Chemoattractant receptors in drug discovery: FPR2 and CCR2-two potential targets

Chemoattractants and chemoattractant receptors have many important functions in multicellular organisms, not least for their role in regulating migration of leukocytes. The receptors are also involved in many pathologies and they have, since they were cloned in the 1990ies, been regarded as attractive targets for development of drugs against cancer, viral infections, and inflammatory/autoimmune diseases. Although chemotactic molecules are very heterogeneous in terms of chemical structure most of them mediate their actions through binding to a G-protein-coupled receptor (GPCR). The formyl peptide receptors (FPRs) are chemoattractant receptors expressed on phagocytes and have important implications in host defense and inflammation as well as in resolution of inflammation. One of the goals was to identify FPR2 specific ligands with similar anti-inflammatory and proresolution properties as the eicosanoid lipoxin A4 previously described. A small compound library was screened for both agonist and antagonists and selected compounds were analyzed with respect to antiinflammatory properties such as inhibition of NADPH oxidase activity and degranulation in primary neutrophils. Receptor selectivity was investigated by compound stimulation and desensitization studies in presence of receptor-specific tools. While the agonist screen resulted in non selective hit compounds with proinflammatory properties, the antagonist screen led to identification of anti-inflammatory agents with different selectivity. Another chemoattractant receptor binds CCL2, a member of the superfamily of chemokines. The function of CCL2 and its receptor (CCR2) is of importance for monocyte recruitment, and together this receptor ligand pair has many implications in inflammation. Accordingly, the importance of the CCL2/CCR2 axis in the recruitment of inflammatory cells and fibroblasts into fibrotic tissues was hypothesized as a mechanism for induction of fibrosis. There is, however, emerging evidence indicating that CCL2 may promote fibrosis also by other mechanisms such as activation of the fibroblast. The myofibroblast, synthesizing large amounts of extracellular matrix components (ECM) in response to profibrotic cytokines and growth factors, is the key cell in fibrosis. The effects of CCL2 on human fibroblasts with respect to ECM expression were studied and it was found that CCL2 might have a more multifaceted role in fibroblast activation than previously described. The truncated form of CCL2, CCL2 (1,9-76), also known as 7ND, is a receptor antagonist and is described to have therapeutic effects in several animal disease models such as bleomycin-induced lung fibrosis. The interaction between the 7ND and the human as well as the murine CCR2 was studied in vitro and data showed that 7ND was an inhibitor of human CCR2 but a very low affinity binder of the murine receptor. 7ND was, however, shown to downregulate fibrotic markers in murine fibroblasts, but in accordance with its inability to bind CCR2 the downregulation was found to be independent of this receptor

Min värld är bättre än din : kosmopoliter och fundamentalister i svensk politik

I denna uppsats har vi studerat Sverigedemokraternas och Feministiskt initiativs officiella Facebooksidor, samt den politiska bloggen Avpixlat, vars politiska tillhörighet kopplas till SD. Syftet med studien är att med utgångspunkt i den dimension om kosmopolitism och fundamentalism som Giddens (2010) diskuterar, undersöka hur Feministiskt initiativ och Sverigedemokraterna på sociala medier hanterar ett alltmer globaliserat och multikulturellt samhälle. Vi har genomfört en kvalitativ innehållsanalys, där vi har granskat inlägg på partiernas respektive sidor. Det har kunnat konkluderas att de båda partierna är framgångsrika i sin användning av de sociala medierna och att en ny politisk dimension här till stor del yttrar sig och ställs på sin spets. De sociala medierna skapar en plattform för de båda partierna att reagera på den förda politiken och de kan således väcka medborgarengagemang och mobilisera sina sympatisörer utifrån sina olika värderingar. Genom att sprida en viss typ av världsbild på sidorna kan de personer som känner sig hemma i dessa, bevara en ontologisk trygghet i en föränderlig värld. De båda partierna utmärker sig i den politiska debatten eftersom deras politiska frågor engagerar medborgarna. Partierna står som varandras motpoler i en ny politisk dimension där mångkultur och tolerans är centrala frågor. Facebook och Avpixlat används för att föra en kamp mellan kosmopolitiska och fundamentalistiska värderingar. Fi reagerar på samhället som intolerant och anser att toleransen måste stärkas genom att förändra existerande strukturer. Detta ska uppnås genom att värna om multikulturella värden och varandras olikheter. Tvärtemot anser SD att samhället är alldeles för tolerant och att Sverige måste bevaras enligt gamla traditioner. Multikulturalismen anses enligt SD vara ett hot mot det svenska samhället och partiet blickar bakåt mot en kollektiv svensk identitet som ideal. Fi kan kopplas till kosmopolitiska, toleranta värderingar och SD till fundamentalistiska, traditionsbevarande värderingar

Glioblastoma and glioblastoma stem cells are dependent on functional MTH1

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer with poor prognosis. Cancer cells are characterized by a specific redox environment that adjusts metabolism to its specific needs and allows the tumor to grow and metastasize. As a consequence, cancer cells and especially GBM cells suffer from elevated oxidative pressure which requires antioxidant-defense and other sanitation enzymes to be upregulated. MTH1, which degrades oxidized nucleotides, is one of these defense enzymes and represents a promising cancer target. We found MTH1 expression levels elevated and correlated with GBM aggressiveness and discovered that siRNA knock-down or inhibition of MTH1 with small molecules efficiently reduced viability of patient-derived GBM cultures. The effect of MTH1 loss on GBM viability was likely mediated through incorporation of oxidized nucleotides and subsequent DNA damage. We revealed that MTH1 inhibition targets GBM independent of aggressiveness as well as potently kills putative GBM stem cells in vitro. We used an orthotopic zebrafish model to confirm our results in vivo and light-sheet microscopy to follow the effect of MTH1 inhibition in GBM in real time. In conclusion, MTH1 represents a promising target for GBM therapy and MTH1 inhibitors may also be effective in patients that suffer from recurring disease

Pharmacological OGG1 inhibition decreases murine allergic airway inflammation

Background and aim: Allergic asthma is a complex inflammatory disease involving type 2 innate lymphoid cells, type 2 T helper cells, macrophages, and eosinophils. The disease is characterized by wheezing, dyspnea, coughing, chest tightness and variable airflow limitation for which there is no cure and is symptomatically treated with inhaled corticosteroids and β2-agonists. Molecular mechanisms underlying its complex pathogenesis are not fully understood. However, 8-oxoguanine DNA glycosylase-1 (OGG1), a DNA repair protein may play a central role, as OGG1 deficiency decreases both innate and allergic inflammation. Methods: Using a murine ovalbumin (OVA) model of allergic airway inflammation we assessed the utility of an inhibitor of OGG1 (TH5487) in this disease context. Cytokines and chemokines, promoting immune cell recruitment were measured using a 23-multiplex assay and Western blotting. Additionally, immune cell recruitment to bronchi was measured using flow cytometry. Histological analyses and immunofluorescent staining were used to confirm immune cell influx and goblet cell hyperplasia of the airways. A PCR array was used to assess asthma-related genes in murine lung tissue following TH5487 treatment. Finally, airway hyperresponsiveness was determined using in vivo lung function measurement. Results: In this study, administration of TH5487 to mice with OVA-induced allergic airway inflammation significantly decreased goblet cell hyperplasia and mucus production. TH5487 treatment also decreased levels of activated NF-κB and expression of proinflammatory cytokines and chemokines resulting in significantly lower recruitment of eosinophils and other immune cells to the lungs. Gene expression profiling of asthma and allergy-related proteins after TH5487 treatment revealed differences in several important regulators, including down regulation of Tnfrsf4, Arg1, Ccl12 and Ccl11, and upregulation of the negative regulator of type 2 inflammation, Bcl6. Furthermore, the gene Clca1 was upregulated following TH5487 treatment, which should be explored further due to its ambiguous role in allergic asthma. In addition, the OVA-induced airway hyperresponsiveness was significantly reduced by TH5487 treatment. Conclusion: Taken together, the data presented in this study suggest OGG1 as a clinically relevant pharmacological target for the treatment of allergic inflammation

Small-molecule-mediated OGG1 inhibition attenuates pulmonary inflammation and lung fibrosis in a murine lung fibrosis model

Interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF) are caused by persistent micro-injuries to alveolar epithelial tissues accompanied by aberrant repair processes. IPF is currently treated with pirfenidone and nintedanib, compounds which slow the rate of disease progression but fail to target underlying pathophysiological mechanisms. The DNA repair protein 8-oxoguanine DNA glycosylase-1 (OGG1) has significant roles in the modulation of inflammation and metabolic syndromes. Currently, no pharmaceutical solutions targeting OGG1 have been utilized in the treatment of IPF. In this study we show Ogg1-targeting siRNA mitigates bleomycin-induced pulmonary fibrosis in male mice, highlighting OGG1 as a tractable target in lung fibrosis. The small molecule OGG1 inhibitor, TH5487, decreases myofibroblast transition and associated pro-fibrotic gene expressions in fibroblast cells. In addition, TH5487 decreases levels of pro-inflammatory mediators, inflammatory cell infiltration, and lung remodeling in a murine model of bleomycin-induced pulmonary fibrosis conducted in male C57BL6/J mice. OGG1 and SMAD7 interact to induce fibroblast proliferation and differentiation and display roles in fibrotic murine and IPF patient lung tissue. Taken together, these data suggest that TH5487 is a potentially clinically relevant treatment for IPF but further study in human trials is required

Development of a chemical probe against NUDT15

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues

Small-molecule inhibitor of OGG1 suppresses pro-inflammatory gene expression and inflammation

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor–α–induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment

Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors