NRF2 in dermatological disorders: Pharmacological activation for protection against cutaneous photodamage and photodermatosis.

The skin barrier and its endogenous protective mechanisms cope daily with exogenous stressors, of which ultraviolet radiation (UVR) poses an imminent danger. Although the skin is able to reduce the potential damage, there is a need for comprehensive strategies for protection. This is particularly important when developing pharmacological approaches to protect against photocarcinogenesis. Activation of NRF2 has the potential to provide comprehensive and long-lasting protection due to the upregulation of numerous cytoprotective downstream effector proteins that can counteract the damaging effects of UVR. This is also applicable to photodermatosis conditions that exacerbate the damage caused by UVR. This review describes the alterations caused by UVR in normal skin and photosensitive disorders, and provides evidence to support the development of NRF2 activators as pharmacological treatments. Key natural and synthetic activators with photoprotective properties are summarized. Lastly, the gap in knowledge in research associated with photodermatosis conditions is highlighted.

Development of anti-inflammatory peptidomimetics based on the structure of human alpha1-antitrypsin.

Human α1-antitrypsin (hAAT) has two distinguishing functions: anti-protease activity and regulation of the immune system. In the present study we hypothesized that those two protein functions are mediated by different structural domains on the hAAT surface. Indeed, such biologically active immunoregulatory sites (not associated with canonical anti-protease activity) on the surface of hAAT were identified by in silico methods. Several peptides were derived from those immunoregulatory sites. Four peptides exhibited impressive biological effects in pharmacological concentration ranges. Peptidomimetic (14) was developed, based on the structure of the most druggable and active peptide. The compound exhibited a potent anti-inflammatory activity in vitro and in vivo. Such a compound could be used as a basis for developing novel anti-inflammatory drug candidates and as a research tool for better understanding hAAT functions.

SH-29 and SK-119 Attenuates Air-Pollution Induced Damage by Activating Nrf2 in HaCaT Cells.

Air pollution has been repeatedly linked to numerous health-related disorders, including skin sensitization, oxidative imbalance, premature extrinsic aging, skin inflammation, and increased cancer prevalence. Nrf2 is a key player in the endogenous protective mechanism of the skin. We hypothesized that pharmacological activation of Nrf2 might reduce the deleterious action of diesel particulate matter (DPM), evaluated in HaCaT cells. SK-119, a recently synthesized pharmacological agent as well as 2,2'-((1E,1'E)-(1,4-phenylenebis(azaneylylidene))bis(methaneylylidene))bis(benzene-1,3,5-triol) (SH-29) were first evaluated in silico, suggesting a potent Nrf2 activation capacity that was validated in vitro. In addition, both compounds were able to attenuate key pathways underlying DPM damage, including cytosolic and mitochondrial reactive oxygen species (ROS) generation, tested by DC-FDA and MitoSOX fluorescent dye, respectively. This effect was independent of the low direct scavenging ability of the compounds. In addition, both SK-119 and SH-29 were able to reduce DPM-induced IL-8 hypersecretion in pharmacologically relevant concentrations. Lastly, the safety of both compounds was evaluated and demonstrated in the ex vivo human skin organ culture model. Collectively, these results suggest that Nrf2 activation by SK-119 and SH-29 can revert the deleterious action of air pollution.

Nano-scale resolution of native retinal rod disk membranes reveals differences in lipid composition.

Photoreceptors rely on distinct membrane compartments to support their specialized function. Unlike protein localization, identification of critical differences in membrane content has not yet been expanded to lipids, due to the difficulty of isolating domain-specific samples. We have overcome this by using SMA to coimmunopurify membrane proteins and their native lipids from two regions of photoreceptor ROS disks. Each sample's copurified lipids were subjected to untargeted lipidomic and fatty acid analysis. Extensive differences between center (rhodopsin) and rim (ABCA4 and PRPH2/ROM1) samples included a lower PC to PE ratio and increased LC- and VLC-PUFAs in the center relative to the rim region, which was enriched in shorter, saturated FAs. The comparatively few differences between the two rim samples likely reflect specific protein-lipid interactions. High-resolution profiling of the ROS disk lipid composition gives new insights into how intricate membrane structure and protein activity are balanced within the ROS, and provides a model for future studies of other complex cellular structures.

Pruritus in psoriasis and atopic dermatitis: current treatments and new perspectives.

Psoriasis and atopic dermatitis (AD) are two common chronic inflammatory skin diseases. Although showing different etiology and clinical manifestations, patients with either disease suffer from low health-related quality of life due to pruritus (dermal itch). Recent studies have revealed that more than 85% of psoriasis patients suffer from pruritus, and it is also the dominating symptom of AD. However, as this is a non-life treating symptom, it was partly neglected for years. In this review, we focus on current findings as well as the impact and potential treatments of pruritus in these two skin diseases. We first distinguish the type of itch based on involved mediators and modulators. This clear delineation between the types of pruritus based on involved receptors and pathways allows for precise treatment. In addition, insights into recent clinical trials aimed to alleviate pruritus by targeting these receptors are presented. We also report about novel advances in combinatorial treatments, dedicated to the type of pruritus linked to a causal disease. Altogether, we suggest that only a focused treatment tailored to the primary disease and the underlying molecular signals will provide fast and sustained relief of pruritus associated with psoriasis or AD.

Endothelial Cell-Derived Triosephosphate Isomerase Attenuates Insulin Secretion From Pancreatic Beta Cells of Male Rats.

Insulin secretion from pancreatic beta cells is tightly regulated by glucose and paracrine signals within the microenvironment of islets of Langerhans. Extracellular matrix from islet microcapillary endothelial cells (IMEC) affect beta-cell spreading and amplify insulin secretion. This study was aimed at investigating the hypothesis that contact-independent paracrine signals generated from IMEC may also modulate beta-cell insulin secretory functions. For this purpose, conditioned medium (CMp) preparations were prepared from primary cultures of rat IMEC and were used to simulate contact-independent beta cell-endothelial cell communication. Glucose-stimulated insulin secretion (GSIS) assays were then performed on freshly isolated rat islets and the INS-1E insulinoma cell line, followed by fractionation of the CMp, mass spectroscopic identification of the factor, and characterization of the mechanism of action. The IMEC-derived CMp markedly attenuated first- and second-phase GSIS in a time- and dose-dependent manner without altering cellular insulin content and cell viability. Size exclusion fractionation, chromatographic and mass-spectroscopic analyses of the CMp identified the attenuating factor as the enzyme triosephosphate isomerase (TPI). An antibody against TPI abrogated the attenuating activity of the CMp while recombinant human TPI (hTPI) attenuated GSIS from beta cells. This effect was reversed in the presence of tolbutamide in the GSIS assay. In silico docking simulation identified regions on the TPI dimer that were important for potential interactions with the extracellular epitopes of the sulfonylurea receptor in the complex. This study supports the hypothesis that an effective paracrine interaction exists between IMEC and beta cells and modulates glucose-induced insulin secretion via TPI-sulfonylurea receptor-KATP channel (SUR1-Kir6.2) complex attenuating interactions.

Advances in Understanding the Initial Steps of Pruritoceptive Itch: How the Itch Hits the Switch.

Pruritoceptive (dermal) itch was long considered an accompanying symptom of diseases, a side effect of drug applications, or a temporary sensation induced by invading pruritogens, as produced by the stinging nettle. Due to extensive research in recent years, it was possible to provide detailed insights into the mechanism of itch mediation and modulation. Hence, it became apparent that pruritus is a complex symptom or disease in itself, which requires particular attention to improve patients' health. Here, we summarize recent findings in pruritoceptive itch, including how this sensation is triggered and modulated by diverse endogenous and exogenous pruritogens and their receptors. A differentiation between mediating pruritogen and modulating pruritogen seems to be of great advantage to understand and decipher the molecular mechanism of itch perception. Only a comprehensive view on itch sensation will provide a solid basis for targeting this long-neglected adverse sensation accompanying numerous diseases and many drug side effects. Finally, we identify critical aspects of itch perception that require future investigation.

Inhibiting the copper efflux system in microbes as a novel approach for developing antibiotics.

Five out of six people receive at least one antibiotic prescription per year. However, the ever-expanding use of antibiotics in medicine, agriculture, and food production has accelerated the evolution of antibiotic-resistant bacteria, which, in turn, made the development of novel antibiotics based on new molecular targets a priority in medicinal chemistry. One way of possibly combatting resistant bacterial infections is by inhibiting the copper transporters in prokaryotic cells. Copper is a key element within all living cells, but it can be toxic in excess. Both eukaryotic and prokaryotic cells have developed distinct copper regulation systems to prevent its toxicity. Therefore, selectively targeting the prokaryotic copper regulation system might be an initial step in developing next-generation antibiotics. One such system is the Gram-negative bacterial CusCFBA efflux system. CusB is a key protein in this system and was previously reported to play an important role in opening the channel for efflux via significant structural changes upon copper binding while also controlling the assembly and disassembly process of the entire channel. In this study, we aimed to develop novel peptide copper channel blockers, designed by in silico calculations based on the structure of CusB. Using a combination of magnetic resonance spectroscopy and various biochemical methods, we found a lead peptide that promotes copper-induced cell toxicity. Targeting copper transport in bacteria has not yet been pursued as an antibiotic mechanism of action. Thus, our study lays the foundation for discovering novel antibiotics.

Novel inhibitors of leukocyte transendothelial migration.

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC50 = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn's disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.

Z-isomerization of retinoids through combination of monochromatic photoisomerization and metal catalysis.

Catalytic Z-isomerization of retinoids to their thermodynamically less stable Z-isomer remains a challenge. In this report, we present a photochemical approach for the catalytic Z-isomerization of retinoids using monochromatic wavelength UV irradiation treatment. We have developed a straightforward approach for the synthesis of Z-retinoids in high yield, overcoming common obstacles normally associated with their synthesis. Calculations based on density functional theory (DFT) have allowed us to correlate the experimentally observed Z-isomer distribution of retinoids with the energies of chemically important intermediates, which include ground- and excited-state potential energy surfaces. We also demonstrate the application of the current method by synthesizing gram-scale quantities of 9-cis-retinyl acetate 9Z-a. Operational simplicity and gram-scale ability make this chemistry a very practical solution to the problem of Z-isomer retinoid synthesis.

Catalytic synthesis of 9-cis-retinoids: mechanistic insights.

The regioselective Z-isomerization of thermodynamically stable all-trans retinoids remains challenging, and ultimately limits the availability of much needed therapeutics for the treatment of human diseases. We present here a novel, straightforward approach for the catalytic Z-isomerization of retinoids using conventional heat treatment or microwave irradiation. A screen of 20 transition metal-based catalysts identified an optimal approach for the regioselective production of Z-retinoids. The most effective catalytic system was comprised of a palladium complex with labile ligands. Several mechanistic studies, including isotopic H/D exchange and state-of-the-art quantum chemical calculations using coupled cluster methods indicate that the isomerization is initiated by catalyst dimerization followed by the formation of a cyclic, six-membered chloropalladate catalyst-substrate adduct, which eventually opens to produce the desired Z-isomer. The synthetic development described here, combined with thorough mechanistic analysis of the underlying chemistry, highlights the use of readily available transition metal-based catalysts in straightforward formats for gram-scale drug synthesis.

Nrf2 Activation by SK-119 Attenuates Oxidative Stress, UVB, and LPS-Induced Damage.

BACKGROUND/AIMS: The Nrf2 signaling pathway plays a pivotal role in neutralizing excess reactive oxygen species formation and therefore enhancing the endogenous cellular protection mechanism. Thus, activating this pathway may provide therapeutic options against oxidative stress-related disorders. We have recently applied a computer-aided drug design approach to the design and synthesis of novel Nrf2 enhancers. The current study was aimed at investigating the potential beneficial impact of (E)-5-oxo-1-(4-((2,4,6-trihydroxybenzylidene)amino)phenyl)pyrrolidine-3-carboxylic acid (SK-119) in skin oxidative damage models. METHODS: SK-119, tested initially in PC-12 cells, attenuated oxidative stress-induced cytotoxicity concomitantly with Nrf2 activation. The potential impact of this compound was evaluated in skin-based disease models both in vitro (HaCaT cells) and ex vivo (human skin organ culture). RESULTS: The data clearly showed the marked anti-inflammatory and photoprotection properties of the compound; SK-119-treated cells or tissues displayed a reduction in cytokine secretion induced by lipopolysaccharides (LPS) in a manner comparable with dexamethasone. In addition, topical application of SK-119 was able to block UVB-induced oxidative stress and attenuated caspase-mediated apoptosis, DNA adduct formation, and the concomitant cellular damage. CONCLUSION: These results indicate that SK-119 is an Nrf2 activator that can be used as a prototype molecule for the development of novel treatments of dermatological disorders related to oxidative stress.

Neuroligin-2-derived peptide-covered polyamidoamine-based (PAMAM) dendrimers enhance pancreatic ß-cells' proliferation and functions.

Pancreatic ß-cell membranes and presynaptic areas of neurons contain analogous protein complexes that control the secretion of bioactive molecules. These complexes include the neuroligins (NLs) and their binding partners, the neurexins (NXs). It has been recently reported that both insulin secretion and the proliferation rates of ß-cells increase when cells are co-cultured with full-length NL-2 clusters. The pharmacological use of full-length protein is always problematic due to its unfavorable pharmacokinetic properties. Thus, NL-2-derived short peptide was conjugated to the surface of polyamidoamine-based (PAMAM) dendrimers. This nanoscale composite improved ß-cell functions in terms of the rate of proliferation, glucose-stimulated insulin secretion (GSIS), and functional maturation. This functionalized dendrimer also protected ß-cells under cellular stress conditions. In addition, various novel peptidomimetic scaffolds of NL-2-derived peptide were designed, synthesized, and conjugated to the surface of PAMAM in order to increase the biostability of the conjugates. However, after being covered by peptidomimetics, PAMAM dendrimers were inactive. Thus, the original peptide-based PAMAM dendrimer is a leading compound for continued research that might provide a unique starting point for designing an innovative class of antidiabetic therapeutics that possess a unique mode of action.

A Small Chaperone Improves Folding and Routing of Rhodopsin Mutants Linked to Inherited Blindness.

The autosomal dominant form of retinitis pigmentosa (adRP) is a blindness-causing conformational disease largely linked to mutations of rhodopsin. Molecular simulations coupled to the graph-based protein structure network (PSN) analysis and in vitro experiments were conducted to determine the effects of 33 adRP rhodopsin mutations on the structure and routing of the opsin protein. The integration of atomic and subcellular levels of analysis was accomplished by the linear correlation between indices of mutational impairment in structure network and in routing. The graph-based index of structural perturbation served also to divide the mutants in four clusters, consistent with their differences in subcellular localization and responses to 9-cis retinal. The stability core of opsin inferred from PSN analysis was targeted by virtual screening of over 300,000 anionic compounds leading to the discovery of a reversible orthosteric inhibitor of retinal binding more effective than retinal in improving routing of three adRP mutants.

Retinal-chitosan Conjugates Effectively Deliver Active Chromophores to Retinal Photoreceptor Cells in Blind Mice and Dogs.

The retinoid (visual) cycle consists of a series of biochemical reactions needed to regenerate the visual chromophore 11-cis-retinal and sustain vision. Genetic or environmental factors affecting chromophore production can lead to blindness. Using animal models that mimic human retinal diseases, we previously demonstrated that mechanism-based pharmacological interventions can maintain vision in otherwise incurable genetic diseases of the retina. Here, we report that after 9-cis-retinal administration to lecithin:retinol acyltransferase-deficient (Lrat-/- ) mice, the drug was rapidly absorbed and then cleared within 1 to 2 hours. However, when conjugated to form chitosan-9-cis-retinal, this prodrug was slowly absorbed from the gastrointestinal tract, resulting in sustainable plasma levels of 9-cis-retinol and recovery of visual function without causing elevated levels, as occurs with unconjugated drug treatment. Administration of chitosan-9-cis-retinal conjugate intravitreally in retinal pigment epithelium-specific 65 retinoid isomerase (RPE65)-deficient dogs improved photoreceptor function as assessed by electroretinography. Functional rescue was dose dependent and maintained for several weeks. Dosing via the gastrointestinal tract in canines was found ineffective, most likely due to peculiarities of vitamin A blood transport in canines. Use of the chitosan conjugate in combination with 11-cis-6-ring-retinal, a locked ring analog of 11-cis-retinal that selectively blocks rod opsin consumption of chromophore while largely sparing cone opsins, was found to prolong cone vision in Lrat-/- mice. Development of such combination low-dose regimens to selectively prolong useful cone vision could not only expand retinal disease treatments to include Leber congenital amaurosis but also the age-related decline in human dark adaptation from progressive retinoid cycle deficiency.

Computer-Aided Design and Synthesis of 1-{4-[(3,4-Dihydroxybenzylidene)amino]phenyl}-5-oxopyrrolidine-3-carboxylic Acid as an Nrf2 Enhancer.

The design and synthesis of a novel nuclear factor erythroid 2-related factor 2 (Nrf2) enhancer is reported. Using a structure-based virtual screening approach, several commercially available compounds were identified as having high probability to interact with the Nrf2-binding pocket in the Kelch-like ECH-associated protein 1 (Keap1). Keap1 is an adaptor protein that recruits Nrf2 to a cullin-3-dependent ubiquitin ligase complex. The identified compounds were tested against rat pheochromocytoma PC-12 cells for their cytoprotective activity, and one compound (SKT359126) demonstrated an Nrf2-mediated cell-protective effect. Based on the structure of SKT359126, 23 novel derivatives were synthesized and evaluated. Of the screened derivatives, 1-{4-[(3,4-dihydroxybenzylidene)amino]phenyl}-5-oxopyrrolidine-3-carboxylic acid demonstrated better activity than the parent molecules in activating the Nrf2 transduction pathway in a dose- and time-dependent manner. This compound represents a promising starting point for the development of therapeutics for the treatment of oxidative-stress-related diseases.

Computer-Aided Design and Synthesis of 1-{4-[(3,4-Dihydroxybenzylidene)amino]phenyl}-5-oxopyrrolidine-3-carboxylic Acid as an Nrf2 Enhancer.

Invited for this month's cover is Prof. Arie Gruzman (Bar-Ilan University) and collaborators who have developed an Nrf2 enhancer. This compound activated the Nrf2 transduction pathway and because of this the translation of dozens of antioxidant cytoprotective proteins in a dose- and time-dependent manner and protected PC-12 cells against oxidative stress. Considering the imbalance between production and elimination of oxidative species involved in the pathophysiology of many human diseases, this compound is a promising starting point for the development of novel therapeutics for the treatment of oxidative-stress-related diseases. Read the full text of the article at 10.1002/cplu.201700539.

Mimicking Neuroligin-2 Functions in ß-Cells by Functionalized Nanoparticles as a Novel Approach for Antidiabetic Therapy.

Both pancreatic ß-cell membranes and presynaptic active zones of neurons include in their structures similar protein complexes, which are responsible for mediating the secretion of bioactive molecules. In addition, these membrane-anchored proteins regulate interactions between neurons and guide the formation and maturation of synapses. These proteins include the neuroligins (e.g., NL-2) and their binding partners, the neurexins. The insulin secretion and maturation of ß-cells is known to depend on their 3-dimensional (3D) arrangement. It was also reported that both insulin secretion and the proliferation rates of ß-cells increase when cells are cocultured with clusters of NL-2. Use of full-length NL-2 or even its exocellular domain as potential ß-cell functional enhancers is limited by the biostability and bioavailability issues common to all protein-based therapeutics. Thus, based on molecular modeling approaches, a short peptide with the potential ability to bind neurexins was derived from the NL-2 sequence. Here, we show that the NL-2-derived peptide conjugates onto innovative functional maghemite (γ-Fe2O3)-based nanoscale composite particles enhance ß-cell functions in terms of glucose-stimulated insulin secretion and protect them under stress conditions. Recruiting the ß-cells' "neuron-like" secretory machinery as a target for diabetes treatment use has never been reported before. Such nanoscale composites might therefore provide a unique starting point for designing a novel class of antidiabetic therapeutic agents that possess a unique mechanism of action.

Design and synthesis of novel protein kinase R (PKR) inhibitors.

Protein kinase RNA-activated (PKR) plays an important role in a broad range of intracellular regulatory mechanisms and in the pathophysiology of many human diseases, including microbial and viral infections, cancer, diabetes and neurodegenerative disorders. Recently, several potent PKR inhibitors have been synthesized. However, the enzyme's multifunctional character and a multitude of PKR downstream targets have prevented the successful transformation of such inhibitors into effective drugs. Thus, the need for additional PKR inhibitors remains. With the help of computer-aided drug-discovery tools, we designed and synthesized potential PKR inhibitors. Indeed, two compounds were found to inhibit recombinant PKR in pharmacologically relevant concentrations. One compound, 6-amino-3-methyl-2-oxo-N-phenyl-2,3-dihydro-1H-benzo[d]imidazole-1-carboxamide, also showed anti-apoptotic properties. The novel molecules diversify the existing pool of PKR inhibitors and provide a basis for the future development of compounds based on PKR signal transduction mechanism.

Activation of PPARδ: from computer modelling to biological effects.

PPARδ is a ligand-activated receptor that dimerizes with another nuclear receptor of the retinoic acid receptor family. The dimers interact with other co-activator proteins and form active complexes that bind to PPAR response elements and promote transcription of genes involved in lipid metabolism. It appears that various natural fatty acids and their metabolites serve as endogenous activators of PPARδ; however, there is no consensus in the literature on the nature of the prime activators of the receptor. In vitro and cell-based assays of PPARδ activation by fatty acids and their derivatives often produce conflicting results. The search for synthetic and selective PPARδ agonists, which may be pharmacologically useful, is intense. Current rational modelling used to obtain such compounds relies mostly on crystal structures of synthetic PPARδ ligands with the recombinant ligand binding domain (LBD) of the receptor. Here, we introduce an original computational prediction model for ligand binding to PPARδ LBD. The model was built based on EC50 data of 16 ligands with available crystal structures and validated by calculating binding probabilities of 82 different natural and synthetic compounds from the literature. These compounds were independently tested in cell-free and cell-based assays for their capacity to bind or activate PPARδ, leading to prediction accuracy of between 70% and 93% (depending on ligand type). This new computational tool could therefore be used in the search for natural and synthetic agonists of the receptor.