Mitochondrial complex III bypass complex I to induce ROS in GPR17 signaling activation in GBM

Guanine nucleotide binding protein (G protein) coupled receptor 17 (GPR17) plays crucial role in Glioblastoma multiforme (GBM) cell signaling and is primarily associated with reactive oxidative species (ROS) production and cell death. However, the underlying mechanisms by which GPR17 regulates ROS level and mitochondrial electron transport chain (ETC) complexes are still unknown. Here, we investigate the novel link between the GPR17 receptor and ETC complex I and III in regulating level of intracellular ROS (ROSi) in GBM using pharmacological inhibitors and gene expression profiling. Incubation of 1321N1 GBM cells with ETC I inhibitor and GPR17 agonist decreased the ROS level, while treatment with GPR17 antagonist increased the ROS level. Also, inhibition of ETC III and activation of GPR17 increased the ROS level whereas opposite function was observed with antagonist interaction. The similar functional role was also observed in multiple GBM cells, LN229 and SNB19, where ROS level increased in the presence of Complex III inhibitor. The level of ROS varies in Complex I inhibitor and GPR17 antagonist treatment conditions suggesting that ETC I function differs depending on the GBM cell line. RNAseq analysis revealed that ~ 500 genes were commonly expressed in both SNB19 and LN229, in which 25 genes are involved in ROS pathway. Furthermore, 33 dysregulated genes were observed to be involved in mitochondria function and 36 genes of complex I-V involved in ROS pathway. Further analysis revealed that induction of GPR17 leads to loss of function of NADH dehydrogenase genes involved in ETC I, while cytochrome b and Ubiquinol Cytochrome c Reductase family genes in ETC III. Overall, our findings suggest that mitochondrial ETC III bypass ETC I to increase ROSi in GPR17 signaling activation in GBM and could provide new opportunities for developing targeted therapy for GBM

Bias-force guided simulations combined with experimental validations towards GPR17 modulators identification

Glioblastoma Multiforme (GBM) is known to be by far the most aggressive brain tumor to affect adults. The median survival rate of GBM patient's is < 15 months, while the GBM cells aggressively develop resistance to chemo- and radiotherapy with their self-renewal capacity which suggests the pressing need to develop novel preventative measures. We have recently proved that GPR17 —an orphan G protein-coupled receptor— is highly expressed on the GBM cell surface and it has a vital role to play in the disease progression. Despite the progress made on GBM downregulation, there still remain difficulties in developing a promising modulator for GPR17, till date. Here, we have performed robust virtual screening combined with biased-force pulling molecular dynamic (MD) simulations to predict high-affinity GPR17 modulators followed by experimental validation. Initially, the database containing 1379 FDA-approved drugs were screened against the orthosteric binding pocket of the GPR17. The external bias-potentials were then applied to the screened hits during the MD simulations which enabled to predict a spectrum of rupture peak force values that were used to select four approved drugs –ZINC000003792417 (Sacubitril), ZINC000014210457 (Victrelis), ZINC000001536109 (Pralatrexate) and ZINC000003925861 (Vorapaxar)– as top hits. The hits selected turns out to demonstrate unique dissociation pathways, interaction pattern, and change in polar network over time. Subsequently the selected hits with GPR17 were measured by inhibiting the forskolin-stimulated cAMP accumulation in GBM cell lines, LN229 and SNB19. The ex vivo validations shows that Sacubitril drug can act as a full agonist, while Vorapaxar functions as a partial agonist for GPR17. The pEC50 of Sacubitril was identified as 4.841 and 4.661 for LN229 and SNB19, respectively. Small interference of the RNA (siRNA)– silenced the GPR17 to further validate the targeted binding of Sacubitril with GPR17. In the current investigation, we have identified new repurposable GPR17 specific drugs which are likely to increase the opportunity to treat orphan deadly diseases.publishedVersionPeer reviewe

Methanodibenzo[b,f][1,5]dioxocins as Novel Glutaminase Inhibitor with Anti-Glioblastoma Potential

Glutamine metabolism is an important hallmark of several cancers with demonstrated antitumor activity in glioblastoma cancer cells (GBM). GBM cells regulate glutamine and use it as a major energy source for their proliferation through the glutaminolysis process. Enzymes, such as glutaminase in glutaminolysis, can be targeted by small-molecule inhibitors, thus exhibiting promising anticancer properties. The resistance to glutaminolysis demands the development of new therapeutic molecules to overcome drug resistance. Herein, we have reported a novel library of constrained methanodibenzo[b,f][1,5]dioxocin derivatives as glutaminase (GLS) inhibitors and their anti-GBM potential. The library consisting of seven molecules was obtained through self-condensation of 2′-hydroxyacetophenones, out of which three molecules, namely compounds 3, 5, and 6, were identified with higher binding energy values ranging between −10.2 and −9.8 kcal/mol with GLS (PDB ID; 4O7D). Pharmacological validation of these compounds also showed a higher growth inhibition effect in GBM cells than the standard drug temozolomide (TMZ). The most promising compound, 6, obeyed Lipinski’s rule of five and was identified to interact with key residues Arg307, Asp326, Lys328, Lys399, and Glu403 of GLS. This compound exhibited the best cytotoxic effect with IC50 values of 63 µM and 83 µM in LN229 and SNB19 cells, respectively. The potential activation of GLS by the best-constrained dibenzo[b,f][1,5]dioxocin in the tested series increased apoptosis via reactive oxygen species production in both GBM cells, and exhibited anti-migratory and anti-proliferative properties over time in both cell lines. Our results highlight the activation mechanism of a dibenzo[b,f][1,5]dioxocin from the structural basis and demonstrate that inhibition of glutaminolysis may facilitate the pharmacological intervention for GBM treatment.publishedVersionPeer reviewe

Cerebral microbleeds and intracranial haemorrhage risk in patients anticoagulated for atrial fibrillation after acute ischaemic stroke or transient ischaemic attack (CROMIS-2):a multicentre observational cohort study

Background: Cerebral microbleeds are a potential neuroimaging biomarker of cerebral small vessel diseases that are prone to intracranial bleeding. We aimed to determine whether presence of cerebral microbleeds can identify patients at high risk of symptomatic intracranial haemorrhage when anticoagulated for atrial fibrillation after recent ischaemic stroke or transient ischaemic attack. Methods: Our observational, multicentre, prospective inception cohort study recruited adults aged 18 years or older from 79 hospitals in the UK and one in the Netherlands with atrial fibrillation and recent acute ischaemic stroke or transient ischaemic attack, treated with a vitamin K antagonist or direct oral anticoagulant, and followed up for 24 months using general practitioner and patient postal questionnaires, telephone interviews, hospital visits, and National Health Service digital data on hospital admissions or death. We excluded patients if they could not undergo MRI, had a definite contraindication to anticoagulation, or had previously received therapeutic anticoagulation. The primary outcome was symptomatic intracranial haemorrhage occurring at any time before the final follow-up at 24 months. The log-rank test was used to compare rates of intracranial haemorrhage between those with and without cerebral microbleeds. We developed two prediction models using Cox regression: first, including all predictors associated with intracranial haemorrhage at the 20% level in univariable analysis; and second, including cerebral microbleed presence and HAS-BLED score. We then compared these with the HAS-BLED score alone. This study is registered with ClinicalTrials.gov, number NCT02513316. Findings: Between Aug 4, 2011, and July 31, 2015, we recruited 1490 participants of whom follow-up data were available for 1447 (97%), over a mean period of 850 days (SD 373; 3366 patient-years). The symptomatic intracranial haemorrhage rate in patients with cerebral microbleeds was 9·8 per 1000 patient-years (95% CI 4·0–20·3) compared with 2·6 per 1000 patient-years (95% CI 1·1–5·4) in those without cerebral microbleeds (adjusted hazard ratio 3·67, 95% CI 1·27–10·60). Compared with the HAS-BLED score alone (C-index 0·41, 95% CI 0·29–0·53), models including cerebral microbleeds and HAS-BLED (0·66, 0·53–0·80) and cerebral microbleeds, diabetes, anticoagulant type, and HAS-BLED (0·74, 0·60–0·88) predicted symptomatic intracranial haemorrhage significantly better (difference in C-index 0·25, 95% CI 0·07–0·43, p=0·0065; and 0·33, 0·14–0·51, p=0·00059, respectively). Interpretation: In patients with atrial fibrillation anticoagulated after recent ischaemic stroke or transient ischaemic attack, cerebral microbleed presence is independently associated with symptomatic intracranial haemorrhage risk and could be used to inform anticoagulation decisions. Large-scale collaborative observational cohort analyses are needed to refine and validate intracranial haemorrhage risk scores incorporating cerebral microbleeds to identify patients at risk of net harm from oral anticoagulation. Funding: The Stroke Association and the British Heart Foundation

Cerebral microbleeds and intracranial haemorrhage risk in patients anticoagulated for atrial fibrillation after acute ischaemic stroke or transient ischaemic attack (CROMIS-2): a multicentre observational cohort study

Summary Background: Cerebral microbleeds are a potential neuroimaging biomarker of cerebral small vessel diseases that are prone to intracranial bleeding. We aimed to determine whether presence of cerebral microbleeds can identify patients at high risk of symptomatic intracranial haemorrhage when anticoagulated for atrial fibrillation after recent ischaemic stroke or transient ischaemic attack. Methods: Our observational, multicentre, prospective inception cohort study recruited adults aged 18 years or older from 79 hospitals in the UK and one in the Netherlands with atrial fibrillation and recent acute ischaemic stroke or transient ischaemic attack, treated with a vitamin K antagonist or direct oral anticoagulant, and followed up for 24 months using general practitioner and patient postal questionnaires, telephone interviews, hospital visits, and National Health Service digital data on hospital admissions or death. We excluded patients if they could not undergo MRI, had a definite contraindication to anticoagulation, or had previously received therapeutic anticoagulation. The primary outcome was symptomatic intracranial haemorrhage occurring at any time before the final follow-up at 24 months. The log-rank test was used to compare rates of intracranial haemorrhage between those with and without cerebral microbleeds. We developed two prediction models using Cox regression: first, including all predictors associated with intracranial haemorrhage at the 20% level in univariable analysis; and second, including cerebral microbleed presence and HAS-BLED score. We then compared these with the HAS-BLED score alone. This study is registered with ClinicalTrials.gov, number NCT02513316. Findings: Between Aug 4, 2011, and July 31, 2015, we recruited 1490 participants of whom follow-up data were available for 1447 (97%), over a mean period of 850 days (SD 373; 3366 patient-years). The symptomatic intracranial haemorrhage rate in patients with cerebral microbleeds was 9·8 per 1000 patient-years (95% CI 4·0–20·3) compared with 2·6 per 1000 patient-years (95% CI 1·1–5·4) in those without cerebral microbleeds (adjusted hazard ratio 3·67, 95% CI 1·27–10·60). Compared with the HAS-BLED score alone (C-index 0·41, 95% CI 0·29–0·53), models including cerebral microbleeds and HAS-BLED (0·66, 0·53–0·80) and cerebral microbleeds, diabetes, anticoagulant type, and HAS-BLED (0·74, 0·60–0·88) predicted symptomatic intracranial haemorrhage significantly better (difference in C-index 0·25, 95% CI 0·07–0·43, p=0·0065; and 0·33, 0·14–0·51, p=0·00059, respectively). Interpretation In patients with atrial fibrillation anticoagulated after recent ischaemic stroke or transient ischaemic attack, cerebral microbleed presence is independently associated with symptomatic intracranial haemorrhage risk and could be used to inform anticoagulation decisions. Large-scale collaborative observational cohort analyses are needed to refine and validate intracranial haemorrhage risk scores incorporating cerebral microbleeds to identify patients at risk of net harm from oral anticoagulation. Funding The Stroke Association and the British Heart Foundation

Mitochondrial complex III bypass complex I to induce ROS in GPR17 signaling activation in GBM

Guanine nucleotide binding protein (G protein) coupled receptor 17 (GPR17) plays crucial role in Glioblastoma multiforme (GBM) cell signaling and is primarily associated with reactive oxidative species (ROS) production and cell death. However, the underlying mechanisms by which GPR17 regulates ROS level and mitochondrial electron transport chain (ETC) complexes are still unknown. Here, we investigate the novel link between the GPR17 receptor and ETC complex I and III in regulating level of intracellular ROS (ROSi) in GBM using pharmacological inhibitors and gene expression profiling. Incubation of 1321N1 GBM cells with ETC I inhibitor and GPR17 agonist decreased the ROS level, while treatment with GPR17 antagonist increased the ROS level. Also, inhibition of ETC III and activation of GPR17 increased the ROS level whereas opposite function was observed with antagonist interaction. The similar functional role was also observed in multiple GBM cells, LN229 and SNB19, where ROS level increased in the presence of Complex III inhibitor. The level of ROS varies in Complex I inhibitor and GPR17 antagonist treatment conditions suggesting that ETC I function differs depending on the GBM cell line. RNAseq analysis revealed that ∼ 500 genes were commonly expressed in both SNB19 and LN229, in which 25 genes are involved in ROS pathway. Furthermore, 33 dysregulated genes were observed to be involved in mitochondria function and 36 genes of complex I-V involved in ROS pathway. Further analysis revealed that induction of GPR17 leads to loss of function of NADH dehydrogenase genes involved in ETC I, while cytochrome b and Ubiquinol Cytochrome c Reductase family genes in ETC III. Overall, our findings suggest that mitochondrial ETC III bypass ETC I to increase ROSi in GPR17 signaling activation in GBM and could provide new opportunities for developing targeted therapy for GBM

Programmed cell death detection methods : a systematic review and a categorical comparison

Programmed cell death is considered a key player in a variety of cellular processes that helps to regulate tissue growth, embryogenesis, cell turnover, immune response, and other biological processes. Among different types of cell death, apoptosis has been studied widely, especially in the field of cancer research to understand and analyse cellular mechanisms, and signaling pathways that control cell cycle arrest. Hallmarks of different types of cell death have been identified by following the patterns and events through microscopy. Identified biomarkers have also supported drug development to induce cell death in cancerous cells. There are various serological and microscopic techniques with advantages and limitations, that are available and are being utilized to detect and study the mechanism of cell death. The complexity of the mechanism and difficulties in distinguishing among different types of programmed cell death make it challenging to carry out the interventions and delay its progression. In this review, mechanisms of different forms of programmed cell death along with their conventional and unconventional methods of detection of have been critically reviewed systematically and categorized on the basis of morphological hallmarks and biomarkers to understand the principle, mechanism, application, advantages and disadvantages of each method. Furthermore, a very comprehensive comparative analysis has been drawn to highlight the most efficient and effective methods of detection of programmed cell death, helping researchers to make a reliable and prudent selection among the available methods of cell death assay. Conclusively, how programmed cell death detection methods can be improved and can provide information about distinctive stages of cell death detection have been discussed.Peer reviewe

Methanodibenzo[b,f][1,5]dioxocins as Novel Glutaminase Inhibitor with Anti-Glioblastoma Potential

Glutamine metabolism is an important hallmark of several cancers with demonstrated antitumor activity in glioblastoma cancer cells (GBM). GBM cells regulate glutamine and use it as a major energy source for their proliferation through the glutaminolysis process. Enzymes, such as glutaminase in glutaminolysis, can be targeted by small-molecule inhibitors, thus exhibiting promising anticancer properties. The resistance to glutaminolysis demands the development of new therapeutic molecules to overcome drug resistance. Herein, we have reported a novel library of constrained methanodibenzo[b,f][1,5]dioxocin derivatives as glutaminase (GLS) inhibitors and their anti-GBM potential. The library consisting of seven molecules was obtained through self-condensation of 2&prime;-hydroxyacetophenones, out of which three molecules, namely compounds 3, 5, and 6, were identified with higher binding energy values ranging between &minus;10.2 and &minus;9.8 kcal/mol with GLS (PDB ID; 4O7D). Pharmacological validation of these compounds also showed a higher growth inhibition effect in GBM cells than the standard drug temozolomide (TMZ). The most promising compound, 6, obeyed Lipinski&rsquo;s rule of five and was identified to interact with key residues Arg307, Asp326, Lys328, Lys399, and Glu403 of GLS. This compound exhibited the best cytotoxic effect with IC50 values of 63 &micro;M and 83 &micro;M in LN229 and SNB19 cells, respectively. The potential activation of GLS by the best-constrained dibenzo[b,f][1,5]dioxocin in the tested series increased apoptosis via reactive oxygen species production in both GBM cells, and exhibited anti-migratory and anti-proliferative properties over time in both cell lines. Our results highlight the activation mechanism of a dibenzo[b,f][1,5]dioxocin from the structural basis and demonstrate that inhibition of glutaminolysis may facilitate the pharmacological intervention for GBM treatment

Methanodibenzo[<i>b</i>,<i>f</i>][1,5]dioxocins as Novel Glutaminase Inhibitor with Anti-Glioblastoma Potential

Glutamine metabolism is an important hallmark of several cancers with demonstrated antitumor activity in glioblastoma cancer cells (GBM). GBM cells regulate glutamine and use it as a major energy source for their proliferation through the glutaminolysis process. Enzymes, such as glutaminase in glutaminolysis, can be targeted by small-molecule inhibitors, thus exhibiting promising anticancer properties. The resistance to glutaminolysis demands the development of new therapeutic molecules to overcome drug resistance. Herein, we have reported a novel library of constrained methanodibenzo[b,f][1,5]dioxocin derivatives as glutaminase (GLS) inhibitors and their anti-GBM potential. The library consisting of seven molecules was obtained through self-condensation of 2′-hydroxyacetophenones, out of which three molecules, namely compounds 3, 5, and 6, were identified with higher binding energy values ranging between −10.2 and −9.8 kcal/mol with GLS (PDB ID; 4O7D). Pharmacological validation of these compounds also showed a higher growth inhibition effect in GBM cells than the standard drug temozolomide (TMZ). The most promising compound, 6, obeyed Lipinski’s rule of five and was identified to interact with key residues Arg307, Asp326, Lys328, Lys399, and Glu403 of GLS. This compound exhibited the best cytotoxic effect with IC50 values of 63 µM and 83 µM in LN229 and SNB19 cells, respectively. The potential activation of GLS by the best-constrained dibenzo[b,f][1,5]dioxocin in the tested series increased apoptosis via reactive oxygen species production in both GBM cells, and exhibited anti-migratory and anti-proliferative properties over time in both cell lines. Our results highlight the activation mechanism of a dibenzo[b,f][1,5]dioxocin from the structural basis and demonstrate that inhibition of glutaminolysis may facilitate the pharmacological intervention for GBM treatment