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Soo-Kyung Kim

Publications and source records attributed to Soo-Kyung Kim.

33 records · Page 2Linked to original sources
PubMed2004

Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor.

The M(3) muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs). To gain insight into the structural mechanisms governing agonist-mediated M(3) receptor activation, we recently developed a genetically modified yeast strain (Saccharomyces cerevisiae) which allows the efficient screening of large libraries of mutant M(3) receptors to identify mutant receptors with altered/novel functional properties. Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to Asp-113 in the rat M(3) muscarinic receptor) which is of fundamental importance for receptor activation. As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast. We then subjected the D113N mutant M(3) receptor to PCR-based random mutagenesis followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor. A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D. When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling. Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells). Interestingly, like Asp-113, Arg-165 and Tyr-250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs. Our data suggest a conformational link between the highly conserved Asp-113, Arg-165, and Tyr-250 residues which is critical for receptor activation.

Alleles↗
PubMed2004

Design and synthesis of 3'-ureidoadenosine-5'-uronamides: effects of the 3'-ureido group on binding to the A3 adenosine receptor.

On the basis of high binding affinity at the A(3) adenosine receptor of 3'-aminoadenosine derivatives with hydrogen bonding donor ability, novel 3'-ureidoadenosine analogues were synthesized from 1,2:5,6-di-O-isopropylidene-d-glucose in order to lead to stronger hydrogen bonding than the corresponding 3'-aminoadenosine derivatives. However, the synthesized 3'-ureidoadenosine analogues were totally devoid of binding affinity, because 3'-urea moiety caused steric and electrostatic repulsions at the binding site of the A(3) adenosine receptor, leading to conformational distortion.

Adenosine↗
PubMed2004

Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position.

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N(6)-substitutions known to enhance human A(3)AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A(1), A(2A), A(2B), and A(3)ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA(3)AR affinity and efficacy in the cases of N(6)-(3-iodobenzyl) and N(6)-(trans-2-phenyl-1-cyclopropyl), for which a full A(3)AR agonist was converted into a selective antagonist; the 2-cyano-N(6)-methyl analogue was a full A(3)AR agonist. The combination of N(6)-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A(1)AR. The environment surrounding the 2-position within the putative A(3)AR binding site was explored using rhodopsin-based homology modeling and ligand docking.

Adenosine↗
PubMed2004

Exploring distal regions of the A3 adenosine receptor binding site: sterically constrained N6-(2-phenylethyl)adenosine derivatives as potent ligands.

We synthesized phenyl ring-substituted analogues of N(6)-(1S,2R)-(2-phenyl-1-cyclopropyl)adenosine, which is highly potent in binding to the human A(3)AR with a Ki value of 0.63 nM. The effects of these structural changes on affinity at human and rat adenosine receptors and on intrinsic efficacy at the hA(3)AR were measured. A 3-nitrophenyl analogue was resolved chromatographically into pure diastereomers, which displayed 10-fold stereoselectivity in A(3)AR binding in favor of the 1S,2R isomer. A molecular model defined a hydrophobic region (Phe168) in the putative A(3)AR binding site around the phenyl moiety. A heteroaromatic group (3-thienyl) could substitute for the phenyl moiety with retention of high affinity of A(3)AR binding. Other related N(6)-substituted adenosine derivatives were included for comparison. Although the N(6)-(2-phenyl-1-cyclopropyl) derivatives were full A(3)AR agonists, several other derivatives had greatly reduced efficacy. N(6)-Cyclopropyladenosine was an A(3)AR antagonist, and adding either one or two phenyl rings at the 2-position of the cyclopropyl moiety restored efficacy. N(6)-(2,2-Diphenylethyl)adenosine was an A(3)AR antagonist, and either adding a bond between the two phenyl rings (N(6)-9-fluorenylmethyl) or shortening the ethyl moiety (N(6)-diphenylmethyl) restored efficacy. A QSAR study of the N(6) region provided a model that was complementary to the putative A(3)AR binding site in a rhodopsin-based homology model. Thus, a new series of high-affinity A(3)AR agonists and related nucleoside antagonists was explored through both empirical and theoretical approaches.

Binding Sites↗
PubMed2004

Structure-activity relationships of thiazole and thiadiazole derivatives as potent and selective human adenosine A3 receptor antagonists.

4-(4-Methoxyphenyl)-2-aminothiazole and 3-(4-methoxyphenyl)-5-aminothiadiazole derivatives have been synthesized and evaluated as selective antagonists for human adenosine A3 receptors. A methoxy group in the 4-position of the phenyl ring and N-acetyl or propionyl substitutions of the aminothiazole and aminothiadiazole templates displayed great increases of binding affinity and selectivity for human adenosine A3 receptors. The most potent A3 antagonist of the present series, N-[3-(4-methoxy-phenyl)-[1,2,4]thiadiazol-5-yl]-acetamide (39) exhibiting a Ki value of 0.79 nM at human adenosine A3 receptors, showed antagonistic property in a functional assay of cAMP biosynthesis involved in one of the signal transduction pathways of adenosine A3 receptors. Molecular modeling study of conformation search and receptor docking experiments to investigate the dramatic differences of binding affinities between two regioisomers of thiadiazole analogues, (39) and (42), suggested possible binding mechanisms in the binding pockets of adenosine receptors.

Adenosine A3 Receptor Antagonists↗
PubMed2004

Murine Asb-17 expression during mouse testis development and spermatogenesis.

In this study we isolated a murine mAsb-17 from mouse testis by RT-PCR using primers designed based on the sequences from the GenBank database. The sequence analysis showed that mAsb-17 encodes a 295 amino acid polypeptide with a molecular weight of approximately 34 kDa containing two ankyrin repeats and one SOCS box. The amino acid sequence of mASB-17 showed 87.5%, 98.3% and 92.9% identity with that of human, rat and dog, respectively. Interestingly, northern blot analysis showed that mAsb-17 was expressed only in the testis. The expression analysis by RT-PCR for mAsb-17 in mouse indicates that mAsb-17 is expressed from the fourth week after birth to adult, with the highest expression in round spermatids. Both northern blot and RT-PCR analyses suggest that mASB-17 may play essential roles in testis development and spermatogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Purine receptors: GPCR structure and agonist design.

An integrated approach to the study of drug-receptor interactions has been applied to adenosine receptors (ARs) and P2Y nucleotide receptors. This approach includes probing the receptor structure through site-directed mutagenesis and molecular modeling, in concert with altering the structure of the agonist ligands. Goals of this structural approach are to generate a testable hypothesis for location of the binding site and subsequently to enable the rational design of new agonists and antagonists. In this manner, receptor subtype selectivity has been increased, and agonists have been converted into partial agonists and antagonists. An approach to receptor engineering (neoceptors) has been explored, in which synthetic small molecule agonists (neoligands) are specifically tailored to activate only receptors in which the putative binding sites have been modified. This orthogonal approach to receptor activation, intended for eventual gene therapy, has been demonstrated for A3 and A2A ARs.

Animals↗
PubMed2004

Preventative effects of rosiglitazone on restenosis after coronary stent implantation in patients with type 2 diabetes.

OBJECTIVE: Despite the popularity of coronary stenting in coronary artery disease (CAD), restenosis remains a challenging clinical problem. This study evaluated the efficacy of rosiglitazone for preventing in-stent restenosis in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: We conducted a prospective, randomized, case-controlled trial involving 95 diabetic patients with CAD who were randomly assigned to either the control or rosiglitazone group (48 and 47 patients, respectively). Quantitative coronary angiography (QCA) was performed at study entry and again at 6-month follow-up. The primary end point was the restenosis rate, which was determined by QCA. RESULTS: Eighty-three patients (45 patients with 55 lesions in the control group and 38 patients with 51 lesions in the rosiglitazone group) completed follow-up angiography. Rosiglitazone treatment for 6 months reduced fasting insulin concentration. The high-sensitivity C-reactive protein concentration was significantly reduced in the rosiglitazone group compared with that in the control group (from 2.92 +/- 1.98 to 0.62 +/- 0.44 mg/l, P < 0.001 vs. from 2.01 +/- 1.33 to 1.79 +/- 1.22 mg/l, P = NS). However, the baseline and follow-up glucose and lipid concentrations were not different between two groups. The rate of in-stent restenosis was significantly reduced in the rosiglitazone group compared with the control group (for stent lesions: 17.6 vs. 38.2%, P = 0.030). The rosiglitazone group had a significantly lower degree of diameter stenosis (23.0 +/- 23.4% vs. 40.9 +/- 31.9%, P = 0.004) compared with the control group. CONCLUSIONS: We demonstrated that treatment with rosiglitazone significantly reduces in-stent restenosis in diabetic patients with CAD who underwent coronary stent implantation.

Adult↗
PubMed2003

Modeling the adenosine receptors: comparison of the binding domains of A2A agonists and antagonists.

A three-dimensional model of the human A(2A) adenosine receptor (AR) and its docked ligands was built by homology to rhodopsin and validated with site-directed mutagenesis and the synthesis of chemically complementary agonists. Different binding modes of A(2A)AR antagonists and agonists were compared by using the FlexiDock automated docking procedure, with manual adjustment. Putative binding regions for the 9H-purine ring in agonist NECA 3 and the 1H-[1,2,4]triazolo[1,5-c]quinazoline ring in antagonist CGS15943 1 overlapped, and the exocyclic amino groups of each were H-bonded to the side chain of N(6.55). For bound agonist, H-bonds formed between the ribose 3'- and 5'-substituents and the hydrophilic amino acids T(3.36), S(7.42), and H(7.43), and the terminal methyl group of the 5'-uronamide interacted with the hydrophobic side chain of F(6.44). Formation of the agonist complex destabilized the ground-state structure of the A(2A)AR, which was stabilized through a network of H-bonding and hydrophobic interactions in the transmembrane helical domain (TM) regions, facilitating a conformational change upon activation. Both flexibility of the ribose moiety, required for the movement of TM6, and its H-bonding to the receptor were important for agonism. Two sets of interhelical H-bonds involved residues conserved among ARs but not in rhodopsin: (1) E13(1.39) and H278(7.43) and (2) D52(2.50), with the highly conserved amino acids N280(7.45) and S281(7.46), and N284(7.49) with S91(3.39). Most of the amino acid residues lining the putative binding site(s) were conserved among the four AR subtypes. The A(2A)AR/3 complex showed a preference for an intermediate conformation about the glycosidic bond, unlike in the A(3)AR/3 complex, which featured an anti-conformation. Hydrophilic amino acids of TMs 3 and 7 (ribose-binding region) were replaced with anionic residues for enhanced binding to amine-derivatized agonists. We identified new neoceptor (T88D)-neoligand pairs that were consistent with the model.

Adenosine A2 Receptor Agonists↗
PubMed2003

Cloning and characterization of rat neuronal apoptosis inhibitory protein cDNA.

The human neuronal apoptosis inhibitory protein (NAIP) gene was originally discovered because of its deletion in infantile spinal muscular atrophy (SMA), a childhood genetic disorder characterized by motor neuron loss and progressive paralysis with muscular atrophy. Although SMA is now known to be caused by deletions of survival motor neuron (SMN), the fact that NAIP is an anti-apoptotic protein is consistent with the NAIP gene modifying SMA severity. Here we report the cloning of a 1.5 kb rat NAIP cDNA fragment which contains BIR-3 (third baculovirus inhibitory repeat) domain. This fragment shows 78% homology to the human NAIP and 86% homology to the murine counterpart. We have investigated the distribution of NAIP mRNA expressing neurons by in situ RT-PCR technique in the rat central nervous system (CNS). Although all of the neurons appeared to express NAIP mRNA ubiquitously, pronounced elevation of NAIP mRNA expression was observed in the areas innervated by glutamatergic neurons after kainic acid (KA) injection. We have raised an anti-rat NAIP antiserum in rabbits using NAIP cDNA and recombinant rat NAIP, and carried out an immunohistological investigation. We observed highly immunoreactive neuronal subpopulations in the retinal ganglion, cerebral cortex, hippocampus, basal forebrain, thalamus, areas of midbrain, Purkinje cells of the cerebellum, and motor neurons in the spinal cord. Increased immunoreactivity of glutamatergic neurons was also observed broadly in the CNS after KA treatment. This study provides additional evidence that expression of mRNA and gene products of NAIP seem to be regulated in response to excessive stimuli or injuries in rat CNS, and these results are compatible with an anti-apoptotic role of NAIP in acute SMA as well as in brain injuries.

Amino Acid Sequence↗
PubMed2003

Cardioselective anti-ischemic ATP-sensitive potassium channel (KATP) openers: benzopyranyl indoline and indole analogues.

This paper describes the design, syntheses, and biological evaluations of novel ATP-sensitive potassium channel (K(ATP)) openers, benzopyranyl indoline and indole derivatives. Among those, two enantiomers of indoline-2-carboxylic ethyl esters (14, 18) showed the best cardioprotective activities both in vitro and in vivo, while their vasorelaxation potencies were very low (concentration for 50% inhibition of vasorelaxation >30 microM). The cardioprotective effect of 14 was completely reversed by 5-hydroxydecanoate, a selective mitochondrial K(ATP) blocker, indicating its provable protective mechanism through the mitochondrial K(ATP) opening. In addition, we performed conformational analyses using 2D-NMR, X-ray crystallography and molecular modeling to study the structure-activity relationships in this series of compounds.

Adenosine Triphosphate↗
PubMed2003

Identification of essential residues involved in the allosteric modulation of the human A(3) adenosine receptor.

We examined the effects on allosteric modulation and ligand binding of the mutation of amino acid residues of the human A(3) adenosine receptor (A(3)AR) that are hypothesized to be near one of three loci: the putative sodium binding site, the putative ligand binding site, and the DRY motif in transmembrane helical domain 3. The effects of three heterocyclic allosteric modulators [the imidazoquinoline 2-cyclopentyl-4-phenylamino-1H-imidazo[4,5-c]quinoline (DU124183), the pyridinylisoquinoline 4-methoxy-N-[7-methyl-3-(2-pyridinyl)-1-isoquinolinyl]benzamide (VUF5455), and the amiloride analog 5-(N,N-hexamethylene)-amiloride] on the dissociation of the agonist radioligand, N(6)- (4-amino-3-[(125)I]iodobenzyl)-5'-N-methylcarboxamidoadenosine, were compared at wild-type (WT) and mutant A(3)ARs. The F182A(5.43) and N274A(7.45) mutations eliminated the allosteric effects of all three modulators but had little effect on agonist binding. The N30A(1.50) and D58N(2.50) mutations abolished the allosteric effects of DU124183 and VUF5455, but not HMA, whereas the D107N(3.49) mutation abolished the effects of DU124183, but not HMA or VUF5455. The T94A(3.36), H95A(3.37), K152A(EL2), W243A(6.48), L244A(6.49), and S247A(6.52) mutations did not influence allosteric effects of the modulators. Sodium ions (100 mM), which modulate agonist binding at a variety of receptors, caused an approximately 80% inhibition of agonist binding in WT A(3)ARs but did not show any effect on D58N(2.50), D107N(3.49), and F182A(5.43) mutant receptors. In contrast, NaCl induced a modest increase of agonist binding in N30A(1.50) and N274A(7.45) mutant receptors. NaCl decreased the dissociation rate of the antagonist radioligand [(3)H]8-ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2.1-i]purin-5-one (PSB-11) at the WT A(3)ARs, but not the D58N(2.50) mutant receptor. The results were interpreted using a rhodopsin-based molecular model of the A(3)AR to suggest multiple binding modes of the allosteric modulators.

Adenosine↗
PubMed2002

Structural determinants of A(3) adenosine receptor activation: nucleoside ligands at the agonist/antagonist boundary.

Mutagenesis of the human A(3) adenosine receptor (AR) suggested that certain amino acid residues contributed differently to ligand binding and activation processes. Here we demonstrated that various adenosine modifications, including adenine substitution and ribose ring constraints, also contributed differentially to these processes. The ligand effects on cyclic AMP production in intact CHO cells expressing the A(3)AR and in receptor binding were compared. Notably, the simple 2-fluoro group alone or 2-chloro in combination with N(6)-substitution dramatically diminished the efficacy of adenosine derivatives, even converting agonist into antagonist. Other affinity-increasing substitutions, including N(6)-(3-iodobenzyl) 4 and the (Northern)-methanocarba 15, also reduced efficacy, except in combination with a flexible 5'-uronamide. 2-Cl-N(6)-(3-iodobenzyl) derivatives, both in the (N)-methanocarba (i.e., of the Northern conformation) and riboside series 18 and 5, respectively, were potent antagonists with little residual agonism. Ring-constrained 2',3'-epoxide derivatives in both riboside and (N)-methanocarba series 13 and 21, respectively, and a cyclized (spiral) 4',5'-uronamide derivative 14 were synthesized and found to be human A(3)AR antagonists. 14 bound potently at both human (26 nM) and rat (49 nM) A(3)ARs. A rhodopsin-based A(3)AR model, containing all domains except the C-terminal region, indicated separate structural requirements for receptor binding and activation for these adenosine analogues. Ligand docking, taking into account binding of selected derivatives at mutant A(3)ARs, featured interactions of TM3 (His95) with the adenine moiety and TMs 6 and 7 with the ribose 5'-region. The 5'-OH group of antagonist N(6)-(3-iodobenzyl)-2-chloroadenosine 5 formed a H-bond with N274 but not with S271. The 5'-substituent of nucleoside antagonists moved toward TM7 and away from TM6. The conserved Trp243 (6.48) side chain, involved in recognition of the classical (nonnucleoside) A(3)AR antagonists but not adenosine-derived ligands, displayed a characteristic movement exclusively upon docking of agonists. Thus, A(3)AR activation appeared to require flexibility at the 5'- and 3'-positions, which was diminished in (N)-methanocarba, spiro, and epoxide analogues, and was characteristic of ribose interactions at TM6 and TM7.

Adenosine↗
PubMed2002

Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor.

Ligand recognition has been extensively explored in G protein-coupled A(1), A(2A), and A(2B) adenosine receptors but not in the A(3) receptor, which is cerebroprotective and cardioprotective. We mutated several residues of the human A(3) adenosine receptor within transmembrane domains 3 and 6 and the second extracellular loop, which have been predicted by previous molecular modeling to be involved in the ligand recognition, including His(95), Trp(243), Leu(244), Ser(247), Asn(250), and Lys(152). The N250A mutant receptor lost the ability to bind both radiolabeled agonist and antagonist. The H95A mutation significantly reduced affinity of both agonists and antagonists. In contrast, the K152A (EL2), W243A (6.48), and W243F (6.48) mutations did not significantly affect the agonist binding but decreased antagonist affinity by approximately 3-38-fold, suggesting that these residues were critical for the high affinity of A(3) adenosine receptor antagonists. Activation of phospholipase C by wild type (WT) and mutant receptors was measured. The A(3) agonist 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine stimulated phosphoinositide turnover in the WT but failed to evoke a response in cells expressing W243A and W243F mutant receptors, in which agonist binding was less sensitive to guanosine 5'-gamma-thiotriphosphate than in WT. Thus, although not important for agonist binding, Trp(243) was critical for receptor activation. The results were interpreted using a rhodopsin-based model of ligand-A(3) receptor interactions.

Amino Acid Sequence↗