• Biomolecules & Therapeutics
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• 제18권2호
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• pp.152-158
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• 2010

Melanin concentrating hormone is a neuropeptide highly expressed in the brain that regulates several physiological functions mediated by receptors in the G-protein coupled receptor family, especially plays an important role in the complex regulation of energy balance and body weight mediated by the melanin concentrating hormone receptor subtype 1 (MCH1). Compelling pharmacological evidence implicating MCH1 signaling in the regulation of food intake and energy expenditure has generated a great deal of interest by pharmaceutical companies as MCH1 antagonists may have potential therapeutic benefit in the treatment of obesity and metabolic syndrome. Although fluorescence-based calcium mobilization assay platform has been one of the most widely accepted tools for receptor research and drug discovery, fluorescence interference and shallow assay window limit their application in high throughput screening and have led to a growing interest in alternative, luminescence-based technologies. Herein, a luminescence-based functional assay system for the MCH1 receptor was developed and validated with the mitochondrial targeted aequorin. Aequorin based functional assay system for MCH1 presented excellent Z' factor (0.8983) and high signal-to-noise ratio (141.9). The nonpeptide MCH1 receptor antagonist, SNAP 7941 and GSK 803430, exhibited $IC_{50}$ values of 0.62 ${\pm}$ 0.11 and 12.29 ${\pm}$ 2.31 nM with excellent correlation coefficient. These results suggest that the aequorin based assay system for MCH1 is a strong alternative to the traditional GPCR related tools such as radioligand binding experiments and fluorescence functional determinations for the compound screening and receptor research.

• Molecules and Cells
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• 제22권2호
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• pp.198-202
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• 2006

The $P2X_7$ receptor, an ATP-gated cation channel, induces cell death in immune cells and is involved in neurodegenerative diseases. Although the receptor plays various roles in these diseases, the cellular mechanisms involved are poorly understood and antagonists are limited. Here, the development of a cell-based assay for human $P2X_7$ receptor is reported. We established permanent lines of HEK 293 cells expressing a high level of $hP2X_7$ receptor. Functional activity of the $hP2X_7$ receptor was confirmed by whole-cell patch recording of ATP-induced ion currents. Prolonged exposure to ATP resulted in death of the $hP2X_7$-expressing HEK 293 cells and this cell death could be quantified. Two known $P2X_7$ antagonists, PPADS and KN-62, blocked ATP-induced death in a concentration-dependent manner. Thus, this assay can be used to screen for new antagonists of $hP2X_7$ receptors.

• Biomolecules & Therapeutics
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• 제19권1호
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• pp.1-8
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• 2011

G-protein coupled receptors (GPCRs) constitute an important class of drug targets and are involved in every aspect of human physiology including sleep regulation, blood pressure, mood, food intake, perception of pain, control of cancer growth, and immune response. Radiometric assays have been the classic method used during the search for potential therapeutics acting at various GPCRs for most GPCR-based drug discovery research programs. An increasing number of diverse small molecules, together with novel GPCR targets identified from genomics efforts, necessitates the use of high-throughput assays with a good sensitivity and specificity. Currently, a wide array of high-throughput tools for research on GPCRs is available and can be used to study receptor-ligand interaction, receptor driven functional response, receptor-receptor interaction,and receptor internalization. Many of the assay technologies are based on luminescence or fluorescence and can be easily applied in cell based models to reduce gaps between in vitro and in vivo studies for drug discovery processes. Especially, cell based models for GPCR can be efficiently employed to deconvolute the integrated information concerning the ligand-receptor-function axis obtained from label-free detection technology. This review covers various platform technologies used for the research of GPCRs, concentrating on the principal, non-radiometric homogeneous assay technologies. As current technology is rapidly advancing, the combination of probe chemistry, optical instruments, and GPCR biology will provide us with many new technologies to apply in the future.

• Toxicological Research
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• 제17권1호
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• pp.65-71
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• 2001

The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. A yeast-based steroid hormone receptor gene trascription assay was previously developed for the evaluation of chemicals with endocrine modulating activity. The yeast transformants used in this assay contain the human estrogen receptor along with the appropriate steroid response elements upstream of the $\beta$-galactosidase reporter gene. We tried to evaluate several natural and synthetic steroids of their potential to interact directly with the steroid receptor. Some putative endocrine disruptors, including nonylphenol, are weakly estrogenic. But the combined treatment oj these chemicals with di-(2-ethylhexyl)phthalate (DEHP) significantly increased the $\beta$-galactosidase activity in the yeast transformant. These results suggest that we also have to consider the synergistic effects of endocrine disruptors. In this study, we showed that yeast-based bioassay is a valuable tool for screening potential endocrine disruptors and quantitative determination of estrogenicity. And the possibility that the estrogen receptor binds multiple environmental chemicals adds another level of complexity to the interaction between the endocrine disruptors and the human hormone system.

• 생약학회지
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• 제37권1호통권144호
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• pp.21-27
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• 2006

Yeast based estrogenicity assay is the simplest and useful for the assay and the discovery of novel estrogenic substances in natural specimens, The estrogen receptor(ER) modulation activity of 50% EtOH extracts of 101 traditional medicinal herbs was assessed using a recombinant yeast assay system with both a human estrogen receptor expression plasmid and a receptor plasmid. Among them, 14 species proved to be active. Pureariae Flos (flower of Puerraria thunbergiana BENTH.) had the highest estrogenic relative potency$(7.75{\times}10^{-3})$ $(EC_{50}=9.39\;{\mu}g/ml)$. The $EC_{50}$ value of $17{\beta}-estradiol$ used as the positive control was $0.073\;{\mu}g/ml)$ (Relative Potency=1.00). There results demonstrated that some of the traditional medical herb may be useful in the therapy of estrogen replacement.

• KSBB Journal
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• 제22권6호
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• pp.426-432
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• 2007

QDs을 기반으로 하는 OsFIA는 매우 빠르고 간단히 수행될 수 있다. 또한 이 분석법은 고체상의 담체나 결합/잔류시약의 분리 등과 같은 여러 과정을 필요로 하지 않으며, 적은 양의 시약으로도 분석이 가능하다. 본 분석법은 높은 감도로 항원을 측정할 수 있으며, 일상적인 분석에도 쉽게 도입될 수 있을 것이다. 선형 범위 내에서 측정 가능한 receptor의 최소농도는 0.05 nM (2.65 ng/mL) 정도이다. 또한, 일반적으로 상용화된 항체를 가치고 수행이 가능하다. 이 OsFIA 분석법은 기존의 실험적 sandwich immunoassay의 효과적인 대안으로 제시된다.

• Journal of Microbiology and Biotechnology
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• 제29권9호
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• pp.1470-1477
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• 2019

${\beta}_2$-adrenergic receptor (${\beta}_2-AR$) was expressed efficiently using Bac-to-Bac Baculovirus Expression System in Sf9 cells as a bio-recognition element for multianalyte screening of ${\beta}$-agonist residues in pork. Sf9 cells were selected as the expression system, and codon optimization of wild-type nucleic acid sequence and time-dependent screening of expression conditions were then carried out for enhancing expression level and biological activity. Under optimum conditions of multiplicity of infection (MOI) = 5 and 48 h post transfection, the protein yield was up to 1.23 mg/ml. After purification by chromatographic techniques, the purified recombinant protein was applied to develop a direct competitive enzyme-linked receptor assay (ELRA) and the efficiency and reliability of the assay was determined. The IC50 values of clenbuterol, salbutamol, and ractopamine were 28.36, 50.70, and $59.57{\mu}g/l$, and clenbuterol showed 47.61% and 55.94% cross-reactivities with ractopamine and salbutamol, respectively. The limit of detection (LOD) was $3.2{\mu}g/l$ and the relevant recoveries in pork samples were in the range of 73.0-91.2%, 69.4-84.6%, and 63.7-80.2%, respectively. The results showed that it had better performance compared with other present nonradioactive receptorbased assays, indicating that the genetically modified ${\beta}_2-AR$ would have great application potential in detection of ${\beta}$-agonist residues.

• 한국산학기술학회논문지
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• 제17권11호
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• pp.640-647
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• 2016

형광 간섭 현상을 최소화시켜 상대적으로 민감한 측정이 가능한 aequorin기반 luminescence기술은 $G_{{\alpha}16}$ 단백질 도입을 통해 세포 내부의 칼슘 이동 신호를 감지하여 G 단백질 결합 수용체(G protein-coupled receptor, GPCR)의 기능 분석을 가능하게 하는 세포 기반 분석 기술로 수용체 및 G 단백질 유전자 전달의 최적화 과정이 필수적이다. 본 연구를 위해 corticotropin releasing factor receptor subtype 2(CRF2) 수용체를 모델 시스템으로 CRF2와 $G_{{\alpha}16}$ 단백질이 구축된 세 가지 안정화 세포주를 제작하였고, 이들을 이용한 서로 다른 세 가지 조건의 임시 발현 세포주에서 작용제(sauvagine)와 길항제(K41498)의 반응성을 분석하여 최적의 유전자 전달 방법을 도출하고자 하였다. 그 결과 sauvagine 및 K41498의 농도에 따른 반응에서 CRF2-$G_{{\alpha}16}$ 안정화 세포주가 임시 발현 세포주보다 10배 이상의 유효신호 비율을 나타내었고(z'=0.77) 임시 발현 세포주의 경우 $G_{{\alpha}16}$의 안정화 발현 이후에 CRF2를 전달하는 경우가 다른 임시 발현 조건보다 2배 이상 높은 효율을 보였다(z'=0.84). 따라서 임시 유전자 전달 기술을 GPCR 세포 기능 분석 시스템에 활용할 경우 $G_{{\alpha}16}$ 단백질에 대한 안정화 세포주를 우선적으로 구축하고, 목표하는 다양한 수용체들을 단계적으로 발현시키는 것이 최선의 방법이라 판단된다.

• Natural Product Sciences
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• 제20권4호
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• pp.281-284
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• 2014

In this study, twenty-one oleanane-type triterpenoid saponins were isolated from a methanol extract of the roots of Pulsatilla koreana. Antagonistic activities were measured in these compounds by the aequorin based cellular functional assay system for the corticotropin releasing factor receptor (CRF1). Of them, compounds 7 - 10 showed the highest degree of CRF1 inhibition further at the concentration of $10{\mu}M$. Moreover, by the analysis based on the structure-activity relationship of isolated saponins, a sugar chain at C-3 and a carboxyl group at C-28, as well as a methyl group at C-23 seems to be key functional elements. To our knowledge, this is the first report on CRF1 inhibition of saponins from P. koreana.

• 한국산학기술학회논문지
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• 제16권11호
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• pp.7575-7581
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• 2015

corticotropin releasing factor(CRF)는 스트레스에 의해 유도되는 신경펩타이드 물질들 중 하나로서 모발의 손실 및 재성장에 영향을 미친다고 광범위하게 제기되어 왔다. 이에 CRF1 수용체 길항제 개발을 위하여 세포 내 칼슘 신호전달 기전을 이용한 스크리닝 시스템을 개발하고 최적화 연구를 수행하고자 하였다. 이를 위하여 에쿼린 모체세포에 CRF1 수용체와 만능 G 단백질인 G${\alpha}$16 유전자를 동시에 발현시켜 안정화 세포주를 구축하였다(HEK293a16/hCRF1). 표준 효현제인 sauvagine의 반응이 임시 발현세포와 비교하여 안정화 세포주에서 농도 의존적 반응 범위가 12배 이상 증가하였으며($EC_{50}:15.21{\pm}1.83nM$), 이에 따라 길항제 스크리닝에 필수적인 안정적인 신호와 높은 용매 허용도를 확보할 수 있었다. CRF1 수용체에 대한 표준 길항제인 antalarmin과 CP154526에 대한 $IC_{50}$ 수치는 각각 $414.1{\pm}5.5$와 $290.7{\pm}1.9nM$로 확인되었는데 냉동보관세포의 경우에도 유사한 결과를 얻었다. 에쿼린 기반 세포 기능실험의 최적화 연구를 통해 구축된 CRF 수용체 안정화 세포주는 모발의 재형성과 관련된 신규의 기능성 화장품 및 조절물질 개발 연구에 적극적으로 활용 가능 할 것으로 판단된다.