Synthesis and Antitumour Evaluation of Tricyclic Indole-2-Carboxamides against Paediatric Brain Cancer Cells.

Antitumour properties of some cannabinoids (CB) have been reported in the literature as early as 1970s, however there is no clear consensus to date on the exact mechanisms leading to cancer cell death. The indole-based WIN 55,212-2 and SDB-001 are both known as potent agonists at both CB1 and CB2 receptors, yet we demonstrate herein that only the former can exert in vitro antitumour effects when tested against a paediatric brain cancer cell line KNS42. In this report, we describe the synthesis of novel 3,4-fused tricyclic indoles and evaluate their functional potencies at both cannabinoid receptors, as well as their abilities to inhibit the growth or proliferation of KNS42 cells. Compared to our previously reported indole-2-carboxamides, these 3,4-fused tricyclic indoles had either completely lost activities, or, showed moderate-to-weak antagonism at both CB1 and CB2 receptors. Compound 23 displayed the most potent antitumour properties among the series. Our results further support the involvement of non-CB pathways for the observed antitumour activities of amidoalkylindole-based cannabinoids, in line with our previous findings. Transcriptomic analysis comparing cells treated or non-treated with compound 23 suggested the observed antitumour effects of 23 are likely to result mainly from disruption of the FOXM1-regulated cell cycle pathways.

Synthesis and antitumour evaluation of indole-2-carboxamides against paediatric brain cancer cells.

Paediatric glioblastomas are rapidly growing, devastating brain neoplasms with an invasive phenotype. Radiotherapy and chemotherapy, which are the current therapeutic adjuvant to surgical resection, are still associated with various toxicity profiles and only marginally improve the course of the disease and life expectancy. A considerable body of evidence supports the antitumour and apoptotic effects of certain cannabinoids, such as WIN55,212-2, against a wide spectrum of cancer cells, including gliomas. In fact, we previously highlighted the potent cytotoxic activity of the cannabinoid ligand 5 against glioblastoma KNS42 cells. Taken together, in this study, we designed, synthesised, and evaluated several indoles and indole bioisosteres for their antitumour activities. Compounds 8a, 8c, 8f, 12c, and 24d demonstrated significant inhibitory activities against the viability (IC50 = 2.34-9.06 µM) and proliferation (IC50 = 2.88-9.85 µM) of paediatric glioblastoma KNS42 cells. All five compounds further retained their antitumour activities against two atypical teratoid/rhabdoid tumour (AT/RT) cell lines. When tested against a medulloblastoma DAOY cell line, only 8c, 8f, 12c, and 24d maintained their viability inhibitory activities. The viability assay against non-neoplastic human fibroblast HFF1 cells suggested that compounds 8a, 8c, 8f, and 12c act selectively towards the panel of paediatric brain tumour cells. In contrast, compound 24d and WIN55,212-2 were highly toxic toward HFF1 cells. Due to their structural resemblance to known cannabimimetics, the most potent compounds were tested in cannabinoid 1 and 2 receptor (CB1R and CB2R) functional assays. Compounds 8a, 8c, and 12c failed to activate or antagonise both CB1R and CB2R, whereas compounds 8f and 24d antagonised CB1R and CB2R, respectively. We also performed a transcriptional analysis on KNS42 cells treated with our prototype compound 8a and highlighted a set of seven genes that were significantly downregulated. The expression levels of these genes were previously shown to be positively correlated with tumour growth and progression, indicating their implication in the antitumour activity of 8a. Overall, the drug-like and selective antitumour profiles of indole-2-carboxamides 8a, 8c, 8f, and 12c substantiate the versatility of the indole scaffold in cancer drug discovery.

Design, synthesis and evaluation of novel indole-2-carboxamides for growth inhibition of Mycobacterium tuberculosis and paediatric brain tumour cells.

The omnipresent threat of tuberculosis (TB) and the scant treatment options thereof necessitate the development of new antitubercular agents, preferably working via a novel mechanism of action distinct from the current drugs. Various studies identified the mycobacterial membrane protein large 3 transporter (MmpL3) as the target of several classes of compounds, including the indole-2-caboxamides. Herein, several indoleamide analogues were rationally designed, synthesised, and evaluated for their antitubercular and antitumour activities. Compound 8g displayed the highest activity (MIC = 0.32 µM) against the drug-sensitive (DS) Mycobacterium tuberculosis (M. tb) H37Rv strain. This compound also exhibited high selective activity towards M. tb over mammalian cells [IC50 (Vero cells) = 40.9 µM, SI = 128], suggesting its minimal cytotoxicity. In addition, when docked into the MmpL3 active site, 8g adopted a binding profile similar to the indoleamide ligand ICA38. A related compound 8f showed dual antitubercular (MIC = 0.62 µM) and cytotoxic activities against paediatric glioblastoma multiforme (GBM) cell line KNS42 [IC50 (viability) = 0.84 µM]. Compound 8f also showed poor cytotoxic activity against healthy Vero cells (IC50 = 39.9 µM). Compounds 9a and 15, which were inactive against M. tb, showed potent cytotoxic (IC50 = 8.25 and 5.04 µM, respectively) and antiproliferative activities (IC50 = 9.85 and 6.62 µM, respectively) against KNS42 cells. Transcriptional analysis of KNS42 cells treated with compound 15 revealed a significant downregulation in the expression of the carbonic anhydrase 9 (CA9) and the spleen tyrosine kinase (SYK) genes. The expression levels of these genes in GBM tumours were previously shown to contribute to tumour progression, suggesting their involvement in our observed antitumour activities. Compounds 9a and 15 were selected for further evaluations against three different paediatric brain tumour cell lines (BT12, BT16 and DAOY) and non-neoplastic human fibroblast cells HFF1. Compound 9a showed remarkable cytotoxic (IC50 = 0.89 and 1.81 µM, respectively) and antiproliferative activities (IC50 = 7.44 and 6.06 µM, respectively) against the two tested atypical teratoid/rhabdoid tumour (AT/RT) cells BT12 and BT16. Interestingly, compound 9a was not cytotoxic when tested against non-neoplastic HFF1 cells [IC50 (viability) = 119 µM]. This suggests that an indoleamide scaffold can be fine-tuned to confer a set of derivatives with selective antitubercular and/or antitumour activities.

Evaluation of Protein Kinase Inhibitors with PLK4 Cross-Over Potential in a Pre-Clinical Model of Cancer.

Polo-like kinase 4 (PLK4) is a cell cycle-regulated protein kinase (PK) recruited at the centrosome in dividing cells. Its overexpression triggers centrosome amplification, which is associated with genetic instability and carcinogenesis. In previous work, we established that PLK4 is overexpressed in pediatric embryonal brain tumors (EBT). We also demonstrated that PLK4 inhibition exerted a cytostatic effect in EBT cells. Here, we examined an array of PK inhibitors (CFI-400945, CFI-400437, centrinone, centrinone-B, R-1530, axitinib, KW-2449, and alisertib) for their potential crossover to PLK4 by comparative structural docking and activity inhibition in multiple established embryonal tumor cell lines (MON, BT-12, BT-16, DAOY, D283). Our analyses demonstrated that: (1) CFI-400437 had the greatest impact overall, but similar to CFI-400945, it is not optimal for brain exposure. Also, their phenotypic anti-cancer impact may, in part, be a consequence of the inhibition of Aurora kinases (AURKs). (2) Centrinone and centrinone B are the most selective PLK4 inhibitors but they are the least likely to penetrate the brain. (3) KW-2449, R-1530 and axitinib are the ones predicted to have moderate-to-good brain penetration. In conclusion, a new selective PLK4 inhibitor with favorable physiochemical properties for optimal brain exposure can be beneficial for the treatment of EBT.

Polo-Like Kinase 4 (PLK4) Is Overexpressed in Central Nervous System Neuroblastoma (CNS-NB).

Neuroblastoma (NB) is the most common extracranial solid tumor in pediatrics, with rare occurrences of primary and metastatic tumors in the central nervous system (CNS). We previously reported the overexpression of the polo-like kinase 4 (PLK4) in embryonal brain tumors. PLK4 has also been found to be overexpressed in a variety of peripheral adult tumors and recently in peripheral NB. Here, we investigated PLK4 expression in NBs of the CNS (CNS-NB) and validated our findings by performing a multi-platform transcriptomic meta-analysis using publicly available data. We evaluated the PLK4 expression by quantitative real-time PCR (qRT-PCR) on the CNS-NB samples and compared the relative expression levels among other embryonal and non-embryonal brain tumors. The relative PLK4 expression levels of the NB samples were found to be significantly higher than the non-embryonal brain tumors (p-value < 0.0001 in both our samples and in public databases). Here, we expand upon our previous work that detected PLK4 overexpression in pediatric embryonal tumors to include CNS-NB. As we previously reported, inhibiting PLK4 in embryonal tumors led to decreased tumor cell proliferation, survival, invasion and migration in vitro and tumor growth in vivo, and therefore PLK4 may be a potential new therapeutic approach to CNS-NB.

A functional screening of the kinome identifies the Polo-like kinase 4 as a potential therapeutic target for malignant rhabdoid tumors, and possibly, other embryonal tumors of the brain.

PURPOSE: Malignant rhabdoid tumors (MRTs) are deadly embryonal tumors of the infancy. With poor survival and modest response to available therapies, more effective and less toxic treatments are needed. We hypothesized that a systematic screening of the kinome will reveal kinases that drive rhabdoid tumors and can be targeted by specific inhibitors. METHODS: We individually mutated 160 kinases in a well-characterized rhabdoid tumor cell line (MON) using lentiviral clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The kinase that most significantly impaired cell growth was further validated. Its expression was evaluated by microarray gene expression (GE) within 111 pediatric tumors, and functional assays were performed. A small molecule inhibitor was tested in multiple rhabdoid tumor cell lines and its toxicity evaluated in zebrafish larvae. RESULTS: The Polo-like kinase 4 (PLK4) was identified as the kinase that resulted in higher impairment of cell proliferation when mutated by CRISPR/Cas9. PLK4 CRISPR-mutated rhabdoid cells demonstrated significant decrease in proliferation, viability, and survival. GE showed upregulation of PLK4 in rhabdoid tumors and other embryonal tumors of the brain. The PLK4 inhibitor CFI-400945 showed cytotoxic effects on rhabdoid tumor cell lines while sparing non-neoplastic human fibroblasts and developing zebrafish larvae. CONCLUSIONS: Our findings indicate that rhabdoid tumor cell proliferation is highly dependent on PLK4 and suggest that targeting PLK4 with small-molecule inhibitors may hold a novel strategy for the treatment of MRT and possibly other embryonal tumors of the brain. This is the first time that PLK4 has been described as a potential target for both brain and pediatric tumors.

Inhibition of polo-like kinase 4 (PLK4): a new therapeutic option for rhabdoid tumors and pediatric medulloblastoma.

Rhabdoid tumors (RT) are highly aggressive and vastly unresponsive embryonal tumors. They are the most common malignant CNS tumors in infants below 6 months of age. Medulloblastomas (MB) are embryonal tumors that arise in the cerebellum and are the most frequent pediatric malignant brain tumors. Despite the advances in recent years, especially for the most favorable molecular subtypes of MB, the prognosis of patients with embryonal tumors remains modest with treatment related toxicity dreadfully high. Therefore, new targeted therapies are needed. The polo-like kinase 4 (PLK4) is a critical regulator of centriole duplication and consequently, mitotic progression. We previously established that PLK4 is overexpressed in RT and MB. We also demonstrated that inhibiting PLK4 with a small molecule inhibitor resulted in impairment of proliferation, survival, migration and invasion of RT cells. Here, we showed in MB the same effects that we previously described for RT. We also demonstrated that PLK4 inhibition induced apoptosis, senescence and polyploidy in RT and MB cells, thereby increasing the susceptibility of cancer cells to DNA-damaging agents. In order to test the hypothesis that PLK4 is a CNS druggable target, we demonstrated efficacy with oral administration to an orthotropic xenograft model. Based on these results, we postulate that targeting PLK4 with small-molecule inhibitors could be a novel strategy for the treatment of RT and MB and that PLK4 inhibitors (PLK4i) might be promising agents to be used solo or in combination with cytotoxic agents.

The F type mitochondrial genome of the scorched mussel: Brachidontes exustus, (Mytiloida, Mytilidae).

The nominal species Brachidontes exustus (Linnaeus, 1758) is a cryptic complex. Long polymerase chain reactions and direct sequencing by primer walking was used to determine the complete F type mitochondrial genome of the Gulf of Mexico clade. The genome is 16,600 bp long and contains a single large unassigned presumptive control region, 13 protein-coding genes, 23 tRNA genes, and 2 rRNA genes, all coded for on the heavy chain. As in many other bivalves, there is the addition of tRNA-Met(AUA). The gene order is different from all other mitogenomes known for the family. The B. exustus mitogenome will contribute to a better understanding of the evolutionary history and phylogenetic relationships of the Mytilidae.