DNA-damaging agents are among the most frequently used anticancer drugs. However, they provide only modest benefit in most cancers. This may be attributed to a genome maintenance network, the DNA damage response (DDR), that recognizes and repairs damaged DNA. ATR is a major regulator of the DDR and an attractive anticancer target. Herein, we describe the discovery of a series of aminopyrazines with potent and selective ATR inhibition. Compound 45 inhibits ATR with a K(i) of 6 nM, shows >600-fold selectivity over related kinases ATM or DNA-PK, and blocks ATR signaling in cells with an IC(50) of 0.42 μM. Using this compound, we show that ATR inhibition markedly enhances death induced by DNA-damaging agents in certain cancers but not normal cells. This differential response between cancer and normal cells highlights the great potential for ATR inhibition as a novel mechanism to dramatically increase the efficacy of many established drugs and ionizing radiation.
Background and purpose: The ATP-gated P2X 7 receptor has been shown to play a role in several inflammatory processes, making it an attractive target for anti-inflammatory drug discovery. We have recently identified a novel set of cyclic imide compounds that inhibited P2X 7 receptor-mediated dye uptake in human macrophage THP-1 cells. In this study the actions and selectivity of one of these compounds, AZ11645373, were characterized. Experimental approach: We measured membrane currents, calcium influx, and YOPRO-1 uptake from HEK cells expressing individual P2X receptors, and YOPRO1 uptake and interleukin-1b release from THP-1 cells in response to ATP and the ATP analogue benzoylbenzoyl ATP (BzATP). Key results: AZ11645373 up to 10 mM, had no agonist or antagonist actions on membrane currents due to P2X receptor activation at human P2X 1 , rat P2X 2 , human P2X 3 , rat P2X 2/3 , human P2X 4 , or human P2X 5 receptors expressed in HEK cells. AZ11645373 inhibited human P2X 7 receptor responses in HEK cells in a non-surmountable manner with K B values ranging from 5 -20 nM, with mean values not significantly different between assays. K B values were not altered by removing extracellular calcium and magnesium. ATP-evoked IL-1b release from lipopolysaccharide-activated THP-1 cells was inhibited by AZ11645373, IC 50 ¼ 90 nM. AZ11645373 was 4 500-fold less effective at inhibiting rat P2X 7 receptor-mediated currents with less than 50% inhibition occurring at 10 mM. Conclusions and implications: AZ11645373 is a highly selective and potent antagonist at human but not rat P2X 7 receptors and will have much practical value in studies of human cells.
A linear synthesis of the indole alkaloid (+/-)-akuammicine (2) was completed by a novel sequence of reactions requiring only 10 steps from commercially available starting materials. The approach features a tandem vinylogous Mannich addition and an intramolecular hetero Diels-Alder reaction to rapidly assemble the pentacyclic heteroyohimboid derivative 8 from the readily available hydrocarboline 6. Oxidation of the E ring of 8 gave the lactone 9 that was converted into deformylgeissoschizine (11). The subsequent elaboration of 11 into 2 was effected by a biomimetically patterned transformation that involved sequential oxidation and base-induced skeletal reorganization. A variation of these tactics was then applied to the synthesis of the C(18) hydroxylated akuammicine derivative 36. Because 36 had previously been converted into strychnine (1) in four steps, its preparation constitutes a concise, formal synthesis of this complex alkaloid.
Choline kinase alpha (ChoKα) is regarded as an attractive cancer target. The enzyme catalyses the formation of phosphocholine (PCho), an important precursor in the generation of phospholipids essential for cell growth. ChoKα has oncogenic properties and is critical for the survival of cancer cells. Overexpression of the ChoKα protein can transform noncancer cells into cells with a cancerous phenotype, and depletion of the ChoKα protein can result in cancer cell death. However, the mechanisms underlying the tumourigenic properties of ChoKα are not fully understood. ChoKα was recently demonstrated to associate with other oncogenic proteins, raising the possibility that a non-catalytic protein scaffolding function drives the tumourigenic properties of ChoKα rather than a catalytic function. In order to differentiate these two roles, we compared the impact on cancer cell survival using two tools specific for ChoKα: (1) small interfering RNA (siRNA) to knockdown the ChoKα protein levels; and (2) compound V-11-0711, a novel potent and selective ChoKα inhibitor (ChoKα IC50 20 nℳ), to impede the catalytic activity. Both treatments targeted the endogenous ChoKα protein in HeLa cells, as demonstrated by a substantial reduction in the PCho levels. siRNA knockdown of the ChoKα protein in HeLa cells resulted in significant cell death through apoptosis. In contrast, compound V-11-0711 caused a reversible growth arrest. This suggests that inhibition of ChoKα catalytic activity alone is not sufficient to kill cancer cells, and leads us to conclude that there is a role for the ChoKα protein in promoting cancer cell survival that is independent of its catalytic activity.
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