TW202327606A - 抗衰老藥的篩選方法及抗衰老藥 - Google Patents
抗衰老藥的篩選方法及抗衰老藥 Download PDFInfo
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Abstract
本發明之目的係提供可有效率地發現新穎抗衰老藥的篩選方法,又,提供一種藉由該篩選方法所得到之抗衰老藥。
本發明係一種阻礙PGAM與Chk1之結合,進一步使老化細胞選擇性地滅絕。具體而言,本發明之抗衰老藥含有選自由mRSK1激酶阻礙劑、Fak激酶阻礙劑、Fak激酶所參與之信號通路的阻礙劑、CDK阻礙劑、鈣拮抗劑、強心配糖體、DNA障礙劑、抗菌劑、極光激酶阻礙劑、類黃酮、PI3K激酶阻礙劑、HDAC阻礙劑、老年性黃斑變性治療藥、p38MAPK阻礙劑、mTOR阻礙劑、酪胺酸激酶阻礙劑、Bc1-2阻礙劑、他汀類藥物、血清素受體拮抗藥、其他激酶阻礙劑、及Nutlin-3b所構成之群組中的至少1種,作為有效成分。
Description
本發明係關於抗衰老藥之篩選方法及藉由該篩選方法所得到的新穎抗衰老藥。
當今,日本為世界上唯一高齡化率超過25%(2014年)的國家。高齡化率之世界平均值雖為8%,但全球高齡化之浪潮正穩定而快速地進展,預測約2050年左右歐洲各國、中國、韓國、泰國、新加坡、伊朗、智利、加拿大等高齡化率將皆超過25%,世界平均值亦將達到18%(WHO預測)。其中,全球高齡化之實際狀態,係與高齡者全面健康增進之同時,多病且多樣之高齡者亦增加。因此,只依據時間軸之「老化」定義在現狀已不合現狀,高齡者之再定義有其必要(2017年日本老年醫學會提議),並且由針對增加之老年性疾病的嶄新對策所致之醫藥品開發成為緊迫的課題。
「日暦年齡」與高齡者之現狀(症狀)不一致之點,亦在基礎老化研究中的細胞老化研究成果中被指出。先前,由於隨著細胞老化的同時,端粒(telomere)(每次細胞分裂變短之染色體末端結構)的長度變短,端粒亦被稱為「老化時鐘」。然而近年來,就端粒非依存性細胞老化而言,發現
「壓力細胞老化」(即使為年輕細胞,亦即,端粒長之細胞也因各種壓力而老化)。該「壓力細胞老化」係作為「抑制致癌之身體防禦機構」(正面效果)而受到注目,但另一方面,亦已知因壓力而老化的細胞係牽連周圍的細胞而造成諸如「慢性炎症」或「癌化」等負面效果。該發現是最新基礎老化研究的成果之一。
具體而言,首先,由Adler氏等人詳細地檢討,判明「老化細胞比年輕細胞不易死亡」的原因之一是獲得了Bc1-2活化等抗細胞凋亡能力(細胞死亡抗性)(非專利文獻1:Adler et al.,2007)。擔任該Bc1-2活化之轉錄因子為NF-κB,然而NF-κB亦為控制炎症之關鍵因子,在老化細胞中亦成為炎症活化的原因。約略同時期,Campisi氏等人發現相較於年輕細胞,老化細胞容易分泌IL-1或IL-6等炎症細胞激素,提出以SASP(Senescence associated secretary phenotype(衰老相關性分泌表型))為名之報告(非專利文獻2:Coppe et al.,2008)。推測源自老化細胞之SASP導致周圍細胞之老化或癌化之促進,以及推測細胞老化為經由「慢性炎症」而誘導個體老化的原因之一。基於以上之認知,「壓力細胞老化」被推想為老化或老年性疾病進展的重要原因之一。
基於上述之認知,「抗衰老」(Senolysis;去除老化細胞)可能成為開發老年性疾病之醫療的新穎途徑。基於上述,亦同時提案「若將成為慢性炎症之重大原因的老化細胞去除,可改善個體老化」的嶄新構想。Darren Baker氏使用如BubR1亞型小鼠(hypomorphic mouse)之顯示早期老化系統的小鼠,並只將老化細胞(屬於老化標記之p16INk4a陽性細胞)選擇性地殺死,而成功地去除遺傳學上之老化細胞,同時亦改善各種臟器的
老化現象。此為由遺傳學上的抗衰老所致之延遲個體小鼠之老化表現型的最早報導(非專利文獻3:Baker et al.,2011)。又,最近發現顯示同樣作用的藥劑(ABT263),其被稱為抗衰老藥(非專利文獻4:Chang et al.,2016)。先前,ABT263係以作為Bc1-2阻礙劑而被開發的抗癌劑(非專利文獻5:Tse et al.,2008),亦與上述之Adler氏等人的見解(Bc1-2亢進為老化細胞之抗細胞凋亡的原因)一致。在小鼠個體中,ABT263處理亦改善老化或動脈硬化(非專利文獻4:Chang et al.,2016、非專利文獻6:Childs et al.,2016)。
抗衰老藥方面亦有上述Bc1-2阻礙劑(ABT263)以外的藥劑的報導。Yi Zhu氏等人(JL Kirkland之團隊),從以老化細胞總轉錄本(transcriptome)解析等情報為基礎進行篩選所得到的46種藥劑,發現如D+Q(達沙替尼(dasanitib)+槲皮素(quercetin))之組合係發揮抗衰老效果。達沙替尼(dasanitib)係作為BCR-ABL等酪胺酸激酶阻礙劑作用的抗癌劑,槲皮素(quercetin)為類黃酮之一種,且為PI3K激酶阻礙劑(非專利文獻7:Zhu et al.,2015)。
又,二個團隊同時發現由強心配糖體所致的抗衰老效果(非專利文獻8:Guerrero et al.,2019、非專利文獻9:Triana-Martinez et al.,2019)。原先,已知強心配糖體係作為藉由阻礙細胞表面之Na+/K+泵浦,而增強心肌細胞的心臟衰竭治療藥。在老化細胞中,觀察到藉由Na+/K+泵浦之阻礙,誘導NOXA(Bc1-2同系物(homolog)),並誘導細胞凋亡(apoptosis)。
另一方面,解糖系代謝意指藉由將細胞攝取之葡萄糖分解,合成所謂ATP之高能量分子之代謝過程的總稱。解糖系代謝中,存在二條途徑:從為其中間產物之丙酮酸合成乳酸的過程,及在粒線體中進行之氧化性磷酸化的過程。前者由於不需要氧呼吸,被稱為嫌氣性解糖系代謝,後者由於藉由粒線體氧呼吸合成ATP,被稱為好氣性解糖系代謝。該解糖系代謝幾乎在所有一般細胞中擔任能量代謝的重要角色,為個體生存上不可或缺者。因此,糖解作用(glycolysis)代謝之異常與人類之疾病密切相關。
若糖解作用代謝降低,成為神經變性疾病、心臟功能降低、糖尿病、肌肉萎縮、貧血等之原因,另一方面,已知糖解作用代謝亢進之病態亦有幾種。就糖解作用代謝亢進之病態而言,例如可列舉癌、缺血(血流降低)、局部炎症等,然而已知特多之癌細胞中,出現糖解作用代謝亢進(瓦氏效果(Warburg effect);非專利文獻10;Warburg O.,Science,1956,124:p.269-270),其成為癌之細胞生物學特徵之一。實際上,現今之臨床現場,利用此性質之FDG(氟去氧葡萄糖)-PET,係對患者體內之腫瘤大小或有無轉移的評估有用且廣泛普及使用。
糖解作用代謝途徑係由10個糖解作用酵素依序反應構成。糖解作用酵素磷酸甘油酸互變酶PGAM係作為糖解作用代謝途徑的第8號之酵素,其係催化從3-磷酸甘油酸酯(3PG)轉變至2-磷酸甘油酸酯(2PG)的反應。本發明人等發現藉由在細胞中強烈地表現PGAM,而誘導糖解作用亢進及氧化壓力減弱效果,而使細胞變得不易老化,另一方面,使PGAM失活時,則誘導壓力老化(非專利文獻11:Kondoh et al.,2005、非專利文獻12:Mikawa et al.,2014)。又,發現在細胞內PGAM與屬於絲胺酸蘇
胺酸蛋白激酶之Chk1結合,然後此物理性結合在糖解作用代謝中扮演重要角色,又,藉由控制此物理性結合可控制糖解作用。再者,亦成功地發現新穎的糖解作用代謝控制物質,該糖解作用代謝控制物質係特異性阻礙癌中之糖解作用代謝的物質,在欲抑制細胞老化之細胞、組織中發揮抗老化效果(anti-aging effect)之物質等(專利文獻1)。
[先前技術文獻]
[專利文獻]
[專利文獻1]日本特開2018-194299號公報
[非專利文獻]
[非專利文獻1]Adler et al., Genes Dev., 2007, 21, 3244-3257
[非專利文獻2]Coppe et al., PLoS Biol., 2008, 6, 2853-2868
[非專利文獻3]Baker et al., Nature, 2011, 479, 232-236
[非專利文獻4]Chang et al., Nat. Med., 2016, 22, 78-83
[非專利文獻5]Tse et al., Cancer Res., 2008, 68, 3421-3428
[非專利文獻6]Childs et al., Science, 2016, 354, 472-477
[非專利文獻7]Zhu et al., Aging Cell, 2015, 14, 644-658
[非專利文獻8]Guerrero et al., Nat Metab., 2019, 1, 1074-1088
[非專利文獻9]Triana-Martinez et al., Nat. Commun., 2019, 10, 4731
[非專利文獻10]Warburg O., Science, 1956, 124, 269-270
[非專利文獻11]Kondoh et al., Cancer Res., 2005, 65, 177-185
[非專利文獻12]Mikawa et al., J. Cell Biol., 2014, 204, 729-745
在如上述之狀況下,本發明之目的係提供一種可有效率地發現新穎抗衰老藥的篩選方法,又提供一種藉由該篩選方法而得到之抗衰老藥。
本發明人等為了解決上述課題而專心研究的結果,確立一種可有效率地發現新穎抗衰老藥之篩選方法,該篩選方法係具有:測定受驗物質之PGAM與Chk1之物理性結合之阻礙活性的初次篩選步驟、及測定選擇性去除老化細胞活性的二次篩選步驟。又,藉由該篩選方法發現許多新穎抗衰老藥。再者,進行PGAM1-Chk1途徑之下游轉錄因子之解析的結果,判明在PGAM1-Chk1結合之下游存在著轉錄因子HIF-2α,HIF-2αsiRNA亦具有抗衰老活性。從此結果可知,除了PGAM1-Chk1結合之控制以外,控制HIF-2α的信號(亦包含乙醯基化或泛素化)的調節亦可經由HIF-2α之失活化而誘導抗衰老。再者,該轉錄因子HIF-2α已知亦朝抑制屬於細胞凋亡誘導基因之BIM的表現的方向作用。亦即,本發明係如下列。
[1]一種抗衰老藥,其係阻礙PGAM與Chk1之結合,更選擇性地使老化細胞滅絕。
[2]如[1]記載之抗衰老藥,其含有選自下列由RSK1激酶阻礙劑、Fak激酶阻礙劑、Fak激酶所參與之信號通路的阻礙劑、CDK阻礙劑、鈣拮抗
劑、強心配糖體、DNA障礙劑、抗菌劑、極光激酶(aurora kinase)阻礙劑、類黃酮(flavonoid)、PI3K激酶阻礙劑、HDAC阻礙劑、老年性黃斑變性治療藥、p38MAPK阻礙劑、mTOR阻礙劑、酪胺酸激酶阻礙劑、Bc1-2阻礙劑、他汀類藥物、血清素受體拮抗藥、其他激酶阻礙劑、及Nutlin-3b所構成之群組中的至少1種作為有效成分。
[3]如[2]記載之抗衰老藥,其中
RSK1激酶阻礙劑為RSK1之反義核酸、RSK1之siRNA、或BI-D1870,
Fak激酶阻礙劑為PF-00562271或PF-431396,
Fak激酶所參與之信號通路的阻礙劑為PHA-665752或CP-100356,
CDK阻礙劑為SNS-032、AZD5438、黃酮吡醇(阿伏西地(Alvocidib))、PHA-793887、AT7519、或PHA-767491,
強心配糖體為海蔥次苷A(Proscillaridin A)或地高辛(digoxin),
DNA障礙劑為多柔比星(Doxorubicin)(阿黴素(Adriamycin))HCl、道諾黴素(Daunorubicin)HCl、泛艾黴素(Epirubicin)HCl、米托蒽醌(Mitoxantrone)2HCl或羥基喜樹鹼(hydroxy camptothecine),
抗菌劑為胺基吖啶(Aminoacridine)、三氯沙(Triclosan)或依沙吖啶乳酸鹽(Ethacridine lactate),
極光激酶阻礙劑為AMG-900或JNJ-7706621,
類黃酮為金黃素(Chrysin),
PI3K激酶阻礙劑為PF-05212384或XL147,
HDAC阻礙劑為曲古抑菌素A(Trichostatin A)(TSA),
老年性黃斑變性治療藥為維替泊芬(Verteporfin),
p38MAPK阻礙劑為SB203580或達馬莫德(Dora mapimod)(BIRB796),
mTOR阻礙劑為AZD8055或KU-0063794,
酪胺酸激酶阻礙劑為來那替尼(Neratinib)、尼達尼布(Vargatef)或NVP-BHG712,
Bc1-2阻礙劑為TW-37,
他汀類藥物為美伐他汀(Mevastatin),
血清素受體拮抗藥為RS-127445,
其他激酶阻礙劑為BMS-345541或CX-4945。
[4]一種抗衰老藥之篩選方法,其係包含下列步驟:
(A)對於受檢物質,測定PGAM與Chk1之結合阻礙活性的步驟;及
(B)對於藉由上述步驟(A)經判斷具有PGAM與Chk1之結合阻礙活性之受驗物質,測定選擇性去除老化細胞活性的步驟。
[5]如[4]記載之篩選方法,其中,在上述步驟(B)中,測定受驗物質對年輕細胞的傷害活性、及對老化細胞之傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質。
[6]一種抗衰老藥,其係具有使HIF-2α失活之作用,更選擇性地滅絕老化細胞。
[7]如[6]記載之抗衰老藥,其係含有HIF-2α阻礙劑作為有效成分。
[8]如[7]記載之抗衰老藥,其中,HIF-2α阻礙劑為HIF-2α之反義核酸或siRNA。
[9]一種抗衰老藥之篩選方法,其係包含下列步驟:
(X)對於受檢物質測定HIF-2α之阻礙活性的步驟;及
(Y)對於藉由上述步驟(X)經判斷具有HIF-2α之阻礙活性的受驗物質,測定選擇性去除老化細胞活性之步驟。
[10]如[9]記載之篩選方法,其中,在上述步驟(Y)中,測定受驗物質對年輕細胞之傷害活性及對老化細胞的傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質。
本發明之抗衰老藥之篩選方法,藉由在測定選擇性去除老化細胞活性的步驟之前,進行包含測定PGAM與Chk1之結合阻礙活性之步驟的初次篩選,而可顯著地提升抗衰老藥之篩選效率。本發明人等發現PGAM與Chk1之結合雖然無法在在年輕正常細胞中觀察到,但在老化細胞中卻顯著地亢進,並藉此引起糖解作用代謝之亢進。由於此,藉由在眾多物質之中,首先篩選具有PGAM與Chk1之結合阻礙活性的物質,可容易地得到可在老化細胞中阻礙PGAM與Chk1之結合的物質,藉由對此等物質測定選擇性去除老化細胞活性,可有效率地發現抗衰老藥。實際上,使用本發明之篩選方法,從安全性經確定之2300餘種既存藥資料庫中,藉由初次篩選搜尋出約200種候選藥,然後進行選擇性去除老化細胞活性試驗,最後成功地鑑定出約40種新穎抗衰老藥。從本發明人等之研究,已確認即使在屬於非端粒依存性細胞老化之壓力細胞老化中,亦與所謂的老化
細胞同樣地,會因PGAM與Chk1之結合而引起糖解作用代謝亢進,根據本發明之篩選方法,可發現不僅對於老年性疾病,亦對於藉由壓力細胞老化而發病之疾病可發揮治療效果的物質。再者,由於發現轉錄因子HIF-2α存在於PGAM1-Chk1結合之下游,故可知藉由控制HIF-2α,亦可誘導抗衰老。基於此新認知,藉由篩選使HIF-2α失活之藥劑亦可發現更有效的抗衰老藥。
圖1係在可藉由他莫昔芬(tamoxifen)藥劑誘導細胞老化的細胞中,可計測PGAM-Chk1結合之實驗系統的說明圖。
圖2係誘導細胞老化時,老化標記蛋白量隨時間變化之圖。
圖3係誘導細胞老化時,屬於老化標記之SA-β-Gal染色及PGAM-Chk1結合隨時間變化之圖。
圖4係誘導細胞老化時,糖解作用代謝隨時間變化的圖。上圖展示乳酸產生的變化,下圖展示糖攝取率的變化。
圖5係展示Nutlin-3a及Nutlin-3b之化學結構的圖。
圖6係展示Nutlin-3a及3b阻礙老化細胞中之PGAM-Chk1結合之效果的圖。
圖7係展示老化細胞由Nutlin-3b之處理所致之PGAM-Chk1結合增強之抑制效果的圖。
圖8係展示Nutlin-3a及3b在老化細胞中之糖解作用代謝抑制效果(糖攝取及乳酸產生)的圖。
圖9係展示由Nutlin-3a及Nutlin-3b所致之癌細胞中之PGAM-Chk1結合之阻礙效果的圖。
圖10係展示Nutlin-3a及3b之p53活化效果之比較的圖。
圖11係展示Nutlin-3a及3b之抗衰老效果之比較的圖。
圖12係展示Nutlin-3a及3b之抗衰老效果之比較的圖。
圖13係展示由Nutlin-3b處理所引起之老化細胞之細胞凋亡的圖。
圖14-1係展示對年老小鼠投予Nutlin-3b之效果的圖。
圖14-2係展示對年老小鼠投予Nutlin-3b之效果的圖。
圖15係展示由RSK激酶阻礙劑所致之Chk1激酶之S280磷酸化之抑制效果的圖。
圖16係展示在年輕細胞及老化細胞中,Chk1激酶之S280A及S280D變異型株中的PGAM-Chk1結合的比較圖。
圖17係展示在老化細胞中,RSK1-Thr573之磷酸化亢進及Chk1-Ser280之磷酸化亢進的圖。
圖18係展示RSK1siRNA處理對於老化細胞之效果的圖。
圖19係展示RSK激酶阻礙劑BI D1870之抗衰老效果的圖。
圖20係展示由RSK1siRNA所致之抗衰老效果的圖。
圖21係展示在年輕細胞及老化細胞中轉錄因子HIF-2α之表現之比較圖。
圖22係展示由Nutlin-3b或RSK激酶阻礙劑BI-D1870處理所致之HIF-2α蛋白減少的圖。
圖23係展示HIF-2αsiRNA處理對於年輕細胞、老化細胞之效果的圖。
圖24係展示HIF-2αsiRNA處理對於年輕細胞、老化細胞之效果的圖。
圖25係展示老化細胞由Nutlin-3b處理所致之BIM基因表現之解析結果圖。
圖26係展示老化細胞由HIF-2α基因敲落(knock down)所致之BIM基因表現之解析結果圖。
圖27係展示由BIM基因之減弱所致之抗衰老之抑制效果的圖。
圖28係展示BIM基因之減弱對於由HIF-2α之減弱所導致之抗衰老的效果的圖。
圖29係概略地展示本發明之篩選方法的圖。
圖30係展示本發明之初次篩選之結果的圖。
圖31-1係展示本發明之二次篩選之結果的圖。
圖31-2係展示本發明之二次篩選之結果的圖。
圖32係展示由本發明之篩選方法所得之抗衰老候補藥的圖。
圖33係展示對老年小鼠之Nutlin-3b投予試驗之時程圖。
圖34係展示Nutlin-3b投予對於肌力(掛線測試(wire hang test))、腎功能標記(血漿BUN、血漿肌酸酐)之效果的圖。
圖35係展示Nutlin-3b投予對於肝功能標記(血漿白蛋白值)及血漿乳酸值之效果的圖。
圖36A係展示組織化學分析Nutlin-3b投予對肝臟之影響之結果的圖。
圖36B係展示Nutlin-3b投予之肝臟中SASP因子(IL6、CXCL1、TNFα及CCL5)mRNA表現之抑制效果的圖。
圖37係展示Nutlin-3b投予對脂肪組織之細胞老化之改善效果的圖(SA-β-Gal染色)。
圖38係展示蛋白酶體阻礙劑MG132阻礙老化細胞之由Nutlin-3b處理所致之HIF-2α蛋白質之表現抑制效果的圖。
圖39係展示在老化細胞之Nutlin-3b處理中HIF-2αmRNA之表現無變動的圖。
圖40係展示位於HIF-2α之N末端,由Chk1所致之磷酸化之共同序列(consensus sequence)的圖。
圖41係展示HIF-2α之S12A變異、S19A變異對於老化細胞中之HIF-2α蛋白質穩定性之影響的圖。
圖42係展示S12A變異、S19A變異對於老化細胞中之HIF-2α泛素化修飾的HIF-2α之影響的圖。
圖43係展示老化細胞中,Chk1依存性HIF-2α之Ser12殘基之磷酸化的圖。
圖44係展示人類纖維母細胞中之PGAM-Chk1相互作用,藉由老化細胞之培養上清液而被增強的圖。
圖45係展示人類纖維母細胞中之HIF-2α之蓄積及該HIF-2α之Ser12之磷酸化、PGAM-Chk1相互作用之增強依存於乳酸濃度的圖。
圖46係展示乳酸對於人類纖維母細胞中之SASP表現、p21表現之影響的圖。
圖47係展示老化細胞中之乳酸受體(GPR81)之表現亢進的圖。
圖48係展示由乳酸受體(GPR81)基因敲落所導致之PGAM-Chk1結合阻礙的圖。
圖49係展示由乳酸處理所導致的RSK、Chk1之磷酸化亢進的圖。
圖50係展示細胞凋亡阻礙劑QVD-Oph,於Nutlin-3b處理下回復老化細胞之生存性的圖。
圖51係展示以Nutlin-3b處理之老化細胞之轉錄組(transcriptome)解析結果的圖。
圖52係展示以Nutlin-3b處理之老化細胞之轉錄組解析結果的圖。
圖53係展示使用ChIPAtlas,將與以Nutlin-3b處理之老化細胞的轉錄組解析結果關係密切之轉錄因子以已報導之ChIP數據組進行探索的圖。
圖54係展示在老化細胞中,藉由Nutlin-3b處理而調降(down regulation)之基因中,被包含於前150位基因中的FOXM1之標的基因的圖。
圖55係展示在老化細胞中,藉由Nutlin-3b處理受到影響的41個細胞凋亡控制相關基因中,前10位之調降基因的圖。
圖56係展示老化細胞中存活素(survivin)之基因敲落之效果的圖。
圖57係展示藉由Nutlin-3b處理受到影響之老化細胞中之mRNA表現的圖。
圖58係展示藉由Nutlin-3b投予所致之FOX家族轉錄因子標的基因之表現變動解析(GSEA解析)結果的圖。
圖59係展示藉由Nutlin-3b投予所致之FOX家族轉錄因子標的基因之表現變動解析(GSEA解析)結果的圖。
圖60係展示藉由Nutlin-3b投予所致之FOX家族轉錄因子之表現變動的圖。
圖61係展示藉由HIF-2α基因敲落所致之FOXM1、存活素之表現降低的圖。
以下,詳細地說明本發明之抗衰老藥之篩選方法及抗衰老藥。
<抗衰老藥之篩選方法>
本發明之抗衰老藥之篩選方法,係一種發現阻礙PGAM與Chk1之結合,且選擇性地滅絕老化細胞之物質的方法,具體而言,該方法包含下列步驟:(A)對於受檢物質測定PGAM與Chk1之結合阻礙活性的步驟;及,(B)對於藉由上述步驟(A)經判斷具有PGAM與Chk1之結合阻礙活性之受驗物質,測定選擇性去除老化細胞活性的步驟。本發明人等發現PGAM與Chk1之結合,雖然無法在年輕正常細胞中觀察到,但在老化細胞中顯著地亢進,並藉此引起糖解作用代謝之亢進。基於此,在眾多物質之中,首先篩選具有PGAM與Chk1之結合阻礙活性的物質,藉此可容易地得到可在老化細胞中阻礙PGAM與Chk1之結合的物質,針對此等物質藉由測定選擇性去除老化細胞活性,可有效率地發現抗衰老藥。
目前不存在細胞老化之普遍性標記,藉由誘導細胞老化之刺激、細胞之種類、時期等而在老化細胞中所表現的標記相異,故在特定老
化細胞時,必須調查複數個細胞老化相關因子。於是,為了使本發明之技術內容明確,如下述般定義本發明中之老化細胞。亦即,在本發明中,老化細胞意指滿足下述中之至少1種條件的細胞:初代細胞之增殖不可逆地停止;正常的細胞功能降低且顯示所謂「細胞質及細胞核擴大或細胞扁平化」的細胞型態變化;SA-bGAL染色陽性(古典細胞老化標記);p16Ink4A上升(細胞老化標記);H2AXγ染色陽性(DNA損傷之標記)。本發明中之老化細胞亦包含因老齡化而老化(所謂的複製老化)的細胞、壓力老化的細胞(受到各種生物學上之壓力(放射線、變異原、氧化壓力等)的細胞)之任一種的概念。就實驗而言,可使用以人工方式施加DNA傷害的早期老化細胞作為老化細胞。另一方面,年輕(正常)細胞意指上述老化細胞以外的細胞,沒有因老齡化而老化或壓力老化的細胞。
在本發明中,抗衰老(senolysis)意指老化細胞之選擇性細胞死亡,將選擇性地滅絕老化細胞的藥劑稱為抗衰老藥。
在本發明中PGAM,意指磷酸甘油酸互變酶,其為糖解作用代謝途徑中10個糖解作用酵素之依序反應中,擔任其第8號反應的酵素,催化從3-磷酸甘油酸酯(3PG)變換成2-磷酸甘油酸酯(2PG)之反應的酵素。PGAM擔任細胞之老化及癌化之接點。PGAM存在PGAM1及PGAM2之二種同功型(isoform)。PGAM1及PGAM2雖可見到組織表現型態相異,然而在胺基酸序列上具有高同源性,研判功能或酵素活性為同等。在序列表中人類PGAM1、PGAM2之胺基酸序列分別以序列編號1、序列編號2表示,cDNA序列分別以序列編號3、序列編號4表示。
在本發明中,Chk1意指絲胺酸蘇胺酸蛋白激酶,為在細胞周期之G2期DNA損傷檢查點之活化中承擔任務的檢查點激酶。Chk1被本發明人等發現在糖解作用代謝中亦擔任重要角色。於序列表中,人類Chk1之胺基酸序列以序列編號5表示,cDNA序列以序列編號6表示。
本發明中之糖解作用代謝,意指藉由將細胞所攝取之葡萄糖分解合成ATP之代謝過程的總稱。糖解作用代謝中,將葡萄糖分解為其中間產物之丙酮酸,並存在合成乳酸之途徑、及於粒線體中進行氧化性磷酸化之途徑之兩種途徑。前者由於不需要有氧呼吸,被稱為無氧糖解作用代謝,後者由於藉由粒線體有氧呼吸合成ATP,被稱為有氧糖解作用代謝。該糖解作用代謝幾乎在通常細胞中擔任能量代謝之重要角色,在個體生存上為不可或缺者。
針對本發明之篩選方法的各步驟進行說明。本發明之篩選方法包含:步驟(A),其係對於受檢物質測定PGAM與Chk1之結合阻礙活性的步驟;以及,步驟(B),其係對於藉由上述步驟(A)判斷具有PGAM與Chk1之結合阻礙活性之受驗物質,測定選擇性去除老化細胞活性的步驟。
〈步驟(A)〉
本步驟係對於受檢物質測定PGAM與Chk1之結合阻礙活性的步驟。在本步驟中,只要是可測定受檢物質之PGAM與Chk1之結合阻礙活性的方法,則可採用任何方法,可列舉例如:於受檢物質存在下及不存在下測定PGAM與Chk1之結合量,從兩結合量之比,算出上述受檢物質之PGAM與Chk1之結合阻礙活性的方法等。此方法較佳為包含下列步驟1)至3)。
1)在包含PGAM及Chk1之試料中添加受檢物質的情況測定PGAM與Chk1之結合量的步驟
2)在不添加上述受檢物質之情況測定PGAM與Chk1之結合量的步驟
3)藉由下述式(i)算出PGAM與Chk1之結合阻礙活性的步驟。
{來自上述步驟2)之測定值-來自上述步驟1)之測定值}/來自上述2)之測定值×100(%)‧‧‧(i)
在本發明之篩選方法的步驟1)中,首先於包含PGAM及Chk1之試料中添加受驗物質,藉此使PGAM及/或Chk1與受驗物質接觸。
此外,本發明之篩選方法中所使用的PGAM及Chk1係如上述。本發明之篩選方法所使用的PGAM及Chk1中,亦包含與上述周知之PGAM及Chk1功能上同等的蛋白質。如此之蛋白質可列舉例如:PGAM及Chk1之變異體、對偶基因(allele)、變種(variant)、同系物(homologue)、PGAM及Chk1之部分肽、與其他蛋白質之融合蛋白質、經由標籤(tag)標識者等,但不以此等為限。
本發明中之PGAM及Chk1的變異體係可列舉:由上述之胺基酸序列中1個或複數個胺基酸經置換、缺失、插入、及/或附加之胺基酸序列所構成的源自天然之蛋白質,且是與由已述之胺基酸序列所構成之蛋白質功能上同等的蛋白質。又,作為PGAM及Chk1之各別的變異體亦可列舉:在嚴苛條件下與由上述鹼基序列所構成之DNA雜交的源自天然之DNA所編碼的蛋白質,且是與由已述之胺基酸序列所構成之蛋白質功能上同等的蛋白質。
在本發明中,變異之胺基酸數無特別限制,但研判通常為30胺基酸以內,較佳為15胺基酸以內,更佳為5胺基酸以內(例如,3胺基酸以內)。在變異之胺基酸殘基中,較佳為變異為胺基酸側鏈之性質被保存的其他胺基酸。例如,胺基酸側鏈之性質可列舉:疏水性胺基酸(A、I、L、M、F、P、W、Y、V)、親水性胺基酸(R、D、N、C、E、Q、G、H、K、S、T)、具有脂肪族側鏈之胺基酸(G、A、V、L、I、P)、具有包含羥基之側鏈的胺基酸(S、T、Y)、具有包含硫原子之側鏈的胺基酸(C、M)、具有包含羧酸及醯胺之側鏈的胺基酸(D、N、E、Q)、具有包含鹼基之側鏈的胺基酸(R、K、H)、具有包含芳香族之側鏈的胺基酸(H、F、Y、W)(括弧內皆表示胺基酸之單字母標記)。已知有具有經修飾之胺基酸序列的多肽仍維持其生物學之活性,該經修飾之胺基酸序列係藉由對於某胺基酸序列之1或複數個胺基酸殘基之缺失、附加及/或經其他胺基酸序列置換而被修飾。
在本發明中,功能上同等意指成為對象之蛋白質係具有與PGAM或Chk1同等之生物學上的功能或生化學上之功能。PGAM之生物學上功能或生化學上功能係作為糖解作用酵素之功能,具體可列舉:催化從3-磷酸甘油酸酯(3PG)變換成2-磷酸甘油酸酯(2PG)之反應的功能。又,Chk1之生物學上功能或生化學上功能可列舉作為絲胺酸蘇胺酸蛋白激酶之功能。
為了調製編碼與目的蛋白質功能上同等之蛋白質的DNA,就所屬技術領域具有通常知識者所熟知之方法而言,可列舉利用雜交技術或聚合酶連鎖反應(PCR)技術的方法。亦即,對所屬技術領域具有通常知識者而言,通常進行以PGAM或Chk1之鹼基序列或其一部分作為探針,又以
與PGAM或Chk1特異性地雜交之寡核苷酸作為引子,將與PGAM或Chk1具有高同源性的DNA單離。本發明之DNA中亦包含將如此般藉由雜交技術或PCR技術可單離之與PGAM或Chk1具有同等功能之蛋白質編碼的DNA。
本發明之篩選方法所使用的成為PGAM及Chk1之來源的生物種類不限定於特定之生物種類。可列舉例如:人類、猴、小鼠、大鼠、天竺鼠、豬、牛、酵母、昆蟲等。
本發明之篩選方法所使用的PGAM及Chk1之狀態無特別限制,例如可為精製之狀態、表現於細胞內之狀態、表現於細胞萃取液內之狀態等。
在本發明之篩選方法中,亦可使用經標籤標識之PGAM及Chk1,如此之經標籤標識的PGAM及Chk1較佳為以針對彼等之抗體、或可特異地結合之樹脂等所存在之物,可列舉例如:FLAG標籤、HA標籤、Myc標籤、His標籤、GST標籤等。又,如後述,PGAM及Chk1亦能經可互相結合的標籤標識。
本發明中之受驗物質無特別限定,可列舉例如:天然化合物、有機化合物、無機化合物、核酸、蛋白質(包含抗體)、肽等單一物質;化合物資料庫(libary)、核酸資料庫、肽資料庫、基因資料庫之表現產物;細胞萃取物、細胞培養上清液、發酵微生物產生物、海洋生物萃取物、植物萃取物、原核細胞萃取物、真核單細胞萃取物或動物細胞萃取物等萃取物等。又,本發明中之受驗物質亦可為此等之混合物。又,此等受驗物質亦可因應所需加以標識而使用。標識可列舉例如:放射標識、螢光標識等。
又,在本發明中,接觸係因應PGAM及Chk1之狀態而進行。例如,若PGAM及Chk1為精製之狀態,可藉由在精製產品中添加受驗物質而進行。又,若為表現於細胞內之狀態或表現於細胞萃取液內的狀態,可分別藉由在細胞之培養液或該細胞萃取液中添加受驗物質,或直接投予至實驗動物而進行。受驗物質為蛋白質時,例如,亦可將包含編碼該蛋白質之DNA的載體導入至正表現著PGAM及Chk1的細胞,或將該載體添加於正表現著PGAM及Chk1之細胞萃取液中而進行。又,例如,亦可利用使用酵母或動物細胞等的雙雜交(two-hybrid)法。
在本發明中,可藉由在包含PGAM及Chk1之試料中添加受驗物質,使PGAM及/或Chk1與受驗物質接觸。又,亦可在包含PGAM或Chk1之任一者的試料中添加受驗物質後,添加未包含之另一者,而分別與受驗物質接觸。
在本發明中,繼而測定PGAM與Chk1之結合量。此外,在本發明之篩選方法的步驟2)中,係成為於包含PGAM及Chk1之試料中不添加受驗物質的情況測定PGAM與Chk1之結合量。具體而言,測定添加受驗物質之情況(使其接觸的情況),及不添加受驗物質之情況(不使其接觸的情況)之PGAM與Chk1之結合量,並於本發明之篩選方法的步驟3)中,比較此等之值。與不添加受驗物質的情況相比,若添加受驗物質之情況中PGAM與Chk1之結合量減少,則可判斷該受驗物質具有阻礙PGAM與Chk1之結合的效果(有PGAM與Chk1之結合阻礙活性)。於是,如此之受驗物質可判定為具有抑制/阻礙糖解作用代謝的效果,具有對於老化細胞之傷害活性。
測定PGAM與Chk1之結合量的方法例如可具體地舉出如下列之方法。從使PGAM2-FLAG及Chk1-myc-His表現載體共表現之細胞的蛋白質萃取液,藉由連接(Conjugate)於蛋白質G瓊脂糖之FLAG標籤抗體,使PGAM2-FLAG蛋白進行免疫沉降。將此時與PGAM2-FLAG蛋白結合並共沉降的Chk1-myc-His蛋白藉由西方墨點法(Western blotting)檢測並定量。或者是,從同樣之蛋白質萃取液,藉由Ni-NTA瓊脂糖珠粒,使Chk1-myc-His蛋白沉降。將此時與Chk1-myc-His蛋白結合並共沉降的PGAM2-FLAG蛋白藉由西方墨點法檢測並定量。
測定PGAM與Chk1之結合量的方法除上述以外,較佳方法亦可列舉例如使用經能互相結合之標籤標識的PGAM及Chk1之方法,具體而言,可列出如利用Nanobit系統的下述方法。Nanobit中,分別以螢光素酶(luciferase)為基礎作成之發光蛋白質的大次單元及小次單元附著於PGAM及Chk1分別作為標籤。此等標籤形成可互相結合之結構。各個次單元不會自行發光,然而藉由與PGAM及Chk1之結合,大次單元及小次單元會合成為完全發光蛋白,藉此產生發光信號。藉由測定此時之發光信號的強度,可以高敏感度且高定量性之方式測定結合量。
〈步驟(B)〉
在本步驟中,針對藉由上述步驟(A)經判斷為具有PGAM與Chk1之結合阻礙活性的受驗物質,測定選擇性去除老化細胞活性,選出選擇性去除老化細胞活性高之受驗物質。選出上述選擇性去除老化細胞活性高之受驗物質的方法,係可舉出測定受驗物質對年輕細胞之傷害活性及對老化細胞之傷害活性,並選出不具有對年輕細胞之傷害活性,且具有對老化細胞
之傷害活性之受驗物質的方法。又,亦可測定受驗物質對年輕細胞之傷害活性及對老化細胞的傷害活性,選出相較於對年輕細胞之傷害活性,對老化細胞之傷害活性顯著較高的受驗物質。
在此,在本發明中,就年輕細胞而言,於小鼠培養細胞中可列舉:小鼠初代纖維母細胞(MEFs;mouse embryonic fibroblasts,或REFs;rat embryonic fibroblasts)、小鼠初代皮膚角質形成細胞等。又,在本發明中,就年輕細胞而言,在人類培養細胞中可列舉:人類胎兒肺纖維母細胞(IMR90或WI-38、TIG、MRC細胞)、人類皮膚初代細胞(NHDF細胞、BJ細胞、NHEK細胞)、正常人類乳腺上皮細胞(NHMEC細胞)、正常人類血管內皮細胞(HUVEC細胞、HAEC細胞、HPAEC細胞、HCAEC細胞)、正常人類前列腺上皮細胞(HPEC)等。此外,依據細胞種類,雖有其增殖速度不同,老化速度亦相異者,然而一般所知,小鼠細胞從最初之建立,繼續培養約1個月成為老化細胞,人類細胞則約3個月成為老化細胞。因此,小鼠細胞從最初之建立繼續培養2-3週以內稱為年輕細胞,人類細胞從最初之建立連續培養1個月以內稱為年輕細胞。另一方面,在本發明中,就老化細胞而言,可列舉培養超過上述繼續培養期間者,若為小鼠為培養3至4週以上者,若為人類為培養1至2個月以上者。再者,小鼠或人類之年輕細胞,亦可藉由誘導細胞老化之壓力(氧化壓力、致癌壓力、DNA傷害壓力等),於短期間轉變成老化細胞。
<抗衰老藥>
如上述,依照本發明之篩選方法可有效率地發現抗衰老藥,本發明人等實際上成功地發現40種以上之新穎抗衰老藥(具有抗衰老活性的化合
物)。又,本發明人等藉由PGAM-Chk1結合之機制解析,發現在PGAM-Chk1結合中,重要的是藉由屬於致癌性Ras變異之下游信號激酶的RSK1激酶,所引起之Chk1激酶的S280磷酸化。因此,根據該新穎知識,亦可確認RSK1激酶阻礙劑係阻礙GAM-Chk1結合,並且具有對老化細胞之選擇性傷害活性,發揮優異的抗衰老效果。
亦即,本發明之抗衰老藥係阻礙PGAM與Chk1之結合,進一步選擇性地滅絕老化細胞的醫藥,含有如此之化合物作為有效成分。具體而言,本發明之抗衰老藥係含有選自由RSK1激酶阻礙劑、Fak激酶阻礙劑、Fak激酶所參與之信號通路的阻礙劑、CDK阻礙劑、鈣拮抗劑、強心配糖體、DNA障礙劑、抗菌劑、極光激酶阻礙劑、類黃酮、PI3K激酶阻礙劑、HDAC阻礙劑、老年性黃斑變性治療藥、p38MAPK阻礙劑、mTOR阻礙劑、酪胺酸激酶阻礙劑、Bc1-2阻礙劑、他汀類藥物、血清素受體拮抗藥、其他激酶阻礙劑、及Nutlin-3b所構成之群組中的至少1種作為有效成分。
(RSK1激酶阻礙劑)
RSK1為RSK(核糖體S6激酶)家族成員,為增殖因子控制型絲胺酸蘇胺酸激酶。RSK1具有2個相異之激酶觸媒結構域(domain),將包含分裂促進因子活化激酶(MAPK)信號傳達途徑成員之各種基質磷酸化。本發明之抗衰老藥所含有的RSK1激酶之阻礙劑只要為阻礙上述RSK1激酶活性的物質,則無特別限定,較佳例可列舉例如:與編碼RSK1之DNA之轉錄產物互補的反義核酸、或siRNA、BI-D1870(CAS編號:501437-28-1)等化合物。
本發明中所用之反義核酸較佳是阻礙轉錄、剪接或轉譯之過程,抑制RSK1基因之表現的反義核酸。反義序列之態樣若設計成與RSK1之mRNA之5’端附近之非轉譯區域互補的反義序列,推測為有效地阻礙基因之轉譯者。然而,亦可使用與編碼區域或3’側之非轉譯區域互補的序列。據此,不只是基因之轉譯區域,亦可使用與非轉譯區域互補的序列。如此,本發明可利用之反義核酸不只包含含有基因之轉譯區域之序列的核酸,亦包含含有非轉譯區域之序列的反義序列的核酸。反義核酸較佳是對於標的基因之轉錄產物具有90%以上,最佳是95%以上的互補性。使用反義序列來有效地阻礙標的基因之表現的反義RNA之長度係沒有特別限制。
本發明中所用的RSK1之siRNA,意指在細胞中未顯示毒性之範圍內由短鏈所構成的雙股RNA,例如可為15至49鹼基對,較佳為15至35鹼基對,更佳為21至30鹼基對。
RSK1之siRNA不需要與RSK1基因完全相同,但具有至少70%以上,較佳為80%以上,更佳為90%以上,最佳為95%以上之序列的同源性。siRNA中RNA彼此配對之雙股RNA部分,不限於完全配對者,亦可包含藉由錯配(mismatch)(對應之鹼基不互補)、凸出(bulge)(沒有對應其中一鏈之鹼基)等造成的不配對部分。在本發明中,dsRNA中RNA彼此配對的雙股RNA區域中,亦可包含凸出及錯配兩者。
在本發明中,RSK1之siRNA可列舉例如包含序列編號7或序列編號8之序列者。此外,本發明中的siRNA中,為了核酸酶分解耐性提高等目的,亦可在3’末端附加dTdT等外伸(overhang)2鹼基。
(Fak激酶阻礙劑)
Fak(黏著斑激酶)為非受體型蛋白質酪胺酸激酶,參與來自介導細胞與細胞外基質接著之整合素(integrin)濃縮接著點的信號傳達。藉由FAK促進之信號,已知參與附著依存性細胞的生存,對回應增殖因子受體或整合素刺激的有效細胞游走非常重要。本發明之抗衰老藥所含有的Fak激酶阻礙劑只要為阻礙上述Fak激酶活性之物質,則無特別限定,較佳例可列舉例如:PF-00562271(CAS No.939791-41-0)、PF-431396(CAS No.717906-29-1)、或此等之鹽或衍生物而與此等具有同等活性的化合物等。
(Fak激酶所參與之信號通路的阻礙劑)
本發明之抗衰老藥所含有的Fak激酶所參與之信號通路的阻礙劑可列舉c-Met阻礙劑、Mdr-1阻礙劑等。本發明之抗衰老藥所含有的c-Met阻礙劑較佳例可列舉:PHA-665752(CAS No.477575-56-7)、或其之鹽或衍生物而與此等具有同等活性的化合物等。又,本發明之抗衰老藥所含有的Mdr-1阻礙劑較佳例可列舉:CP-100356(CAS No.142715-48-8)、或其鹽或衍生物而與此等具有與其同等活性的化合物等。
(CDK阻礙劑)
週期蛋白(cyclin)依存性激酶(cyclin-dependent kinase:CDK),為於控管細胞周期之核內激酶群中,與週期蛋白結合後才具有活性之蛋白質。在細胞周期之不同時期存在被活化之複數種CDK。活性係藉由CDK自身之酪胺酸、蘇胺酸殘基之磷酸化及脫磷酸化來控制。本發明之抗衰老藥所含有的CDK阻礙劑係阻礙、抑制如此之CDK活性的物質,較佳例可列舉例如:SNS-032(CDK2/7/9阻礙劑;CAS No.345627-80-7)、AZD5438(CDK1/2/9阻礙劑;CAS No.602306-29-6)、黃酮吡醇(Alvocidib)
(CDK1/2/4/6/9阻礙劑;CAS No.146426-40-6)、PHA-793887(CDK7/9阻礙劑;CAS No.718630-59-2)、AT7519(CDK1/2/4/6/9阻礙劑;CAS No.844442-38-2)、PHA-767491(CDK7/9阻礙劑;CAS No.942425-68-5)、或此等之鹽或衍生物而與此等具有同等活性的化合物等。
(鈣拮抗劑)
鈣拮抗劑為與細胞膜上之鈣通道結合,阻礙鈣離子流入細胞內的藥劑。本發明之抗衰老藥所含有的鈣拮抗劑之較佳例可列舉例如:馬尼地平(Manidipine)(CAS No.89226-50-6)、洛美利2HCl(Lomerizine 2HCl)(CAS No.101477-54-7)、或此等之鹽或衍生物而與此等具有同等活性的化合物等。
(強心配糖體)
強心配糖體意指針對伴隨心房撲動(atrial flutter)、心房顫動(atrial fibrillation)等心室上頻脈或浮腫的鬱血性心臟衰竭或心律不整所用之類固醇配糖體的總稱,然而本發明之抗衰老藥所含有的強心配糖體之較佳例可列舉例如:海蔥次苷A(CAS No.466-06-8)、地高辛(digoxin)(CAS No.20830-75-5)、或此等之鹽或衍生物而與此等具有同等之活性的化合物等。
(DNA傷害型抗癌劑)
DNA傷害型抗癌劑意指藉由對癌細胞之DNA造成傷害,而選擇性地使癌細胞導向死亡之類型之抗癌劑的總稱,本發明之抗衰老藥所含有的DNA傷害型抗癌劑之下述較佳例可列舉例如:多柔比星(Doxorubicin)(阿黴素(Adriamycin))HCl(CAS No.25316-40-9)、道諾黴素(Daunorubicin)
HCl(CAS No.23541-50-6)、泛艾黴素HCl(Epirubicin HCl)(CAS No.56390-09-1)、米托蒽醌(Mitoxantrone 2HCl)(CAS No.70476-82-3)、羥基喜樹鹼(hydroxy camptothecine)(CAS No.64439-81-2)、或此等之鹽或衍生物而與此等具有同等之活性的化合物等。
(抗菌劑)
本發明之抗衰老藥所含有的抗菌劑之較佳例可列舉例如:胺基吖啶(CAS No.90-45-9)、三氯沙(CAS No.3380-34-5)、依沙吖啶乳酸鹽(TAZ活化;CAS No.1837-57-6)、或此等之鹽或衍生物而與此等具有同等之活性的化合物等。
(極光激酶阻礙劑)
極光激酶為在人類中被AURKA基因編碼的酵素,且為絲胺酸/蘇胺酸激酶家族之成員。正常之細胞增殖中,在有絲分裂及減數分裂之過程中,不可或缺的是極光激酶適當地發揮功能。已知極光激酶係藉由1處或複數處之磷酸化而被活化,其活性在細胞周期之G2期移行至M期時達到高峰。本發明之抗衰老藥所含有的極光激酶阻礙劑之較佳例可列舉例如:AMG-900(CAS No.945595-80-2)、JNJ-7706621(CDK1、2、Aurora A、B阻礙;CAS No.443797-96-4)、或此等之鹽或衍生物而與此等具有同等之活性的化合物等。
(Nutlin-3b)
Nutlin-3b(CAS No.675576-97-3)為Nutlin-3a之光學異構物。本發明之抗衰老藥較佳為含有Nutlin-3b(CAS No.675576-97-3)之物。
上述以外,由於下列化合物亦具有抗衰老活性,因此此等化合物、或此等之鹽或衍生物而與此等具有同等之抗衰老活性的化合物,亦可採用為本發明之抗衰老藥的有效成分:類黃酮(金黃素;CAS No.480-40-0)、PI3K激酶阻礙劑(PF-05212384;CAS No.1197160-78-3、XL147;CAS No.934526-89-3)、HDAC阻礙劑(曲古抑菌素A(TSA);CAS No.58880-19-6)、老年性黃斑變性治療藥(維替泊芬;CAS No.129497-78-5)、p38MAPK阻礙劑(SB203580;CAS No.152121-47-6、達馬莫德(BIRB 796);CAS No.285983-48-4)、mTOR阻礙劑(AZD8055;CAS No.1009298-09-2、KU-0063794;CAS No.938440-64-3)、酪胺酸激酶阻礙劑(來那替尼(HER2、EGFR阻礙);CAS No.698387-09-6、尼達尼布;CAS No.656247-17-5、NVP-BHG712(C-raf、C-src阻礙);CAS No.940310-85-0)、Bc1-2阻礙劑(TW-37;CAS No.877877-35-5)、他汀類藥物(Mevastatin;CAS No.73573-88-3)、血清素受體拮抗藥(RS-127445;CAS No.199864-87-4)、其他激酶阻礙劑(BMS-345541;CAS No.445430-58-0、CX-4945;CAS No.1009820-21-6)。
本發明之抗衰老藥,除上述有效成分外,亦可含有下列製劑上、藥理學上或生理學上可容許的一種或二種以上做為其他任意成分:載體、賦形劑、黏合劑、崩解劑、潤滑劑、分散劑、界面活性劑、塑化劑、懸浮劑、乳化劑、稀釋劑、緩衝劑、抗氧化劑、細菌抑制劑等。又,亦可包含其他低分子量之多肽、血清白蛋白、明膠或免疫球蛋白等蛋白質、胺基酸、多糖及單糖等糖類或碳水化物、糖醇。進一步亦可因應所需含有其他有效成分。
本發明之抗衰老藥,可藉由醫藥品、醫藥部外品、食品等領域中先前周知的任意方法製造。在其製造過程中,亦可將上述有效成分、其他任意成分以周知之任何方法添加/混合。
在製為注射用之水溶液的情況,可列舉例如:生理食鹽水、包含葡萄糖或其他輔助藥之等張液,例如,D-山梨醇、D-甘露糖、D-山梨醇、氯化鈉,亦可併用適當的溶解輔助劑,例如:醇(乙醇等)、多元醇(丙二醇、PEG等)、非離子性界面活性劑(Polysorbate 80、HCO-50)等。因應所需亦可進一步含有稀釋劑、溶解輔助劑、pH調整劑、無痛化劑、含硫還原劑、抗氧化劑等。
又,亦可封入微膠囊(羥基甲基纖維素、明膠、聚[甲基丙烯酸甲酯]等微膠囊),或製成膠體藥物輸送系統(脂質體、白蛋白微球、微乳液、奈米粒子及奈米膠囊等)。再者,將藥劑製成緩釋性之藥劑的方法亦為周知,可適用於本發明。
在將本發明之抗衰老藥作為人類或其他動物之醫藥使用的情況,除將此等物質本身直接對患者投予以外,亦可依照周知之製劑學上的方法製劑化進行投予。在進行製劑化之情況,亦可添加上述記載之製劑上容許的成分。
本發明中之所有藥劑,能以醫藥品之形式投予,可藉由經口或非經口方式對全身或局部進行投予。例如,可選擇點滴等靜脈內注射、肌肉內注射、腹腔內注射、皮下注射、栓劑、灌腸、經口性腸溶劑等,可依照患者之年齡、症狀而選擇適當的投予方法。有效投予量,每一次每1kg體重是在0.001mg至100mg之範圍內選擇。或者是,對每一患者,可選擇
0.1至1000mg,較佳為0.1至50mg之投予量。具體而言,每1kg體重於1個月(4週),投予0.1mg至40mg,較佳1mg至20mg,以1次或分數次投予,例如2次/週、1次/週、1次/2週、1次/4週等投予時程,藉由點滴等靜脈內注射、皮下注射、經口投予等方法進行投予。投予時程亦可一邊觀察投予後之狀態及觀察血液檢查值之動向,一邊以將投予間隔如從2次/週或1次/週延長至1次/2週、1次/3週、1次/4週等之方式來調整。
<抗衰老藥2>
進一步進行PGAM1-Chk1途徑之下游轉錄因子之解析的結果,判明轉錄因子HIF-2α存在於PGAM1-Chk1結合之下游,且發現HIF-2αsiRNA亦具有抗衰老活性。由此,可知除了控制PGAM1-Chk1結合以外,調節控制HIF-2α之信號(亦包含乙醯基化或泛素化),亦可經由HIF-2α之失活而誘導抗衰老。再者,亦明白該轉錄因子HIF-2α,係朝抑制屬於細胞凋亡誘導基因之BIM之表現的方向進行作用。基於此等結果,本發明亦包含具有使HIF-2α失活之作用,並進一步選擇性地滅絕老化細胞的抗衰老藥。具體而言,以含有HIF-2α阻礙劑作為有效成分之抗衰老藥。
HIF-2α為轉錄因子HIF-1α之功能性同系物,於進化上被保存。HIF-家族蛋白為細胞或組織暴露於低氧狀況時被活化的轉錄因子。在低氧狀況中,由於不能進行粒線體有氧呼吸,細胞無法依存於糖解作用代謝,產生能量。在低氧環境中,藉由HIF蛋白之活化,糖解作用代謝mRNA亢進,而可亢進糖解作用代謝(Iyer et al.,Genes and Development,1998,Vol12,p149-62)。在通常之氧環境中,轉錄因子HIF蛋白為容易受到泛素化修飾而被蛋白分解的不穩定蛋白,另一方面,在低氧狀況中,HIF之泛
素化受到阻礙,蛋白穩定化,使活性亢進。本發明之抗衰老藥所含有的HIF-2α阻礙劑若為阻礙上述HIF-2α功能的物質則沒有特別限定,較佳例可列舉例如:與編碼HIF-2α之DNA之轉錄產物互補的反義核酸、或siRNA(例如,序列編號9、序列編號10)等。此外,本發明中所用之反義核酸、siRNA,可直接將上述之RSK1激酶阻礙劑之項的說明中,RSK1替換為HIF-2α而適用。
上述<抗衰老藥>中之與一般醫藥共通之內容的說明,亦可適用於<抗衰老藥2>中。
<抗衰老藥之篩選方法2>
再者,本發明亦包含一種抗衰老藥之篩選方法,其係包含下列步驟:(X)對於受檢物質,測定HIF-2α之阻礙活性的步驟;及(Y)對於藉由上述步驟(X)經判斷具有HIF-2α之阻礙活性的受驗物質,測定選擇性去除老化細胞活性的步驟。在上述步驟(Y)中,其特徵為測定受驗物質對年輕細胞之傷害活性及對老化細胞的傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質,具體之說明可直接適用上述<抗衰老藥之篩選方法>中的(B)步驟之說明。此外,在上述(X)步驟中,HIF-2α之阻礙活性意指不僅包含阻礙HIF-2α之作用的活性,亦包含抑制HIF-2α之表現的活性。
<抗衰老藥3>
如上述,Nutlin-3b具有優異之抗衰老活性。本發明人等進一步進行關於Nutlin-3b之研究時,發現CCNB1、CCNB2、PLK1、ASPM、CEP55、BIRC5(存活素;Survivin)於老化細胞中表現亢進,藉由Nutlin-3b處理而
抑制表現。尤其是,BIRC5(存活素;Survivin)之亢進特別顯著。從此結果可謂,對於CCNB1、CCNB2、PLK1、ASPM、CEP55、BIRC5(存活素;Survivin)之抑制劑,尤其是對於BIRC5(存活素;Survivin)之抑制劑,係具有優異的抗衰老活性。CCNB1、CCNB2、PLK1、ASPM、CEP55、BIRC5之抑制劑可列舉如:與編碼此等之DNA之轉錄產物互補的反義核酸、或siRNA等。基於此等結果,本發明亦包含具有使CCNB1、CCNB2、PLK1、ASPM、CEP55、及/或BIRC5失活之作用,且進一步選擇性地滅絕老化細胞的抗衰老藥。具體而言,以含有CCNB1、CCNB2、PLK1、ASPM、CEP55、及/或BIRC5之阻礙劑作為有效成分的抗衰老藥。
上述<抗衰老藥>中之與一般醫藥共通之內容的說明亦可適用於<抗衰老藥3>。
<抗衰老藥之篩選方法3>
再者,本發明亦包含一種抗衰老藥之篩選方法,其係包含下列步驟:(X)對於受檢物質,測定CCNB1、CCNB2、PLK1、ASPM、CEP55、及/或BIRC5之阻礙活性的步驟;及(Y)對於藉由上述步驟(X)經判斷為具有CCNB1、CCNB2、PLK1、ASPM、CEP55、及/或BIRC5之阻礙活性的受驗物質,測定選擇性去除老化細胞活性之步驟。在上述步驟(Y)中,其特徵為測定受驗物質對年輕細胞之傷害活性及對老化細胞的傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質,具體說明,可直接適用上述<抗衰老藥之篩選方法>中的(B)步驟之說明。此外,上述(X)步驟中,CCNB1、CCNB2、PLK1、ASPM、CEP55、及/或BIRC5之阻礙活性,意指不僅阻礙CCNB1、CCNB2、PLK1、ASPM、CEP55、
及/或BIRC5之作用的活性,亦包含抑制CCNB1、CCNB2、PLK1、ASPM、CEP55、及/或BIRC5之表現的活性。
<抗衰老藥4>
從對於Nutlin-3b之進一步研究,明白特定之FOX家族基因之表現深度參與由Nutlin-3b所致的抗衰老。FOXM1、FOXF1、FOXC1、FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、FOXN1,係藉由Nutlin-3b處理而減少表現。尤其是,FOXM1之表現減少顯著。又,FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、FOXJ2,係藉由Nutlin-3b處理而表現亢進。尤其是,FOXO1、FOXP1之表現亢進顯著。從此結果可謂,控制上述特定之FOX家族基因之表現的物質,尤其是控制包含FOXM1、FOXO1之FOXO家族、及包含FOXP1之FOXP家族之表現的物質,特別是使FOXM1表現減少之物質,使包含FOXO1之FOXO家族、及包含FOXP1之FOXP家族之表現亢進的物質,係具有優異之抗衰老活性。
例如,使FOXM1等表現減少之物質可列舉:與編碼FOXM1等之DNA之轉錄產物互補的反義核酸、或siRNA等。亦即,本發明亦包含具有使FOXM1、FOXF1、FOXC1、FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、及/或FOXN1,尤其是FOXM1失活之作用,且進一步選擇性地滅絕老化細胞的抗衰老藥。具體而言,以FOXM1、FOXF1、FOXC1、FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、及/或FOXN1之阻礙劑,尤其是FOXM1之阻礙劑作為有效成分的抗衰老藥。
再者,本發明亦包含:具有使FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、及/或FOXJ2,尤其是包含FOXO1之FOXO家族及/或包含FOXP1之FOXP家族活化的作用,並進一步選擇性地滅絕老化細胞的抗衰老藥。具體而言,以FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、及/或FOXJ2,尤其是包含FOXO1之FOXO家族、及/或包含FOXP1之FOXP家族的活化劑,作為有效成分的抗衰老藥。
關於上述之<抗衰老藥>中之與一般醫藥共通之內容的說明,亦可適用於<抗衰老藥4>。
<抗衰老藥之篩選方法4>
再者,本發明亦包含一種抗衰老藥之篩選方法,其係包含下列步驟:(X)對於受檢物質,測定FOXM1、FOXF1、FOXC1、FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、及/或FOXN1之阻礙活性的步驟;及(Y)對於藉由上述步驟(X)經判斷具有FOXM1、FOXF1、FOXC1、FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、及/或FOXN1之阻礙活性的受驗物質,測定選擇性去除老化細胞活性之步驟。在上述步驟(Y)中,其特徵為測定受驗物質對年輕細胞之傷害活性及對老化細胞的傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質,具體而言,可直接適用上述<抗衰老藥之篩選方法>中的(B)步驟之說明。此外,在上述(X)步驟中,FOXM1、FOXF1、FOXC1、FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、及/或FOXN1之阻礙活性,意指不僅阻礙FOXM1、FOXF1、FOXC1、
FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、及/或FOXN1之作用的活性,亦包含抑制FOXM1、FOXF1、FOXC1、FOXL1、FOXL2、FOXD1、FOXB2、FOXI2、及/或FOXN1之表現的活性。
再者,本發明亦包含一種抗衰老藥之篩選方法,其係包含下列步驟:(X)對於受檢物質,測定FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、及/或FOXJ2之亢進活性的步驟,及(Y)對於藉由上述步驟(X)經判斷具有FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、及/或FOXJ2之亢進活性的受驗物質,測定選擇性去除老化細胞活性之步驟。在上述步驟(Y)中,其特徵為測定受驗物質對年輕細胞之傷害活性及對老化細胞的傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質,具體而言,可直接適用上述<抗衰老藥之篩選方法>中的(B)步驟之說明。再者,在上述(X)步驟中,FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、及/或FOXJ2之亢進活性,意指不僅使FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、及/或FOXJ2之作用亢進的活性,亦包含使FOXO1、FOXP1、FOXH1、FOXF2、FOXO4、FOXO3、FOXJ3、FOXQ1、FOXK2、FOXN3、FOXO6、FOXK1、FOXP4、FOXN2、及/或FOXJ2之表現亢進的活性。
[實施例]
藉由下列實施例具體地說明本發明,然而本發明不是受實施例所限定者。
1. 於老化細胞之糖解作用亢進及PGAM-Chk1結合之亢進
已知在通常細胞中,藉由致癌性Ras變異,誘導早期細胞老化(Serrano et al.Cell 1997,Vol.88,Issue 5,593-602)。又,已報導於細胞老化中糖解作用代謝亢進之事(Goldstein and Trieman,1975;Goldstein et al.,1982)。因此,驗證老化細胞中之PGAM-Chk1結合。具體而言,在MCR5細胞(源自人類胎兒肺之正常二倍體纖維母細胞)中,導入附加Nano-Bit標籤之PGAM及Chk1激酶基因,製作出可穩定地表現的細胞株。在此MCR5細胞之年輕細胞(連續培養1個月以內)中導入RasER基因。RasER蛋白係藉由他莫昔芬(tamoxifen)藥劑(4OHT)之投予,而可將Ras基因活化的融合蛋白。如圖1所示,在完成之對MCR5-RasER細胞投予他莫昔芬後,經時地觀察老化表現型,並且亦可同時地觀察藉由Nano-Bit系統所致的PGAM-Chk1結合。如圖2所示,在對該細胞中投予他莫昔芬後,從4日後開始觀察到老化標記之變化(核片層蛋白B1(Lamin B1)之減少、p53蓄積、p21蓄積)。在圖3顯示PGAM-Chk1結合之結果,在屬於細胞老化之指標的SA-βGal染色中,於第8日至第16日可見到明顯的陽性率上升。使用該細胞,分別計測年輕細胞及老化細胞中,各個細胞內由Nano-Bit所產生的螢光素酶發光信號。又,回收培養基,藉由糖消耗量評估及乳酸產生計測來評估各個細胞內之糖解作用代謝效率。乳酸值,係將每小時培養基中分泌之乳酸量(μmol/h)利用以細胞總蛋白質量校正所得的值(μmol/mg/h)而計測算出,並以與Day0之實測值的相對值來表示。糖消費,係將每小時從培養基
中減少之葡萄糖量(μmol/h),利用以細胞總蛋白質量校正所得的值(μmol/mg/h)而計測算出,並以與Day0之實測值的相對值來表示。分別藉由葡萄糖檢定套組(Glucose assay kit)(BioVision)(圖4之下圖)及乳酸檢定套組(Lactate assay kit)(BioVision)(圖4之上圖),計測糖及乳酸。
以上,老化細胞中,如圖3所示,觀察到由Nano-Bit所致之螢光素酶發光信號的顯著增加(縱軸),可確認PGAM-Chk1結合亢進。又,如圖4所示,可知在老化細胞中,乳酸產生及糖之攝取效率同時上升。
2. 老化細胞中由PGAM-Chk1結合阻礙所致的抗衰老-1
檢討已知為PGAM-Chk1結合阻礙劑之Nutlin-3a、以及屬於其光學異構物之Nutlin-3b的抗衰老效果。此外,Nutlin-3a係從西格瑪奧瑞奇公司(Sigma-Aldrich公司)購入,Nutlin-3b係從開曼化學公司(Cayman chemical公司)購入。
已知藉由屬於PGAM-Chk1結合阻礙劑之Nutlin-3a,可觀察到老化細胞之細胞凋亡。亦即,研判藉由阻礙老化細胞中之PGAM-Chk1結合,可誘導老化細胞之細胞凋亡。然而,已知Nutlin-3a將p53活化(Vassilev et al.,2004,Science 303,844-848;以下稱為「Vassilev et al.,2004」),對年輕細胞、老化細胞兩者之增殖/生存均有負面作用,故不被稱為抗衰老藥。另一方面,吾等發現Nutlin-3a以外之其他PGAM-Chk1結合阻礙劑。其為屬於Nutlin-3a之光學異構物之Nutlin-3b(圖5)。Nutlin-3b係作為阻礙p53與Mdm2之結合的化學物質,與Nutlin-3a被一起開發的抗癌劑中之一種。然而,關於p53-Mdm2結合阻礙活性,Nutlin-3a與
Nutlin-3b有很大差距,與Nutlin-3a相比,已報導Nutlin-3b僅有約100分之1的活性(Vassilev et al.,2004)。
首先,藉由屬於PGAM-Chk1結合阻礙劑之Nutlin-3a及3b,確認於老化細胞中亦阻礙PGAM-Chk1結合,導致糖解作用代謝降低。於以下說明具體之方法及結果。
人類正常纖維母細胞、IMR90細胞係從JCRB細胞銀行取得。對於IMR90細胞之年輕細胞(連續培養1個月以內),藉由逆轉錄病毒導入致癌性Ras變異,獲得壓力老化細胞。將所得到之老化細胞藉由濃度20μM或40μM之Nutlin-3a或3b處理48小時。然後,將此等細胞進行6小時之MG132處理,進行蛋白萃取。對於所得到之蛋白萃取液,藉由抗Chk1抗體,進行免疫沉降。將所得到之免疫沉降物,使用藉由抗PGAM抗體的西方墨點法,評估PGAM-Chk1之結合。其結果,判定在老化細胞中,Nutlin-3a及3b能以相同程度進行PGAM-Chk1阻礙(圖6)。
又,對於IMR90細胞,導入附加Nano-Bit標籤之PGAM及Chk1激酶基因,製作出可穩定表現的細胞株。將此IMR90細胞之年輕細胞(連續培養1個月以內),以100uM之依托泊苷(etoposide)處理48小時,獲得老化細胞。使用所獲得之上述IMR90細胞之年輕細胞、老化細胞,以濃度相異(20μM或40μM)的Nutlin-3b處理48小時,計測各個細胞內之由Nano-Bit所致的螢光素酶發光信號。其結果,於老化細胞中觀察到的PGAM-Chk1結合之增強,係藉由Nutlin-3b處理而濃度依存性地抑制(圖7)。
繼而,將IMR90細胞之年輕細胞(連續培養1個月以內)以100μM依托泊苷處理48小時,獲得老化細胞。將所得到之老化細胞,以濃度20μM或40μM之Nutlin-3a或3b處理48小時。Nutlin處理後,回收培養基,進行糖消耗量評估及乳酸產生計測。乳酸值,係藉由每小時於培養基中分泌之乳酸量(μmol/h)利用以細胞總蛋白質量校正所得之值(μmol/mg/h)而計測算出,並以與Day0之實測值的相對值來表示。糖消費,係藉由每小時從培養基中減少之葡萄糖量(μmol/h)利用以細胞總蛋白質量校正所得的值(μmol/mg/h)而計測算出,並以與Day0之實測值的相對值來表示。分別藉由葡萄糖檢定套組(BioVision)(圖8之上圖)及乳酸酯檢定套組(BioVision)(圖8之下圖)計測糖及乳酸。其結果,在老化細胞中,判明藉由Nutlin-3a或3b濃度依存性地抑制糖消費及乳酸產生之任一者,確認到糖解作用代謝被抑制。
如上述,首先,可確認藉由屬於PGAM-Chk1結合阻礙劑的Nutlin-3a及3b,即使於老化細胞中亦阻礙PGAM-Chk1結合,導致糖解作用代謝降低。又,老化細胞與癌細胞之代謝上共通點有PGAM-Chk1結合依存性之糖解作用代謝亢進,亦判明該結合阻礙係在兩者中皆誘導糖解作用代謝降低。
繼而,關於癌細胞中之PGAM-Chk1結合阻礙效果,發現Nutlin-3a與Nutlin-3b幾乎同等(圖9)。於以下說明具體的試驗方法及結果。
對癌細胞株H1299導入附加Nano-Bit標籤的PGAM及Chk1激酶基因,製作出可穩定表現的細胞株。將所得到之細胞分別以濃度40μM
之Nutlin-3a或3b處理12、24、48小時。使用處理後之細胞,計測各個細胞內由Nano-Bit所產生的螢光素酶發光信號。其結果,藉由Nutlin-3a或3b處理,觀察到藉由Nano-Bit所產生的螢光素酶發光信號是處理時間依存性地降低(縱軸),確認到PGAM-Chk1結合抑制(圖9)。
然而,關於在年輕細胞中之p53活化,Nutlin-3a及Nutlin-3b的作用大為不同,前者使p53充分地活化,另一方面,後者完全無法使p53活化(圖10)。此點與已報導(Vassilev et al.,2004)之「關於p53-Mdm2結合阻礙活性,Nutlin-3a與Nutlin-3b有很大差距」的見解一致。於以下說明具體之試驗方法及結果。
將IMR90細胞之年輕細胞(連續培養1個月以內),用20μM之Nutlin-3a或3b處理48小時。處理後,從細胞萃取蛋白,進行西方墨點法。P53或其下游因子p21之蛋白量,係藉由Nutlin-3a處理而顯著地上升;另一方面,藉由Nutlin-3b處理則幾乎無變化(圖10)。
繼而,比較Nutlin-3a及3b對年輕細胞及老化細胞之效果。其結果,發現Nutlin-3b係與Nutlin-3a不同,在年輕細胞中不呈現毒性,另一方面,於老化培養細胞中誘導選擇性之細胞凋亡(抗衰老)(圖11)。又,Nutlin-3b所致之抗衰老效果係濃度依存性(圖12),藉由細胞凋亡蛋白酶3(caspase 3)斷片化之西方墨點法,可確認老化細胞之細胞凋亡(圖13)。於以下說明具體的試驗方法及結果。
將致癌性變異Ras基因以逆轉錄病毒感染導入至IMR90細胞之年輕細胞(連續培養1個月以內),而獲得老化細胞。對IMR90之年輕細胞、老化細胞,分別進行72小時之40μM之Nutlin-3a處理、40μM之
Nutlin-3b處理。對照群係只進行DMSO處理。展示有代表性之顯微鏡下的細胞觀察之影像(圖11)。在Nutlin-3a中,於年輕細胞及老化細胞中皆同樣地觀察到細胞死亡、細胞減少,另一方面,在Nutlin-3b處理中,觀察到抗衰老現象(年輕細胞正常,但老化細胞選擇性地細胞死亡)。
繼而,對上述之年輕細胞、老化細胞分別進行96小時之濃度相異的Nutlin-3b處理,評估細胞死亡。在20或40μM之濃度之Nutlin-3b中,確認到顯著的抗衰老效果(圖12)。
對IMR90細胞之年輕細胞(連續培養1個月以內),將致癌性變異Ras基因藉由逆轉錄病毒感染導入,獲得壓力老化細胞。對年輕細胞、老化細胞分別進行72小時之濃度相異的Nutlin-3b處理,將細胞死亡藉由斷片化細胞凋亡蛋白酶3之西方墨點法進行評估。在年輕細胞中,即使以Nutlin-3b處理亦不引起細胞凋亡蛋白酶3之斷片化。另一方面,在老化細胞中,藉由Nutlin-3b處理,觀察到濃度依存性地細胞凋亡蛋白酶3的斷片化(圖13)。
繼而,檢討對老年小鼠投予Nutlin-3b的效果。具體而言,準備出生後90週之C57BL/6的高齡小鼠16隻。分為2群(對照群8隻、藥劑群8隻),將每週一次之腹腔內注射投予持續進行2個月。對照群係注射生理食鹽水,藥劑群係將Nutlin-3b以11.63mg/kg之濃度投予。最後之投予結束1週後,將全部小鼠解剖,從各內臟萃取RNA。將老化標記或炎症標記藉由RTPCR評估。將其結果示於圖14-1及14-2。如圖14-1及14-2所示,於藥劑投予群(Nutlin-3b)確認到老化標記或炎症標記之減少。與先
前的藥ABT263同樣地(Chang et al.,Nat.Med.,2016),藉由Nutlin-3b投予確認到各種老化標記減少。
3. PGAM-Chk1激酶結合之機制解析
致癌變異型Ras為最早發現的癌基因之一,於胰癌、肺癌、大腸癌等中的頻率非常高(分別為90%、30%、50%)。本發明人等在表現致癌性Ras變異之癌細胞中,發現PGAM-Chk1激酶結合被誘導,而維持癌的糖解作用代謝亢進(瓦氏效果(Warburg effect))(Mikawa et al.,2020,iScience 23,101306;以下稱為「Mikawa et al.,2020」)。Chk1激酶為絲胺酸蘇胺酸蛋白激酶,係在細胞周期之G2期,DNA損傷檢查點之活化中承擔任務的檢查點激酶。依據上述見解,發現Chk1在糖解作用代謝中亦擔任重要角色(Mikawa et al.,2020)。PGAM-Chk1激酶結合可在表現致癌性Ras變異之癌細胞中觀察到,另一方面,在不表現致癌性Ras變異的通常細胞中則未觀察到(Mikawa et al.,2020)。
將由致癌性Ras變異所致之信號通路進一步詳細調查的結果,判明出重要的是存在於其下游之MEK激酶及RSK激酶(Mikawa et al.,2020)。對癌細胞投予各個阻礙劑(U126及BI-D1870)時,PGAM-Chk1激酶結合被阻礙,糖解作用代謝降低(Mikawa et al.,2020)。已知RSK激酶係將Chk1激酶之S280殘基進行直接磷酸化控制(Li et al.,2012,Mol Biol Cell 23,1582-1592)。吾等確認即使在H1299細胞,藉由RSK激酶阻礙劑亦抑制Chk1激酶之S280磷酸化(圖15)。具體而言,將癌細胞株H1299細胞以濃度10μM之MEK激酶阻礙劑(U126)處理48小時。回收處理後之細胞,蛋白萃取後,進行西方墨點法。Chk1激酶之蛋白量無變化,可確認
其S280磷酸化被抑制。又,吾等製成Chk1激酶之S280A變異型。在此未能磷酸化之變異型(Chk1-S280A)中,發現於老化細胞中PGAM-Chk1激酶結合被阻礙(圖16左)。相反地,在疑似磷酸化之變異型(Chk1-S280D)中,發現於年輕細胞中PGAM-Chk1激酶結合亢進(圖16右)。具體而言,對IMR90細胞導入附加Nano-Bit標籤的PGAM及Chk1激酶基因,製作出可穩定表現之細胞株。此時,將野生型Chk1、S280A變異型、或S280D變異型之Chk1的3種類分別導入至細胞中。對該細胞中藉由逆轉錄病毒感染導入致癌性變異Ras基因,獲得壓力老化細胞。使用所得到之老化細胞,計測各個細胞內由Nano-Bit所致的螢光素酶發光信號。其結果,於S280A變異中,觀察到由Nano-Bit產生的螢光素酶發光信號降低(縱軸),確認到PGAM-Chk1結合抑制(圖16左)。在未導入致癌性變異Ras基因之年輕細胞,藉由S280D變異亦確認到由Nano-Bit產生的螢光素酶發光信號亢進(圖16右)。從以上所述,判明由屬於致癌性Ras變異之下游信號激酶的RSK激酶所致的Chk1激酶之S280磷酸化,係對PGAM-Chk1激酶結合很重要。
再者,在由致癌性Ras變異所產生之老化細胞中,發現RSK1-Thr573磷酸化亢進,Chk1-Ser280磷酸化亦亢進(圖17)。又,可知藉由RSKsiRNA處理,老化細胞中之PGAM-Chk1結合被阻礙(圖18)。於以下說明具體的試驗方法及結果。
對於IMR90細胞之年輕細胞(連續培養1個月以內),藉由逆轉錄病毒感染導入致癌性變異Ras基因,獲得壓力老化細胞。回收所得到之年輕細胞、老化細胞,蛋白萃取後,進行西方墨點法。在老化細胞中,
Chk1激酶之S280磷酸化或RSK1激酶之Thr573磷酸化亢進,Chk1及RSK1激酶被活化(圖17)。
繼而,與上述同樣地使用IMR90細胞,藉由致癌性變異Ras基因導入,準備壓力老化細胞。對於老化細胞,進行RSK1siRNA(序列編號7)或對照群之轉染,進行RSK1之基因敲落。回收所得到之細胞,進行蛋白萃取。對於所得到之蛋白萃取液,藉由抗Chk1抗體,進行免疫沉降。將所得到之免疫沉降物,使用由抗PGAM抗體所致的西方墨點法,評估PGAM-Chk1之結合。其結果,判明在老化細胞中,藉由RSK1之基因敲落,Chk1激酶之S280磷酸化降低,可進行PGAM-Chk1阻礙(圖18)。
繼而,驗證由RSK激酶阻礙所產生的抗衰老效果。在由RSK激酶阻礙劑BID1870(圖19)或RSK1siRNA所致之基因敲落(圖20)中,與Nutlin-3b同樣地觀察到抗衰老效果。由RSK阻礙劑所致之抗衰老為濃度依存性(圖19),由siRNA所致之抗衰老效果,與其同等或效率更佳(圖20)。於以下說明具體的試驗方法及結果。
對於IMR90細胞之年輕細胞(連續培養1個月以內),藉由逆轉錄病毒感染導入致癌性變異Ras基因,獲得壓力老化細胞。對年輕細胞、老化細胞分別進行96小時之濃度相異的RSK激酶阻礙劑BI-D1870之處理,評估細胞死亡。藉由10μM或20μM之濃度的BI-D1870確認到顯著之抗衰老效果(圖19)。
對IMR90細胞之年輕細胞(連續培養1個月以內)藉由逆轉錄病毒感染導入致癌性變異Ras基因,獲得壓力老化細胞。分別對於IMR90之年輕細胞、老化細胞進行RSK1siRNA(標的序列相異之2種類
siRNA#1(序列編號7)或#2(序列編號8))或對照群的轉染,以進行RSK1之基因敲落。展示代表性之顯微鏡下之細胞觀察的影像(圖20上圖)。在RSK1之基因敲落中,觀察到抗衰老現象(年輕細胞為正常,而老化細胞選擇性地細胞死亡)(圖20下圖)。
對IMR90細胞之年輕細胞(連續培養1個月以內)導入致癌性Ras,誘導出老化細胞(OIS)。藉由西方墨點法比較在IMR90之年輕細胞、老化細胞中,參與糖解作用控制之轉錄因子(HIF-2α,HIF-1α,E2F1,c-Myc)的表現。於老化細胞中,確認到屬於老化之指標的核片層蛋白B1(Lamin B1)顯著降低。將其結果示於圖21。如圖21所示,可知在老化細胞中,引起HIF-2α之顯著蓄積。
又,對於同樣經不同濃度的Nutlin3b處理或RSK激酶阻礙劑BI-D1870進行96小時處理的年輕細胞、老化細胞,進行轉錄因子HIF-2α之西方墨點法。其結果,在Nutlin3b或BI-D1870處理群中,觀察到HIF-2α蛋白減少(圖22之上圖及下圖)。
對IMR90細胞之年輕細胞(連續培養1個月以內)導入致癌性Ras,誘導出老化細胞(OIS)。對於IMR90之年輕細胞、老化細胞,轉染HIF-2α特異性標的序列的2種相異siRNA(序列編號9、序列編號10),進行HIF-2α基因敲落。然後,評估細胞死亡時,在使用上述HIF-2α特異性標的序列的2種相異siRNA,使HIF-2α基因敲落之老化細胞中,觀察到選擇性的細胞死亡(抗衰老)。將結果示於圖23、24。
4. PGAM1-Chk1途徑之下游轉錄因子的解析
對IMR90細胞之年輕細胞(連續培養1個月以內)導入致癌性Ras,誘導出老化細胞(OIS)。對上述老化細胞進行48小時之Nutlin-3b處理,解析屬於細胞凋亡誘導基因之BIM基因的表現(圖25)。如圖25所示,確認到老化細胞藉由Nutlin-3b處理,細胞凋亡誘導基因BIM之表現上升。
對IMR90細胞之年輕細胞(連續培養1個月以內)導入致癌性Ras,誘導出老化細胞(OIS)。對上述老化細胞,轉染HIF-2α特異性siRNA(序列編號9),使HIF-2α基因敲落。然後,解析為細胞凋亡誘導基因之BIM基因的表現(圖26)。如圖26所示,確認藉由老化細胞之HIF-2α基因敲落,細胞凋亡誘導基因BIM的表現上升。
對IMR90細胞之年輕細胞(連續培養1個月以內)導入致癌性Ras,誘導出老化細胞(OIS)。對於IMR90之年輕細胞、老化細胞,轉染BIM特異性siRNA,使BIM基因敲落。然後,進行96小時之Nutlin-3b處理,確認細胞死亡(圖27)。如圖27所示,可知藉由Nutlin-3b處理所致之抗衰老,係因BIM基因敲落而被解除。
對IMR90細胞之年輕細胞(連續培養1個月以內)導入致癌性Ras,誘導出老化細胞(OIS)。在IMR90之年輕細胞、老化細胞中,分別使用特異性siRNA(序列編號9、序列編號10),將HIF-2α單獨、或HIF-2α與BIM兩者進行基因敲落,確認細胞死亡(圖28)。如圖28所示,可知藉由HIF-2α基因敲落所致之抗衰老,係因BIM基因敲落而被解除。
5. 抗衰老藥之篩選
將上述數據進行綜合判斷,推測若以PGAM-Chk1結合作為指標,可進一步鑑定強力的抗衰老藥。因此,以PGAM-Chk1結合可視化作為指標,
實施使用了Cell-base NanoBiT系統的PGAM-Chk1結合阻礙高通量(HTP(high throughput))篩選。具體係依照下述方法進行。在NanoBiT中,對PGAM及Chk1分別附加以螢光素酶為基礎所製成之發光蛋白質的大次單元及小次單元而分別作為標籤。此等標籤可互相結合而形成一結構。各個次單元本身雖不發光,然而藉由PGAM及Chk1之結合,大次單元及小次單元會合,則藉由形成完整的發光蛋白而發出發光信號。藉由測定此時之發光信號強度,可以高敏感度且高定量性之方式測定結合量。此時,在如Nutlin之阻礙PGAM及Chk1之結合的物質的,存在下,發光信號降低。因此,以該發光信號之減少作為指標,可從藥劑資料庫中篩選出阻礙PGAM與Chk1結合的化合物。
在接受日本京都大學醫學研究支援中心之協助下,使用該PGAM-Chk1結合可視化Cell-baseNanoBiT系統,以安全性評估獲得確定的既有藥資料庫(2300餘)作為對象,進行PGAM-Chk1結合阻礙HTP篩選,於初次篩選獲得約200強之結合阻礙候選藥(圖29、圖30)。
再者,於第二次篩選中,以針對通常細胞之抗衰老活性作為指標。發現具有抗衰老活性且與ABT263機構相異的約40種抗衰老藥(圖29)。於以下說明具體的試驗方法及結果。
在二次篩選中,比較年輕IMR90細胞及老化IMR90細胞之藥劑感受性。老化IMR90細胞係藉由誘導致癌性Ras變異而製成。在96孔培養盤中,分別播種年輕細胞及老化細胞,2日後,將從初次篩選所得到之候選藥以5μM及10μM的2階段濃度處理。72小時後,將存活之細胞以福馬林固定,用結晶紫染色。然後,將各孔之結晶紫以2% Triton X100
萃取,並定量(圖31-1、圖31-2)。對於年輕細胞及老化細胞,分別將溶劑(DMSO)處理細胞作為陰性對照,將相對於陰性對照的生存率,依照下列式算出。進一步,比較年輕細胞及老化細胞之藥劑感受性,作為抗衰老活性進行評估(年輕細胞生存率/老化細胞生存率)。再者,陽性之基準為抗衰老活性為1.14以上。
生存率(%)=(樣本/陰性對象)×100
抗衰老活性=年輕細胞生存率/老化細胞生存率
如上述,於目前的時點,將包含Nutlin-3b及RSK激酶阻礙劑BI-D1870以及二次篩選所獲得的藥劑整理於圖32,成功地鑑定出40個以上之抗衰老藥。於二次篩選所獲得之此等新穎抗衰老藥係可依據功能分類,大約分為7組。
6. 對老年小鼠投予Nutlin-3b之效果的檢討2
進一步詳細地評估在老年小鼠中Nutlin-3b之生體(in vivo)效果。將試驗時程示於圖33。將20個月齡之C57BL/6小鼠(日本傑克遜實驗室)分為2群,在Nutlin-3b投予群方面,將懸浮於溶劑(PBS:PEG400=50:50)之Nutlin-3b,以11.62mg/kg進行腹腔內投予,每週1次,共計3個月。在媒液(vehicle)群方面,在相同時程只投予溶劑。其結果,在Nutlin-3b投予群中,觀察到身體狀態有幾項改善。除了肌力(掛線測試)、屬於腎功能指標之血漿BUN、血漿肌酸酐之外,屬於肝功能指標的血漿白蛋白值及乳酸值亦藉由投予Nutlin-3b而改善(圖34、35)。肝臟之組織化學分析,係顯示在高齡小鼠中蓄積的p21陽性細胞,在以Nutlin-3b處理的高齡小鼠中有
意義地降低(圖36A)。在老化組織中顯示巨噬細胞浸潤的F4/F80陽性細胞,亦藉由Nutlin-3b處理而減少,與數種SASP因子(IL6、CXCL1、TNFα及CCL5)之減少一致。又,對於各群之脂肪組織,進行屬於細胞老化指標之β半乳糖苷酶(senescence-associated β-galactosidase,SA β-Gal)染色時,於Nutlin-3b投予群中,細胞老化之程度獲得改善(圖37)。
7. PGAM1-Chk1途徑之下游轉錄因子的解析(HIF-2α之由Chk1所致之控制)
對IMR90細胞之年輕細胞(連續培養1個月以內)導入致癌性Ras,誘導出老化細胞(OIS)。將該老化細胞進行Nutlin-3b處理,HIF-2α蛋白質之表現受到抑制,然而藉由以蛋白酶體阻礙劑MG132處理,卻使HIF-2α蛋白質濃度顯著地回復(圖38)。不過,對其mRNA之影響比較少(圖39),此暗示在Nutlin-3b處理中,可能參與對於HIF-2α之泛素化。已知藉由將泛素化部位附近磷酸化,可誘導泛素化(Hagai et al.,2012)。位於HIF-2α之N末端之由Chk1所致之磷酸化的共同序列(K/R-K/R-x-x-S/T)被保存,該等被命名為Motif1(包含Ser12殘基)及Motif2(包含Ser19殘基)(圖40,序列編號77至81)。該等之中,HIF-2α之Ser12,若依據全球磷酸化蛋白質(https://www.phosphosite.org(Hornbecket al.,2015)之數據庫,亦有報導其他細胞型被磷酸化。HIF-2α之S12A變異體,於老化細胞之泛素化方面非常弱,然而S19A變異體則非如此(圖41、42)。一貫地,Ser12殘基之磷酸化,係依存於Chk1而在老化細胞中被驗出(圖43)。此等數據,暗示HIF-2α為PGAM-Chk1相互作用之老化標的。
8. 老化細胞中糖解作用亢進之生理意義的檢討
為了探討老化之糖解作用亢進的生理學上意義,調製由老化細胞及非老化細胞所構成之馴化培養基,並添加初期繼代時的人類纖維母細胞。PGAM-Chk1相互作用係藉由來自老化細胞之培養上清液增強,而對照群並未增強(圖44)。由此研判在來自老化細胞之培養上清液中,包含使PGAM-Chk1相互作用增強的成分。由於研判作為如此之成分的候補為乳酸,因而對於人類纖維母細胞進行乳酸處理。其結果,觀察到乳酸濃度依存性地HIF-2α的蓄積及其之Ser12的磷酸化、PGAM-Chk1相互作用的增強(圖45)。此結果,與老年小鼠之血清及老化細胞之培養上清液中上升之乳酸程度的結果一致。又,乳酸雖然刺激SASP表現,但p21之程度與對照無差異(圖46)。再者,針對乳酸之細胞表面受體,即GPR81,雖於老化細胞中上調(up-regulated)(圖47),但其基因敲落卻使乳酸所導致的PGAM-Chk1相互作用增強消失(圖48)。已知GPR81信號傳達,係部分地參與包含Ras途徑(Ohno et al.,2018)之致癌模組。在老化細胞或乳酸處理細胞中,觀察到存在於Ras途徑下游的RSK1激酶被活化,並伴隨作為RSK標的已知之Chk1之Ser280(Li氏等,2012年)被磷酸化(圖49)。
9. 由Survivin(存活素)表現抑制所產生之抗衰老效果的檢討
觀察細胞凋亡阻礙劑QVD-Oph在Nutlin-3b處理下,回復老化細胞之生存性(圖50)。又,為了解明PGAM-Chk1結合阻礙所產生之抗衰老效果的分子機構,實施轉錄組解析(圖51)。將在老化細胞中藉由Nutlin-3b處理而減少2倍以上表現的1,168個基因(圖52)、及美國國家生物技術資訊中心(National Center for Biotechnology Information)(NCBI)所報告的ChIP(染色質免疫沉澱(chromatin immunoprecipitation))情報,藉由濃縮
解析進行比較檢討。後者之情報,係由藉由903個抗原(轉錄因子或表觀遺傳調節劑(epigenetic regulator))所致之12,655個ChIP數據組所構成。其結果,在5,078個ChIP數據組中,與藉由Nutlin-3b處理而減少2倍以上表現的基因群之間顯示10個以上之重複基因,21個數據組顯示最高值(4倍以上)之富集倍數(fold enrichment)(FE)。在此等21個數據組之中,12組為由FOXM1所致之ChIP數據,最高濃縮分數雖為8.77(圖53),但亦包含其他數個因子(HDAC2、MYBL2、E2F4、RBL2/p130、Rad21)。又,藉由Nutlin-3b而被控制在3.45倍以上之前150個基因之中,約26.0%(39基因)係作為FOXM1標的被報導(圖54)(Lam et al.,2013)。
關於Nutlin-3b處理老化細胞之微陣列數據組,分析被分類為細胞凋亡過程之調節的202個基因(GO:0042981)。藉由Nutlin-3b處理受到影響的41個基因之中,列出前10個調降基因(圖55)。屬於IAP蛋白質之一的存活素(Survivin)/BIRC5,在抗細胞凋亡家族之中最顯著地被調降(down-regulated)(約30倍)。一貫地,Survivin(存活素)蛋白質,在經Nutlin-3b處理的老化細胞中調降。實際上,由於老化細胞中之存活素的基因敲落,係對細胞凋亡誘導中等程度之抗衰老(圖56),因而暗示由Nutlin-3b所產生之抗衰老效果,係經由HIF2-FOXM1-存活素途徑的抑制來達成。
10. 藉由FOXM1之Survivin(存活素)表現控制的檢討
除了Survivin(存活素)之外,對於包含細胞周期調節劑(CCNB1、B2及D1)及有絲分裂關連基因(PLK1、ASPM、及CEP55)(Lamet al.,2013)之FoxM1基因以外之轉錄標的之mRNA,亦發現藉由Nutlin-3b處理而造成有意義之影響(圖57)。微陣列數據之GESA(基因組(gene set)濃縮分析),
顯示FoxM1標的有意義地被調降(FDRq值小於0.001)(圖58、59)。Fox家族之中,Foxo蛋白質深度參與老化過程。又,FOX轉錄家族之成員中,FoxM1 mRNA係藉由Nutlin-3b而被最劇烈地調降(圖60)。再者,老化細胞中,藉由HIF-2α之失活,FoxM1蛋白質被調降,然而在年輕繼代細胞中無此情形(圖61),與「FoxM1係由HIF-2α控制轉錄」的研判一致(Bai et al.,2019)。
將本實施例中所使用之引子整理於下列表中(序列編號11至76)。
[產業上之可利用性]
本發明之抗衰老藥之篩選方法,藉由在測定選擇性去除老化細胞活性的步驟之前,進行包含測定PGAM與Chk1之結合阻礙活性之步驟的初次篩選,可使抗衰老藥之篩選的效率顯著地提高。本發明人等發現PGAM與Chk1之結合,雖然在年輕正常細胞中無法被觀察到,但在老化細胞中卻顯著地亢進,並藉其引起糖解作用代謝之亢進。基於此,藉由在眾多物質之中,首先篩選具有PGAM與Chk1之結合阻礙活性的物質,可容易地得到在老化細胞中,能阻礙PGAM與Chk1之結合的物質,對於彼等,藉由測定選擇性去除老化細胞活性,可有效率地發現抗衰老藥。實際上,使用本發明之篩選方法,從安全性確定之2300餘種已存藥資料庫中,藉由初次篩選搜尋出約200種候選藥,然後進行選擇性去除老化細胞活性試驗,最後成功地鑑定出約40種新穎抗衰老藥。從本發明人等之研究,已確認即使在為端粒非依存性細胞老化之壓力細胞老化中,亦與所謂老化細胞同樣地,發生由PGAM與Chk1之結合所引起的糖解作用代謝亢進,若依照本發明之篩選方法,可發現不僅對於老年性疾病,對於因壓力細胞老化而發病之疾病,亦可發揮治療效果的物質。
TW202327606A_111133922_SEQL.xml
Claims (10)
- 一種抗衰老藥,其係阻礙PGAM與Chk1之結合,更選擇性地滅絕老化細胞。
- 如請求項1所述之抗衰老藥,其係含有選自由RSK1激酶阻礙劑、Fak激酶阻礙劑、Fak激酶所參與之信號通路的阻礙劑、CDK阻礙劑、鈣拮抗劑、強心配糖體、DNA障礙劑、抗菌劑、極光激酶(aurora kinase)阻礙劑、類黃酮(flavonoid)、PI3K激酶阻礙劑、HDAC阻礙劑、老年性黃斑變性治療藥、p38MAPK阻礙劑、mTOR阻礙劑、酪胺酸激酶阻礙劑、Bc1-2阻礙劑、他汀類藥物、血清素受體拮抗藥、其他激酶阻礙劑、及Nutlin-3b所構成之群組中的至少1種作為有效成分。
- 如請求項2所述之抗衰老藥,其中,RSK1激酶阻礙劑為RSK1之反義核酸、RSK1之siRNA、或BI-D1870;Fak激酶阻礙劑為PF-00562271或PF-431396;Fak激酶所參與之信號通路的阻礙劑為PHA-665752或CP-100356;CDK阻礙劑為SNS-032、AZD5438、黃酮吡醇(Flavopiridol)(阿伏西地(Alvocidib))、PHA-793887、AT7519、或PHA-767491;強心配糖體為海蔥次苷A(Proscillaridin A)或地高辛(digoxin);DNA障礙劑為多柔比星(Doxorubicin)(阿黴素(Adriamycin))HCl、道諾黴素(Daunorubicin)HCl、泛艾黴素(Epirubicin)HCl、米托蒽醌(Mitoxantrone)2HCl或羥基喜樹鹼(hydroxy camptothecine);抗菌劑為胺基吖啶、三氯沙或依沙吖啶乳酸鹽(Ethacridine lactate);極光激酶阻礙劑為AMG-900或JNJ-7706621;類黃酮為金黃素(Chrysin);PI3K激酶阻礙劑為PF-05212384或XL147;HDAC阻礙劑為曲古抑菌素A(Trichostatin A(TSA));老年性黃斑變性治療藥為維替泊芬(Verteporfin);p38MAPK阻礙劑為SB203580或達馬莫德(Doramapimod)(BIRB796);mTOR阻礙劑為AZD8055或KU-0063794;酪胺酸激酶阻礙劑為來那替尼(Neratinib)、尼達尼布(Vargatef)或NVP-BHG712;Bc1-2阻礙劑為TW-37;他汀類藥物為美伐他汀(Mevastatin);血清素受體拮抗藥為RS-127445;其他激酶阻礙劑為BMS-345541或CX-4945。
- 一種抗衰老藥之篩選方法,其係包含下列步驟:(A)對於受檢物質測定PGAM與Chk1之結合阻礙活性的步驟、及(B)對於藉由上述步驟(A)被判斷為具有PGAM與Chk1之結合阻礙活性之受驗物質,測定選擇性去除老化細胞活性的步驟。
- 如請求項4所述之篩選方法,係在上述步驟(B)中,測定受驗物質對年輕細胞的傷害活性及對老化細胞之傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質。
- 一種抗衰老藥,其係具有使HIF-2α失活之作用,更選擇性地滅絕老化細胞。
- 如請求項6所述之抗衰老藥,其係含有HIF-2α阻礙劑作為有效成分。
- 如請求項7所述之抗衰老藥,其中,HIF-2α阻礙劑為HIF-2α之反義核酸或siRNA。
- 一種抗衰老藥之篩選方法,其係包含下列步驟:(X)對於受檢物質測定HIF-2α之阻礙活性的步驟,及(Y)對於藉由上述步驟(X)判定具有HIF-2α之阻礙活性的受驗物質,測定選擇性去除老化細胞活性之步驟。
- 如請求項9所述之篩選方法,其係在上述步驟(Y)中,測定受驗物質對年輕細胞之傷害活性及對老化細胞的傷害活性,選出對年輕細胞不具有傷害活性,且對老化細胞具有傷害活性的受驗物質。
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