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Amyotrophic lateral sclerosis (ALS) is a progressive neurological disorder with currently no cure. Central to the cellular dysfunction associated with this fatal proteinopathy is the accumulation of unfolded/misfolded superoxide dismutase 1 (SOD1) in various subcellular locations. The molecular mechanism driving the formation of SOD1 aggregates is not fully understood but numerous studies suggest that aberrant aggregation escalates with folding instability of mutant apoSOD1. Recent advances on combining organelle-targeting therapies with the anti-aggregation capacity of chemical chaperones have successfully reduce the subcellular load of misfolded/aggregated SOD1 as well as their downstream anomalous cellular processes at low concentrations (micromolar range). Nevertheless, if such local aggregate reduction directly correlates with increased folding stability remains to be explored. To fill this gap, we synthesized and tested here the effect of 9 ER-, mitochondria- and lysosome-targeted chemical chaperones on the folding stability of truncated monomeric SOD1 (SOD1bar) mutants directed to those organelles. We found that compound ER-15 specifically increased the native state stability of ER-SOD1bar-A4V, while scaffold compound FDA-approved 4-phenylbutyric acid (PBA) decreased it. Furthermore, our results suggested that ER15 mechanism of action is distinct from that of PBA, opening new therapeutic perspectives of this novel chemical chaperone on ALS treatment. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the degeneration of motor neurons. Mutations in the superoxide dismutase (SOD1) gene, causing protein misfolding and aggregation, were suggested as the pathogenic mechanisms involved in familial ALS cases. In the present study, we investigated the potential therapeutic effect of C4 and C5, two derivatives of the chemical chaperone 4-phenylbutyric acid (4-PBA). By combining in vivo and in vitro techniques, we show that, although C4 and C5 successfully inhibited amyloid aggregation of recombinant mutant SOD1 in a dose-dependent manner, they failed to suppress the accumulation of misfolded SOD1. Moreover, C4 or C5 daily injections to SOD1G93A mice following onset had no effect on either the accumulation of misfolded SOD1 or the neuroinflammatory response in the spinal cord and, consequently, failed to extend the survival of SOD1G93A mice or to improve their motor symptoms. Finally, pharmacokinetic (PK) studies demonstrated that high concentrations of C4 and C5 reached the brain and spinal cord but only for a short period of time. Thus, our findings suggest that use of such chemical chaperones for ALS drug development may need to be optimized for more effective results. [ABSTRACT FROM AUTHOR]

Herein we report on a simplified synthesis of scarcely explored, terminally disubstituted electron‐poor alkylidene cyclopent‐2‐en‐4‐ones through uncommon olefination. Secondary sulfones, activated by electron‐withdrawing groups at the adjacent carbon atom, undergo K2CO3‐promoted coupling with 4‐acyloxy‐ and 4‐tert‐butyldimethylsilyloxycyclopent‐2‐en‐1‐ones giving directly, or after a separate dehydrosulfinylation step, alkylidene cyclopent‐2‐en‐4‐ones. A plausible mechanism for these transformations is proposed. Initially, β‐arylsulfonyl esters as well as their acetyl or nitrile analogues are allylated by cyclopentenone derivatives via a tandem Michael addition of α‐sulfonyl carbanions followed by proton migration and retro‐Michael‐type O‐nucleofuge elimination. The primary allylation products are formed as two diastereomers whose configuration and conformation were elucidated using single crystal X‐ray diffraction and NMR spectroscopy. Regardless of stereochemistry, both sets of diastereomers are subjected to Zaitsev‐type retro‐Michael vinylogous dehydrosulfinylation under either basic or thermal silica gel promoted conditions resulting in E/Z‐alkylidene cyclopent‐2‐en‐4‐ones. In these reactions activated sulfones serve as bearing electron‐withdrawing group alkylidene anion‐radical synthons, whereas 4‐oxy‐substituted cyclopentenones represent cyclopent‐2‐en‐4‐one cation‐radical surrogates. [ABSTRACT FROM AUTHOR]

The C2-WW-HECT-domain E3 ubiquitin ligase SMURF2 emerges as an important regulator of diverse cellular processes. To date, SMURF2-specific modulators were not developed. Here, we generated and investigated a set of SMURF2-targeting synthetic peptides and peptidomimetics designed to stimulate SMURF2's autoubiquitination and turnover via a disruption of the inhibitory intramolecular interaction between its C2 and HECT domains. The results revealed the effects of these molecules both in vitro and in cellulo at the nanomolar concentration range. Moreover, the data showed that targeting of SMURF2 with either these modifiers or SMURF2-specific shRNAs could accelerate cell growth in a cell-context-dependent manner. Intriguingly, a concomitant cell treatment with a selected SMURF2-targeting compound and the DNA-damaging drug etoposide markedly increased the cytotoxicity produced by this drug in growing cells. Altogether, these findings demonstrate that SMURF2 can be druggable through its self-destructive autoubiquitination, and inactivation of SMURF2 might be used to affect cell sensitivity to certain anticancer drugs. [ABSTRACT FROM AUTHOR]

The role of human prostatic acid phosphatase (PAcP, P15309|PPAP HUMAN) in prostate cancer was investigated using a new proteomics tool termed signal sequence swapping (replacement of domains from the native cleaved amino terminal signal sequence of secretory/membrane proteins with corresponding regions of functionally distinct signal sequence subtypes). This manipulation preferentially redirects proteins to different pathways of biogenesis at the endoplasmic reticulum (ER), magnifying normally difficult to detect subsets of the protein of interest. For PAcP, this technique reveals three forms identical in amino acid sequence but profoundly different in physiological functions, subcellular location, and biochemical properties. These three forms of PAcP can also occur with the wildtype PAcP signal sequence. Clinical specimens from patients with prostate cancer demonstrate that one form, termed PLPAcP, correlates with early prostate cancer. These findings confirm the analytical power of this method, implicate PLPAcP in prostate cancer pathogenesis, and suggest novel anticancer therapeutic strategies [ABSTRACT FROM AUTHOR]

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mm range) required to mediate their effect. One of the most common chemical chaperones is 4‐phenylbutyric acid (4‐PBA). Despite its unfavorable pharmacokinetic properties, 4‐PBA was approved as a drug to treat ornithine cycle diseases. Here, we report that 2‐isopropyl‐4‐phenylbutanoic acid (5) has been found to be 2–10‐fold more effective than 4‐PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism‐linked Arg451Cys Neuroligin3 (R451C NLGN3). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Protein kinases are among the most important regulators of cellular homeostasis, regulating many aspects of the cell life cycle through phosphorylation. The threonine/serine kinase cAMP-dependent protein kinase (PKA) is a major intracellular effector for a variety of bioactive molecules and hormones that work via a cAMP signaling pathway. In this work we report the discovery of a peptide and its simple peptidomimetic derivatives, which can activate the PKA catalytic unit. The direct binding of these new compounds to PKA and their resulting and activation mode were demonstrated by cellular (cardiomyocytes) and in vitro methods. Activation of PKA in cardiomyocytes after myocardial infarction might be a promising therapeutic strategy to protect the heart from ischemia and reperfusion. In addition, small-molecule PKA activators may prove useful as pharmacological tools for studying transduction mechanisms related to PKA. Several small molecules, which can activate a regulatory PKA unit, were developed. However, no small molecules that can activate a PKA catalytic unit were described in the scientific literature. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The phenol-diamide compound, 5-(tert-butyl)-2-hydroxy-N1,N3-bis(2-hydroxyethyl)isophthalamide (1OH), is water-soluble, non-cytotoxic, and capable of both, trapping ROS species and chelating Cu(ii) and Fe(iii) ions; these combined properties confer a protective effect against ROS induced cell death. [ABSTRACT FROM AUTHOR]

Drugs currently used for treating Parkinson's disease patients provide symptomatic relief without altering the neurodegenerative process. Our aim was to examine the possibility of using DJ-1 (PARK7), as a novel therapeutic target for Parkinson's disease. We designed a short peptide, named ND-13. This peptide consists of a 13 amino acids segment of the DJ-1-protein attached to 7 amino acids derived from TAT, a cell penetrating protein. We examined the effects of ND-13 using in vitro and in vivo experimental models of Parkinson's disease. We demonstrated that ND-13 protects cultured cells against oxidative and neurotoxic insults, reduced reactive oxygen species accumulation, activated the protective erythroid-2 related factor 2 system and increased cell survival. ND-13 robustly attenuated dopaminergic system dysfunction and in improved the behavioral outcome in the 6-hydroxydopamine mouse model of Parkinson's disease, both in wild type and in DJ-1 knockout mice. Moreover, ND-13 restored dopamine content in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model. These findings validate DJ-1 as a promising therapeutic target in Parkinson's disease and identify a novel peptide with clinical potential, which may be significant for a broader range of neurological diseases, possibly with an important impact for the neurosciences. [ABSTRACT FROM AUTHOR]

Riluzole is the only approved ALS drug. Riluzole influences several cellular pathways, but its exact mechanism of action remains unclear. Our goal was to study the drug's influence on the glucose transport rate in two ALS relevant cell types, neurons and myotubes. Stably transfected wild-type or mutant G93A human SOD1 NSC-34 motor neuron-like cells and rat L6 myotubes were exposed to riluzole. The rate of glucose uptake, translocation of glucose transporters to the cell's plasma membrane and the main glucose transport regulatory proteins' phosphorylation levels were measured. We found that riluzole increases the glucose transport rate and up-regulates the translocation of glucose transporters to plasma membrane in both types of cells. Riluzole leads to AMPK phosphorylation and to the phosphorylation of its downstream target, AS-160. In conclusion, increasing the glucose transport rate in ALS affected cells might be one of the mechanisms of riluzole's therapeutic effect. These findings can be used to rationally design and synthesize novel anti-ALS drugs that modulate glucose transport in neurons and skeletal muscles. [ABSTRACT FROM AUTHOR]

The one-pot three-component preparation of novel selenium-containing spiroketals with potential biological activity is described. When in situ prepared selenium dichloride is reacted with either propargyl or homopropargyl alcohols in the presence of acetone, two competitive reactions take place, namely 1,2-addition to the triple bond with the formation of the corresponding functionalized divinyl selenides in a completely regio- and stereospecific manner and selenylation at the α-position of the ketone, leading to the novel selenium-containing spiroketals. An alternative approach to the synthesis of the selenium-containing spiroketals with substantial increase of the chemical yield involves the reaction of alkynyl acetals with SeCl2. The structure of ( 4E,11E)-4,11-bis(chloromethylene)-1,8-dioxa-5,12-diselenaspiro[6.6]tridecane was unambiguously confirmed by X-ray analysis. These novel selenium-containing spiroketals were tested for regulation of glucose uptake in L6 myotubes. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1) antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS). This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I). Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis. [ABSTRACT FROM AUTHOR]

Objective: Vascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid-metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator-activated receptor (PPAR) delta was investigated.Research Design and Methods: Effects of 4-HDDE and PPARdelta on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions.Results: Using GW501516 (PPARdelta agonist) and GSK0660 (PPARdelta antagonist), we discovered that high-glucose-induced downregulation of the glucose transport system in VECs is mediated by PPARdelta. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARdelta in cells overexpressing human PPAR (hPPAR)delta but not hPPARalpha, -gamma1, or -gamma2. Moreover, silencing of PPARdelta prevented high-glucose-dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARdelta to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay.Conclusions: Collectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARdelta. [ABSTRACT FROM AUTHOR]

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The deleterious action of ultraviolet radiation is summarized. • UV-induced damage is aggravated in light sensitive disorders. • Pharmacological activation of NRF2 can prevent photodamage and photocarcinogenesis. • The limitations of targeting NRF2 is presented. [ABSTRACT FROM AUTHOR]

The serine/threonine kinase, GSK-3, is a promising drug discovery target for treating multiple pathological disorders. Most GSK-3 inhibitors that were developed function as ATP competitive inhibitors, with typical limitations in specificity, safety and drug-induced resistance. In contrast, substrate competitive inhibitors (SCIs), are considered highly selective, and more suitable for clinical practice. The development of SCIs has been largely neglected in the past because the ambiguous, undefined nature of the substrate-binding site makes them difficult to design. In this study, we used our previously described structural models of GSK-3 bound to SCI peptides, to design a pharmacophore model and to virtually screen the "drug-like" Zinc database (~6.3 million compounds). We identified leading hits that interact with critical binding elements in the GSK-3 substrate binding site and are chemically distinct from known GSK-3 inhibitors. Accordingly, novel GSK-3 SCI compounds were designed and synthesized with IC50 values of~1–4 μM. Biological activity of the SCI compound was confirmed in cells and in primary neurons that showed increased β-catenin levels and reduced tau phosphorylation in response to compound treatment. We have generated a new type of small molecule GSK-3 inhibitors and propose to use this strategy to further develop SCIs for other protein kinases. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The rate of glucose influx to skeletal muscles is determined primarily by the number of functional units of glucose transporter-4 (GLUT4) in the myotube plasma membrane. The abundance of GLUT4 in the plasma membrane is tightly regulated by insulin or contractile activity, which employ distinct pathways to translocate GLUT4-rich vesicles from intracellular compartments. Various studies have indicated that GLUT4 intrinsic activity is also regulated by conformational changes and/or interactions with membrane components and intracellular proteins in the vicinity of the plasma membrane. Here we show that the non-metabolizable glucose analog 3- O -methyl- d -glucose (MeGlc) augmented the rate of hexose transport into myotubes by increasing GLUT4 intrinsic activity without altering the content of the transporter in the plasma membrane. This effect was not a consequence of ATP depletion or hyperosmolar stress and did not involve Akt/PKB or AMPK signal transduction pathways. MeGlc reduced the inhibitory potency (increased K i ) of indinavir, a selective inhibitor of GLUT4, in a dose-dependent manner. Kinetic analyses indicate that MeGlc induced changes in GLUT4 or GLUT4 complexes within the plasma membrane, which enhanced the hexose transport activity and reduced the potency of indinavir inhibition. Finally, we present a simple kinetic analysis for screening and discovering low molecular weight compounds that augment GLUT4 activity. [ABSTRACT FROM AUTHOR]