A simple end-point assay for calcium channel activity

Chandrika, Arunkumar Renganathan; Steephan, Mathew; Nair, Rajeevkumar Raveendran; Devi, Suma Priya Sudarsana; Kumar, Manish; Paul, Soumya; Mayadevi, Madhavan; Omkumar, Ramakrishnapillai V.

doi:10.1016/j.ceca.2018.05.009

Cited by 5 publications

(16 citation statements)

References 48 publications

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“…The effect of MTDLs on NMDAR activity was evaluated using a cell-based assay system, in which the appearance of fluorescent punctate pattern occurred during the activation of NMDAR by its agonists and calcium (Ca 2+ ) ( Fig. 2E (i)) [66]. The punctate appearance was not observed when NMDAR was treated with its agonists in the absence of Ca 2+ ( Fig.…”

Section: Improved Antagonistic Potential Of Tacrine Derived Mtdls Towmentioning

confidence: 99%

“…To check the effect of MTDLs on NMDAR activity a cell-based assay system that works on the basis of protein-protein interaction between NMDAR and α-CaMKII was used [66]. The plasmids coding for NMDAR subunits, GluN1 and GluN2B, and α-CaMKII tagged with GFP (GFP-α-CaMKII) were co-transfected into HEK-293 cells and the activity of GluN2B containing NMDAR was detected.…”

Section: Effect Of Mtdls On Nmda Receptor Activitymentioning

confidence: 99%

“…The plasmids for mammalian expression carrying the cDNAs for GluN1, GluN2B and GFP-α-CaMKII were prepared from bacterial cell using QIAGEN Midi Kit (Qiagen, USA). The plasmids were quantified using NanoDrop 2000 and their purity was checked on 1% agarose gel as reported previously [66]. These plasmids were further co-transfected into pre-grown HEK Final values were obtained from three independent experiments.…”

Section: Preparation and Transfection Of Plasmids Coding For Glun1 Gmentioning

confidence: 99%

“…In order to study the effect of MTDLs on the interaction of GFP-α-CaMKII and GluN2B, HEK-293 cells stably expressing GFP-α-CaMKII and a construct having the GluN2B motif which binds to α-CaMKII, tagged with mitochondrial localization signal (MLS-NR2B) was used [66]. Since there are no functional NMDAR channels in the membrane, GFP-α-CaMKII was activated by Ca 2+ influx through ionomycin, a Ca 2+ ionophore.…”

Section: Effect Of Mtdls On Interaction Between Gfp-α-camkii and Glun2bmentioning

confidence: 99%

“…This reduction in punctae would not be due to the blocking of NMDAR channel activity. To test whether the compounds have any effect on the interaction of GluN2B and α-CaMKII, an experiment was carried out using HEK-293 cells stably expressing GFP-α-CaMKII and GluN2B sequence [66]. Formation of perinuclear punctae by treatment with ionomycin and Ca 2+ , is indicative of interaction between CaMKII and GluN2B.…”

Section: Mtdls Do Not Interfere With the Interaction Between Glun2b And α-Camkiimentioning

confidence: 99%

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Neuroprotective derivatives of tacrine that target NMDA receptor and acetyl cholinesterase - Design, synthesis and biological evaluation

Remya

Vijayan

Reddy

et al. 2021

Preprint

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The complex and multifactorial nature of neuropsychiatric diseases demands multi-target drugs that can intervene with various sub-pathologies underlying disease progression. Targeting the impairments in cholinergic and glutamatergic neurotransmissions with small molecules has been suggested as one of the potential disease-modifying approaches for Alzheimer’s disease (AD). Tacrine, apotent inhibitor of acetylcholinesterase (AChE) is the first FDA approved drug for the treatment of AD. Tacrine is also a low affinity antagonist of N-methyl-D-aspartate receptor (NMDAR). However, tacrine was withdrawn from its clinical use later due to its hepato-toxicity. With an aim to develop novel high affinity multi-target directed ligands (MTDLs) against AChE and NMDAR, with reduced hepatotoxicity, we performed in silico structure-based modifications on tacrine, chemical synthesis of the derivatives and in vitro validation of their activities. Nineteen such derivatives showed inhibition with IC50 values in the range of 18.53±2.09 to 184.09±19.23 nM against AChE and 0.27±0.05 to 38.84±9.64 μM against NMDAR. Some of the selected compounds also protected rat primary cortical neurons from glutamate induced excitotoxicity. Two of the tacrine derived MTDLs, 201 and 208 exhibited in vivo efficacy in rats by protecting against behavioral impairment induced by administration of the excitotoxic agent, monosodium glutamate. Additionally, several of these synthesized compounds also exhibited promising inhibitory activities against butyrylcholinesterase and β-secretase. Given the therapeutic potential of MTDLs in disease-modifying therapy, our studies revealed several promising MTDLs of which 201 appears to be a potential candidate for immediate preclinical and clinical evaluations.

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Section: Improved Antagonistic Potential Of Tacrine Derived Mtdls Towmentioning

confidence: 99%

Section: Effect Of Mtdls On Nmda Receptor Activitymentioning

confidence: 99%

Section: Preparation and Transfection Of Plasmids Coding For Glun1 Gmentioning

confidence: 99%

Section: Effect Of Mtdls On Interaction Between Gfp-α-camkii and Glun2bmentioning

confidence: 99%

Section: Mtdls Do Not Interfere With the Interaction Between Glun2b And α-Camkiimentioning

confidence: 99%

See 3 more Smart Citations

Neuroprotective derivatives of tacrine that target NMDA receptor and acetyl cholinesterase - Design, synthesis and biological evaluation

Remya

Vijayan

Reddy

et al. 2021

Preprint

Self Cite

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Lobeline: A multifunctional alkaloid modulates cholinergic and glutamatergic activities

et al. 2023

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Developing drugs for Alzheimer's disease (AD) is an extremely challenging task due to its devastating pathology. Previous studies have indicated that natural compounds play a crucial role as lead molecules in the development of drugs. Even though, there are remarkable technological advancements in the isolation and synthesis of natural compounds, the targets for many of them are still unknown. In the present study, lobeline, a piperidine alkaloid has been identified as a cholinesterase inhibitor through chemical similarity assisted target fishing method. The structural similarities between lobeline and donepezil, a known acetylcholinesterase (AChE) inhibitor encouraged us to hypothesize that lobeline may also exhibit AChE inhibitory properties. It was further confirmed by in silico, in vitro and biophysical studies that lobeline could inhibit cholinesterase. The binding profiles indicated that lobeline has a higher affinity for AChE than BChE. Since excitotoxicity is one of the major pathological events associated with AD progression, we also investigated the neuroprotective potential of lobeline against glutamate mediated excitotoxicity in rat primary cortical neurons. The cell based NMDA receptor (NMDAR) assay with lobeline suggested that neuroprotective potential of lobeline is mediated through the blockade of NMDAR activity.

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