摘要:以第二型 DNA 拓撲異構酶(type II DNA topoisomerases, TOP2s)為標的(target)之抗癌藥物(如etoposide、doxorubicin 及mitoxantrone 等)是癌症化學治療的主要成份之一,此類藥物可藉由引發具有細胞毒性的DNA 斷裂而導致癌細胞的死亡。雖然這些藥物的療效十分顯著,但用藥後的副作用(如後天白血病以及心臟毒性等)使得治療時仍有安全性的疑慮。最近的研究結果顯示,副作用的成因在於這些藥物會同時作用於-及-型之人類TOP2 同功酶(hTOP2和hTOP2):藥物與hTOP2的交互作用可殺死癌細胞,而與hTOP2的結合則可能是引起副作用的主要原因。因此,開發以hTOP2為標靶的、具有同功酶專一性的藥物確有其臨床上的重要性。此外,治療時也常篩選出TOP2基因帶有突變而有藥物抗性的癌細胞。為了解決上述藥物副作用與抗藥性在治療時衍生的問題,本研究的主要目的在於提供藥物開發時所需的重要結構資訊,以輔助新型抗癌藥物的研發。鑑於蛋白質結構資料庫(Protein Data Bank)中現有的TOP2 結構皆來自於酵母菌及若干細菌,以致於人類hTOP2和hTOP2間主要的結構差異以及抗藥性的分子機轉仍存有許多不明之處。為提供hTOP2與hTOP2其藥物作用區(DNA-binding and cleavagecore; DBCC)確切的結構訊息,本研究將解析hTOP2_DBCC或hTOP2_DBCC與DNA和現有抗癌藥物形成之三重複合體結構。此外、我們亦將建立一個結構解析平台以快速分析各種新藥先導化合物(lead compounds)與DNA 和hTOP2_DBCC 或hTOP2_DBCC 的交互作用方式。為達成新藥開發的目標,我們擬訂了以下兩個具體的研究方向:目標一、建立一個結構解析平台以快速分析hTOP2_DBCC 或hTOP2_DBCC 與DNA和各種抗癌藥物或新藥先導化合物形成之三重複合體結構。本實驗室已在此研究方向上獲得重要的進展(請參閱C012-1 表中所敘述的“初步研究成果”)。我們成功的判定hTOP2_DBCC 與一段雙股DNA 和抗癌藥物etoposide 形的三重複合體結構(解析度達到2.16 Å),基於此結果我們進一步建立了可以成功將etoposide 置換為其他抗癌藥物的實驗流程,並成功得到了mitoxantrone 與doxorubicin結合後的晶體結構。這個關鍵技術將被用於探討新藥先導化合物與TOP2 的交互作用方式,我們亦將用類似的方法研究對hTOP2_DBCC 進行分析。目標二、建立高可信度的藥效基團結構(pharmacophore models)以輔助藥物研發。由“目標一”中獲得的結構將可提供藥物結合區的立體形狀、極性分佈、結構彈性、以及參與藥物結合的關鍵胺基酸與DNA 鹼基對,此外、目前已成功判定的結構亦顯示hTOP2和hTOP2間的差異,這些訊息將使建構中之藥效基團結構具有極高的可信度,可應用於新藥的研發。綜合以上敘述可知:本研究計畫的方向明確,且已獲得關鍵的初步結果,因此預期本研究工作應能順利進行,並可望為新一代抗癌藥物的研發提供重要且豐富的結構訊息。
Abstract: Drugs targeting type II DNA topoisomerases (TOP2s) are among the most widely prescribedchemotherapeutic agents for treating a variety of cancers, including lymphomas, leukemias,sarcomas, gastric cancer, testicular cancer, and small cell lung cancer. By modulating the DNAcleavage activity of TOP2, these drugs exert cell-killing effects by promoting the formation ofdouble-stranded DNA breaks to initiate the cell death pathways. Although proven to be effective,serious side effects such as therapy-related secondary leukemia and cardiotoxicity hamper theirlong-term usage, a drawback likely attributed to the simultaneous drug-targeting of both TOP2isoforms, hTOP2 and hTOP2. Recent studies have suggested that targeting of hTOP2 issufficient for the antitumor activity, whereas hTOP2-poisoning may be linked to side effects.Moreover, drug-resistant tumor cells carrying mutant TOP2 allels usually emerged during treatment.To overcome these problems, it would be clinically desirable to have new and preferablyisoform-specific targeting agents. The overall goal of this cooperative project aims on elucidatingthe functional and drug-interacting distinction between human hTOP2 and hTOP2, thereforeproviding a rational for developing isoform-specific targeting therapy for future clinical use.Due to the lack of any experimentally determined structure of the drug-targeting domains ofthe human TOP2s (hTOP2_DBCC and hTOP2_DBCC, in which DBCC stands forDNA-binding and cleavage core), molecular basis for the drug-resistant mutations andisoform-specific TOP2-drug interactions have remained uncharacterized. To facilitate drugdevelopment and to evaluate the interactions between new lead compounds and human TOP2s, thissub-project is aimed to establish a platform for efficient structural characterization of the ternarycomplexes formed by TOP2, DNA, and either existing drugs or newly identified lead compounds.Structural insights obtained by this sub-project will not only provide a potential rational for thedevelopment of isoform-specific targeting agents, but will also guides the refinement of new leadcompounds. Toward this goal, the following two specific aims were proposed:Aim 1. Establish the platform for high-throughput structural analysis of ternary complexes formedby hTOP2, DNA, and antitumor drugs/new lead compounds.Aim 2. Constructing highly reliable isoform-specific pharmacophore models based on highresolution structures of the ternary complexes.As described in the “Previous Achievements” section in C012-1, we have obtained highresolution crystal structures of the DNA-binding and cleavage core of the hTOP2 in complexeswith DNA and three widely prescribed anticancer drugs (etoposide, mitoxantrone, and doxorubicin),representing the first observations of TOP2 ternary cleavage complexes stabilized by anticancerdrugs. Most importantly, we have established a procedure by which etoposide can be removed andreplaced by different TOP2-targeting compounds. Given our strong preliminary results, I firmlybelieve that both specific aims will be accomplished successfully. We should be able to formulatemolecular codes useful for the development of new generation of isoform-specific TOP2-targetinganticancer drugs, which has the potential to complement or even replace the widely used ones inclinical chemotherapy.