-heterocyclic carbene (NHC) catalyzed reactions, in which ß-unsubstituted conjugate acceptors undergo homoenolate formation and C-C bond formation twice, have been developed. Specifically, the all-carbon (5+1) annulations give a range of mono- and bicyclic cyclohexanones (31 examples). In the first family of annulations, ß-unsubstituted acrylates tethered to a divinyl ketone undergo cycloisomerization, providing hexahydroindenes and tetralins. In the second, partially untethered substrates undergo an intermolecular (5+1) annulation involving dimerization followed by cycloisomerization. While enantioselectivity was not possible with the former, the latter proved viable, allowing cyclohexanones to be produced with high levels of enantiopurity (most >95:5 e.r.) and exclusive diastereoselectivity (>20:1 d.r.). Derivatizations and mechanistic studies are also reported.

Enantioselective N-Heterocyclic Carbene Catalysis that Exploits Imine Umpolung.

The catalytic umpolung of imines remains an underdeveloped approach to reaction discovery. Herein we report an enantioselective aza-Stetter reaction that proceeds via imine umpolung using N-heterocyclic carbene catalysis. The reaction proceeds with high levels of enantioselectivity (all ≥96:4 er) and good generality (21 examples). Mechanistic studies are reported and are consistent with turnover-limiting addition of the NHC to the imine.

Biocatalytic hydrogen atom transfer: an invigorating approach to free-radical reactions.

Initiating and terminating free-radical reactionis via hydrogen atom transfer (HAT) is an attractive means of avoiding substrate prefunctionalization. Small molecule catalysts and reagents, however, struggle to execute this fundamental step with useful levels of diastereoselectivity and enantioselectivity. In contrast, nature often carries out HAT with exquisite levels of selectivity for even electronically unactivated carbon-hydrogen bonds. By understanding how enzymes exploit and control this fundamental step, new strategies can be developed to address several long-standing challenges in free-radical reactions. This review will cover recent discoveries in biocatalysis that exploit a HAT mechanism to either initiate or terminate novel one-electron reactions.

Enantioselective N-Heterocyclic Carbene Catalyzed Cyclopentene Synthesis via the ß-Azolium Ylide.

Herein we report the cycloisomerization of electron-poor 1,5-dienes via the ß-azolium ylide to give enantioenriched cyclopentenes. The reaction is mediated by a chiral N-heterocyclic carbene (NHC) catalyst, exploits readily available substrates, has good generality (17 examples), and displays excellent enantioselectivity (mostly >94:6). Studies demonstrating the viability of a related dynamic kinetic resolution are reported, as are those with alternate tethers and derivatizations.

Enantioselective N-Heterocyclic Carbene Catalysis by the Umpolung of α,ß-Unsaturated Ketones.

N-Heterocyclic carbene-catalyzed formation of ß-anionic intermediates from enones has been employed in the enantioselective synthesis of 2-aryl propionates. The reaction was achievable using a homochiral 4-MeOC6H4 morpholinone catalyst allowing the first example of enantioselective catalysis by umpolung of α,ß-unsaturated ketones. The reaction is high yielding, and shows robustness with reasonable generality. A mechanism is proposed in which the enantiodetermining protonation is achieved using either hexafluoroisopropanol or the formed naphthol product.