A simple end-point assay for calcium channel activity.

Cellular calcium signaling events are transient. Hence they are observed in real time using fluorescence imaging or electrophysiological methods that require sophisticated instrumentation and specialized skills. For high throughput assays simple and inexpensive techniques are desirable. Many calcium channels that serve as drug targets have subtypes arising from diverse subunit combinations. These need to be targeted selectively for achieving efficacy and for avoiding side effects in therapies. This in turn increases the number of calcium channels that act as drug targets. We report a novel method for intracellular calcium sensing that utilizes the calcium dependent stable interaction between CaM kinase II (CaMKII) and its ligands such as the NMDA receptor subunit GluN2B. The CaMKII-GluN2B complex formed persists as a memory of the transient increase in calcium. In a cell-based assay system GFP-α-CaMKII expressed in the cytosol responds to calcium by translocating towards GluN2B sequence motif exogenously expressed on mitochondria or endoplasmic reticulum. The resulting punctate fluorescence pattern serves as the signal for intracellular calcium release. The pattern is stable, unaffected by sample processing and is observable without real time imaging. The activities of calcium channel proteins heterologously expressed in HEK-293 cells were detected with specificity using this technique. A calcium sensor vector and a calcium sensor cell line were developed as tools to perform this technique. This technique being simple and less expensive could significantly facilitate high throughput screening in calcium channel drug discovery.

Hesperetin and Naringenin sensitize HER2 positive cancer cells to death by serving as HER2 Tyrosine Kinase inhibitors.

AIM: Aberrant human epidermal growth factor receptor-2 (HER2) expression and constitutive mutant activation of its tyrosine kinase domain account for tumor aggression and therapy resistance in many types of cancers with major share in breast cancer cases. HER2 specific treatment modalities still face challenges owing to the side effects and acquired resistance of available therapeutics. Recently, the anti-proliferative and pro-apoptotic potential of phytochemicals, especially of flavonoids have become increasingly appreciated as powerful chemo preventive agents. Consequently, the major goal of our study is to identify flavonoids capable of inhibiting HER2 Tyrosine Kinase (HER2-TK) activity and validate their anti-tumor activity against HER2 positive tumors. MAIN METHODS: Molecular docking studies for identifying flavonoids binding at HER2 kinase domain, ADP-Glo™ Kinase Assay for determining kinase activity, MTT assay to measure growth inhibition, various apoptotic assays and cell cycle analysis by FACS were performed. KEY FINDINGS: Among the flavonoids screened, Naringenin (NG) and Hesperetin (HP) possessed high glide scores from molecular docking studies of enzyme-inhibitor mode. The interaction analysis revealed their ability to establish stable and strong interaction at the ATP binding site of HER2-TK. These compounds also inhibited in vitro HER2-TK activity suggesting their role as HER2 inhibitors. The study also unraveled the anti-proliferative, pro-apoptotic and anti-cancerous activity of these flavonoids against HER2 positive breast cancer cell line. SIGNIFICANCE: The study identified two citrus fruit flavonoids, NG and HP as HER2-TK inhibitors and this is the first report on their potential to target preferentially and sensitize HER2 positive cancer cells to cell death.

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II.

Ca(2+) influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca(2+)/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo. Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca(2+)/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein-alpha-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione-S-transferase pull-down assay. Thr286-autophosphorylated alpha-CaMKII or the autophosphorylation mimicking mutant, T286D-alpha-CaMKII, binds NR2B sequence independent of Ca(2+)/calmodulin unlike native wild-type alpha-CaMKII. We show enhancement of this binding by Ca(2+)/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca(2+)/calmodulin-independent binding whereas it allows the Ca(2+)/calmodulin-dependent binding of alpha-CaMKII in vitro. Similarly, the autonomously active mutants, T286D-alpha-CaMKII and F293E/N294D-alpha-CaMKII, exhibited Ca(2+)-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.