Specific Activator Of Protein Kinase Research Articles

Downregulation of nuclear receptor-binding SET domain protein 1 induces proinflammatory cytokine expression via mitogen-activated protein kinase pathways in U87MG cells

Haploinsufficiency of the nuclear receptor binding SET domain-containing protein 1 gene (NSD1) leads to a neurodevelopmental disorder known as Sotos syndrome (SOTOS). This study investigated the effects of NSD1 knockdown in glial cells. U87MG glioma cells were transfected with siRNA targeting NSD1, which resulted in morphological changes characteristic of activated astrocytes. These activated phenotypes were accompanied by specific activation of mitogen-activated protein kinase (MAPK) signaling pathways, particularly those mediated by p38 MAPK and c-Jun N-terminal kinase (JNK). Transcriptome analysis showed increased expression of proinflammatory cytokine genes, particularly interleukin (IL)-1α, IL-1β, and IL-6, following NSD1 knockdown. Treatment with MAPK inhibitors significantly reduced the cytokine induction caused by NSD1 knockdown, with the p38 MAPK inhibitor being more effective than the JNK inhibitor. These findings provide new insights into the role of NSD1 loss in neurological dysfunctions associated with SOTOS.

Abstract 1676: A continuous kinetic assay to quantitate specific protein kinase activity in unfractionated cell lysates

Abstract Protein kinases form a network of signaling pathways and are modified by multiple post-translational modifications, including phosphorylation, which regulate the kinase enzymatic activity, protein complexes formation, and its cellular location. Given this rich target complexity, >30% of all drug development is focused on kinases. However, these target only 15% of the human kinome and are primarily just one type (ATP-competitive) that can readily result in off-target inhibition and the development of resistance during cancer treatment. Most kinase inhibitor development is carried out in vitro assays with an epitope-tagged recombinant kinase. As a result, efficacy in patients can be limited due to poor inhibitor performance under physiological conditions (e.g., mM ATP and kinases being complexed with other proteins). In contrast, in vitro assays using unfractionated cell lysates provide a close approximation of the native kinase environment, including the complexity of interactions with other cellular components. Being able to selectively quantify the enzymatic activity of all protein kinases in crude cell or tissue homogenates would enable an improved understanding of kinase signaling biology, drug development, and personalized medicine. However, despite the large amount of literature on kinase assays, only a fraction is compatible with unfractionated lysates since most peptide substrates are not selective for a given kinase. To that end, we utilized a continuous assay format coupled with high-throughput Sox-sensor-peptide library synthesis using physiological sequences to identify selective sensor peptide substrates for multiple high-profile kinases implicated in cancer. As an example, we identified a highly selective sensor peptide for ERK1 and 2 mitogen-activated protein kinases (MAPKs) involved in pro-growth/oncogenic signaling. We demonstrated selectivity initially with the recombinant enzymes for ERK1/2 and a panel of other related MAPKs or CMGC kinases. Next, we extended the selectivity screen to 392 kinases using a large panel kinome profiling service. The screen also demonstrated high selectivity for ERK1/2 isoforms. We then tested our best sensor peptide substrate for ERK1/2 using unfractionated lysates from the HEK293T cell line with ERK2 overexpression and demonstrated high activity that could be increased 50-fold by the addition of recombinant MEK1 or 2, the upstream kinase that activates ERK1 and 2, and completely blocked with the addition of an ERK2-selective inhibitor. These results demonstrate a method to systematically generate a selective sensor peptide for ERK1/2 to continuously monitor this kinase activity in crude cell or tissue lysates. This poster will fully describe the methods and results pertaining to ERK-selective sensor peptide development and validation, and the ability to extend to other kinases of interest. Citation Format: Venkatesh Nemmara, Susan Cornell-Kennon, Zhibing Lu, Gabriela Pikul, Zacchary Belisle, Matthew Hakar, Jefferson Chin, Erik Schaefer, Earl May. A continuous kinetic assay to quantitate specific protein kinase activity in unfractionated cell lysates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1676.

Differentiated modulation of signaling molecules AMPK and SIRT1 in experimentally drug-induced hepatocyte injury.

Currently available medicines have little to offer in terms of supporting the regeneration of injured hepatic cells. Previous experimental studies have shown that resveratrol and metformin, less specific activators of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1), can effectively attenuate acute liver injury. The aim of this experimental study was to elucidate whether modulation of AMPK and SIRT1 activity can modify drug/paracetamol (APAP)-induced hepatocyte damage in vitro. Primary rat hepatocytes were pretreated with mutual combinations of specific synthetic activators and inhibitors of SIRT1 and AMPK and followed by a toxic dose of APAP. At the end of cultivation, medium samples were collected for biochemical analysis of alanine-aminotransferase and nitrite levels. Hepatocyte viability, thiobarbituric reactive substances, SIRT1 and AMPK activity and protein expression were also assessed. The harmful effect of APAP was associated with decreased AMPK and SIRT1 activity and protein expression alongside enhanced oxidative stress in hepatocytes. The addition of AMPK activator (AICAR) or SIRT1 activator (CAY10591) significantly attenuated the deleterious effects of AMPK inhibitor (Compound C) on the hepatotoxicity of APAP. Furthermore, CAY10591 but not AICAR markedly decreased the deleterious effect of APAP in combination with SIRT1 inhibitor (EX-527). Our findings demonstrate that decreased AMPK activity is associated with the hepatotoxic effect of APAP which can be significantly attenuated by the administration of a SIRT1 activator. These findings suggest that differentiated modulation of AMPK and SIRT1 activity could therefore provide an interesting and novel therapeutic opportunity in the future to combat hepatocyte injury.

Transcriptomic Profiling Reveals a Role for TREM-1 Activation in Enterovirus D68 Infection-Induced Proinflammatory Responses.

Increasing cases related to the pathogenicity of Enterovirus D68 (EV-D68) have made it a growing worldwide public health concern, especially due to increased severe respiratory illness and acute flaccid myelitis (AFM) in children. There are currently no vaccines or medicines to prevent or treat EV-D68 infections. Herein, we performed genome-wide transcriptional profiling of EV-D68-infected human rhabdomyosarcoma (RD) cells to investigate host-pathogen interplay. RNA sequencing and subsequent experiments revealed that EV-D68 infection induced a profound transcriptional dysregulation of host genes, causing significantly elevated inflammatory responses and altered antiviral immune responses. In particular, triggering receptor expressed on myeloid cells 1 (TREM-1) is involved in highly activated TREM-1 signaling processes, acting as an important mediator in EV-D68 infection, and it is related to upregulation of interleukin 8 (IL-8), IL-6, IL-12p70, IL-1β, and tumor necrosis factor alpha (TNF-α). Further results demonstrated that NF-κB p65 was essential for EV-D68-induced TREM-1 upregulation. Moreover, inhibition of the TREM1 signaling pathway by the specific inhibitor LP17 dampened activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade, suggesting that TREM-1 mainly transmits activation signals to phosphorylate p38 MAPK. Interestingly, treatment with LP17 to inhibit TREM-1 inhibited viral replication and infection. These findings imply the pathogenic mechanisms of EV-D68 and provide critical insight into therapeutic intervention in enterovirus diseases.

Chronic bryostatin-1 rescues autistic and cognitive phenotypes in the fragile X mice

Fragile X syndrome (FXS), an X-chromosome linked intellectual disability, is the leading monogenetic cause of autism spectrum disorder (ASD), a neurodevelopmental condition that currently has no specific drug treatment. Building upon the demonstrated therapeutic effects on spatial memory of bryostatin-1, a relatively specific activator of protein kinase C (PKC)ε, (also of PKCα) on impaired synaptic plasticity/maturation and spatial learning and memory in FXS mice, we investigated whether bryostatin-1 might affect the autistic phenotypes and other behaviors, including open field activity, activities of daily living (nesting and marble burying), at the effective therapeutic dose for spatial memory deficits. Further evaluation included other non-spatial learning and memory tasks. Interestingly, a short period of treatment (5 weeks) only produced very limited or no therapeutic effects on the autistic and cognitive phenotypes in the Fmr1 KO2 mice, while a longer treatment (13 weeks) with the same dose of bryostatin-1 effectively rescued the autistic and non-spatial learning deficit cognitive phenotypes. It is possible that longer-term treatment would result in further improvement in these fragile X phenotypes. This effect is clearly different from other treatment strategies tested to date, in that the drug shows little acute effect, but strong long-term effects. It also shows no evidence of tolerance, which has been a problem with other drug classes (mGluR5 antagonists, GABA-A and -B agonists). The results strongly suggest that, at appropriate dosing and therapeutic period, chronic bryostatin-1 may have great therapeutic value for both ASD and FXS.

Epithelial-Immune Cell Interactions for Drug Discovery in Chronic Obstructive Pulmonary Disease.

New studies of chronic obstructive pulmonary disease (COPD) are revealing the key role of airway epithelial cells and innate immune cells in the initiation, exacerbation, and progression of airway disease. An emerging scheme focuses on expansion of airway progenitor epithelial cells that feed forward for a type 2 immune response and consequent IL-13-driven mucus production that is linked to the morbidity and mortality of COPD. Analysis of human airway progenitor epithelial cells and airway tissue shows that IL-13 signaling to MUC5AC mucin gene expression relies on specific activation of mitogen-activated protein kinase 13, providing a druggable target for attenuating mucus production in the setting of viral infection and other inhaled stimuli of airway inflammation. Moreover, structure-based drug design is delivering highly potent, selective, and nontoxic small-molecule kinase inhibitors of mitogen-activated protein kinase 13 that offer a therapeutic strategy to downregulate excess mucus production to a physiological level and thereby achieve a precision medicine solution to the major health care problem of COPD and related airway diseases.

CGMP Binding Domain D Mediates a Unique Activation Mechanism in Plasmodium falciparum PKG.

cGMP-dependent protein kinase from Plasmodium falciparum ( PfPKG) plays a crucial role in the sexual as well as the asexual proliferation of this human malaria causing parasite. However, function and regulation of PfPKG are largely unknown. Previous studies showed that the domain organization of PfPKG significantly differs from human PKG ( hPKG) and indicated a critical role of the cyclic nucleotide binding domain D (CNB-D). We identified a novel mechanism, where the CNB-D controls activation and regulation of the parasite specific protein kinase. Here, kinase activity is not dependent on a pseudosubstrate autoinhibitory sequence (IS), as reported for human PKG. A construct lacking the putative IS and containing only the CNB-D and the catalytic domain is inactive in the absence of cGMP and can efficiently be activated with cGMP. On the basis of structural evidence, we describe a regulatory mechanism, whereby cGMP binding to CNB-D induces a conformational change involving the αC-helix of the CNB-D. The inactive state is defined by a unique interaction between Asp597 of the catalytic domain and Arg528 of the αC-helix. The same arginine (R528), however, stabilizes cGMP binding by interacting with Tyr480 of the phosphate binding cassette (PBC). This represents the active state of PfPKG. Our results unveil fundamental differences in the activation mechanism between PfPKG and hPKG, building the basis for the development of strategies for targeted drug design in fighting malaria.

OncoPPi-informed discovery of mitogen-activated protein kinase kinase 3 as a novel binding partner of c-Myc.

Mitogen-activated protein kinase kinase 3 (MKK3) is a dual threonine/tyrosine protein kinase that regulates inflammation, proliferation and apoptosis through specific phosphorylation and activation of the p38 MAPK. However, the role of MKK3 beyond p38-signaling remains elusive. Recently, we reported a protein-protein interaction (PPI) network of cancer-associated genes, termed OncoPPi, as a resource for the scientific community to generate new biological models. Analysis of the OncoPPi connectivity identified MKK3 as one of the major hub proteins in the network. Here, we show that MKK3 interacts with a large number of proteins critical for cell growth and metabolism, including the major oncogenic driver MYC. Multiple complementary approaches were employed to demonstrate the direct interaction of MKK3 with MYC in vitro and in vivo. Computational modeling and experimental studies mapped the interaction interface to the MYC helix-loop-helix domain and a novel 15-residue MYC-binding motif in MKK3 (MBM). The MBM in MKK3 is distinct from the known binding sites for p38 or upstream kinases. Functionally, MKK3 stabilized MYC protein, enhanced its transcriptional activity and increased expression of MYC-regulated genes. The defined MBM peptide mimicked the MKK3 effect in promoting MYC activity. Together, the exploration of OncoPPi led to a new biological model in which MKK3 operates by two distinct mechanisms in cellular regulation through its phosphorylation of p38 and its activation of MYC through protein-protein interaction.

Measurement of Phosphorylated ERK As a Prognostic and Predictive Marker for MEK Inhibition in Pediatric B-Lymphoblastic Leukemia: A Pilot Study

Abstract Background: Outcomes for patients with relapsed B- acute lymphoblastic leukemia (B-ALL) remain suboptimal. We and others have identified the Mitogen activated protein kinase/ Extracellular signal regulated kinase (MAPK/ERK) pathway as a primary driver of drug resistance in pediatric B-ALL. Constitutive activation of ERK has been found to be an independent predictor of complete response and is associated with other poor prognostic factors in adults with B-ALL. Mutations that affect activation of the Ras/Raf/MEK/ERK pathway have been found in up to 40% of children with newly diagnosed and relapsed ALL and these mutations correlate with early relapse and poor overall survival. However, we have demonstrated MAPK activation in the absence of Ras pathway mutations, suggesting alternative mechanisms of activation. Hypothesis: We hypothesize that activation of the MAPK pathway drives drug resistance in pediatric B-ALL. Activation of this pathway at diagnosis may portend a high likelihood of relapse and inhibition of the MAPK pathway may restore chemosensitivity to conventional agents. Methods: For our initial pilot study, a total of 24 patient samples from initial diagnosis obtained from Children's Oncology Group cell bank and from NYU, were thawed and placed either in serum-free media or media containing Trametinib (MEK1/2 inhibitor) at 1µM for 4 hours. Samples were then stimulated with Phorbol 12-myristate 13-acetate (PMA), a specific activator of Protein Kinase C, which activates MEK via Ras. Samples were then fixed with 4% paraformaldehyde, permeabilized with 95% methanol, and stained for Caspase 3, CD10, phosphorylated ERK (p-ERK), and p-AKT. All samples were simultaneously processed on the BD LSRII HTS and analyzed on FlowJo software (Treestar, V.10). Viable leukemic blasts were identified as CD10 positive and Caspase 3 negative cells, which were gated on for further analysis. Median fluorescence intensity (MFI) for phosphorylated proteins was measured, expressed as a ratio to isotype controls and compared between samples. MAPK activation was defined by maximal p-ERK levels upon stimulation with PMA. Inhibition was quantified as percentage decrease in maximal p-ERK levels (upon PMA stimulation) after treatment with Trametinib. Targeted Sanger sequencing of commonly mutated exons of Ras pathway genes (NRAS, KRAS, FLT3 and PTPN11) was performed on 14 samples based on availability of DNA. Results: Of our 24 samples, 4 samples were excluded from analysis due to poor viability (<40% live cells), giving an assay success rate of 83.3%. We were able to successfully measure p-ERK in all 20 viable patient samples and also measure levels of baseline p-AKT in 16 samples. A wide range of p-ERK levels and MAPK activation was observed within patient samples (median activation ratio 12.3, range 4.4-44.7) and 7 samples showed markedly higher levels of activation with ratios between 21.9 - 44.78. Levels of inhibition by Trametinib ranged from 0-82.2%, with a median of 43%. Greater degree of MAPK activation was found to correlate strongly with higher levels of inhibition by Trametinib (overall correlation coefficient 0.72). In 2 samples, the levels of inhibition by Trametinib seemed lower than anticipated for levels of MAPK activation, but both of these samples showed higher than average levels of p-AKT (p-AKT to isotype ratio of 12.3-12.9, median of 2.4 in rest of the samples), suggesting a possible alternative mechanism of activation via the PI3K/AKT signaling pathway. DNA was obtained for Sanger sequencing in 14 of the 20 viable patient samples. Three of the 14 samples had point mutations in Ras pathway genes of which two had mutations in exon 2 of NRAS while one had a mutation in exon 14 of FLT3. Two of the seven patient samples with the highest MAPK activation had Ras mutations, which is consistent with our prior data showing that additional mechanisms of pathway activation are operative. Conclusion: We have successfully established a phospho-flow protocol that enables the measurement of MAPK activation in real time in patient samples. Based on our pilot data, we also conclude that MAPK activation in itself serves as a better predictive marker for susceptibility to MAPK inhibition, as opposed to Ras pathway mutation status alone. Ongoing investigation will allow correlation with outcome and the assay could serve in the future to identify those patients who might benefit from MEK inhibition or other nodes in the pathway. Disclosures Loh: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Heparan sulfate hexasaccharide selectively inhibits cancer stem cells self-renewal by activating p38 MAP kinase.

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/β mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.

The phosphodiesterase 4 inhibitor roflumilast augments the Th17-promoting capability of dendritic cells by enhancing IL-23 production, and impairs their T cell stimulatory activity due to elevated IL-10

Phosphodiesterase 4 (PDE4) inhibitors serve to prevent degradation of the intracellular second messenger cAMP, resulting in broad anti-inflammatory effects on different cell types including immune cells. Agents that elevate cAMP levels via activation of adenylate cyclase have been shown to imprint a Th17-promoting capacity in dendritic cells (DCs). Therefore, we studied the potential of therapeutically relevant PDE inhibitors to induce a pronounced Th17-skewing capacity in DCs. Here we show that mouse bone marrow-derived (BM-) DCs when treated with the PDE4 inhibitor roflumilast (ROF, trade name: Daxas) in the course of stimulation with LPS (ROF-DCs) evoked elevated IL-17 levels in cocultured allogeneic T cells. In addition, as compared with control settings, levels of IFN-γ remained unaltered, while contents of Th2 cytokines (IL-5, IL-10) were diminished. ROF enhanced expression of the Th17-promoting factor IL-23 in BM-DCs. In line, neutralizing antibodies specific for IL-23 or IL-6 when applied to DC/T cell cocultures partially inhibited the IL17-promoting effect of ROF-DCs. Furthermore, ROF-DCs displayed a markedly diminished allogeneic T cell stimulatory capacity due to enhanced production of IL-10, which was restored upon application of IL-10 specific neutralizing antibody to DC/T cell cocultures. Both the IL-17-inducing and impaired T cell stimulatory capacity of BM-DCs were mimicked by a specific activator of protein kinase A, while stimulation of EPACs (exchange proteins of activated cAMP) did not yield such effects. Taken together, our findings suggest that PDE4 inhibitors aside from their broad overall anti-inflammatory effects may enhance the Th17-polarizing capacity in DCs as an unwanted side effect.

Local expression of SOD1G93A mutant protein triggers neuromuscular junction dismantlement

The alteration of Reactive Oxygen Species (ROS) homeostasis plays a causal role in several chronic pathology such as aging and neurodegenerative diseases like Amyotrophic Lateral Sclerosis (ALS). Although it is recognized that axon and synapses are first cellular sites of degeneration in ALS disease, controversy exists on whether pathological events initially begin at the NMJs and then, in a dying back phenomena, contribute to motor neuron degeneration. Moreover, the precise molecular mechanisms of pathology-associated deterioration in neuromuscular system have remained elusive (1). Here we provide evidences that muscle specific accumulation of SOD1G93A in the transgenic mice model MLC/SOD1G93A (2) induces mitochondria dysfunction and triggers NMJ dismantlement. Further, we demonstrate that treatment of MLC/SOD1G93A mice with Trolox, a potent antioxidant, is sufficient to rescue mitochondria and NMJ defects in the MLC/SOD1G93A mice, stabilizing muscle-nerve connection. The analysis of potential molecular mechanisms that mediate the toxic activity of SOD1 revealed the activation of specific Protein Kinase as a downstream player of NMJ dismantlement. Overall our data demonstrate that muscle specific expression of SOD1G93A mutation causes mitochondrial impairment and NMJ dismantlement, suggesting that muscle defects and NMJs alteration precede motor neuron degeneration rather than resulting from it.

Monitoring of apoptosis in 3D cell cultures by FRET and light sheet fluorescence microscopy.

Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure.

Assessing GPCR and G protein signaling to the nucleus in live cells using fluorescent biosensors.

G protein-coupled receptor (GPCR) signaling cascades regulate a wide variety of cellular processes and feature prominently in many cardiovascular pathologies. As such they represent major drug targets and discovering novel aspects of GPCR signaling provide important opportunities to identify additional potential therapeutic approaches to reverse or prevent cardiac remodeling and failure. Monitoring cellular trafficking of signaling components and specific protein kinase activities using fluorescent biosensors has provided key insight into stress/GPCR-induced kinase signaling networks and their effect on cardiac gene expression. Herein we describe the protocols for the expression, visualization (by confocal microscopy), and interpretation of data obtained with such biosensors expressed in adult cardiomyocytes. Our focus is on the cellular trafficking of class II histone deacetylases (i.e., HDAC5) and on the FRET sensor (Camui) for calmodulin-dependent protein kinase II (CaMKII).

The role of c‐Jun in controlling the EPAC1‐dependent induction of the SOCS3 gene in HUVECs

The cyclic AMP sensor, EPAC1, activates AP1-mediated transcription in HUVECs. Correspondingly, induction of the SOCS3 minimal promoter by EPAC1 requires a single AP1 site that constitutively binds phosphorylated (Ser63) c-Jun in DNA-pull-down assays. c-Jun (Ser63) becomes further phosphorylated following cyclic AMP stimulation and specific activation of protein kinase A (PKA), but not through selective activation of EPAC1. Moreover, despite a requirement for c-Jun for SOCS3 induction in fibroblasts, phospho-null c-Jun (Ser63/73Ala) had little effect on SOCS3 induction by cyclic AMP in HUVECs. AP1 activation and SOCS3 induction by EPAC1 in HUVECs therefore occur independently of c-Jun phosphorylation on Ser63.

Discovery of N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-sulfonamide derivatives as novel protein kinase and angiogenesis inhibitors for the treatment of cancer: Synthesis and biological evaluation. Part III

A novel series of N-(3-((7H-purin-6-yl)thio)-4-hydroxynaphthalen-1-yl)-sulfonamides were designed and synthesized. Biological characterization revealed that several compounds exerted enhanced anti-proliferative activity against human umbilical vein endothelial cells (HUVECs) and several cancer cell lines and high specific protein kinase and angiogenesis inhibitory activities. Compared with our previously synthesized compounds, the substitution of sulfonamide structure for amide fragment played an essential role for the advance of inhibitory activities. In addition, the replacement of 1H-1,2,4-triazole ring by 7H-purine did not result in obvious decrease of inhibition efficacy, indicating that the sulfonamide structure contributes even more to the inhibition efficacy than the 1H-1,2,4-triazole ring. Among these compounds, compound 9n demonstrated comparable in vitro antiangiogenic activities to pazopanib in both HUVEC tube formation assay and the rat thoracic aorta rings (TARs) test. Meanwhile, compound 9n was identified to inhibit Akt1 (IC50=1.73μM) and Abl tyrosine kinase (IC50=1.53μM) effectively.

C1q‐tumour necrosis factor‐related protein 8 (CTRP8) is a novel interaction partner of relaxin receptor RXFP1 in human brain cancer cells

We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain.

Discovery of 4-amino-2-(thio)phenol derivatives as novel protein kinase and angiogenesis inhibitors for the treatment of cancer: Synthesis and biological evaluation. Part II

A novel series of 4-amino-2-(thio)phenol derivatives were well synthesized. The preliminary biological test revealed that several compounds displayed high specific protein kinase and angiogenesis inhibitory activities compared with previous work mainly because of the substitution of sulfonamide structure for amide fragment. Among which, compound 5i was identified to inhibit protein kinase B/AKT (IC50 = 1.26 μM) and ABL tyrosine kinase (IC50 = 1.50 μM) effectively. Meanwhile, compound 5i demonstrated competitive in vitro antiangiogenic activities to Pazopanib in both human umbilical vein endothelial cell (HUVEC) tube formation assay and the rat thoracic aorta rings test.

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

Activated hepatic stellate cells mediate the differentiation of macrophages

Liver macrophages play integral roles in both the progression and resolution of hepatic inflammation and fibrosis, comprising opposing functions that largely coincide with the activation state of nearby hepatic stellate cells (HSC). While cross-talk between HSC and macrophages may be essential at various stages of inflammation and fibrogenesis, many facets of this interaction have yet to be thoroughly explored. Here, we examine the potential roles of HSC-derived signaling molecules as mediators of liver macrophage differentiation. Human peripheral blood mononuclear cells (PBMC) were differentiated to macrophages in the presence or absence of cultured HSC-derived conditioned media. The phenotype of resulting macrophages was characterized by examination of cell surface marker expression, antigen-presenting capabilities and cytokine secretion. Conditioned media from activated human HSC promoted the differentiation of a unique set of macrophages that differed in morphology and function from both classical (M1) and alternative (M2) macrophages, expressing increased levels of CD14 and CD16, as well as a distinct interleukin (IL)-6(high) /IL-10(low) /transforming growth factor (TGF)-β(high) expression profile. These macrophages expressed high levels of CD206, CD209, CD80 and human leukocyte antigen DR, though no significant increases in antigen presentation were apparent. HSC-derived macrophages exhibited specific activation of p38 mitogen-activated protein kinase, and inhibition of this activation by p38 inhibitors during differentiation effectively reversed increases in IL-6 and TGF-β. The present results suggest that HSC-derived signaling molecules promote differentiation of liver macrophages with both pro-inflammatory and profibrotic functions. Furthermore, these effects appear to be mediated, at least partially, in a p38-dependent manner.