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Hyewhon Rhim

Publications and source records attributed to Hyewhon Rhim.

At least 19 recordsLinked to original sources
PubMed2006

The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn.

The human 5-HT(6) receptor (5-HT(6)R) is one of the latest cloned receptors among the known 5-HT receptors. Its abundant distribution in the limbic region, which participates in the control of mood and emotion and is involved in nervous system diseases such as depression and Alzheimer disease, has caused it to generate much interest. However, the cellular mechanisms of 5-HT(6)R are poorly understood. In the present study we found, using a yeast two-hybrid assay, that the carboxyl-terminal region of 5-HT(6)R interacts with the Fyn-tyrosine kinase. We also determined using a glutathione S-transferase pulldown assay that this interaction was mediated through the SH3 domain of Fyn and confirmed this by co-immunoprecipitation assays in two different transfected cell lines as well as in adult rat brains. Immunocyto(histo)chemistry also showed prominent co-localization between 5-HT(6)R and Fyn in transfected cells and a similar distribution between 5-HT(6)R and Fyn in the rat brain. Based on this interaction, we further examined the modulation of 5-HT(6)R by Fyn and vice versa. In addition, we demonstrated that the activation of 5-HT(6)R activated the extracellular signal-regulated kinase1/2 via an Fyn-dependent pathway. These findings suggest that Fyn may play an important role in 5-HT(6)R- mediated signaling pathways in the central nervous system.

Affect↗
PubMed2006

Lead discovery and optimization of T-type calcium channel blockers.

A series of compounds were designed as T-type calcium channel blocker containing 6 or 5 pharmacophore features from structure-based virtual screening. To optimize the suggested structure, over 130 derivatives were synthesized and their inhibitory activities on T-type calcium channel were assayed using in vitro screening system with alpha1(G) and alpha1(H) clones. For the compounds with higher activities in FDSS assay system, the efficacy was measured by patch-clamp method. Among the library with 5 features, alkaneamide derivatives (7b, 9j, 11b, 11g, 11h) with 4-arylsubstituted piperazine showed better IC(50) values than Mibefradil.

Alkanes↗
PubMed2006

3D pharmacophore based virtual screening of T-type calcium channel blockers.

Virtual screening of the commercial databases was done by using a three dimensional pharmacophore previously developed for T-type calcium channel blockers using CATALYSTtrade mark program. Biological evaluation of 25 selected virtual hits resulted in the discovery of a highly potent compound VH04 with IC(50) value of 0.10 microM, eight times as potent as the known selective T-type calcium channel blocker, mibefradil. Search for similar compounds yielded several hits with micro-molar IC(50) values and high T-type calcium channel selectivity. Based on the structure of the virtual hits, small molecule libraries with novel scaffolds were designed, synthesis and biological evaluation of which are currently in progress. This result shows a successful example of ligand based drug discovery of potent T-type calcium channel blockers.

Animals↗
PubMed2006

Synthesis and biological evaluation of novel T-type calcium channel blockers.

3,4-Dihydroquinazoline analogues substituted by N-methyl-N-(5-pyrrolidinopentyl)amine at the 2-position were synthesized and their blocking effects were evaluated for T- and N-type calcium channels. Compound 11b (KYS05080), compared to mibefradil (IC50=1.34+/-0.49 microM), was about 5-fold potent (IC50=0.26+/-0.01 microM) for T-type calcium channel (alpha1G) blocking and its selectivity of T/N-type was also improved (7.5 versus 1.4 of mibefradil).

Animals↗
PubMed2006

Novel T-type calcium channel blockers: dioxoquinazoline carboxamide derivatives.

T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains. Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium channel blockers. A small molecule library of dioxoquinazoline carboxamide derivatives containing 155 compounds was designed, synthesized, and biologically evaluated for T-type calcium channel blocking activity. Among those compounds synthesized, the compound 1n shows the most potent T-type calcium current blocking activity with an IC(50) value of 1.52 microM, which is comparable to that of mibefradil.

Amides↗
PubMed2006

Dyrk1A phosphorylates alpha-synuclein and enhances intracellular inclusion formation.

Lewy bodies (LBs) are pathological hallmarks of Parkinson disease (PD) but also occur in Alzheimer disease (AD) and dementia of LBs. Alpha-synuclein, the major component of LBs, is observed in the brain of Down syndrome (DS) patients with AD. Dyrk1A, a dual specificity tyrosine-regulated kinase (Dyrk) family member, is the mammalian ortholog of the Drosophila minibrain (Mnb) gene, essential for normal postembryonic neurogenesis. The Dyrk1A gene resides in the human chromosome 21q22.2 region, which is associated with DS anomalies, including mental retardation. In this study, we examined whether Dyrk1A interacts with alpha-synuclein and subsequently affects intracellular alpha-synuclein inclusion formation in immortalized hippocampal neuronal (H19-7) cells. Dyrk1A selectively binds to alpha-synuclein in transformed and primary neuronal cells. Alpha-synuclein overexpression, followed by basic fibroblast growth factor-induced neuronal differentiation, resulted in cell death. We observed that accompanying cell death was increased alpha-synuclein phosphorylation and intracytoplasmic aggregation. In addition, the transfection of kinase-inactive Dyrk1A or Dyrk1A small interfering RNA blocked alpha-synuclein phosphorylation and aggregate formation. In vitro kinase assay of anti-Dyrk1A immunocomplexes demonstrated that Dyrk1A could phosphorylate alpha-synuclein at Ser-87. Furthermore, aggregates formed by phosphorylated alpha-synuclein have a distinct morphology and are more neurotoxic compared with aggregates composed of unmodified wild type alpha-synuclein. These findings suggest alpha-synuclein inclusion formation regulated by Dyrk1A, potentially affecting neuronal cell viability.

Cell Survival↗
PubMed2006

Morpholin-2-one derivatives as novel selective T-type Ca2+ channel blockers.

Morpholin-2-one-5-carboxamide derivatives were prepared by using the one-pot Ugi multicomponent reaction and evaluated for blocking effects on T- and N-type Ca(2+) channels. Among them, compound 5i produced the highest potency (IC(50)=0.45+/-0.02 microM), while compounds 5d, 5f, 5k, 5n, 5o, and 6m produced relatively high potency as well as selectivity on T-type Ca(2+) channels. These novel scaffolds showed potent and selective T-type Ca(2+) channel blocking activities.

Animals↗
PubMed2006

Growth inhibition of human cancer cells in vitro by T-type calcium channel blockers.

This paper describes the preliminary biological results that novel T-type calcium channel blockers inhibit the growth of human cancer cells by blocking calcium influx into the cell, based on unknown mechanism on the cell cycle responsible for cellular proliferation. Among the selected compounds from compound library, compound 9c (KYS05041) was identified to be nearly equipotent with Cisplatin against some human cancers in the micromolar range.

Antineoplastic Agents↗
PubMed2006

Cellular action of cholecystokinin-8S-mediated excitatory effects in the rat periaqueductal gray.

The peptide cholecystokinin (CCK) is one of the major neurotransmitters modulating satiety, nociception, and anxiety behavior. Although many behavioral studies showing anti-analgesic and anxiogenic actions of CCK have been reported, less is known about its cellular action in the central nervous system (CNS). Therefore, we examined the action of CCK in rat dorsolateral periaqueductal gray (PAG) neurons using slice preparations and whole-cell patch-clamp recordings. Application of CCK-8S produced an inward current accompanied by increased spontaneous synaptic activities. The CCK-8S-induced inward current (I(CCK)) was recovered after washout and reproduced by multiple exposures. Current-voltage plots revealed that I(CCK) reversed near the equilibrium potential for K(+) ions with a decreased membrane conductance. When several K(+) channel blockers were used, application of CdCl(2), TEA, or apamin significantly reduced I(CCK). I(CCK) was also significantly reduced by the CCK(2) receptor antagonist, L-365,260, while it was not affected by the CCK(1) receptor antagonist, L-364,718. Furthermore, we examined the effects of CCK-8S on miniature excitatory postsynaptic currents (mEPSCs) in order to determine the mechanism of CCK-mediated increase on synaptic activities. We found that CCK-8S increased the frequency of mEPSCs, but had no effect on mEPSC amplitude. This presynaptic effect persisted in the presence of CdCl(2) or Ca(2+)-free bath solution, but was completely abolished by pre-treatment with BAPTA-AM, thapsigargin or L-365,260. Taken together, our results indicate that CCK can excite PAG neurons at both pre- and postsynaptic loci via the activation of CCK(2) receptors. These effects may be important for the effects of CCK on behavior and autonomic function that are mediated via PAG neurons.

Animals↗
PubMed2006

20(S)-ginsenoside Rh2, a newly identified active ingredient of ginseng, inhibits NMDA receptors in cultured rat hippocampal neurons.

Most herbal medicines that are orally administrated have been known to be metabolized before they are absorbed from the gastrointestinal tract. We, therefore, examined the effects of 20(S)-ginsenosides Rb1, Rg1 and Rg3, the three most commonly studied ginsenosides in the central nervous system, and their main metabolites on NMDA receptors using fura-2-based digital imaging and perforated whole-cell patch-clamp techniques. Among the nine ginsenosides tested, 20(S)-ginsenoside Rh2 (20(S)-Rh2) along with 20(S)-ginsenoside Rg3 (20(S)-Rg3) produced the highest inhibitory effect in cultured hippocampal neurons. Although 20(S)-Rg3 and 20(S)-Rh2 selectively targeted NMDA receptors with similar potency, they produced additive effects and seemed to modulate different NMDA receptor regulatory sites. As a competitive antagonist, 20(S)-Rh2 seems to inhibit the receptor via its interaction with polyamine-binding sites, and 20(S)-Rg3 does so using glycine-binding sites. Therefore, these results suggest that the treatment of 20(S)-Rh2, a newly identified active ingredient of ginseng, might be a novel preventive candidate in treating neurodegenerative disorders.

Animals↗
PubMed2006

Synthesis and SAR studies of a novel series of T-type calcium channel blockers.

For the novel, potent, and selective T-type Ca2+ channel blockers, a series of sulfonamido-containing 3,4-dihydroquinazoline derivatives were prepared and evaluated for their blocking actions on T- and N-type Ca2+ channels. Among them, 9c (KYS05064, IC50 = 0.96 +/- 0.22 microM) was found to be as potent as Mibefradil and also showed the highest selectivity for T-type Ca2+ channel with no effect on N-type Ca2+ channel.

Calcium Channel Blockers↗
PubMed2006

Cloning and heterologous expression of a Ca2+-activated chloride channel isoform from rat brain.

We previously reported the cloning of a calcium-activated chloride channel (CLCA) from rat brain (Biochem. Biophys. Res. Commun., 334, 569-576 (2005)), which we designated rbCLCA1. We further showed that rbCLCA1 is expressed in the central nervous system and peripheral organs, and may be functionally expressed in mammalian HEK293 cells. In the present study, we report the successful cloning of a second CLCA from rat cerebrum (designated rbCLCA2), using reverse transcription-PCR (RT-PCR) with primers specific for rbCLCA1. We have begun to clone this cDNA based on the rbCLCA1-like sequence. The full-length rbCLCA2 cDNA, obtained via 5' and 3' rapid amplification of cDNA ends (RACE), is 2900 bp long and encodes a putative polypeptide of 905 amino acids having at least two major transmembrane domains and showing 85.2% identity to rbCLCA1. RT-PCR analysis revealed that, similar to rbCLCA1, rbCLCA2 was predominantly expressed in the rat cerebrum, cerebellum, kidney, stomach, spinal cord, lung and small intestine, but not in the heart, large intestine, liver, orand spleen. Whole-cell patch clamp studies in HEK293 cells transiently co-transfected with expression vectors encoding rbCLCA2 and EGFP allowed us to identify the presence of niflumic acid (a CLCA channel blocker)-sensitive and voltage-dependent chloride currents in cells expressing rbCLCA2 but not EGFP alone. Treatment of these cells with ionomycin, a Ca2+ ionophore, significantly increased the novel currents in cells expressing rbCLCA2 and EGFP, but not those expressing EGFP alone, indicating that activation of the rbCLCA2 current is Ca2+-dependent. In sum, we herein report the cloning of a second member of the rbCLCA family from rat brain and its functional expression in vitro, thus adding to our knowledge of anion channels and facilitating future exploration of brain and other organ physiology.

Amino Acid Sequence↗
PubMed2006

Ginseng saponins diminish adverse vascular effects associated with chronic methionine-induced hyperhomocysteinemia.

Recent studies have shown that Panax ginseng has a variety of beneficial effects on the cardiovascular systems. Homocysteine (Hcy), which is derived from L-methionine (Met), has been closely associated with the increased risk of cardiovascular diseases. In the present study, we examined whether in vivo long-term administration of ginseng saponins (GS), active ingredients of Panax ginseng, attenuate adverse vascular effects associated with chronic Met-induced hyperhomocysteinemia (H-Hcy). We found that plasma Hcy level, which was measured after 30 and 60 d, in GS (100 mg/kg)+Met co-administration group was significantly reduced when it was compared with Met alone treatment group. We could also observe the alleviation of endothelial damages of aortic artery vessels in GS (100 mg/kg)+Met co-administration group compared with Met alone treatment group. We compared aortic vasocontractile and vasodilatory responses between Met alone and GS (100 mg/kg)+Met co-treatment groups. We found that norepinephrine-induced vasocontractile responses were greatly decreased in GS (100 mg/kg)+Met co-treatment group and that carbachol-induced dilatory responses were greatly enhanced in GS (100 mg/kg)+Met co-administration groups as compared with Met alone treatment group. The present results indicate that in vivo long-term administration of GS attenuates adverse vascular effects associated with chronic Met-induced H-Hcy in rats.

Animals↗
PubMed2005

Parkin ubiquitinates and promotes the degradation of RanBP2.

Parkinson disease (PD) is a common neurodegenerative disorder, which involves the deterioration of dopaminergic neurons in the pars compacta of the substantia nigra. The etiology of PD is still unknown, but recent identification of mutations in familial cases of PD has advanced the understanding of the molecular mechanisms of this neurological disease. Mutations in the parkin gene, which encodes for ubiquitin-protein ligase (E3), have been implicated in autosomal recessive juvenile Parkinsonism, an early onset and common familial form of PD. Here we reported that Parkin selectively binds to RanBP2, which is localized in the cytoplasmic filament of the nuclear pore complex and belongs to the small ubiquitin-related modifier E3 ligase family. We also demonstrated that RanBP2 becomes a target for Parkin E3 ubiquitin-ligase and is processed via Parkin-mediated ubiquitination and subsequent proteasomal degradation. Furthermore, Parkin controls the intracellular levels of sumoylated HDAC4, as a result of the ubiquitination and degradation of RanBP2. Our findings suggested that the intracellular levels of RanBP2 and its functional activity may be modulated by Parkin-mediated ubiquitination and proteasomal pathways.

Amino Acid Motifs↗
PubMed2005

T-type calcium channel trigger p21ras signaling pathway to ERK in Cav3.1-expressed HEK293 cells.

We constructed a new cell line which stably expressed Cav3.1 and Kir2.1 subunits in HEK293 cells (HEK293/Cav3.1/Kir2.1) in order to investigate the unknown cellular signaling pathways of T-type voltage-dependent calcium channels. The new cell line has a stable resting membrane potential and can activate T-type Ca(2+) channels by KCl-mediated depolarization. We showed that Cav3.1 activation resulted in the level of p21(ras)-GTP in the cells being rapidly decreased during the first 2 min, and then recovering between 2 min and 15 min. The kinetics of ERK activation following Cav3.1 stimulation was also investigated. ERK activation was decreased from 2 min to 5 min after KCl stimulation, which means that Cav3.1 activation reduced ERK activity in the very early stages of activation. In addition, similar results for Cav3.1 activation were also shown in the case of Sos1, Grb2, and Shc, which means that Cav3.1 activation triggers p21(ras) and that this signal is transferred to ERK by Sos1, Grb2, and Shc.

Blotting, Western↗
PubMed2005

Characteristics of ginsenoside Rg3-mediated brain Na+ current inhibition.

We demonstrated previously that ginsenoside Rg(3) (Rg(3)), an active ingredient of Panax ginseng, inhibits brain-type Na(+) channel activity. In this study, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced Na(+) channel inhibition. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on Na(+) currents (I(Na)) in Xenopus laevis oocytes expressing wild-type rat brain Na(V)1.2 alpha and beta1 subunits, or mutants in the channel entrance, the pore region, the lidocaine/tetrodotoxin (TTX) binding sites, the S4 voltage sensor segments of domains I to IV, and the Ile-Phe-Met inactivation cluster. In oocytes expressing wild-type Na(+) channels, Rg(3) induced tonic and use-dependent inhibitions of peak I(Na). The Rg(3)-induced tonic inhibition of I(Na) was voltage-dependent, dose-dependent, and reversible, with an IC(50) value of 32 +/- 6 microM. Rg(3) treatment produced a 11.2 +/- 3.5 mV depolarizing shift in the activation voltage but did not alter the steady-state inactivation voltage. Mutations in the channel entrance, pore region, lidocaine/TTX binding sites, or voltage sensor segments did not affect Rg(3)-induced tonic blockade of peak I(Na). However, Rg(3) treatment inhibited the peak and plateau I(Na) in the IFMQ3 mutant, indicating that Rg(3) inhibits both the resting and open states of Na(+) channel. Neutralization of the positive charge at position 859 of voltage sensor segment domain II abolished the Rg(3)-induced activation voltage shift and use-dependent inhibition. These results reveal that Rg(3) is a novel Na(+) channel inhibitor capable of acting on the resting and open states of Na(+) channel via interactions with the S4 voltage-sensor segment of domain II.

Animals↗
PubMed2005

Synthesis and biological activity of 3,4-dihydroquinazolines for selective T-type Ca2+ channel blockers.

We have synthesized 3,4-dihydroquinazoline derivatives for the potent and selective T-type Ca(2+) channel blockers and evaluated for their inhibitory activities against two subtypes T-type Ca(2+) channels and N-type Ca(2+) channels. Among them, 5b (KYS05044, IC(50)=0.56+/-0.10 microM) was identified as potent T-type Ca(2+) channel blocker with in vitro selectivity profile at meaningful level (T/N-type, SI=>100).

Calcium Channel Blockers↗