Conserved gatekeeper methionine regulates the binding and access of kinase inhibitors to ATP sites of MAP2K1, 4, and 7: Clues for developing selective inhibitors.

Mitogen-activated protein kinase kinases (MAP2Ks) 1, 4, and 7 are potential targets for treating various diseases. Here, we solved the crystal structures of MAP2K1 and MAP2K4 complexed with covalent inhibitor 5Z-7-oxozeaenol (5Z7O). The elucidated structures showed that 5Z7O was non-covalently bound to the ATP binding site of MAP2K4, while it covalently attached to cysteine at the DFG-1 position of the deep ATP site of MAP2K1. In contrast, we previously showed that 5Z7O covalently binds to MAP2K7 via another cysteine on the solvent-accessible edge of the ATP site. Structural analyses and molecular dynamics calculations indicated that the configuration and mobility of conserved gatekeeper methionine located at the central ATP site regulated the binding and access of 5Z7O to the ATP site of MAP2Ks. These structural features provide clues for developing highly potent and selective inhibitors against MAP2Ks. Abbreviations: ATP, adenosine triphosphate; FDA, Food and Drug Administration; MAP2Ks, mitogen-activated protein kinase kinases; MD, molecular dynamics; NSCLC, non-small cell lung cancer; 5Z7O, 5Z-7-oxozeaenol; PDB, protein data bank; RMSD, root-mean-square deviation.

Nanoscale synthesis and affinity ranking.

Most drugs are developed through iterative rounds of chemical synthesis and biochemical testing to optimize the affinity of a particular compound for a protein target of therapeutic interest. This process is challenging because candidate molecules must be selected from a chemical space of more than 1060 drug-like possibilities 1 , and a single reaction used to synthesize each molecule has more than 107 plausible permutations of catalysts, ligands, additives and other parameters 2 . The merger of a method for high-throughput chemical synthesis with a biochemical assay would facilitate the exploration of this enormous search space and streamline the hunt for new drugs and chemical probes. Miniaturized high-throughput chemical synthesis3-7 has enabled rapid evaluation of reaction space, but so far the merger of such syntheses with bioassays has been achieved with only low-density reaction arrays, which analyse only a handful of analogues prepared under a single reaction condition8-13. High-density chemical synthesis approaches that have been coupled to bioassays, including on-bead 14 , on-surface 15 , on-DNA 16 and mass-encoding technologies 17 , greatly reduce material requirements, but they require the covalent linkage of substrates to a potentially reactive support, must be performed under high dilution and must operate in a mixture format. These reaction attributes limit the application of transition-metal catalysts, which are easily poisoned by the many functional groups present in a complex mixture, and of transformations for which the kinetics require a high concentration of reactant. Here we couple high-throughput nanomole-scale synthesis with a label-free affinity-selection mass spectrometry bioassay. Each reaction is performed at a 0.1-molar concentration in a discrete well to enable transition-metal catalysis while consuming less than 0.05 milligrams of substrate per reaction. The affinity-selection mass spectrometry bioassay is then used to rank the affinity of the reaction products to target proteins, removing the need for time-intensive reaction purification. This method enables the primary synthesis and testing steps that are critical to the invention of protein inhibitors to be performed rapidly and with minimal consumption of starting materials.

Diminished PLK2 Induces Cardiac Fibrosis and Promotes Atrial Fibrillation.

[Figure: see text].

Identification of a novel target site for ATP-independent ERK2 inhibitors.

Extracellular signal-regulated kinase 2 (ERK2) controls vital physiological processes involving proliferation and differentiation and is a drug target molecule for many diseases such as cancers. In silico screening focusing on an allosteric site that plays a crucial role in substrate anchoring conferred an ERK2 inhibitor (compound 1). However, a competitive binding assay indicated that compound 1 did not bind to the allosteric site. Here, the crystal structure of ERK2 in complex with compound 1 revealed a novel binding site. This finding demonstrates the feasibility of developing new types of ERK2 inhibitors.

The ERK phosphorylation levels in the amygdala predict anxiety symptoms in humans and MEK/ERK inhibition dissociates innate and learned defensive behaviors in rats.

We demonstrate that the rate of extracellular signal-related kinase phosphorylation (P-ERK1,2/Total-ERK1,2) in the amygdala is negatively and independently associated with anxiety symptoms in 23 consecutive patients with drug-resistant mesial temporal lobe epilepsy that was surgically treated. In naive Wistar rats, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala correlates negatively with innate anxiety-related behavior on the elevated plus maze (n = 20) but positively with expression of defensive-learned behavior (i.e., freezing) on Pavlovian aversive (fear) conditioning (n = 29). The microinfusion of ERK1/2 inhibitor (FR180204, n = 8-13/group) or MEK inhibitor (U0126, n = 8-9/group) into the basolateral amygdala did not affect anxiety-related behavior but impaired the evocation (anticipation) of conditioned-defensive behavior (n = 9-11/group). In conclusion, the P-ERK1,2/Total-ERK1,2 ratio in the amygdala predicts anxiety in humans and the innate anxiety- and conditioned freezing behaviors in rats. However, the ERK1/2 in the basolateral AMY is only required for the expression of defensive-learned behavior. These results support a dissociate ERK-dependent mechanism in the amygdala between innate anxiety-like responses and the anticipation of learned-defensive behavior. These findings have implications for understanding highly prevalent psychiatric disorders related to the defensive circuit manifested by anxiety and fear. HIGHLIGHTS: The P-ERK1,2/Total-ERK1,2 ratio in the amygdala (AMY) correlates negatively with anxiety symptoms in patients with mesial temporal lobe epilepsy. The P-ERK1,2/Total-ERK1,2 in the amygdala correlates negatively with the anxiety-like behavior and positively with freezing-learned behavior in naive rats. ERK1,2 in the basolateral amygdala is required for learned-defensive but not for the anxiety-like behavior expression in rats.

Carbon dioxide inhibits COVID-19-type proinflammatory responses through extracellular signal-regulated kinases 1 and 2, novel carbon dioxide sensors.

Mitogen-activated protein kinase (MAPK) signalling pathways are crucial for developmental processes, oncogenesis, and inflammation, including the production of proinflammatory cytokines caused by reactive oxygen species and upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. There are no drugs that can effectively prevent excessive inflammatory responses in endothelial cells in the lungs, heart, brain, and kidneys, which are considered the main causes of severe coronavirus disease 2019 (COVID-19). In this work, we demonstrate that human MAPKs, i.e. extracellular signal-regulated kinases 1 and 2 (ERK1/2), are CO2 sensors and CO2 is an efficient anti-inflammatory compound that exerts its effects through inactivating ERK1/2 in cultured endothelial cells when the CO2 concentration is elevated. CO2 is a potent inhibitor of cellular proinflammatory responses caused by H2O2 or the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2. ERK1/2 activated by the combined action of RBD and cytokines crucial for the development of severe COVID-19, i.e. interferon-gamma (IFNγ) and tumour necrosis factor-α (TNFα), are more effectively inactivated by CO2 than by dexamethasone or acetylsalicylic acid in human bronchial epithelial cells. Previously, many preclinical and clinical studies showed that the transient application of 5-8% CO2 is safe and effective in the treatment of many diseases. Therefore, our research indicates that CO2 may be used for the treatment of COVID-19 as well as the modification of hundreds of cellular pathways.

Calpain activation mediates microgravity-induced myocardial abnormalities in mice via p38 and ERK1/2 MAPK pathways.

The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1 Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47 phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47 phox , and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47 phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47 phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.

Ritanserin, a potent serotonin 2A receptor antagonist, represses MEK/ERK signalling pathway to restore PAX6 production and function in aniridia-like cellular model.

Aniridia is a panocular inherited rare eye disease linked to heterozygous mutations on the PAX6 gene, which fail to properly produce sufficient protein essential for normal eye development and function. Most of the patients suffer from aniridia-related keratopathy, a progressive opacification of the cornea. There is no effective treatment for this blinding disease. Here we screen for small compounds and identified Ritanserin, a serotonin 2A receptor antagonist, that can rescue PAX6 haploinsufficiency of mutant limbal cells, defective cell migration and PAX6-target gene expression. We further demonstrated that Ritanserin activates PAX6 production through the selective inactivation of the MEK/ERK signaling pathway. Our data strongly suggest that repurposing this therapeutic molecule could be effective in preventing or treating existing blindness by restoring corneal transparency.

Lithium chloride inhibits the migration and invasion of osteosarcoma cells by blocking nuclear translocation of phospho-Erk.

Lithium chloride (LiCl) is an important mood-stabilizing therapeutic agent for bipolar disorders, which has also been shown to inhibit cancer cell metastasis. Investigations of LiCl-induced signaling have focused mainly on extracellular signal regulated kinase 1/2 (ERK1/2) and glycogen synthase kinase 3 (GSK-3). However, little is known about the differences in cellular activities resulting from specific signaling via each of these pathways. In this study, we investigated the difference in responses between the Wnt/ß-catenin and ERK pathways by LiCl or epidermal growth factor (EGF) treatment of osteosarcoma cells. In particular, we analyzed the mechanisms responsible for differences in cell mobility and cell proliferation when pERK or ß-catenin is activated. In osteosarcoma cells treated with LiCl or EGF, active ß-catenin and p-ERK protein levels were significantly increased compared to those in the control group. However, in wound healing and transwell invasion assays, U2OS and SaOS2 cell migration was significantly reduced by LiCl treatment but increased by EGF treatment. In addition, the proliferation of U2OS cells was reduced by LiCl treatment but increased by EGF treatment. Using immunofluorescence microscopy, we observed nuclear accumulation of phosphorylated ERK (pERK) with EGF treatment, but pERK was restricted to the perinuclear area with LiCl treatment. These results were confirmed using immunoblot assays after subcellular fractionation. Together, these data suggest that LiCl interferes with the translocation of pERK from the cytoplasm to the nucleus.

Extracellular signal-regulated kinases 2 (Erk2) and Erk5 in the central nervous system differentially contribute to central sensitization in male mice.

Nervous systems are designed to become extra sensitive to afferent nociceptive stimuli under certain circumstances such as inflammation and nerve injury. How pain hypersensitivity comes about is key issue in the field since it ultimately results in chronic pain. Central sensitization represents enhanced pain sensitivity due to increased neural signaling within the central nervous system (CNS). Particularly, much evidence indicates that underlying mechanism of central sensitization is associated with the change of spinal neurons. Extracellular signal-regulated kinases have received attention as key molecules in central sensitization. Previously, we revealed the isoform-specific function of extracellular signal-regulated kinase 2 (Erk2) in spinal neurons for central sensitization using mice with Cre-loxP-mediated deletion of Erk2 in the CNS. Still, how extracellular signal-regulated kinase 5 (Erk5) in spinal neurons contributes to central sensitization has not been directly tested, nor is the functional relevance of Erk5 and Erk2 known. Here, we show that Erk5 and Erk2 in the CNS play redundant and/or distinct roles in central sensitization, depending on the plasticity context (cell types, pain types, time, etc.). We used male mice with Erk5 deletion specifically in the CNS and found that Erk5 plays important roles in central sensitization in a formalin-induced inflammatory pain model. Deletion of both Erk2 and Erk5 leads to greater attenuation of central sensitization in this model, compared to deletion of either isoform alone. Conversely, Erk2 but not Erk5 plays important roles in central sensitization in neuropathic pain, a type of chronic pain caused by nerve damage. Our results suggest the elaborate mechanisms of Erk signaling in central sensitization.

Inhibition of BRAF and ERK1/2 has synergistic effects on thyroid cancer growth in vitro and in vivo.

Mutations in the BRAF gene are highly prevalent in thyroid cancer. However, the response rate of thyroid tumors to BRAF-directed therapies has been mixed. Increasingly, combination therapies inhibiting the MAPK pathway at multiple nodes have shown promise. Recently developed ERK1/2 inhibitors are of interest for use in combination therapies as they have the advantage of inhibiting the most downstream node of the MAPK pathway, therefore preventing pathway reactivation. Here, we examined the effect of combined BRAF inhibition (dabrafenib) and ERK1/2 inhibition (SCH772984) on the growth and survival of a panel of BRAF-mutant thyroid cancer cell lines using in vitro and in vivo approaches. We found that resistance due to MAPK pathway reactivation occurs quickly with single-agent BRAF inhibition, but can be prevented with combined BRAF and ERK1/2 inhibition. Combined inhibition also results in synergistic growth inhibition, decreased clonogenic survival, and enhanced induction of apoptosis in a subset of BRAF-mutant thyroid cancer cells. Finally, combined inhibition of BRAF and ERK1/2 results in enhanced inhibition of tumor growth in an anaplastic thyroid cancer in vivo model. These results provide key rationale to pursue combined BRAF and ERK1/2 inhibition as an alternative therapeutic strategy for BRAF-mutant advanced thyroid cancer patients.

A kinome-wide RNAi screen identifies ERK2 as a druggable regulator of Shank3 stability.

Neurons are sensitive to changes in the dosage of many genes, especially those regulating synaptic functions. Haploinsufficiency of SHANK3 causes Phelan-McDermid syndrome and autism, whereas duplication of the same gene leads to SHANK3 duplication syndrome, a disorder characterized by neuropsychiatric phenotypes including hyperactivity and bipolar disorder as well as epilepsy. We recently demonstrated the functional modularity of Shank3, which suggests that normalizing levels of Shank3 itself might be more fruitful than correcting pathways that function downstream of it for treatment of disorders caused by alterations in SHANK3 dosage. To identify upstream regulators of Shank3 abundance, we performed a kinome-wide siRNA screen and identified multiple kinases that potentially regulate Shank3 protein stability. Interestingly, we discovered that several kinases in the MEK/ERK2 pathway destabilize Shank3 and that genetic deletion and pharmacological inhibition of ERK2 increases Shank3 abundance in vivo. Mechanistically, we show that ERK2 binds Shank3 and phosphorylates it at three residues to promote its poly-ubiquitination-dependent degradation. Altogether, our findings uncover a druggable pathway as a potential therapeutic target for disorders with reduced SHANK3 dosage, provide a rich resource for studying Shank3 regulation, and demonstrate the feasibility of this approach for identifying regulators of dosage-sensitive genes.

Inhibition of the ERK1/2-ubiquitous calpains pathway attenuates experimental pulmonary fibrosis in vivo and in vitro.

Idiopathic pulmonary fibrosis (IPF) is a fibrotic lung disease with poor prognosis. Epithelial-mesenchymal transition (EMT) has been reported to play an important role in IPF. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) cascade, which regulates EMT and oncogenesis, has been implicated in the pathogenesis of IPF. Calpains, Ca2+-dependent cysteine proteinases that mediate controlled proteolysis of many specific substrates including epithelial cell marker E-cadherin, participate in organ fibrosis. Calpain-1 and calpain-2 of calpain family are ubiquitous calpains. ERK1/2 signaling stimulates the ubiquitous calpains activity in cancer development, but whether ERK1/2 signaling mediates the ubiquitous calpains activity in pulmonary fibrosis is unknown. Here we investigated whether inhibition of ERK1/2 signaling and the ubiquitous calpains attenuated experimental pulmonary fibrosis and examined the potential mechanism. Our results showed that inhibition of ERK1/2 signaling and the ubiquitous calpains both attenuated bleomycin (BLM)-induced lung fibrosis in mice. Inhibition of ERK1/2 signaling downregulated the expression of calpain-1 and calpain-2 in vivo and in vitro. We detected decreased E-cadherin expression and increased calpain-1 expression in IPF patients. Inhibition of ERK1/2 signaling and the ubiquitous calpains both suppressed the development of EMT in vivo and in vitro. Our study indicated that inhibition of the ERK1/2-ubiquitous calpains pathway protected pulmonary fibrosis from BLM, possibly via inhibition of EMT. Therefore, targeting ubiquitous calpains may be a potential strategy to attenuate IPF.

Methylene blue ameliorates brain edema in rats with experimental ischemic stroke via inhibiting aquaporin 4 expression.

Brain edema is a common and serious complication of ischemic stroke with limited effective treatment. We previously reported that methylene blue (MB) attenuated ischemic brain edema in rats, but the underlying mechanisms remained unknown. Aquaporin 4 (AQP4) in astrocytes plays a key role in brain edema. We also found that extracellular signal-regulated kinase 1/2 (ERK1/2) activation was involved in the regulation of AQP4 expression in astrocytes. In the present study, we investigated whether AQP4 and ERK1/2 were involved in the protective effect of MB against cerebral edema. Rats were subjected to transient middle cerebral artery occlusion (tMCAO), MB (3 mg/kg, for 30 min) was infused intravenously through the tail vein started immediately after reperfusion and again at 3 h after ischemia (1.5 mg/kg, for 15 min). Brain edema was determined by MRI at 0.5, 2.5, and 48 h after tMCAO. The decreases of apparent diffusion coefficient (ADC) values on diffusion-weighted MRI indicated cytotoxic brain edema, whereas the increase of T2 MRI values reflected vasogenic brain edema. We found that MB infusion significantly ameliorated cytotoxic brain edema at 2.5 and 48 h after tMCAO and decreased vasogenic brain edema at 48 h after tMCAO. In addition, MB infusion blocked the AQP4 increases and ERK1/2 activation in the cerebral cortex in ischemic penumbra at 48 h after tMCAO. In a cell swelling model established in cultured rat astrocyte exposed to glutamate (1 mM), we consistently found that MB (10 µM) attenuated cell swelling, AQP4 increases and ERK1/2 activation. Moreover, the ERK1/2 inhibitor U0126 (10 µM) had the similar effects as MB. These results demonstrate that MB improves brain edema and astrocyte swelling, which may be mediated by the inhibition of AQP4 expression via ERK1/2 pathway, suggesting that MB may be a potential choice for the treatment of brain edema.

LY3214996 relieves acquired resistance to sorafenib in hepatocellular carcinoma cells.

Background: Sorafenib, an oral multi-kinase inhibitor of rapidly accelerated fibrosarcoma; vascular endothelial growth factor receptor-2/3, platelet-derived growth factor receptor, c-Kit, and Flt-3 signaling, is approved for treatment of advanced hepatocellular carcinoma (HCC). However, the benefit of sorafenib is often diminished because of acquired resistance through the reactivation of ERK signaling in sorafenib-resistant HCC cells. In this work, we investigated whether adding LY3214996, a selective ERK1/2 inhibitor, to sorafenib would increase the anti-tumor effectiveness of sorafenib to HCC cells. Methods: The Huh7 cell line was used as a cell model for treatment with sorafenib, LY3214996, and their combination. Phosphorylation of the key kinases in the Ras/Raf/MAPK and PI3K/Akt pathways, protein expression of the cell cycle, and apoptosis migration were assessed with western blot. MTT and colony-formation assays were used to evaluate cell proliferation. Wound-healing assay was used to assess cell migration. Cell cycle and apoptosis analyses were conducted with flow cytometry. Results: LY3214996 decreased phosphorylation of the Ras/Raf/MAPK and PI3K/Akt pathways, including p-c-Raf, p-P90RSK, p-S6K and p-eIF4EBP1 activated by sorafenib, despite increased p-ERK1/2 levels. LY3214996 increased the anti-proliferation, anti-migration, cell-cycle progression, and pro-apoptotic effects of sorafenib on Huh7R cells. Conclusions: Reactivation of ERK1/2 appears to be a molecular mechanism of acquired resistance of HCC to sorafenib. LY3214996 combined with sorafenib enhanced the anti-tumor effects of sorafenib in HCC. These findings form a theoretical basis for trial of LY3214996 combined with sorafenib as second-line treatment of sorafenib-resistant in advanced HCC.

Extracellular signal-regulated kinase 1/2 regulates NAD metabolism during acute kidney injury through microRNA-34a-mediated NAMPT expression.

Prior studies have established the important role of extracellular signal-regulated kinase 1/2 (ERK1/2) as a mediator of acute kidney injury (AKI). We demonstrated rapid ERK1/2 activation induced renal dysfunction following ischemia/reperfusion (IR)-induced AKI and downregulated the mitochondrial biogenesis (MB) regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in mice. In this study, ERK1/2 regulation of cellular nicotinamide adenine dinucleotide (NAD) and PGC-1α were explored. Inhibition of ERK1/2 activation during AKI in mice using the MEK1/2 inhibitor, trametinib, attenuated renal cortical oxidized NAD (NAD+) depletion. The rate-limiting NAD biosynthesis salvage enzyme, NAMPT, decreased following AKI, and this decrease was prevented by ERK1/2 inhibition. The microRNA miR34a decreased with the inhibition of ERK1/2, leading to increased NAMPT protein. Mice treated with a miR34a mimic prevented increases in NAMPT protein in the renal cortex in the presence of ERK1/2 inhibition. In addition, ERK1/2 activation increased acetylated PGC-1α, the less active form, whereas inhibition of ERK1/2 activation prevented an increase in acetylated PGC-1α after AKI through SIRT1 and NAD+ attenuation. These results implicate IR-induced ERK1/2 activation as an important contributor to the downregulation of both PGC-1α and NAD+ pathways that ultimately decrease cellular metabolism and renal function. Inhibition of ERK1/2 activation prior to the initiation of IR injury attenuated decreases in PGC-1α and NAD+ and prevented kidney dysfunction.

Mesenchymal stem cell-derived exosomal miR-21a-5p promotes M2 macrophage polarization and reduces macrophage infiltration to attenuate atherosclerosis.

Atherosclerosis (AS) is the main pathological basis for ischemic cardiovascular and cerebrovascular diseases. Mesenchymal stem cell (MSC)-derived exosomes have the potential to alleviate AS, while the underlying mechanism remains unclear. Here, we aimed to investigate the mechanism of MSC-derived exosomes in AS. The AS mouse model was prepared by feeding ApoE-/- mice with high-fat diet. AS mice were administered with MSC-derived exosomes, and the atherosclerotic plaque area was analyzed by Oil Red O staining. Mouse RAW264.7 macrophages were incubated with MSC-derived exosomes. The macrophage infiltration, macrophage proportion, and cell migration were estimated by immunohistochemistry, flow cytometry, or Transwell assay. The relationship between miR-21a-5p and kruppel-like factor 6 (KLF6) or extracellular signal-regulated protein kinases 2 (ERK2) was verified by luciferase reporter assay. We found that MSC-derived exosomes promoted M2 polarization of macrophages and reduced plaque area and macrophage infiltration in AS mice. miR-21a-5p overexpression caused an increase of M2 macrophages in RAW264.7 cells and led to a decrease in migration of RAW264.7 cells. Moreover, both KLF6 and ERK2 are the targets of miR-21a-5p. MSC-derived exosomes containing miR-21a-5p promoted M2 polarization of RAW264.7 cells by suppressing KLF6 expression. MSC-derived exosomes containing miR-21a-5p inhibited migration of RAW264.7 cells through inhibiting the ERK1/2 signaling pathway. In conclusion, MSC-derived exosomes containing miR-21a-5p promote macrophage polarization and reduce macrophage infiltration by targeting KLF6 and ERK1/2 signaling pathways, thereby attenuating the development of AS. Thus, MSC-derived exosomes may be a promising treatment for AS.

Standardized Extract of Asparagus officinalis Stem Attenuates SARS-CoV-2 Spike Protein-Induced IL-6 and IL-1ß Production by Suppressing p44/42 MAPK and Akt Phosphorylation in Murine Primary Macrophages.

Excessive host inflammation following infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with severity and mortality in coronavirus disease 2019 (COVID-19). We recently reported that the SARS-CoV-2 spike protein S1 subunit (S1) induces pro-inflammatory responses by activating toll-like receptor 4 (TLR4) signaling in macrophages. A standardized extract of Asparagus officinalis stem (EAS) is a unique functional food that elicits anti-photoaging effects by suppressing pro-inflammatory signaling in hydrogen peroxide and ultraviolet B-exposed skin fibroblasts. To elucidate its potential in preventing excessive inflammation in COVID-19, we examined the effects of EAS on pro-inflammatory responses in S1-stimulated macrophages. Murine peritoneal exudate macrophages were co-treated with EAS and S1. Concentrations and mRNA levels of pro-inflammatory cytokines were assessed using enzyme-linked immunosorbent assay and reverse transcription and real-time polymerase chain reaction, respectively. Expression and phosphorylation levels of signaling proteins were analyzed using western blotting and fluorescence immunomicroscopy. EAS significantly attenuated S1-induced secretion of interleukin (IL)-6 in a concentration-dependent manner without reducing cell viability. EAS also markedly suppressed the S1-induced transcription of IL-6 and IL-1ß. However, among the TLR4 signaling proteins, EAS did not affect the degradation of inhibitor κBα, nuclear translocation of nuclear factor-κB p65 subunit, and phosphorylation of c-Jun N-terminal kinase p54 subunit after S1 exposure. In contrast, EAS significantly suppressed S1-induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) and Akt. Attenuation of S1-induced transcription of IL-6 and IL-1ß by the MAPK kinase inhibitor U0126 was greater than that by the Akt inhibitor perifosine, and the effects were potentiated by simultaneous treatment with both inhibitors. These results suggest that EAS attenuates S1-induced IL-6 and IL-1ß production by suppressing p44/42 MAPK and Akt signaling in macrophages. Therefore, EAS may be beneficial in regulating excessive inflammation in patients with COVID-19.

Betulin, a Newly Characterized Compound in Acacia auriculiformis Bark, Is a Multi-Target Protein Kinase Inhibitor.

The purpose of this work is to investigate the protein kinase inhibitory activity of constituents from Acacia auriculiformis stem bark. Column chromatography and NMR spectroscopy were used to purify and characterize betulin from an ethyl acetate soluble fraction of acacia bark. Betulin, a known inducer of apoptosis, was screened against a panel of 16 disease-related protein kinases. Betulin was shown to inhibit Abelson murine leukemia viral oncogene homolog 1 (ABL1) kinase, casein kinase 1ε (CK1ε), glycogen synthase kinase 3α/ß (GSK-3 α/ß), Janus kinase 3 (JAK3), NIMA Related Kinase 6 (NEK6), and vascular endothelial growth factor receptor 2 kinase (VEGFR2) with activities in the micromolar range for each. The effect of betulin on the cell viability of doxorubicin-resistant K562R chronic myelogenous leukemia cells was then verified to investigate its putative use as an anti-cancer compound. Betulin was shown to modulate the mitogen-activated protein (MAP) kinase pathway, with activity similar to that of imatinib mesylate, a known ABL1 kinase inhibitor. The interaction of betulin and ABL1 was studied by molecular docking, revealing an interaction of the inhibitor with the ABL1 ATP binding pocket. Together, these data demonstrate that betulin is a multi-target inhibitor of protein kinases, an activity that can contribute to the anticancer properties of the natural compound and to potential treatments for leukemia.

Role of MAPK ERK1/2 and p38 in the Regulation of Secretory Functions of Different Populations of Neuroglia in Ethanol-Induced Neurodegeneration.

We studied the participation of ERK1/2 and p38 in secretion of neurotrophic growth factors by various types of neuroglia under conditions of in vitro and in vivo modeled ethanol-induced neurodegeneration. The inhibitory role of these protein kinases in the production of neurotrophins by intact astrocytes and the absence of their participation in the regulation of functions of oligodendrocytes and microglial cells were shown. Under conditions of ethanol neurotoxicity, the role of ERK1/2 and p38 in the production of growth factors by glial elements was significantly changed. Neurodegeneration modeled in vitro led to inversion of the role of both protein kinases in the secretion of neurotrophins by astroglia and inhibition of the cytokine-synthesizing function of oligodendrocytes and microglial cells by ERK1/2 and p38. In mice receiving ethanol per os for a long time (as well as in cells in vitro exposed to ethanol), mitogen-activated kinases stimulated the function of astrocytes and inhibited the production of growth factors by microglial cells. At the same time, chronic alcoholization was accompanied by the appearance of the stimulating role of ERK1/2 and p38 in the implementation of the secretory function by oligodendrocytes.