A dual fluorescence protein expression system detects cell cycle dependent protein noise.

Inherently identical cells exhibit significant phenotypic variation. It can be essential for many biological processes and is known to arise from stochastic, 'noisy', gene expression that is determined by intrinsic and extrinsic components. It is now obvious that the noise varies as a function of inducer concentration. However, its fluctuation over the cell cycle is limited. Applying dual colour fluorescence protein reporter system, Cyan Fluorescent Protein (CFP) and Yellow fluorescent protein (YFP) tagged multi-copy plasmids, we determine variation of the noise components over the phases in lac promoter induced by Isopropyl ß-D-1-thiogalactopyranoside (IPTG) and in presence of additional Magnesium, Mg2+ ion. We, also, estimate the how such system deviates from observations of single-copy plasmid. Found 25 % difference between multi-copy system and single-copy system clarifies that observed noise is considerable and estimates population behaviour during the cell cycle. We show that total variation in cells induced with IPTG is determined by higher extrinsic than intrinsic noise. It increases from Lag to Exponential phase and decreases from Retardation to Stationary phase. By observing slow and fast dividing cells, we show that 5 mM Mg2+ increases population homogeneity compared to 2.5 mM Mg2+ in the environment. The experimental data obtained using dual colour fluorescence protein reporter system demonstrates that protein expression noise, depending on intra cellular ionic concentration, is tightly controlled by phase of the cell.

Design, synthesis and anticancer evaluation of novel arylhydrazones of active methylene compounds.

Nerve growth factor (NGF) and its receptor, tropomyosin kinase receptor kinase type A (TrkA) is emerging as an important target for Glioblastoma (GBM) treatment. TrkA is the cancer biomarker majorly involved in tumor invasion and migration into nearby normal tissue. However, currently, available Trk inhibitors exhibit many adverse effects in cancer patients, thus demanding a novel class of ligands to regulate Trk signaling. Here, we exploited the role of TrkA (NTRK1) expression from the 651 datasets of brain tumors. RNA sequence analysis identified overexpression of NTRK1 in GBM, recurrent GBM as well in Oligoastrocytoma patients. Also, TrkA expression tends to increase over the higher grades of GBM. TrkA protein targeting hydrazone derivatives, R48, R142, and R234, were designed and their mode of interaction was studied using molecular docking and dynamic simulation studies. Ligands' stability and binding assessment reveals R48, 2 2-(2-(2-hydroxy-4-nitrophenyl) hydrazineylidene)-1-phenylbutane-1,3-dione, as a potent ligand that interacts well with TrkA's hydrophobic residues, Ile, Phe, Leu, Ala, and Val. R48- TrkA exhibits stable binding potentials with an average RMSD value <0.8 nm. R48 obeyed Lipinski's rule of five and possessed the best oral bioavailability, suggesting R48 as a potential compound with drug-likeness properties. In-vitro analysis also revealed that R48 exhibited a higher cytotoxicity effect for U87 GBM cells than TMZ with the IC50 value of 68.99 µM. It showed the lowest percentage of cytotoxicity to the non-cancerous TrkA expressing MEF cells. However, further SiRNA analysis validates the non-specific binding of R48, necessitating structural alteration for the development of R48-based TrkA inhibitor for GBM therapeutics.

A novel EGFR inhibitor, HNPMI, regulates apoptosis and oncogenesis by modulating BCL-2/BAX and p53 in colon cancer.

BACKGROUND AND PURPOSE: Colorectal cancer (CRC) is the second most lethal disease, with high mortality due to its heterogeneity and chemo-resistance. Here, we have focused on the epidermal growth factor receptor (EGFR) as an effective therapeutic target in CRC and studied the effects of polyphenols known to modulate several key signalling mechanisms including EGFR signalling, associated with anti-proliferative and anti-metastatic properties. EXPERIMENTAL APPROACH: Using ligand- and structure-based cheminformatics, we developed three potent, selective alkylaminophenols, 2-[(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl]phenol (THTMP), 2-[(1,2,3,4-tetrahydroquinolin-1-yl)(4-methoxyphenyl)methyl]phenol (THMPP) and N-[2-hydroxy-5-nitrophenyl(4'-methylphenyl)methyl]indoline (HNPMI). These alkylaminophenols were assessed for EGFR interaction, EGFR-pathway modulation, cytotoxic and apoptosis induction, caspase activation and transcriptional and translational regulation. The lead compound HNPMI was evaluated in mice bearing xenografts of CRC cells. KEY RESULTS: Of the three alkylaminophenols tested, HNPMI exhibited the lowest IC50 in CRC cells and potential cytotoxic effects on other tumour cells. Modulation of EGFR pathway down-regulated protein levels of osteopontin, survivin and cathepsin S, leading to apoptosis. Cell cycle analysis revealed that HNPMI induced G0/G1 phase arrest in CRC cells. HNPMI altered the mRNA for and protein levels of several apoptosis-related proteins including caspase 3, BCL-2 and p53. HNPMI down-regulated the proteins crucial to oncogenesis in CRC cells. Assays in mice bearing CRC xenografts showed that HNPMI reduced the relative tumour volume. CONCLUSIONS AND IMPLICATIONS: HNPMI is a promising EGFR inhibitor for clinical translation. HNPMI regulated apoptosis and oncogenesis by modulating BCL-2/BAX and p53 in CRC cell lines, showing potential as a therapeutic agent in the treatment of CRC.

Benzenesulfonamide Analogs: Synthesis, Anti-GBM Activity and Pharmacoprofiling.

The tropomyosin receptor kinase A (TrkA) family of receptor tyrosine kinases (RTKs) emerge as a potential target for glioblastoma (GBM) treatment. Benzenesulfonamide analogs were identified as kinase inhibitors possessing promising anticancer properties. In the present work, four known and two novel benzenesulfonamide derivatives were synthesized, and their inhibitory activities in TrkA overexpressing cells, U87 and MEF cells were investigated. The cytotoxic effect of benzenesulfonamide derivatives and cisplatin was determined using trypan blue exclusion assays. The mode of interaction of benzenesulfonamides with TrkA was predicted by docking and structural analysis. ADMET profiling was also performed for all compounds to calculate the drug likeness property. Appropriate QSAR models were developed for studying structure-activity relationships. Compound 4-[2-(4,4-dimethyl-2,6-dioxocyclohexylidene)hydrazinyl]-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfon-amide (AL106) and 4-[2-(1,3-dioxo-1,3-dihydro-2H-inden-2-ylidene)hydrazinyl]-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide (AL107) showed acceptable binding energies with the active sites for human nerve growth factor receptor, TrkA. Here, AL106 was identified as a potential anti-GBM compound, with an IC50 value of 58.6 µM with a less toxic effect in non-cancerous cells than the known chemotherapeutic agent, cisplatin. In silico analysis indicated that AL106 formed prominent stabilizing hydrophobic interactions with Tyr359, Ser371, Ile374 and charged interactions with Gln369 of TrkA. Furthermore, in silico analysis of all benzenesulfonamide derivatives revealed that AL106 has good pharmacokinetics properties, drug likeness and toxicity profiles, suggesting the compound may be suitable for clinical trial. Thus, benzenesulfonamide analog, AL106 could potentially induce GBM cell death through its interaction with TrkA and might be an attractive strategy for developing a drug targeted therapy to treat glioblastoma.

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.

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.

Structural analysis of human G-protein-coupled receptor 17 ligand binding sites.

The human G protein coupled membrane receptor (GPR17), the sensor of brain damage, is identified as a biomarker for many neurological diseases. In human brain tissue, GPR17 exist in two isoforms, long and short. While cryo-electron microscopy technology has provided the structure of the long isoform of GPR17 with Gi complex, the structure of the short isoform and its activation mechanism remains unclear. Recently, we theoretically modeled the structure of the short isoform of GPR17 with Gi signaling protein and identified novel ligands. In the present work, we demonstrated the presence of two distinct ligand binding sites in the short isoform of GPR17. The molecular docking of GPR17 with endogenous (UDP) and synthetic ligands (T0510.3657, MDL29950) found the presence of two distinct binding pockets. Our observations revealed that endogenous ligand UDP can bind stronger in two different binding pockets as evidenced by glide and autodock vina scores, whereas the other two ligand's binding with GPR17 has less docking score. The analysis of receptor-UDP interactions shows complexes' stability in the lipid environment by 100 ns atomic molecular dynamics simulations. The amino acid residues VAL83, ARG87, and PHE111 constitute ligand binding site 1, whereas site 2 constitutes ASN67, ARG129, and LYS232. Root mean square fluctuation analysis showed the residues 83, 87, and 232 with higher fluctuations during molecular dynamics simulation in both binding pockets. Our findings imply that the residues of GPR17's two binding sites are crucial, and their interaction with UDP reveals the protein's hidden signaling and communication properties. Furthermore, this finding may assist in the development of targeted therapies for the treatment of neurological diseases.

Signaling landscape of mitochondrial non-coding RNAs.

Human mitochondria are the vital cell organelle acting as a storehouse of energy generation and diverse regulatory functions. Mitochondrial DNA comprises 93% coding region and 7% non-coding regions, in which the non-coding region hypothesized as responsible for signaling is our specific interest. Here, we explored the unknown functions of mitochondrial non-coding RNAs by studying their respective signaling pathways. We retrieved conserved motifs of interactions from known experimental protein-RNA complexes to model unknown mitochondrial ncRNA sequences. Our results provide the ncRNAs list and show their involvement in four crucial pathways, such as (i) Processing of Capped Intron-Containing Pre-mRNA, (ii) Spliceosome, (iii) Spliceosomal assembly, and (iv) RNA Polymerase II Transcription, respectively. The interactome analysis revealed that the SRSF2 and U2AF2 proteins interact with ncRNAs. Further, we have simulated the selected ncRNA-protein complexes in cell-like environmental conditions and found them stable in terms of energetics. Through our study, we have identified an apparent interaction of mitochondrial ncRNAs with proteins and their role in critical signaling pathways, providing new insights into mitochondrial ncRNA-dependent gene regulation.Communicated by Ramaswamy H. Sarma.

Marine halophyte derived polyphenols inhibit glioma cell growth through mitogen-activated protein kinase signaling pathway.

Plants that are pharmacologically significant require intensive phytochemical characterization for bioactive profiling of the compounds, which has enabled their safe use in ayurvedic medicine. The present study is focused on the phytochemical analyses, quantitative estimation and profiling of secondary metabolites of leaf extract, as well as the antioxidant and cytotoxic activity of the potent halophytes such as Avicennia marina, Ceriops tagal, Ipomoea pes-caprae, and Sonneratia apetala. The in vitro antioxidant property was investigated using DPPH, ferric reducing antioxidant capacity (FRAP) assay. Bioactive compounds such as phenols, flavonoids, saponin and alkaloids were quantitatively estimated from the extracts of A.marina, C.tagal, I.pes-capra and S.apetala, which possessed higher phenol content than the other studied halophytes. The extracts at 200 µg/ml revealed higher antioxidant activity than the standard ascorbic acid and it functions as a powerful oxygen free radical scavenger with 77.37%, 75.35% and 72.84% for S.apetala, I.pes-caprae and C.tagal respectively and with least IC50 for I.pes-caprae (11.95 µg/ml) followed by C.tagal (49.94 µg/ml). Cell viability and anti-proliferative activity of different polyphenolic fractions of C.tagal (CT1 and CT2) and I.pes-caprae fraction (IP) against LN229, SNB19 revealed Ipomoea as the promising anti-cytotoxic fraction. IP-derived polyphenols was further subjected to apoptosis, migration assay, ROS and caspase - 3 and - 7 to elucidate its potentiality as a therapeutic drug. IP-polyphenols was found to have higher percentage of inhibition than the CT1 and CT2 polyphenols of C.tagal on comparison with TMZ. All the above-mentioned in-vitro analysis further validated the ability of IP-polyphenols inducing cell death via ROS-mediated caspase dependent pathway. Further, proteomic and phospho-proteomic analysis revealed the potential role of IP-polyphenols in the regulation of cell proliferation through MMK3, p53, p70 S6 kinase and RSK1 proteins involved in mitogen-activated protein kinase signaling pathway. Our analysis confirmed the promising role of I.pes-caprae derived polyphenols as an anti-metastatic compound against GBM cells.

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.

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.

P2Y1 agonist HIC in combination with androgen receptor inhibitor abiraterone acetate impairs cell growth of prostate cancer.

P2Y receptors belong to the large superfamily of G-protein-coupled receptors and play a crucial role in cell death and survival. P2Y1 receptor has been identified as a marker for prostate cancer (PCa). A previously unveiled selective P2Y1 receptor agonist, the indoline-derived HIC (1-(1-((2-hydroxy-5-nitrophenyl)(4-hydroxyphenyl)methyl)indoline-4-carbonitrile), induces a series of molecular and biological responses in PCa cells PC3 and DU145, but minimal toxicity to normal cells. Here, we evaluated the combinatorial effect of HIC with abiraterone acetate (AA) targeted on androgen receptor (AR) on the inhibition of PCa cells. Here, the presence of HIC and AA significantly inhibited cell proliferation of PC3 and DU145 cells with time-dependent manner as a synerfistic combination. Moreover, it was also shown that the anticancer and antimetastasis effects of the combinratorial drugs were noticed through a decrease in colony-forming ability, cell migration, and cell invasion. In addition, the HIC + AA induced apoptotic population of PCa cells as well as cell cycle arrest in G1 progression phase. In summary, these studies show that the combination of P2Y1 receptor agonist, HIC and AR inhibitor, AA, effectively improved the antitumor activity of each drug. Thus, the combinatorial model of HIC and AA should be a novel and promising therapeutic strategy for treating prostate cancer.

GPR17 signaling activation by CHBC agonist induced cell death via modulation of MAPK pathway in glioblastoma.

AIM: Glioblastoma multiforme (GBM) is the most common and aggressive primary adult brain tumor. GBM is characterized by a heterogeneous population of cells that are resistant to chemotherapy. Recently, we have synthesized CHBC, a novel indole derivative targeted to GBM biomarker G-protein-coupled receptor 17 and inhibitor of GBM cells. In this study, CHBC was further investigated to characterize the efficiency of this agonist at the molecular level and its underlying mechanism in GBM cell death induction. MATERIALS AND METHODS: The effect of CHBC and TMZ was determined using time dependent inhibitor assay in glioblastoma cells, LN229 and SNB19. Drug induced cell cycle arrest was measured using PI staining followed by image analysis. The induction of apoptosis and mechanism of action of CHBC was studied using apoptosis, caspase 3/7 and mitochondrial membrane permeability assays. Modulation of the key genes involved in MAPK signaling pathway was also measured using immunoblotting array. KEY FINDINGS: The inhibitory kinetic study has revealed that CHBC inhibited SNB19 and LN229 cell growth in a time-dependent manner. Furthermore, CHBC with the IC50 of 85 µM, mediated cell death through an apoptosis mechanism in both studied cell lines. The study also has revealed that CHBC targets GPR17 leading to the induction of apoptosis via the activation of Caspase 3/7 and dysfunction of mitochondrial membrane potential. In addition, CHBC treatment led to marked G2/M cell cycle arrest. The protein array has confirmed the anticancer effect of CHBC by the disruption of the mitogen-activated protein kinase pathway (MAPK). SIGNIFICANCE: Taken together, these results demonstrated that CHBC induced G2/M cell cycle arrest and apoptosis by disrupting MAPK signaling in human glioblastoma cells. This study concludes that CHBC represent a class of compounds for treating glioblastoma.

Transcriptomic analysis of glioblastoma multiforme providing new insights into GPR17 signaling communication.

Glioblastoma Multiforme (GBM) is one of the most aggressive malignant tumors in the central nervous system, which arises due to the failure or crosstalk in the signaling networks. GPR17, an orphan G protein-coupled receptor is anticipated to be associated with the biology of the GBM disease progression. In the present study, we have identified the differential expressions of around 170 genes along with GPR17 through the RNA-Seq analysis of 169 GBM samples. Coordinated expression patterns of all other gene products with this receptor were analysed using gene ontology and protein-protein interaction data. Several crucial signaling components and genes that play a significant role in tumor progression have been identified among which GPR17 was found to be significantly interacting with about 30 different pathways. High-throughput molecular docking of GPR17 by homology-based model against differentially expressed proteins, showed effective recognition and binding of PX, SH3, and Ig-like domains besides Gi protein. Pathways of PI3, Src, Ptdn, Ras, cytoplasmic tyrosine kinases, phospholipases, nexins and other proteins possessing these structural domains are identified as critical signaling components of the complex GBM signaling network. Our findings also provide a mechanistic insight of GPR17-T0510-3657 interaction, which potentially regulates the interaction of PX domain and helical mPTS recognition domain-containing proteins. Overall, our results demonstrate that GPR17 mediated signaling networks could be used as a therapeutic target for GBM. Communicated by Ramaswamy H. Sarma.

Molecular modeling and dynamics studies of the synthetic small molecule agonists with GPR17 and P2Y1 receptor.

The human Guanine Protein coupled membrane Receptor 17 (hGPR17), an orphan receptor that activates uracil nucleotides and cysteinyl leukotrienes is considered as a crucial target for the neurodegenerative diseases. Yet, the detailed molecular interaction of potential synthetic ligands of GPR17 needs to be characterized. Here, we have studied a comparative analysis on the interaction specificity of GPR17-ligands with hGPR17 and human purinergic G protein-coupled receptor (hP2Y1) receptors. Previously, we have simulated the interaction stability of synthetic ligands such as T0510.3657, AC1MLNKK, and MDL29951 with hGPR17 and hP2Y1 receptor in the lipid environment. In the present work, we have comparatively studied the protein-ligand interaction of hGPR17-T0510.3657 and P2Y1-MRS2500. Sequence analysis and structural superimposition of hGPR17 and hP2Y1 receptor revealed the similarities in the structural arrangement with the local backbone root mean square deviation (RMSD) value of 1.16 Å and global backbone RMSD value of 5.30 Å. The comparative receptor-ligand interaction analysis between hGPR17 and hP2Y1 receptor exposed the distinct binding sites in terms of geometrical properties. Further, the molecular docking of T0510.3657 with the hP2Y1 receptor have shown non-specific interaction. The experimental validation also revealed that Gi-coupled activation of GPR17 by specific ligands leads to the adenylyl cyclase inhibition, while there is no inhibition upon hP2Y1 activation. Overall, the above findings suggest that T0510.3657-GPR17 binding specificity could be further explored for the treatment of numerous neuronal diseases. Communicated by Ramaswamy H. Sarma.

Molecular interaction study of novel indoline derivatives with EGFR-kinase domain using multiple computational analysis.

Epidermal growth factor receptors are constitutively overexpressed in breast cancer cells, which in turn stimulate many downstream signaling pathways that are involved in many carcinogenic processes. This makes EGFR a striking target for cancer therapy. This study focuses on the EGFR kinase domain inactivation by novel synthesized indoline derivatives. The compounds used are N-(2-hydroxy-5-nitrophenyl (4'-methyl phenyl) methyl) indoline (HNPMI), alkylaminophenols - 2-((3,4-Dihydroquinolin-1(2H)-yl) (p-tolyl) methyl) phenol (THTMP) and 2-((1, 2, 3, 4-Tetrahydroquinolin-1-yl) (4 methoxyphenyl) methyl) phenol (THMPP). To get a clear insight into the molecular interaction of EGFR and the three compounds, we have used ADME/Tox prediction, Flexible docking analysis followed by MM/GB-SA, QM/MM analysis, E-pharmacophore mapping of the ligands and Molecular dynamic simulation of protein-ligand complexes. All three compounds showed good ADME/Tox properties obeying the rules of drug-likeliness and showed high human oral absorption. Molecular docking was performed with the compounds and EGFR using Glide Flexible docking mode. This showed that the HNPMI was best among the three compounds and had interactions with key residue Lys 721. The protein-ligand complexes were stable when simulated for 100 ns using Desmond software. The interactions were further substantiated using QM/MM analysis and MM-GB/SA analysis in which HNPMI was scored as the best molecule. All the analyses were carried out with a reference molecule-Gefitinib which is a known standard inhibitor of EGFR. Thus, the study elucidates the potential role of the indoline derivatives as an anti-cancer agent against breast cancer by effectively inhibiting EGFR.Communicated by Ramaswamy H. Sarma.

Functional characterization of HIC, a P2Y1 agonist, as a p53 stabilizer for prostate cancer cell death induction.

Background: (1-(2-hydroxy-5-nitrophenyl)(4-hydroxyphenyl)methyl)indoline-4-carbonitrile (HIC), an agonist of the P2Y1 receptor (P2Y1R), induces cell death in prostate cancer cells. However, the molecular mechanism behind the inhibition of HIC in prostate cancer remains elusive. Methods & results: Here, to outline the inhibitory role of HIC on prostate cancer cells, PC-3 and DU145 cell lines were treated with the respective IC50 concentrations, which reduced cell proliferation, adherence properties and spheroid formation. HIC was able to arrest the cell cycle at G1/S phase and also induced apoptosis and DNA damage, validated by gene expression profiling. HIC inhibited the prostate cancer cells' migration and invasion, revealing its antimetastatic ability. P2Y1R-targeted HIC affects p53, MAPK and NF-κB protein expression, thereby improving the p53 stabilization essential for G1/S arrest and cell death. Conclusion: These findings provide an insight on the potential use of HIC, which remains the mainstay treatment for prostate cancer.

Targeting Orphan G Protein-Coupled Receptor 17 with T0 Ligand Impairs Glioblastoma Growth.

Glioblastoma, an invasive high-grade brain cancer, exhibits numerous treatment challenges. Amongst the current therapies, targeting functional receptors and active signaling pathways were found to be a potential approach for treating GBM. We exploited the role of endogenous expression of GPR17, a G protein-coupled receptor (GPCR), with agonist GA-T0 in the survival and treatment of GBM. RNA sequencing was performed to understand the association of GPR17 expression with LGG and GBM. RT-PCR and immunoblotting were performed to confirm the endogenous expression of GPR17 mRNA and its encoded protein. Biological functions of GPR17 in the GBM cells was assessed by in vitro analysis. HPLC and histopathology in wild mice and an acute-toxicity analysis in a patient-derived xenograft model were performed to understand the clinical implication of GA-T0 targeting GPR17. We observed the upregulation of GPR17 in association with improved survival of LGG and GBM, confirming it as a predictive biomarker. GA-T0-stimulated GPR17 leads to the inhibition of cyclic AMP and calcium flux. GPR17 signaling activation enhances cytotoxicity against GBM cells and, in patient tissue-derived mesenchymal subtype GBM cells, induces apoptosis and prevents proliferation by stoppage of the cell cycle at the G1 phase. Modulation of the key genes involved in DNA damage, cell cycle arrest, and in several signaling pathways, including MAPK/ERK, PI3K-Akt, STAT, and NF-κB, prevents tumor regression. In vivo activation of GPR17 by GA-T0 reduces the tumor volume, uncovering the potential of GA-T0-GPR17 as a targeted therapy for GBM treatment. Conclusion: Our analysis suggests that GA-T0 targeting the GPR17 receptor presents a novel therapy for treating glioblastoma.

Alkylaminophenol and GPR17 Agonist for Glioblastoma Therapy: A Combinational Approach for Enhanced Cell Death Activity.

Drug resistance and tumor heterogeneity limits the therapeutic efficacy in treating glioblastoma, an aggressive infiltrative type of brain tumor. GBM cells develops resistance against chemotherapeutic agent, temozolomide (TMZ), which leads to the failure in treatment strategies. This enduring challenge of GBM drug resistance could be rational by combinatorial targeted therapy. Here, we evaluated the combinatorial effect of phenolic compound (2-(3,4-dihydroquinolin-1(2H)-yl)(p-tolyl)methyl)phenol (THTMP), GPR17 agonist 2-({5-[3-(Morpholine-4-sulfonyl)phenyl]-4-[4-(trifluoromethoxy)phenyl]-4H-1,2,4-triazol-3-yl}sulfanyl)-N-[4-(propan-2-yl)phenyl]acetamide (T0510.3657 or T0) with the frontline drug, TMZ, on the inhibition of GBM cells. Mesenchymal cell lines derived from patients' tumors, MMK1 and JK2 were treated with the combination of THTMP + T0, THTMP + TMZ and T0 + TMZ. Cellular migration, invasion and clonogenicity assays were performed to check the migratory behavior and the ability to form colony of GBM cells. Mitochondrial membrane permeability (MMP) assay and intracellular calcium, [Ca2+]i, assay was done to comprehend the mechanism of apoptosis. Role of apoptosis-related signaling molecules was analyzed in the induction of programmed cell death. In vivo validation in the xenograft models further validates the preclinical efficacy of the combinatorial drug. GBM cells exert better synergistic effect when exposed to the cytotoxic concentration of THTMP + T0, than other combinations. It also inhibited tumor cell proliferation, migration, invasion, colony-forming ability and cell cycle progression in S phase, better than the other combinations. Moreover, the combination of THTMP + T0 profoundly increased the [Ca2+]i, reactive oxygen species in a time-dependent manner, thus affecting MMP and leading to apoptosis. The activation of intrinsic apoptotic pathway was regulated by the expression of Bcl-2, cleaved caspases-3, cytochrome c, HSP27, cIAP-1, cIAP-2, p53, and XIAP. The combinatorial drug showed promising anti-tumor efficacy in GBM xenograft model by reducing the tumor volume, suggesting it as an alternative drug to TMZ. Our findings indicate the coordinated administration of THTMP + T0 as an efficient therapy for inhibiting GBM cell proliferation.

Molecular interaction of HIC, an agonist of P2Y1 receptor, and its role in prostate cancer apoptosis.

Prostate cancer is a heterogeneous, slow growing asymptomatic cancer that predominantly affects man. A purinergic G-protein coupled receptor, P2Y1R, is targeted for its therapeutic value since it plays a crucial role in many key molecular events of cancer progression and invasion. Our previous study demonstrated that indoline derivative, 1 ((1-(2-Hydroxy-5-nitrophenyl) (4-hydroxyphenyl) methyl)indoline-4­carbonitrile; HIC), stimulates prostate cancer cell (PCa) growth inhibition via P2Y1R. However, the mode of interaction of P2Y1R with HIC involved in this process remains unclear. Here, we have reported the molecular interactions of HIC with P2Y1R. Molecular dynamics simulation was performed that revealed the stable specific binding of the protein-ligand complex. In vitro analysis has shown increased apoptosis of PCa-cells, PC3, and DU145, upon specific interaction of P2Y1R-HIC. This was further validated using siRNA analysis that showed a higher percentage of apoptotic cells in PCa-cells transfected with P2Y-siRNA-MRS2365 than P2Y-siRNA-HIC treatment. Decreased mitochondrial membrane potential (MMP) activity and reduced glutathione (GSH) level show their role in P2Y1R-HIC mediated apoptosis. These in silico and in vitro results confirmed that HIC could induce mitochondrial apoptotic signaling through the P2Y1R activation. Thus, HIC being a potential ligand upon interaction with P2Y1R might have therapeutic value for the treatment of prostate cancer.