Targeting alternative splicing as a new cancer immunotherapy-phosphorylation of serine arginine-rich splicing factor (SRSF1) by SR protein kinase 1 (SRPK1) regulates alternative splicing of PD1 to generate a soluble antagonistic isoform that prevents T cell exhaustion.

BACKGROUND: Regulation of alternative splicing is a new therapeutic approach in cancer. The programmed cell death receptor 1 (PD-1) is an immunoinhibitory receptor expressed on immune cells that binds to its ligands, PD-L1 and PD-L2 expressed by cancer cells forming a dominant immune checkpoint pathway in the tumour microenvironment. Targeting this pathway using blocking antibodies (nivolumab and pembrolizumab) is the mainstay of anti-cancer immunotherapies, restoring the function of exhausted T cells. PD-1 is alternatively spliced to form isoforms that are either transmembrane signalling receptors (flPD1) that mediate T cell death by binding to the ligand, PD-L1 or an alternatively spliced, soluble, variant that lacks the transmembrane domain. METHODS: We used PCR and western blotting on primary peripheral blood mononuclear cells (PBMCs) and Jurkat T cells, IL-2 ELISA, flow cytometry, co-culture of melanoma and cholangiocarcinoma cells, and bioinformatics analysis and molecular cloning to examine the mechanism of splicing of PD1 and its consequence. RESULTS: The soluble form of PD-1, generated by skipping exon 3 (∆Ex3PD1), was endogenously expressed in PBMCs and T cells and prevents cancer cell-mediated T cell repression. Multiple binding sites of SRSF1 are adjacent to PD-1 exon 3 splicing sites. Overexpression of phosphomimic SRSF1 resulted in preferential expression of flPD1. Inhibition of SRSF1 phosphorylation both by SRPK1 shRNA knockdown and by a selective inhibitor, SPHINX31, resulted in a switch in splicing to ∆Ex3PD1. Cholangiocarcinoma cell-mediated repression of T cell IL-2 expression was reversed by SPHINX31 (equivalent to pembrolizumab). CONCLUSIONS: These results indicate that switching of the splicing decision from flPD1 to ∆Ex3PD1 by targeting SRPK1 could represent a potential novel mechanism of immune checkpoint inhibition in cancer.

Serine-arginine-rich protein kinase-1 inhibition for the treatment of diabetic retinopathy.

Angiogenic VEGF isoforms are upregulated in diabetic retinopathy (DR), driving pathological growth and fluid leakage. Serine-arginine-rich protein kinase-1 (SRPK1) regulates VEGF splicing, and its inhibition blocks angiogenesis. We tested the hypothesis that SRPK1 is activated in diabetes, and an SRPK1 inhibitor (SPHINX31) switches VEGF splicing in DR and prevents increased vascular permeability into the retina. SRPK1 was activated by high glucose (HG), in a PKC-dependent manner, and was blocked by SPHINX31. HG induced release of SRSF1 from the nuclear speckles, which was also SRPK1 dependent, and increased retinal pigment epithelial (RPE) monolayer admittance, which was reversed by SRPK1 inhibition (P < 0.05). Diabetes increased retinal permeability and thickness after 14 days which was blocked by treatment with SPHINX31 eye drops (P < 0.0001). These results show that SRPK1 inhibition, administered as an eye drop, protected the retinal barrier from hyperglycemia-associated loss of integrity in RPE cells in vitro and in diabetic rats in vivo. A clinical trial of another SRPK1 inhibitor has now been initiated in patients with diabetic macular edema.NEW & NOTEWORTHY VEGF-A165b splicing is induced by hyperglycemia through PKC-mediated activation of SRPK1 in RPE cells, increasing their permeability and angiogenic capability. SRPK1 inhibitors can be given as eye drops to reduce retinal permeability and edema in diabetic retinopathy.

Polyamine-Conjugated Nitroxides Are Efficacious Inhibitors of Oxidative Reactions Catalyzed by Endothelial-Localized Myeloperoxidase.

The heme enzyme myeloperoxidase (MPO) is a key mediator of endothelial dysfunction and a therapeutic target in cardiovascular disease. During inflammation, MPO released by circulating leukocytes is internalized by endothelial cells and transcytosed into the subendothelial extracellular matrix of diseased vessels. At this site, MPO mediates endothelial dysfunction by catalytically consuming nitric oxide (NO) and producing reactive oxidants, hypochlorous acid (HOCl) and the nitrogen dioxide radical (•NO2). Accordingly, there is interest in developing MPO inhibitors that effectively target endothelial-localized MPO. Here we studied a series of piperidine nitroxides conjugated to polyamine moieties as novel endothelial-targeted MPO inhibitors. Electron paramagnetic resonance analysis of cell lysates showed that polyamine conjugated nitroxides were efficiently internalized into endothelial cells in a heparan sulfate dependent manner. Nitroxides effectively inhibited the consumption of MPO's substrate hydrogen peroxide (H2O2) and formation of HOCl catalyzed by endothelial-localized MPO, with their efficacy dependent on both nitroxide and conjugated-polyamine structure. Nitroxides also differentially inhibited protein nitration catalyzed by both purified and endothelial-localized MPO, which was dependent on •NO2 scavenging rather than MPO inhibition. Finally, nitroxides uniformly inhibited the catalytic consumption of NO by MPO in human plasma. These studies show for the first time that nitroxides effectively inhibit local oxidative reactions catalyzed by endothelial-localized MPO. Novel polyamine-conjugated nitroxides, ethylenediamine-TEMPO and putrescine-TEMPO, emerged as efficacious nitroxides uniquely exhibiting high endothelial cell uptake and efficient inhibition of MPO-catalyzed HOCl production, protein nitration, and NO oxidation. Polyamine-conjugated nitroxides represent a versatile class of antioxidant drugs capable of targeting endothelial-localized MPO during vascular inflammation.

Pharmacology of Modulators of Alternative Splicing.

More than 95% of genes in the human genome are alternatively spliced to form multiple transcripts, often encoding proteins with differing or opposing function. The control of alternative splicing is now being elucidated, and with this comes the opportunity to develop modulators of alternative splicing that can control cellular function. A number of approaches have been taken to develop compounds that can experimentally, and sometimes clinically, affect splicing control, resulting in potential novel therapeutics. Here we develop the concepts that targeting alternative splicing can result in relatively specific pathway inhibitors/activators that result in dampening down of physiologic or pathologic processes, from changes in muscle physiology to altering angiogenesis or pain. The targets and pharmacology of some of the current inhibitors/activators of alternative splicing are demonstrated and future directions discussed.

TRAIL signals through the ubiquitin ligase MID1 to promote pulmonary fibrosis.

BACKGROUND: Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has previously been demonstrated to play a pro-inflammatory role in allergic airways disease and COPD through the upregulation of the E3 ubiquitin ligase MID1 and the subsequent deactivation of protein phosphatase 2A (PP2A). METHODS: Biopsies were taken from eight IPF patients presenting to the Second Affiliated Hospital of Jilin University, China between January 2013 and February 2014 with control samples obtained from resected lung cancers. Serum TRAIL, MID1 protein and PP2A activity in biopsies, and patients' lung function were measured. Wild type and TRAIL deficient Tnfsf10-/- BALB/c mice were administered bleomycin to induce fibrosis and some groups were treated with the FTY720 analogue AAL(s) to activate PP2A. Mouse fibroblasts were treated with recombinant TRAIL and fibrotic responses were assessed. RESULTS: TRAIL in serum and MID1 protein levels in biopsies from IPF patients were increased compared to controls. MID1 levels were inversely associated while PP2A activity levels correlated with DLco. Tnfsf10-/- and mice treated with the PP2A activator AAL(s) were largely protected against bleomycin-induced reductions in lung function and fibrotic changes. Addition of recombinant TRAIL to mouse fibroblasts in-vitro increased collagen production which was reversed by PP2A activation with AAL(s). CONCLUSION: TRAIL signalling through MID1 deactivates PP2A and promotes fibrosis with corresponding lung function decline. This may provide novel therapeutic targets for IPF.

Concise Total Synthesis of Dioncophylline†E through an ortho-Arylation Strategy.

The first total synthesis of the potent antimalarial 7,3'-linked naphthylisoquinoline alkaloid dioncophylline E (1) has been completed. The synthesis proceeds in 12 steps (longest linear sequence) and in 15 % overall yield. Key transformations include an ortho-arylation of a naphthol with an aryllead triacetate to construct the sterically hindered biaryl bond, and a three-step sequence to stereoselectively generate the trans-1,3-dimethyl-1,2,3,4-tetrahydroisoquinoline moiety.

Development of novel PP2A activators for use in the treatment of acute myeloid leukaemia.

AAL(S), the chiral deoxy analog of the FDA approved drug FTY720, has been shown to inhibit proliferation and apoptosis in several cancer cell lines. It has been suggested that it does this by activating protein phosphatase 2A (PP2A). Here we report the synthesis of new cytotoxic analogs of AAL(S) and the evaluation of their cytotoxicity in two myeloid cell lines, one of which is sensitive to PP2A activation. We show that these analogs activate PP2A in these cells supporting the suggested mechanism for their cytotoxic properties. Our findings identify key structural motifs required for anti-cancer effects.

Synthesis and neuroprotective activity of dictyoquinazol A and analogues.

A flexible and efficient synthesis of the neuroprotective alkaloid, dictyoquinazol A, is reported. Several structural analogues of the target molecule were produced, and the neuroprotective activity of this series of compounds was investigated using three different cell-based models of stroke. Several of the new compounds were found to have superior activity compared to the natural product. This work has established a new molecular scaffold that holds promise for a novel pharmaceutical treatment for stroke.

MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity.

BACKGROUND: Steroid-resistant asthma is a major clinical problem that is linked to activation of innate immune cells. Levels of IFN-γ and LPS are often increased in these patients. Cooperative signaling between IFN-γ/LPS induces macrophage-dependent steroid-resistant airway hyperresponsiveness (AHR) in mouse models. MicroRNAs (miRs) are small noncoding RNAs that regulate the function of innate immune cells by controlling mRNA stability and translation. Their role in regulating glucocorticoid responsiveness and AHR remains unexplored. OBJECTIVE: IFN-γ and LPS synergistically increase the expression of miR-9 in macrophages and lung tissue, suggesting a role in the mechanisms of steroid resistance. Here we demonstrate the role of miR-9 in IFN-γ/LPS-induced inhibition of dexamethasone (DEX) signaling in macrophages and in induction of steroid-resistant AHR. METHODS: MiRNA-9 expression was assessed by means of quantitative RT-PCR. Putative miR-9 targets were determined in silico and confirmed in luciferase reporter assays. miR-9 function was inhibited with sequence-specific antagomirs. The efficacy of DEX was assessed by quantifying glucocorticoid receptor (GR) cellular localization, protein phosphatase 2A (PP2A) activity, and AHR. RESULTS: Exposure of pulmonary macrophages to IFN-γ/LPS synergistically induced miR-9 expression; reduced levels of its target transcript, protein phosphatase 2 regulatory subunit B (B56) δ isoform; attenuated PP2A activity; and inhibited DEX-induced GR nuclear translocation. Inhibition of miR-9 increased both PP2A activity and GR nuclear translocation in macrophages and restored steroid sensitivity in multiple models of steroid-resistant AHR. Pharmacologic activation of PP2A restored DEX efficacy and inhibited AHR. MiR-9 expression was increased in sputum of patients with neutrophilic but not those with eosinophilic asthma. CONCLUSION: MiR-9 regulates GR signaling and steroid-resistant AHR. Targeting miR-9 function might be a novel approach for the treatment of steroid-resistant asthma.

Synthesis and biological evaluation of analogs of AAL(S) for use as ceramide synthase 1 inhibitors.

A convergent synthesis to access hydrophobic tail analogs and head group modifications of AAL(S) is described. The analogs synthesised were evaluated for their ability to inhibit ceramide synthase 1 and for their cytotoxicity in K562 cells. Our results have identified inhibitors which are non-cytotoxic yet maintain CerS1 inhibition.

Salmeterol attenuates chemotactic responses in rhinovirus-induced exacerbation of allergic airways disease by modulating protein phosphatase 2A.

BACKGROUND: ß-Agonists are used for relief and control of asthma symptoms by reversing bronchoconstriction. They might also have anti-inflammatory properties, but the underpinning mechanisms remain poorly understood. Recently, a direct interaction between formoterol and protein phosphatase 2A (PP2A) has been described in vitro. OBJECTIVE: We sought to elucidate the molecular mechanisms by which ß-agonists exert anti-inflammatory effects in allergen-driven and rhinovirus 1B-exacerbated allergic airways disease (AAD). METHODS: Mice were sensitized and then challenged with house dust mite to induce AAD while receiving treatment with salmeterol, formoterol, or salbutamol. Mice were also infected with rhinovirus 1B to exacerbate lung inflammation and therapeutically administered salmeterol, dexamethasone, or the PP2A-activating drug (S)-2-amino-4-(4-[heptyloxy]phenyl)-2-methylbutan-1-ol (AAL[S]). RESULTS: Systemic or intranasal administration of salmeterol protected against the development of allergen- and rhinovirus-induced airway hyperreactivity and decreased eosinophil recruitment to the lungs as effectively as dexamethasone. Formoterol and salbutamol also showed anti-inflammatory properties. Salmeterol, but not dexamethasone, increased PP2A activity, which reduced CCL11, CCL20, and CXCL2 expression and reduced levels of phosphorylated extracellular signal-regulated kinase 1 and active nuclear factor κB subunits in the lungs. The anti-inflammatory effect of salmeterol was blocked by targeting the catalytic subunit of PP2A with small RNA interference. Conversely, increasing PP2A activity with AAL(S) abolished rhinovirus-induced airway hyperreactivity, eosinophil influx, and CCL11, CCL20, and CXCL2 expression. Salmeterol also directly activated immunoprecipitated PP2A in vitro isolated from human airway epithelial cells. CONCLUSIONS: Salmeterol exerts anti-inflammatory effects by increasing PP2A activity in AAD and rhinovirus-induced lung inflammation, which might potentially account for some of its clinical benefits.

Tumor necrosis factor-related apoptosis-inducing ligand regulates hallmark features of airways remodeling in allergic airways disease.

Allergic asthma is a complex disease characterized by acute inflammation of the airways that over time leads to the development of significant structural changes termed remodeling. TNF-related apoptosis-inducing ligand (TRAIL) has an important regulatory role in acute allergic airways inflammation through up-regulation of the E3 ubiquitin ligase Midline-1 (MID-1), which limits protein phosphatase 2A (PP2A) activity and downstream dephosphorylation of proinflammatory signaling molecules. The relevance of TRAIL in the development of airways remodeling has yet to be determined. In this study, the lungs of wild-type (WT) BALB/c and Tnfsf10 knockout (TRAIL-/-) mice were chronically exposed to ovalbumin (OVA) for 12 weeks to induce hallmark features of chronic allergic airways disease, including airways hyperreactivity (AHR), subepithelial collagen deposition, goblet cell hyperplasia, and smooth muscle hypertrophy. TRAIL-/- mice were largely protected from the development of AHR and peribronchial eosinophilia and had reduced levels of mast cells in the airways. This correlated with lower levels of cytokines, including IL-4, -5, -10, and -13, and with lower levels of proinflammatory chemokines from cultured cells isolated from the draining lymph nodes. TRAIL-/- mice were also protected from the characteristic features of airways remodeling, including peribronchial fibrosis, smooth muscle hypertrophy, and mucus hypersecretion, which correlated with reduced TGF-ß1 levels in the lungs. MID-1 expression was reduced in TRAIL-/- mice and up-regulated in allergic WT mice. Raising PP2A activity using 2-amino-4-(4-heptyloyphenol)-2-methylbutan-1-ol in allergic WT mice reduced eosinophilia, TGF-ß1, and peribronchial fibrosis. This study shows that TRAIL promotes airways remodeling in an OVA-induced model of chronic allergic airways disease. Targeting TRAIL and its downstream proinflammatory signaling pathway involving PP2A may be of therapeutic benefit in reducing the hallmark features of airways remodeling observed in chronic allergic airways inflammation.

Marine natural products: synthetic aspects.

Covering: January to December 2010. Previous review: Nat. Prod. Rep., 2011, 28, 269. An overview of marine natural products synthesis during 2010 is provided. As with earlier installments in this series, the emphasis is on total syntheses of molecules of contemporary interest, new total syntheses, and syntheses that have resulted in structure confirmation or stereochemical assignments.

Vascular endothelial growth factor isoforms differentially protect neurons against neurotoxic events associated with Alzheimer's disease.

Alzheimer's disease (AD) is the most common cause of dementia, the chronic and progressive deterioration of memory and cognitive abilities. AD can be pathologically characterised by neuritic plaques and neurofibrillary tangles, formed by the aberrant aggregation of ß-amyloid and tau proteins, respectively. We tested the hypothesis that VEGF isoforms VEGF-A165a and VEGF-A165b, produced by differential splice site selection in exon 8, could differentially protect neurons from neurotoxicities induced by ß-amyloid and tau proteins, and that controlling expression of splicing factor kinase activity could have protective effects on AD-related neurotoxicity in vitro. Using oxidative stress, ß-amyloid, and tau hyperphosphorylation models, we investigated the effect of VEGF-A splicing isoforms, previously established to be neurotrophic agents, as well as small molecule kinase inhibitors, which selectively inhibit SRPK1, the major regulator of VEGF splicing. While both VEGF-A165a and VEGF-A165b isoforms were protective against AD-related neurotoxicity, measured by increased metabolic activity and neurite outgrowth, VEGF-A165a was able to enhance neurite outgrowth but VEGF-A165b did not. In contrast, VEGF-A165b was more effective than VEGF-A165a in preventing neurite "dieback" in a tau hyperphosphorylation model. SRPK1 inhibition was found to significantly protect against neurite "dieback" through shifting AS of VEGFA towards the VEGF-A165b isoform. These results indicate that controlling the activities of the two different isoforms could have therapeutic potential in Alzheimer's disease, but their effect may depend on the predominant mechanism of the neurotoxicity-tau or ß-amyloid.

Mitochondrial electron transport chain, ceramide, and coenzyme Q are linked in a pathway that drives insulin resistance in skeletal muscle.

Insulin resistance (IR) is a complex metabolic disorder that underlies several human diseases, including type 2 diabetes and cardiovascular disease. Despite extensive research, the precise mechanisms underlying IR development remain poorly understood. Previously we showed that deficiency of coenzyme Q (CoQ) is necessary and sufficient for IR in adipocytes and skeletal muscle (Fazakerley et al., 2018). Here, we provide new insights into the mechanistic connections between cellular alterations associated with IR, including increased ceramides, CoQ deficiency, mitochondrial dysfunction, and oxidative stress. We demonstrate that elevated levels of ceramide in the mitochondria of skeletal muscle cells result in CoQ depletion and loss of mitochondrial respiratory chain components, leading to mitochondrial dysfunction and IR. Further, decreasing mitochondrial ceramide levels in vitro and in animal models (mice, C57BL/6J) (under chow and high-fat diet) increased CoQ levels and was protective against IR. CoQ supplementation also rescued ceramide-associated IR. Examination of the mitochondrial proteome from human muscle biopsies revealed a strong correlation between the respirasome system and mitochondrial ceramide as key determinants of insulin sensitivity. Our findings highlight the mitochondrial ceramide-CoQ-respiratory chain nexus as a potential foundation of an IR pathway that may also play a critical role in other conditions associated with ceramide accumulation and mitochondrial dysfunction, such as heart failure, cancer, and aging. These insights may have important clinical implications for the development of novel therapeutic strategies for the treatment of IR and related metabolic disorders.

A fluorescent assay for ceramide synthase activity.

The sphingolipids are a diverse family of lipids with important roles in membrane compartmentalization, intracellular signaling, and cell-cell recognition. The central sphingolipid metabolite is ceramide, formed by the transfer of a variable length fatty acid from coenzyme A to a sphingoid base, generally sphingosine or dihydrosphingosine (sphinganine) in mammals. This reaction is catalyzed by a family of six ceramide synthases (CerS1-6). CerS activity is usually assayed using either radioactive substrates or LC-MS/MS. We describe a CerS assay with fluorescent, NBD-labeled sphinganine as substrate. The assay is readily able to detect endogenous CerS activity when using amounts of cell or tissue homogenate protein that are lower than those reported for the radioactive assay, and the Michaelis-Menten constant was essentially the same for NBD-sphinganine and unlabeled sphinganine, indicating that NBD-sphinganine is a good substrate for these enzymes. Using our assay, we confirm that the new clinical immunosuppressant FTY720 is a competitive inhibitor of CerS activity, and show that inhibition requires the compound's lipid tail and amine headgroup. In summary, we describe a fluorescent assay for CerS activity that circumvents the need to use radioactive substrates, while being more accessible and cheaper than LC-MS based assays.

Preclinical and clinical evaluation of German-sourced ONC201 for the treatment of H3K27M-mutant diffuse intrinsic pontine glioma.

BACKGROUND: Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan; however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound-ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc. METHODS: Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017-2020) was analyzed. RESULTS: GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months (P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not (P = .012). CONCLUSIONS: This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug; however, GsONC201 was never used as a monotherapy.

Elimination of a hydroxyl group in FTY720 dramatically improves the phosphorylation rate.

The new immunosuppressant FTY720 (fingolimod), an analog of the endogenous lipid sphingosine, induces transient lymphopenia through the sequestration of lymphocytes in secondary lymphoid organs. Phosphorylation of FTY720 by sphingosine kinase 2 (SphK2) yields the active metabolite FTY720-phosphate (FTY-P), which induces lymphopenia through agonism of the sphingosine 1-phosphate receptor S1P(1) on endothelial cells and lymphocytes. Dephosphorylation of circulating FTY-P creates an equilibrium between FTY720 and its phosphate, and results with human patients indicate that phosphorylation of FTY720 could be rate limiting for efficacy. We report that the FTY720 derivative 2-amino-4-(4-heptyloxyphenyl)-2-methylbutanol [AAL(R)] is phosphorylated much more rapidly than FTY720 in cultured human cells and whole blood. The K(cat) for AAL(R) with recombinant SphK2 is 8-fold higher than for FTY720, whereas the K(m) for the two substrates is very similar, indicating that the increased rate of phosphorylation results from faster turnover by SphK2 rather than a higher binding affinity. Consequently, treating cells with AAL(R), but not FTY720, triggers an apoptotic pathway that is dependent on excessive intracellular accumulation of long-chain base phosphates. In agreement with the in vitro results, phosphorylation of AAL(R) is more complete than that of FTY720 in vivo (mice), and AAL(R) is a more potent inducer of lymphopenia. These differences may be magnified in humans, because phosphorylation of FTY720 is much less efficient in humans compared with rodents. Our results suggest that AAL(R) is a better tool than FTY720 for in vivo studies with S1P analogs and would probably be a more effective immunosuppressant than FTY720.

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

Enhancing tristetraprolin activity reduces the severity of cigarette smoke-induced experimental chronic obstructive pulmonary disease.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a progressive disease that causes significant mortality and morbidity worldwide and is primarily caused by the inhalation of cigarette smoke (CS). Lack of effective treatments for COPD means there is an urgent need to identify new therapeutic strategies for the underlying mechanisms of pathogenesis. Tristetraprolin (TTP) encoded by the Zfp36 gene is an anti-inflammatory protein that induces mRNA decay, especially of transcripts encoding inflammatory cytokines, including those implicated in COPD. METHODS: Here, we identify a novel protective role for TTP in CS-induced experimental COPD using Zfp36aa/aa mice, a genetically modified mouse strain in which endogenous TTP cannot be phosphorylated, rendering it constitutively active as an mRNA-destabilising factor. TTP wild-type (Zfp36 +/+) and Zfp36aa/aa active C57BL/6J mice were exposed to CS for four days or eight weeks, and the impact on acute inflammatory responses or chronic features of COPD, respectively, was assessed. RESULTS: After four days of CS exposure, Zfp36aa/aa mice had reduced numbers of airway neutrophils and lymphocytes and mRNA expression levels of cytokines compared to wild-type controls. After eight weeks, Zfp36aa/aa mice had reduced pulmonary inflammation, airway remodelling and emphysema-like alveolar enlargement, and lung function was improved. We then used pharmacological treatments in vivo (protein phosphatase 2A activator, AAL(S), and the proteasome inhibitor, bortezomib) to promote the activation and stabilisation of TTP and show that hallmark features of CS-induced experimental COPD were ameliorated. CONCLUSION: Collectively, our study provides the first evidence for the therapeutic potential of inducing TTP as a treatment for COPD.