• 한국미생물·생명공학회지
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• 제31권2호
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• pp.117-123
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• 2003

공시균인 Saccha. erythraea IFO 13426으로부터 VB-C에 의한 erythromycin 생산 유도능이 시사된 바 있으므로, 공시균으로부터 VB-C와 특이적으로 결합하는 autoregulators 및 receptor gene을 탐색하여, EM의 생산 조절 기구를 규명하고자 하였다. 탐색의 일환으로 기존의 Streptomyce속 receptor gene의 공통배열을 primer로 이용하여 PCR을 수행하였고, 예상 크기인 120bp의 단편을 pUC19 vector에 ligation하여 E. coli DH5$\alpha$에 형질전환한 후, plasmid를 분리하여 BamHI을 처리하여 2% agarose gel에 전기영동한 결과, pUC19 (2.7kbp)외에 receptor gene PCR 산물이 120bp위치에 존재하는 것을 확인하였다. 형질전환된 plasmid로 PCR을 수행하여 염기배열을 결정한 후 해석한 결과 Streptomyces sp. 유래의 receptor gene과 유사함을 확인하였다. 따라서 Saccha. erythraea IFO 13426에는 항생물질인 erythromycin의 생산에 관여한다고 추정되는 autoregulator receptor protein을 코드하는 유전자가 존재할 것으로 예상되어 120 bp의 PCR product를 probe로 이용하여 Southern 및 colony hybridization을 통하여 3.2 kbp의 SacI 단편을 가지는 plasmid(pESG)를 제작하였고, 이를 sequencing한 결과, autoregulator receptor protein 유전자가 KpnI과 SalI을 포함하는 영역에 존재한다는 것을 알 수 있었으며 이를 EsgR이라 명명하였다. 유전자 해석 결과, EsgR은 205개의 아미노산으로 구성되어 있으며, 이는 기존의 autoregulator receptor proteins과 비교시 30%이상의 상동성을 나타내었으며, 기존의 autoregulator receptor prorein들이 하부의 항생물질 생합성 유전자들의 제어를 위해 보유하고 있는 helix-turn-helix DNA binding motif를 EsgR이 보유하고 있는 점에서, EsgR은 Saccha. erythraea가 보유하는 autoregulator receptor protein을 code하는 유전자로 추정되었다.

• 통합자연과학논문집
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• 제3권3호
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• pp.143-147
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• 2010

Since it is of great importance to know how a ligand binds to a receptor, there have been a lot of efforts to improve the quality of prediction of docking poses. Earlier efforts were focused on improving search algorithm and scoring function in a docking program resulting in a partial improvement with a lot of variations. Although these are basically very important and essential, more tangible improvements came from the reduction of search space. In a normal docking study, the approximate active site is assumed to be known. After defining active site, scoring functions and search algorithms are used to locate the expected binding pose within this search space. A good search algorithm will sample wisely toward the correct binding pose. By careful study of receptor structure, it was possible to prioritize sub-space in the active site using "receptor-based pharmacophores" or "hot spots". In a sense, these techniques reduce the search space from the beginning. Further improvements were made when the bound ligand structure is available, i.e., the searching could be directed by molecular similarity using ligand information. This could be very helpful to increase the accuracy of binding pose. In addition, if the biological activity data is available, docking program could be improved to the level of being useful in affinity prediction for a series of congeneric ligands. Since the number of co-crystal structures is increasing in protein databank, "Ligand-Guided Docking" to reduce the search space would be more important to improve the accuracy of docking pose prediction and the efficiency of virtual screening. Further improvements in this area would be useful to produce more reliable docking programs.

• Bulletin of the Korean Chemical Society
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• 제34권11호
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• pp.3429-3443
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• 2013

Chemokine receptor (CCR2) is a G protein-coupled receptor that contains seven transmembrane helices. Recent pharmaceutical research has focused on the antagonism of CCR2 and candidate drugs are currently undergoing clinical studies for the treatment of diseases like arthritis, multiple sclerosis, and type 2 diabetes. In this study, we analyzed the time dependent behavior of CCR2 docked with a potent 4-azetidinyl-1-aryl-cyclohexane (4AAC) derivative using molecular dynamics simulations (MDS) for 20 nanoseconds (ns). Homology modeling of CCR2 was performed and the 4AAC derivative was docked into this binding site. The docked model of selected conformations was then utilized to study the dynamic behavior of the 4AAC enzyme complexes inside lipid membrane. MDS of CCR2-16b of 4AAC complexes allowed us to refine the system since binding of an inhibitor to a receptor is a dynamic process and identify stable structures and better binding modes. Structure activity relationships (SAR) for 4AAC derivatives were investigated and reasons for the activities were determined. Probable binding pose for some CCR2 antagonists were determined from the perspectives of binding site. Initial modeling showed that Tyr49, Trp98, Ser101, Glu291, and additional residues are crucial for 4AAC binding, but MDS analysis showed that Ser101 may not be vital. 4AAC moved away from Ser101 and the hydrogen bonding between 4AAC and Ser101 vanished. The results of this study provide useful information regarding the structure-based drug design of CCR2 antagonists and additionally suggest key residues for further study by mutagenesis.

• 대한약리학회지
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• 제30권1호
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• pp.49-57
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• 1994

Oxomemazine이 muscarinic receptor subtypes에 대하여 선택성을 가지는지에 관한 지견을 얻고자, 대뇌, 심실 및 회장 muscarinic receptor에 대한 oxomemazine의 결합성질을 조사, 비교하였다. $[^3H]QNB$ 포화결합실험 결과 세 조직의 muscarinic receptor는 $[^3H]QNB$에 대해서는 affinity가 약 60pM인 단일 receptor인 것으로 추정되었다. 대뇌에서 pirenzepine과 oxomemazine의 $[^3H]QNB$ 결합억제에 대한 Hill coefficient는 각각 0.67 및 0.8로서 대뇌에는 이들 약물에 대하여 affinity가 서로 다른 두 종류의 muscarinic receptor subtypes가 존재하는 것으로 나타났으며, pirenzepine에 대한 high $affinity(M_1)$와 $low affinity(M_2)$ receptor 및 oxomemazine에 대한 high $affinity(O_H)$와 $low\;affinity (O_L)$ receptor의 분포비는 약 60:40 및 40:60이었고, $M_1$과 $M_2$ receptor에 대한 pirenzepine의 $K_i$치는 16nM 및 431 nM, $O_H$와 $O_L$, receptor에 대한 oxomemazine 의 $K_i$치는 80nM 및 1350nM이었다. 그러나 심실과 회장에서 이들 약물의 $[^3H]QNB$ 결합억제에 대한 Hill coefficient는 1에 가까웠다. 심실과 회장 muscarinic receptor에 대한 pirenzepine의 $K_i$치는 850nM 및 250nM, oxomemazine의 $K_i$치는 1460nM 및 670nM로서 대피에서 이들 약물의 low affinity receptor에 대한 $K_i$치에 가까웠다. 즉, muscarinic receptor에 대한 affinity면에서 oxomemazine은 pirenzepine과 같이 대뇌에서 가장 높았으며, 회장에 대해서는 중등도였고, 심실에서 가장 낮았다. 이로 보아 oxomemazine은 $M_1\;receptor$에 선택성이 있는 것으로 추정된다.

• 대한약리학회지
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• 제32권3호
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• pp.311-321
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• 1996

$M_1$과 $M_2$ 무스카린성 수용체의 두 번째 transmembrane domain의 C-말단에는 leucine(L), tyrosine(Y), threonine(T)로 구성된 3중체(triplet)가 있다. 이 3중체는 $M_2$ 무스카린성 수용체에서는 두 번째 transmembrane domain과 첫 번째 세포외 고리사이의 연접부위에서 LYT-LYT의 반복구조로 존재하며 $M_1$ 무스카린성 수용체에서는 흥미롭게도 LYT-TYL의 역상구조로 존재한다. 본 연구에서는 site-directed mutagenesis방법을 사용하여 이와 같은 특이한 구조적차이가 두 subtype의 수용체의 기능상 차이와 관련한 역할을 가지고 있는지를 확인하고자 하였다. $M_1$ 수용체에서는 LYTTYL서열을 $M_2$ 수용체의 서열에 해당하는 LYTLYT로 mutation시켰으며 $M_2$ 수용체에서는 LYTLYT8서열을 $M_1$ 수용체의 서열에 해당하는 LYTTYL로 mutation시켰다. 이와같은 mutation은 $M_1$과 $M_2$ 수용체에서 효능제 carbachol의 수용체 결합친화력에 유의한 변화를 주지 않았다. 또한 $M_1$ 수용체에서의 mutation은 cyclic AMP 증가작용에 대한 coupling은 변화시키지 않고 phosphoinositides (PI) hydrolysis 촉진작용과 세포내 $Ca^{2+}$ 농도 상승을 현저히 증가시켰다. 또한 $M_2$ 수용체에서의 mutation은 adenylate cyclase 억제에 대한 coupling은 변화시키지 않고 PI hydrolysis 촉진을 약간 증가 시켰다. 이상의 결과는 $M_1$과 $M_2$ 수용체에서 LYTTYL/LYTLYT 아미노산 서열의 차이는 두 수용체의 PI hydrolysis에 대한 coupling을 조절하는 역할을 하지만, 두 수용체 사이에서 ligand 결합과 신호전달계의 차이를 구분하는데 중요한 역할을 하지는 않는다.

• 한국동물생명공학회지
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• 제34권4호
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• pp.267-271
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• 2019

This study aimed to investigate the function of the constitutively activating mutation D540G on eel FSHR activity by in vitro functional studies. Site-directed mutagenesis was carried out to generate the D-to-G mutation at position 540 of the pcDNA3-eel FSHR construct. Vectors expressing either wild type or mutant receptor were transfected into Chinese hamster ovary (CHO-K1) cells. The functional characteristics of both the wild type and mutant receptors were analyzed by a cAMP assay. cAMP accumulation was highly increased in cells transfected with the D540G mutant receptor in a dose-dependent manner. Of note, basal cAMP levels were remarkably increased (~13.1-fold) with expression of this mutant when compared to wild type receptor. These findings suggest that the D540G mutation in the eel FSHR may contribute to ovulation during eel sex maturation as well as play a pivotal role in inducing FSHR activity.

• Biomolecules & Therapeutics
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• 제3권3호
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• pp.192-198
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• 1995

The affinity of mequitazine, a non-sedating antihistamine, for muscarinic receptors was evaluated in the guinea-pig ventricle and ileum by in vitro binding techniques and functional studies. In binding studies, [$^3$H]quinuclidinyl benzilate (QNB) identified a single class of muscarinic receptors with similar apparent $K_{D}$ value of about 100 pM in two tissues. Mequitazine inhibited [$^3$H]QNB binding to muscarinic receptors competitively. Analysis of the mequitazine inhibition curve of [$^3$H]QNB binding to ventricular microsome and ileal homogenate indicated the presence of a single homogeneous binding site with Ki value of 25 nM and 18 nM, respectively. In functional studies, mequitazine caused parallel rightward shifts of concentration-response curves for carbachol and histamine in the isolated guinea-pig ileum. The slope values obtained from Schild plot analysis for the antagonistic action of mequitazine on muscarinic and histamine $H_1$-receptors were not significantly different from unity. The p $A_2$values of mequitazine for muscarinic and histamine $H_1$-receptors were about 7.6 ( $K_{M}$= 25.1 nM) and 8.88 ( $K_{H}$= 1.32 nM), respectively. These results indicate that the muscarinic receptor blocking action of mequitazine is 15 times less potent than the $H_1$receptor blocking action, but high concentration of this drug may cause the peripheral muscarinic receptor blocking effect.t.t.t.

• The Korean Journal of Physiology and Pharmacology
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• 제5권5호
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• pp.423-431
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• 2001

Some recent investigations revealed that vasodilatory action of adenosine is mainly not mediated by surface A2 receptor and suggested the existence of an intracellular action site. In the present study, we tried to differentiate intracellular from extracellular site of adenosine action in the regulation of coronary flow. In perfused rabbit hearts, concentration-response curve of coronary flow to exogenous adenosine was constructed in the presence or absence of dipyridamole, an inhibitor of transmembrane purine transport. Inhibition of cellular adenosine uptake by dipyridamole suppressed the increase of flow rate while enhancing the decrease in heart rate induced by exogenous adenosine. In another series of experiments, perfused rabbit hearts were subjected to energy deprivation in order to increase the production of endogenous adenosine. Energy deprivation along with dipyridamole administration resulted in higher coronary flow rate. Lower perfusate adenosine concentration was observed along with higher tissue adenosine content in this group. These results implied that coronary flow rate is determined not by interstitial adenosine concentration but by intracellular activity of adenosine. To confirm the effects of dypiridamole in vivo, direct measurement of interstitial adenosine concentration by mycrodialysis along with the assay of intracellular adenosine content was performed after intranenous dipyridamole administration. After dipyridamole infusion, intracellular adenosine content was markedly increased while interstitial adenosine concentration was not altered. In another series of experiments, the right shift of concentration-response curve of adenosine-induced vasodilation by 8-phenyltheophilline, a representative adenosine receptor antagonist, was mostly abolished by prior administration of prazosin, indicating that the influence of 8-PT on the adenosine action is not attributed to the inhibition of A2 receptor but related to the suppression of ${\alpha}-adrenoceptor$ activation. From these results, we concluded that adenosine acts intracellularly to regulate the coronary blood flow.

• Biomolecules & Therapeutics
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• 제27권6호
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• pp.584-590
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• 2019

Luteolin, a widespread flavonoid, has been known to have neuroprotective activity against various neurologic diseases such as epilepsy, and Alzheimer's disease. However, little information is available regarding the hypnotic effect of luteolin. In this study, we evaluated the hypnotic effect of luteolin and its underlying mechanism. In pentobarbital-induced sleeping mice model, luteolin (1, and 3 mg/kg, p.o.) decreased sleep latency and increased the total sleep time. Through electroencephalogram (EEG) and electromyogram (EMG) recording, we demonstrated that luteolin increased non-rapid eye movement (NREM) sleep time and decreased wake time. To evaluate the underlying mechanism, we examined the effects of various pharmacological antagonists on the hypnotic effect of luteolin. The hypnotic effect of 3 mg/kg of luteolin was not affected by flumazenil, a GABAA receptorbenzodiazepine (GABAAR-BDZ) binding site antagonist, and bicuculine, a GABAAR-GABA binding site antagonist. On the other hand, the hypnotic effect of 3 mg/kg of luteolin was almost completely blocked by caffeine, an antagonist for both adenosine A1 and A2A receptor (A1R and A2AR), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), an A1R antagonist, and SCH-58261, an A2AR antagonist. From the binding affinity assay, we have found that luteolin significantly binds to not only A1R but also A2AR with $IC_{50}$ of 1.19, $0.84{\mu}g/kg$, respectively. However, luteolin did not bind to either BDZ-receptor or GABAAR. From these results, it has been suggested that luteolin has hypnotic efficacy through A1R and A2AR binding.

• The Korean Journal of Physiology and Pharmacology
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• 제23권3호
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• pp.191-201
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• 2019

The transient receptor potential canonical (TRPC) 5 channel, known as a nonselective cation channel, has a crucial role in calcium influx. TRPC5 has been reported to be activated by muscarinic receptor activation and extracellular pH change and inhibited by the protein kinase C pathway. Recent studies have also suggested that TRPC5 is extracellularly activated by englerin A (EA), but the mechanism remains unclear. The purpose of this study is to identify the EA-interaction sites in TRPC5 and thereby clarify the mechanism of TRPC5 activation. TRPC5 channels are over-expressed in human embryonic kidney (HEK293) cells. TRPC5 mutants were generated by site-directed mutagenesis. The whole-cell patch-clamp configuration was used to record TRPC5 currents. Western analysis was also performed to observe the expression of TRPC5 mutants. To identify the EA-interaction site in TRPC5, we first generated pore mutants. When screening the mutants with EA, we observed the EA-induced current increases of TRPC5 abolished in K554N, H594N, and E598Q mutants. The current increases of other mutants were reduced in different levels. We also examined the functional intactness of the mutants that had no effect by EA with TRPC5 agonists, such as carbachol or $GTP{\gamma}S$. Our results suggest that the three residues, Lys-554, His-594, and Glu-598, in TRPC5 might be responsible for direct interaction with EA, inducing the channel activation. We also suggest that although other pore residues are not critical, they could partly contribute to the EA-induced channel activation.