近日科學(xué)家們發(fā)現(xiàn)了一種小分子可顯著延長(zhǎng)人類細(xì)胞的晝夜節(jié)律，其作用遠(yuǎn)遠(yuǎn)強(qiáng)過目前已知的任何藥物及基因操作方法,。研究人員證實(shí)這種小分子可使人類細(xì)胞的晝夜循環(huán)延長(zhǎng)10小時(shí)以上,，同時(shí)還可延長(zhǎng)斑馬魚的晝夜節(jié)律,。研究論文發(fā)表在12月14日的 PLoS Biology雜志上。這是研究人員第一次應(yīng)用大規(guī)?；衔锖Y查的方法開展晝夜節(jié)律研究,。新技術(shù)可幫助研究人員揭示出生物鐘的機(jī)制，并篩選出治療晝夜節(jié)律紊亂的候選藥物,。
地球上幾乎所有的生物體其晝夜節(jié)律都可隨著它們的行為,、生理及新陳代謝而發(fā)生改變。機(jī)體內(nèi)生物鐘異常不僅會(huì)導(dǎo)致睡眠障礙,，還與心血管疾病,、癌癥和許多代謝性疾病相關(guān)。盡管目前科學(xué)家們已發(fā)現(xiàn)了多個(gè)生物鐘基因,，證實(shí)這些基因編碼的轉(zhuǎn)錄因子組成的網(wǎng)絡(luò)可使得與生物晝夜節(jié)律相關(guān)的成千上萬的基因呈節(jié)律性表達(dá),。但是目前科學(xué)家們對(duì)于生物鐘系統(tǒng)的組成仍知之甚少。
為了鑒別出可影響生物鐘功能的其他因子,，加州大學(xué)圣地亞哥分校的分子生物學(xué)家Steve Kay及同事們決定采用一種獨(dú)特的方法即通過化合物篩選的方法檢測(cè)他們手中約10萬種小分子對(duì)于生物鐘周期的影響,。
在試驗(yàn)中研究人員利用了一種特殊的人類細(xì)胞系。這種細(xì)胞系是研究人員從前開發(fā)的一種分析系統(tǒng),，當(dāng)細(xì)胞的生物鐘打開時(shí)細(xì)胞就會(huì)發(fā)出亮光,。然后研究人員將12萬種結(jié)構(gòu)不同的功能未知的化合物分別處理這些細(xì)胞，并觀察哪些細(xì)胞非同步發(fā)光,。
盡管有大量的化合物都影響了細(xì)胞的生物鐘,，然而有一個(gè)化合物特別引起了研究人員的注意。研究人員發(fā)現(xiàn)這個(gè)化合物可以以劑量依賴性方式延長(zhǎng)細(xì)胞的周期,。研究人員接著又合成了大約40個(gè)該復(fù)合物的相關(guān)衍生物,，并證實(shí)其中一個(gè)效應(yīng)更強(qiáng)，可延長(zhǎng)周期超過10個(gè)小時(shí),。“這遠(yuǎn)遠(yuǎn)超過任何一個(gè)單基因突變所能達(dá)到的效應(yīng),，”Kay在給 The Scientist雜志的郵件中說道。研究人員將這個(gè)分子命名為“longdaysin”,。
此外研究人員還在斑馬魚體內(nèi)檢測(cè)了longdaysin的效應(yīng),，并發(fā)現(xiàn)了與人類細(xì)胞培養(yǎng)物中看到的類似的周期延長(zhǎng)效應(yīng)，證實(shí)這個(gè)化合物確實(shí)能夠有效影響生物鐘周期,。
在檢測(cè)longdaysin的作用機(jī)理過程中,，研究人員證實(shí)longdaysin是通過作用于三種蛋白質(zhì)激酶CKIδ, CKIα, 和ERK2而影響生物鐘周期的,。除了CKIδ是晝夜節(jié)律系統(tǒng)的一個(gè)已知的組件，CKIα 和 ERK2均是第一次被鑒定為生物鐘基因,，表明這種化合物篩查方法是一種用于鑒別新的生物鐘組件的有效方法,。
“我們的化學(xué)探針的優(yōu)勢(shì)在于它能夠針對(duì)多個(gè)靶點(diǎn)，從而揭示出了兩個(gè)與生物鐘有關(guān)的新激酶,，”Kay說：“現(xiàn)在我們能夠明確地進(jìn)行多種生物鐘化合物篩查,，這將推動(dòng)我們揭示更多的生物鐘新功能。”（生物谷Bioon.com）
生物谷推薦原文出處：
PLoS Biology DOI: 10.1371/journal.pbio.1000559
High-Throughput Chemical Screen Identifies a Novel Potent Modulator of Cellular Circadian Rhythms and Reveals CKIα as a Clock Regulatory Kinase
Tsuyoshi Hirota1,2, Jae Wook Lee2,3, Warren G. Lewis1,2, Eric E. Zhang1,2, Ghislain Breton1, Xianzhong Liu2, Michael Garcia2, Eric C. Peters2, Jean-Pierre Etchegaray4, David Traver1, Peter G. Schultz2,3, Steve A. Kay1*
1 Division of Biological Sciences, University of California San Diego, La Jolla, California, United States of America, 2 Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America, 3 Department of Chemistry, The Scripps Research Institute, La Jolla, California, United States of America, 4 Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America
Abstract
The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ~120,000 uncharacterized compounds on human cells containing a circadian reporter. The analysis identified a small molecule that potently lengthens the circadian period in a dose-dependent manner. Subsequent analysis showed that the compound also lengthened the period in a variety of cells from different tissues including the mouse suprachiasmatic nucleus, the central clock controlling behavioral rhythms. Based on the prominent period lengthening effect, we named the compound longdaysin. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. Combined with siRNA-mediated gene knockdown, we identified the protein kinases CKIδ, CKIα, and ERK2 as targets of longdaysin responsible for the observed effect on circadian period. Although individual knockdown of CKIδ, CKIα, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIα in the clock mechanism and found that CKIα-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIδ. Longdaysin treatment inhibited PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIα as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species.
