Unveiling the molecular basis of lobeline's allosteric regulation of NMDAR: insights from molecular modeling.

Neurological and psychiatric disorders contribute significantly to the global disease burden, adversely affecting the quality of life for both patients and their families. Impaired glutamatergic signaling is considered to be a major cause for most of the neurological and psychiatric disorders. Glutamate receptors are over activated in excitotoxic conditions, leading to dysregulation of Ca2+ homeostasis, triggering the production of free radicals and oxidative stress, mitochondrial dysfunction and eventually cell death. Excitotoxicity primarily results from the overactivity of NMDARs, a subtype of ionotropic glutamate receptors, due to their pronounced Ca2+ permeability and conductance characteristics. NMDAR antagonists are suggested to have therapeutic use as they can prevent excitotoxicity. Our previous studies demonstrated lobeline, an alkaloid, exerts neuroprotective action in excitotoxic conditions by blocking NMDAR. However, the atomic level interactions of lobeline with NMDAR was not characterized yet. Structural comparison of lobeline with a known NMDAR antagonist ifenprodil, followed by molecular docking and dynamics simulations revealed that lobeline could bind to the ifenprodil binding site i.e., in the heterodimer interface of GluN1-GluN2B subunits and exert ifenprodil like activities. By in silico structure guided modifications on lobeline and subsequent free energy calculations, we propose putative NMDAR antagonists derived from lobeline.

Assays for L-type voltage gated calcium channels.

Voltage gated calcium channels (VGCCs) are pursued as drug targets for neurodegenerative and cardiovascular diseases. High throughput drug screening targeting VGCCs depends on patch-clamp electrophysiology or fluorophore-based calcium imaging that requires powerful equipment and specialized expertise thus leading to cost escalation. Moreover, VGCC needs to be transfected into cell lines such as HEK-293. We report the presence of L-type VGCC (L-VGCC) subunit proteins, Cav1.2, α2δ and ß in HEK-293 cells and the application of simple methods for its assay. Endogenous expression of the channel in HEK-293 cells overcomes the need for transfection. L-VGCC in HEK-293 cells was activated either by the agonist, BayK8644 or by KCl-mediated depolarization. Activity was detected using the calcium sensing probe, GCaMP6m by live imaging. L-VGCC activity induced enhancement in GCaMP6m fluorescence returned to baseline corresponding to channel-closure. Activity was also shown using a methodology involving end-point detection of the calcium dependent interaction of α-CaMKII with NMDA receptor subunit GluN2B sequence. This methodology further simplifies the assay as it eliminates the need for real time imaging. Activation was blocked by the specific L-type VGCC antagonist, nifedipine. Finding the protein and activity of L-VGCC in HEK-293 cells offers commercially viable assays for drug screening.

Role of Ca2+/Calmodulin-Dependent Protein Kinase Type II in Mediating Function and Dysfunction at Glutamatergic Synapses.

Glutamatergic synapses harbor abundant amounts of the multifunctional Ca2+/calmodulin-dependent protein kinase type II (CaMKII). Both in the postsynaptic density as well as in the cytosolic compartment of postsynaptic terminals, CaMKII plays major roles. In addition to its Ca2+-stimulated kinase activity, it can also bind to a variety of membrane proteins at the synapse and thus exert spatially restricted activity. The abundance of CaMKII in glutamatergic synapse is akin to scaffolding proteins although its prominent function still appears to be that of a kinase. The multimeric structure of CaMKII also confers several functional capabilities on the enzyme. The versatility of the enzyme has prompted hypotheses proposing several roles for the enzyme such as Ca2+ signal transduction, memory molecule function and scaffolding. The article will review the multiple roles played by CaMKII in glutamatergic synapses and how they are affected in disease conditions.

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

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, a potent 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 hepatotoxicity. 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 - 184.09 ± 19.23 nM against AChE and 0.27 ± 0.05 - 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 activitiy against butyrylcholinesterase. MTDL-201 was also devoid of hepatotoxicity in vivo. Given the therapeutic potential of MTDLs in disease-modifying therapy, our studies revealed several promising MTDLs among which 201 appears to be a potential candidate for immediate preclinical evaluations.

Chemical similarity assisted search for acetylcholinesterase inhibitors: Molecular modeling and evaluation of their neuroprotective properties.

Alzheimer's disease (AD) is an obstinate and progressive neurodegenerative disorder, mainly characterized by cognitive decline. Increasing number of AD patients and the lack of promising treatment strategies demands novel therapeutic agents to combat various disease pathologies in AD. Recent progresses in understanding molecular mechanisms in AD helped researchers to streamline the various therapeutic approaches. Inhibiting acetylcholinesterase (AChE) activity has emerged as one of the potential treatment strategies. The present study discusses the identification of two potent AChE inhibitors (ZINC11709541 and ZINC11996936) from ZINC database through conventional in silico approaches and their in vitro validations. These inhibitors have strong preferences towards AChE than butyrylcholinesterase (BChE) and didn't evoke any significant reduction in the cell viability of HEK-293 cells and primary cortical neurons. Furthermore, promising neuroprotective properties has also been displayed against glutamate induced excitotoxicity in primary cortical neurons. The present study proposes two potential drug lead compounds for the treatment of AD, that can be used for further studies and preclinical evaluation.

Cellular calcium signaling in the aging brain.

Aging in the biological system is an irreversible process. In the initial stages of lifespan aging improves survival skills of an organism while in the later stages aging reduce the survival skills. Aging is associated with changes in several cellular and molecular functions among which calcium signaling is a prominent one. Calcium signaling is essential for many vital functions of the brain and even minor impairments in calcium signaling can lead to deleterious consequences including neuronal death. Calcium signaling proteins are pursued as promising drug targets for many aging related diseases. This review attempts to summarize changes in calcium signaling in the brain as a result of aging.

Novel tacrine derivatives exhibiting improved acetylcholinesterase inhibition: Design, synthesis and biological evaluation.

A novel series of twenty four tacrine derivatives were designed and synthesised. Among these, thirteen were taken for the acetylcholinesterase (AChE) inhibition studies. Three compounds such as 4c, 6c and 6f were found to possess significant AChE inhibitory properties with IC50 values 12.97 ± 0.47 nM, 5.17 ± 0.24 nM and 7.14 ± 0.78 nM respectively. In silico docking studies revealed that these compounds can bind strongly in the active site of the enzyme and prevent enzyme-substrate interactions. On binding, the substituted groups were oriented either towards the peripheral anionic site (PAS) (Pocket A) or towards a hydrophobic cavity (pocket B) located near the active site. The cytotoxicity and hepatotoxicity of the compounds were tested using HEK-293 and HepG2 cell lines respectively. The compound 4c did not show any significant decrease in the cell viability even at a concentration of 300 µM indicating that its cytotoxicity and hepatotoxicity are significantly lesser compared to tacrine, due to the chemical modification. Based on the available results, it can be suggested that the compound 4c might be a potential drug lead compound with AChE inhibitory activity. However, further pharmacokinetic studies are necessary to comment on the efficacy of the compound as a drug for AD.

New insights of superoxide dismutase inhibition of pyrogallol autoxidation.

Autoxidation of pyrogallol in alkaline medium is characterized by increases in oxygen consumption, absorbance at 440 nm, and absorbance at 600 nm. The primary products are H2O2 by reduction of O2 and pyrogallol-ortho-quinone by oxidation of pyrogallol. About 20 % of the consumed oxygen was used for ring opening leading to the bicyclic product, purpurogallin-quinone (PPQ). The absorbance peak at 440 nm representing the quinone end-products increased throughout at a constant rate. Prolonged incubation of pyrogallol in alkali yielded a product with ESR signal. In contrast the absorbance peak at 600 nm increased to a maximum and then declined after oxygen consumption ceased. This represents quinhydrone charge-transfer complexes as similar peak instantly appeared on mixing pyrogallol with benzoquinones, and these were ESR-silent. Superoxide dismutase inhibition of pyrogallol autoxidation spared the substrates, pyrogallol, and oxygen, indicating that an early step is the target. The SOD concentration-dependent extent of decrease in the autoxidation rate remained the same regardless of higher control rates at pyrogallol concentrations above 0.2 mM. This gave the clue that SOD is catalyzing a reaction that annuls the forward electron transfer step that produces superoxide and pyrogallol-semiquinone, both oxygen radicals. By dismutating these oxygen radicals, an action it is known for, SOD can reverse autoxidation, echoing the reported proposal of superoxide:semiquinone oxidoreductase activity for SOD. The following insights emerged out of these studies. The end-product of pyrogallol autoxidation is PPQ, and not purpurogallin. The quinone products instantly form quinhydrone complexes. These decompose into undefined humic acid-like complexes as late products after cessation of oxygen consumption. SOD catalyzes reversal of autoxidation manifesting as its inhibition. SOD saves catechols from autoxidation and extends their bioavailability.

Curcumin is an inhibitor of calcium/calmodulin dependent protein kinase II.

Calcium/calmodulin dependent protein kinase II (CaMKII) is involved in the mechanisms underlying higher order brain functions such as learning and memory. CaMKII participates in pathological glutamate signaling also, since it is activated by calcium influx through the N-methyl-d-aspartate type glutamate receptor (NMDAR). In our attempt to identify phytomodulators of CaMKII, we observed that curcumin, a constituent of turmeric and its analogs inhibit the Ca(2+)-dependent and independent kinase activities of CaMKII. We further report that a heterocyclic analog of curcumin I, (3,5-bis[ß-(4-hydroxy-3-methoxyphenyl)ethenyl]pyrazole), named as pyrazole-curcumin, is a more potent inhibitor of CaMKII than curcumin. Microwave assisted, rapid synthesis of curcumin I and its heterocyclic analogues is also reported.

Regulation of phosphorylation at Ser(1303) of GluN2B receptor in the postsynaptic density.

Neuronal N-methyl-D-aspartate subtype of ionotropic glutamate receptor (NMDAR) that plays essential roles in excitatory synaptic transmission is regulated by phosphorylation. However, the kinases and phosphatases involved in this regulation are not completely known. We show that the GluN2B subunit of NMDAR is phosphorylated at Ser(1303) by protein kinase C (PKC) and is dephosphorylated by protein phosphatase 1 (PP1), but not protein phosphatase 2A (PP2A) in isolated postsynaptic density (PSD). Although PSD is known to harbor PKC, PP1 and PP2A, their ability to regulate phosphorylation of GluN2B-Ser(1303) would depend on the accessibility of GluN2B-Ser(1303) to these proteins. Since PSD preparation is likely to maintain the organization of its component proteins as inside neurons, accessibility of kinases and phosphatases to GluN2B-Ser(1303)in vivo would be addressed by experiments using this system. Using an antibody specific for the phosphorylated state of GluN2B-Ser(1303) we demonstrate that PP1 is the major phosphatase in rat brain PSD that can dephosphorylate the GluN2B-Ser(1303) endogenous to PSD. We also show that PKC present in PSD can phosphorylate GluN2B-Ser(1303). The events reported here might be important in regulating GluN2B-Ser(1303) phosphorylation in vivo.

Growth of gold nanoparticles in human cells.

Gold nanoparticles of 20-100 nm diameter were synthesized within HEK-293 (human embryonic kidney), HeLa (human cervical cancer), SiHa (human cervical cancer), and SKNSH (human neuroblastoma) cells. Incubation of 1 mM tetrachloroaurate solution, prepared in phosphate buffered saline (PBS), pH 7.4, with human cells grown to approximately 80% confluency yielded systematic growth of nanoparticles over a period of 96 h. The cells, stained due to nanoparticle growth, were adherent to the bottom of the wells of the tissue culture plates, with their morphology preserved, indicating that the cell membrane was intact. Transmission electron microscopy of ultrathin sections showed the presence of nanoparticles within the cytoplasm and in the nucleus, the latter being much smaller in dimension. Scanning near field microscopic images confirmed the growth of large particles within the cytoplasm. Normal cells gave UV-visible signatures of higher intensity than the cancer cells. Differences in the cellular metabolism of cancer and noncancer cells were manifested, presumably in their ability to carry out the reduction process.

The C-terminus of CaMKII is truncated when expressed in E. coli.

The neuronal enzyme Calcium/calmodulin dependent protein kinase type II (CaMKII) is a key molecule in biochemical events necessary for learning and memory. The alpha-subunit of CaMKII expressed in E. coli as well as in insect cells shows similar catalytic behavior [Praseeda, M., Pradeep, K. K., Krupa, A., Sri Krishna, S., Leena, S., Rajeev Kumar, R., John Cheriyan, Mayadevi, M., Srinivasan, N., and Omkumar, R. V. (2003) Biochem. J. In Press]. The association domain of the enzyme has been crystallized in its native multimeric form after expression in E. coli [Hoelz, A., Nairn, A. C. and Kuriyan, J. (2003) Molecular Cell 11, 1241]. However a major truncation product accompanies the full-length protein when expressed in E. coli. We show by epitope labeling and immunoblotting that the truncation occurs at the C-terminal half of the protein so that the N-terminal catalytic domain is complete in the truncated product. This supports the use of the preparation of alpha-CaMKII expressed in E. coli for studies on functions of the catalytic site. Our data will also be helpful in designing modified prokaryotic expression systems for CaMKII devoid of the trun-cation product, which are easier to use compared to the insect cell system.

Interaction of peptide substrate outside the active site influences catalysis by CaMKII.

The interaction of calcium/calmodulin-dependent protein kinase II (CaMKII) with the NR2B subunit of N-methyl-D-aspartate-type glutamate receptor is thought to be one of the important events leading to synaptic plasticity. CaMKII binds NR2B by its catalytic site and by the autophosphorylation site binding pocket (APBP), a non-catalytic site. Mutagenesis of Glu-236, a residue in the APBP of CaMKII that is likely to be interacting with NR2B, influences phosphorylation of NR2B. The phosphorylation of syntide-2, a classical catalytic site substrate of CaMKII, is influenced to a much lesser extent by this mutation. Taken together these results indicate that interaction of NR2B at the non-catalytic site of CaMKII influences catalysis. Our data suggest that kinetic models of peptide substrate phosphorylation by CaMKII should incorporate the non-catalytic mode of binding of peptides that is dependent on the sequence of the peptide.

Sequence determinants on the NR2A and NR2B subunits of NMDA receptor responsible for specificity of phosphorylation by CaMKII.

Calcium/calmodulin-dependent protein kinase type II (CaMKII) and NMDA-type glutamate receptor (NMDAR) are neuronal proteins involved in learning and memory. CaMKII binds to the NR2B subunit of NMDAR in more than one mode, a stable association involving a noncatalytic site on CaMKII and an enzyme-substrate mode of interaction by its catalytic site. The latter binding results in phosphorylation of serine-1303 on NR2B. We have investigated this binding by studying the kinetics of phosphorylation of synthetic peptides harboring nested sequences of the phosphorylation site motif. We find that residues 1292-1297 of NR2B enhance the affinity of the catalytic site-mediated binding of CaMKII to the minimal phosphorylation site motif, 1298-1308 of NR2B, as evident from measurements of K(m) values for phosphorylation. However, CaMKII shows decreased affinity towards the closely related NR2A subunit due to an -Ile-Asn- motif present as a natural insertion in the analogous sequence on NR2A.

Identification of a phosphorylation site for calcium/calmodulindependent protein kinase II in the NR2B subunit of the N-methyl-D-aspartate receptor.

The N-methyl-D-aspartate (NMDA) subtype of excitatory glutamate receptors plays critical roles in embryonic and adult synaptic plasticity in the central nervous system. The receptor is a heteromultimer of core subunits, NR1, and one or more regulatory subunits, NR2A-D. Protein phosphorylation can regulate NMDA receptor function (Lieberman, D. N., and Mody, I. (1994) Nature 369, 235-239; Wang, Y. T., and Salter, M. W. (1994) Nature 369, 233-235; Wang, L. -Y., Orser, B. A., Brautigan, D. L., and MacDonald, J. F. (1994) Nature 369, 230-232). Here we identify a major phosphorylation site on subunit NR2B that is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), an abundant protein kinase located at postsynaptic sites in glutamatergic synapses. For the initial identification of the site, we constructed a recombinant fusion protein containing 334 amino acids of the C terminus of the NR2B subunit and phosphorylated it with CaM kinase II in vitro. By peptide mapping, automated sequencing, and mass spectrometry, we identified the major site of phosphorylation on the fusion protein as Ser-383, corresponding to Ser-1303 of full-length NR2B. The Km for phosphorylation of this site in the fusion protein was approximately 50 nM, much lower than that of other known substrates for CaM kinase II, suggesting that the receptor is a high affinity substrate. We show that serine 1303 in the full-length NR2B and/or the cognate site in NR2A is a major site of phosphorylation of the receptor both in the postsynaptic density fraction and in living hippocampal neurons.

Phosphorylation of Ser871 impairs the function of His865 of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase.

The attenuation of catalytic activity that accompanies phosphorylation of Ser871 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) reflects primarily the introduction of negative charge (Omkumar, R. V., Darnay, B. G., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 6810-6814). To investigate how a negative charge at position 871 attenuates activity, we phosphorylated wild-type and mutant HMG-CoA reductases and assayed reduction of the putative intermediate mevaldehyde to mevalonate. We observed attenuated activity when the phosphorylated wild-type enzyme was assayed in the presence or absence of coenzyme A, but not when assayed in the presence of desthio-CoA. These observations recall the behavior of mutant enzyme H865Q, for which coenzyme A inhibits, whereas desthio-CoA stimulates mevaldehyde reduction (Frimpong, K. F., and Rodwell, V. W. (1994) J. Biol. Chem. 269, 11478-11483). Catalysis of mevaldehyde reduction by mutant enzyme H865Q was unaffected by phosphorylation. By contrast, mutant enzymes H860Q and H868Y, in which nearby, but noncatalytic, histidines had been mutated, exhibited wild-type behavior upon phosphorylation. We conclude that the introduction of negative charge at position 871 impairs the function of His865, presumably by a specific electrostatic interaction. We propose a novel mechanism by which phosphorylation regulates activity. Phosphorylation of the terminal serine of the consensus AGxLV(K/R)SHMxxNRS motif of eukaryotic HMG-CoA reductases attenuates activity by impairing the ability of the catalytic histidine to protonate the CoAS- anion formed during the reductive deacylation of HMG-CoA to mevaldehyde.

Modulation of Syrian hamster 3-hydroxy-3-methylglutaryl-CoA reductase activity by phosphorylation. Role of serine 871.

Attenuation of Syrian hamster 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) activity by in vitro phosphorylation was studied using AMP-activated protein kinase and wild-type and mutant forms of HMG-CoA reductase. The only residue of the wild-type enzyme phosphorylated was Ser871. Substrates protected against kinase-mediated attenuation of activity, consistent with substrate-induced conformational changes at the C-terminal region. Although close to the catalytic histidine His865, Ser871 appears to play no direct role in catalysis or substrate recognition. Mutant enzymes S871A, S871H, S871N, and S871Q exhibited from 62-106% of wild-type activity and had wild-type Km values for HMG-CoA and NADPH. Replacement of Ser871 by aspartate or glutamate, but not by glutamine, asparagine, histidine, or tyrosine, severely attenuated activity. Attenuation of catalytic activity that accompanies phosphorylation thus appears to result primarily from the introduction of negative charge, not merely steric hindrance. Other than the wild-type enzyme, only mutant enzyme S871T was phosphorylated, and phosphorylation was accompanied by attenuation of activity. The AMP-activated kinase thus can also phosphorylate threonyl residues.

On the involvement of intramolecular protein disulfide in the irreversible inactivation of 3-hydroxy-3-methylglutaryl-CoA reductase by diallyl disulfide.

Treatment with diallyl disulfide, a constituent of garlic oil, irreversibly inactivated microsomal and a soluble 50 kDa form of HMG-CoA reductase. No radioactivity was found to be protein-bound on treating the soluble enzyme with [35S]diallyl disulfide, indicating the absence of the mixed disulfide of the type allyl-S-S-protein. SDS-PAGE and Western blot analyses of the diallyl-disulfide-treated protein showed no traces of the dimer of the type protein-S-S-protein, but clearly indicated BME-reversible increased mobility, as expected of an intramolecular protein disulfide. The sulfhydryl groups, as measured by alkylation with iodo[2-14C]acetic acid, were found to decrease in the diallyl-disulfide-treated enzyme protein. Tryptic peptide analysis also gave support for the possible presence of disulfide-containing peptides in such a protein. It appears that diallyl disulfide inactivated HMG-CoA reductase by forming an internal protein disulfide that became inaccessible for reduction by DTT, and thereby retaining the inactive state of the enzyme.

Irreversible inactivation of 3-hydroxy-3-methylglutaryl-CoA reductase by H2O2.

A concentration-dependent inactivation of 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase was found on preincubation of rat liver microsomal preparations with H2O2 and at lower concentrations in the presence of KCN which inhibited the contaminating catalase. The inactivation was not affected in the presence quenchers of hydroxyl radicals and singlet oxygen and was also obtained when H2O2 was added during the reaction. HMG-CoA, but not NADPH, partially protected the enzyme from H2O2-inactivation. Even at high concentration DTT was unable to reverse this inactivation. The soluble 50 kDa-enzyme was similarly inactivated by H2O2, and the tryptic-digest of the inactivated protein indicated the presence of a disulfide-containing peptide. The results support the view that H2O2 by directly acting on the catalytic domain possibly converts an active thiol group to an inaccessible disulfide and irreversibly inactivates HMG-CoA reductase.

Feedback-type inhibition of activity of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ubiquinone.

Accompanying the decrease in serum cholesterol and increase in concentration of ubiquinone in liver and its microsomes, the activity, but not the protein, of HMG-CoA reductase decreased in ubiquinone-supplemented rats. A soluble 58-kDa preparation of HMG-CoA reductase was partially inhibited on addition of ubiquinone indicating a possible feedback type of action.