建立專一性第二型拓樸異構酶亞型藥物篩選系統

Abstract

人類第二型拓樸異構酶(hTop2)是目前臨床化療藥物主要針對的蛋白質，這類的化療藥在臨床上很有效，而且廣泛被使用在許多癌症上，像是血癌、乳癌、攝護腺癌… …等等。雖然這些藥物很有效，但是嚴重的副作用像是心毒性、藥物引起的癌症等等，都大大的降低這些藥物在臨床上的使用。而人類第二型拓樸異構酶分成兩種亞型: hTop2α 和hTop2β。這兩種亞型負責調控不同的細胞功能，hTop2α 主要負責細胞複製和分裂而且在癌細胞會大量表現；hTop2β 則負責細胞基因表現的調控而且在所有細胞中表現量都差不多。目前許多研究指出，藥物針對hTop2α與化療療效有關，而針對hTop2β則與副作用息息相關。但不幸的是，目前現行的臨床藥物並沒有辦法區分hTop2α 和hTop2β，因此開發能夠區分這兩種亞型的藥物是很急迫地而且是目前臨床上需要的。我們實驗室目前有兩線藥物開發，一個是Mitoxantrone 的衍生物另一個是Etoposide 的衍生物。透過細胞外的實驗，Mitoxantrone 衍生物871和Etoposide 衍生物7882, 78871, 78769R, 78769S，可以區分兩種亞型，而其中7882、78871可以主要藉由hTop2α 造成DNA的斷裂。除了藥物開發外，我們也建立可以區分這兩種亞型的篩選系統，7882, 78871在這個篩選系統中也呈現他們比較會形成hTop2α cleavable complex。因此我們認為Etoposide 的衍生物可以比較針對hTop2α而且可能具有較低的副作用，有成為新一代化療藥物的潛力。

Human typeⅡ topoisomerase has been proved to be an excellent target of chemotherapy. Current clinical drugs like Doxorubicin (DOX), Etoposide (VP-16) are hTop2-targeting drugs. They are very effective and has been widely used in many kinds of cancer for many years. Although they are effective, the severe side effects like cardiotoxicity and secondary malignancy have also been reported and have been studied well. Human typeⅡ topoisomerase has two isozyme, hTop2α and hTop2β respectively. Because of their regulating different cellular functions, the results of targeting hTop2α and hTop2β are very different. Targeting hTop2α, which up regulated in cancer, is responsible for the efficacy of treatment and targeting hTop2β, which expression level remains a constant level in all kinds of cell, takes charge of side effects. Unfortunately, current clinical drugs do not have good isozyme selectivity. Therefore discovering potential isozyme selectivity agents is urgent and un-met need. In addition to discovering new agents, establishing a screening system for those potential chemotherapy agents is also important. Our lab is developing two lines of new chemotherapy agents, one comes from Mitoxantrone and the other comes from Etoposide. Through conducting in-vitro relaxation assay, using purified recombinant hTop2α and hTop2β, Compound 871, a derivative from Mitoxantrone, and compound 7882, 78871, 78769R, 78769S, derivatives from Etoposide, have good isozyme selectivity, which can target hTop2α more than hTop2β. In cleavage assay, we demonstrate that compound 7882, 78871 can cause DNA damage mainly through hTop2α. Besides, those compounds do not have strong ability of DNA intercalating, which indicates that those agents could have lower unwanted side effects cause from direct DNA damage. We also establish a screening system for those agents by biophysical measurement. We find Etoposide derivatives, 7882, 78871 and 78769R could form hTop2αcc more than hTop2βcc and are better than Etoposide and Teniposide, which are current clinical drugs. Taking all together, our Etoposide derivatives might be excellent new chemotherapy agents, which can preferentially target hTop2α and might have lower side effects.