Heterocyclic scaffolds as promising anticancer agents against tumours of the central nervous system: Exploring the scope of indole and carbazole derivatives

Tumours of the central nervous system are intrinsically more dangerous than tumours at other sites, and in particular, brain tumours are responsible for 3% of cancer deaths in the UK. Despite this, research into new therapies only receives 1% of national cancer research spend. The most common chemotherapies are temozolomide, procarbazine, carmustine, lomustine and vincristine, but because of the rapid development of chemoresistance, these drugs alone simply aren’t sufficient for long-term treatment. Such poor prognosis of brain tumour patients prompted us to research new treatments for malignant glioma, and in doing so, it became apparent that aromatic heterocycles play an important part, especially the indole, carbazole and indolocarbazole scaffolds. This review highlights compounds in development for the treatment of tumours of the central nervous system which are structurally based on the indole, carbazole and indolocarbazole scaffolds, under the expectation that it will highlight new avenues for research for the development of new compounds to treat these devastating neoplasms

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

HIV-1 virological synapse formation enhances infection spread by dysregulating Aurora Kinase B.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity

Decision tree used to identify proteins involved in HIV cell to cell spread.

Initial screening was performed using producer cell mixing with TZM-BL target cells (Fig 4A). Potential hits were confirmed by further analysis as shown. Compounds were rejected if either producer or target cells viability was negatively affected by chemical treatment (Fig 4B). Next, potential hits were rejected if chemical treatment inhibited cell free virion infection of target cells (Fig 4D). Finally, if inhibitors enhanced syncytia formation, they were classified as a specific class of hit that affected the fusogenic activity of Env and selected for further study (Fig 5). (EPS)</p

AURKB regulation of HIV spread through virological synapse requires the C-terminal domain of Env.

Schematic of HIV-1 virological synapse. Cytoplasmic AURKB exerts an unknown negative effect on HIV-1 spread through the virological synapse which reduces the fusion activity of the HIV-1 Env protein. HIV-1 overcomes this through the CTD of HIV-1 Env which induces premature nuclear localization of AURKB to the CPC.</p

Volcano plots.

Fold changes and statistical significance of the (A) protein abundance changes at 5 min, (B) protein abundance changes at 60 min, (C) protein phosphorylation changes at 5 min, and (D) protein phosphorylation changes at 60 min. Statistical test results from the same time point and different multiplexes are displayed in the same volcano plot. Flat portions in some q-value distributions are due to the q-value being defined as the minimum false discovery rate that can be achieved when including a phosphopeptide or protein as significant. Because q-values depend on the number of statistical tests performed and the number of peptides quantified is different in each multiplex, the range of -log10 q-values differs for each multiplex. (EPS)</p

Phosphorylation changes after VS formation.

HIV-1 spreads efficiently through direct cell-to-cell transmission at virological synapses (VSs) formed by interactions between HIV-1 envelope proteins (Env) on the surface of infected cells and CD4 receptors on uninfected target cells. Env-CD4 interactions bring the infected and uninfected cellular membranes into close proximity and induce transport of viral and cellular factors to the VS for efficient virion assembly and HIV-1 transmission. Using novel, cell-specific stable isotope labeling and quantitative mass spectrometric proteomics, we identified extensive changes in the levels and phosphorylation states of proteins in HIV-1 infected producer cells upon mixing with CD4+ target cells under conditions inducing VS formation. These coculture-induced alterations involved multiple cellular pathways including transcription, TCR signaling and, unexpectedly, cell cycle regulation, and were dominated by Env-dependent responses. We confirmed the proteomic results using inhibitors targeting regulatory kinases and phosphatases in selected pathways identified by our proteomic analysis. Strikingly, inhibiting the key mitotic regulator Aurora kinase B (AURKB) in HIV-1 infected cells significantly increased HIV activity in cell-to-cell fusion and transmission but had little effect on cell-free infection. Consistent with this, we found that AURKB regulates the fusogenic activity of HIV-1 Env. In the Jurkat T cell line and primary T cells, HIV-1 Env:CD4 interaction also dramatically induced cell cycle-independent AURKB relocalization to the centromere, and this signaling required the long (150 aa) cytoplasmic C-terminal domain (CTD) of Env. These results imply that cytoplasmic/plasma membrane AURKB restricts HIV-1 envelope fusion, and that this restriction is overcome by Env CTD-induced AURKB relocalization. Taken together, our data reveal a new signaling pathway regulating HIV-1 cell-to-cell transmission and potential new avenues for therapeutic intervention through targeting the Env CTD and AURKB activity.</div

Producer cell phosphopeptides identified after VS formation.

Phosphopeptides identified by protein group, UniProt ID, representative name, and phosphorylation isoform. Columns show log2 fold changes and q-values in phosphopeptide level in experiments 1 and 2 at 5 and 60 minutes relative to 0 min controls. True in the Significant column means the phosphopeptide met criteria of a q-value ≤ 0.1 and a fold change ≥ 1.5 in magnitude in at least one experiment and time point. (XLSX)</p

Interaction with soluble CD4 HIV Env is sufficient to relocalize AURKB.

(A) Jurkat cells were co-transfected with plasmids encoding the indicated HIV envelope, GFP-AURKB and mCherry-CENBP. 24 hours post transfection, cells were incubated with purified IgG or soluble IgG-CD4 fusion protein for 20 minutes, fixed, mounted and imaged at 60X by confocal microscopy. Treatment with the soluble CD4 fusion protein caused HIV to relocalized to CENBP adjacent foci. (B) Schematic for quantitation of CD4 induced AURKB localization and cell cycle changes. Jurkat cells were co-transfected with plasmids encoding the indicated HIV envelope, along with mAzurite-Histone H2B to mark the transfected cells and a plasmid that encodes the 2-color PIP-Fucci system than uses a YFP-PIP protein to mark G1 cells, an mCherry-Geminin to mark S-phase cells. G2/M phase cells are dual positive. 24 hours post transfection, cells were incubated with purified IgG or soluble IgG-CD4 fusion protein for 20 minutes, fixed, stained for endogenous AURKB with a far-red secondary (alexafluor 647), mounted and imaged at 60X by confocal microscopy. 10 independent fields were counted, and transfected cells were scored for CD4 induced changes to the cell cycle stage with (C) WT or (D) ΔCTD envelope. (E) Total AURKB localization at nuclear puncta by WT or (F) ΔCTD envelope. The data shown are the average mean values obtained from three independent experiments with a minimum of 550 cells counted per condition between experiments. Error bars indicate the standard deviation of the data in all panels. P-values were calculated using a standard Student’s t-test and significant changes of IgG treated control cells to soluble CD4 treated cells are indicated.</p

Kinase and phosphatase inhibitors used in spread assay.

Kinase and phosphatase inhibitors used in spread assay.</p