人類第二型拓樸異構酶β亞型與新穎小分子藥物形成之切割複合體之結構解析

Abstract

拓樸異構酶是一群普遍存在於古生菌、細菌及真核生物的蛋白。當DNA進行複製、轉錄、或染色體重組等作用時，其雙股螺旋結構發生解旋而引起的拓樸構型問題，如超螺旋、連鎖、扭結等結構的形成，將使得上述作用無法進行或導致基因無法表達，甚至可能引發細胞凋亡。拓樸異構酶則能有效解決拓樸構型問題，使DNA代謝正常運作。依照演化親緣關係與催化機制的不同，拓樸異構酶可分為兩大型：第ㄧ型酵素催化DNA單股斷裂，而第二型酵素可使DNA雙股同時斷裂，使DNA拓樸結構得以發生改變，且在反應完成之後將斷裂之DNA重新接回，以確保DNA結構的完整性。第二型酵素又可再分成IIA及IIB兩種子群，在哺乳類細胞中，其IIA型酵素(Top2)包含⍺及β兩種亞型，本篇研究的目標蛋白為人類第二型拓樸異構酶β亞型 (hTop2β)。真核生物的Top2為同質二聚體所構成，結構中包含三個由二聚體交互作用介面組成、可關閉或開啟的閘門 (gate)，依序為N端閘門 (N-gate)、DNA閘門 (DNA-gate)、C端閘門 (C-gate)。目前所認為的作用機制是透過”雙閘門機制” (two-gate mechanism)，一段稱為G-segment的雙股DNA由N-gate進入後會被酵素活性中心的酪胺酸 (tyrosine) 切割，使一段稱為T-segment的DNA可以由此切口通過，再經由C-gate離開。 鑒於Top2對於生物體不可或缺的特性，長久以來便是許多抗癌藥物的標的。能夠抑制Top2的小分子藥物依其作用機制的不同可分為兩大類：毒化劑 (poison)及催化抑制劑 (catalytic inhibitor)，poison會穩定Top2及DNA形成的切割複合體，造成DNA雙股斷裂，對於細胞有極高的毒性而得名。Catalytic inhibitor會抑制酵素作用，將蛋白鎖在反應中間態的構型而阻斷反應，對於細胞相對安全性較高、但藥效較差。雖然這些以Top2為作用標靶的藥物已在臨床上應用多年，然而，嚴重的副作用問題如急性骨髓性白血病 (acute myeloid leukemia; AML)、心臟毒性，以及日益增加的抗藥性問題仍待解決。因此與我們合作的義大利研究團隊Professor Marco De Vivo，利用藥效基團混成技術 (pharmacophore hybridization strategy)，將臨床上常用的etoposide的E-ring和merbarone的thiobarbituric core組合，開發出一個名為ARN16267的新穎小分子化合物，能夠有效抑制Top2的功能並能誘發DNA雙股斷裂，後續在E-ring的meta位置用不同結構取代，得到三個抑制活性最好的衍生物，分別命名為3f、3g、3i，本研究的主要目的即在揭示此三藥物的作用機轉。 先前本實驗室已經解析出hTop2β與DNA及抗癌藥物etoposide所形成之切割複合體的結構 (PDB code 3QX3)，發現etoposide主要是和Top2之DNA結合及切割的活性區域 (DNA binding and cleavage core; DBCC) 發生交互作用，且DBCC相較於全長hTop2β更易於純化及結晶，因此本研究希望透過結構解析的方式，來探討新藥和hTop2β DBCC的交互作用，進而了解藥物的作用機制，以輔助新藥開發。目前已利用高純度hTop2β進行DNA切割活性分析 (cleavage assay)，確認三種藥物 (3f、3g、3i) 的確兼具毒化及抑制活性，並透過熱遷移實驗 (thermal shift assay)，確認藥物和酵素及DNA會發生交互作用。接著透過氣相擴散法進行晶體培養，利用共結晶的方式，目前分別獲得hTop2β DBCC和兩種藥物的二元複合體之晶體，惟X-射線繞射解析度僅達7.86 Å及6.93 Å，尚無法進行結構解析。此外、亦透過浸潤 (soaking) 法，以hTop2β DBCC-DNA-etoposide的晶體為起始，嘗試將晶體中的etoposide置換成3f、3g、3i，目前已得到3f結合於hTop2β DBCC-DNA切割複合體的晶體，解析度約為10 Å，晶體排列屬於P3空間群。然而，由於晶體偏小且X-射線繞射解析度不佳，晶體仍待再優化。未來希望透過微調長晶條件，來得到更立體、品質更佳的晶體，以利用X-射線繞射法得到更高解析度的數據，解析出新藥和hTop2β的完整交互作用，闡明此類新穎化合物的作用機制。

Topoisomerases are essential enzymes specialized in resolving DNA topological problems resulted from cellular DNA transactions, such as replication, transcription, recombination, and chromosome segregation. Specifically, the unwinding of DNA double helix during these processes will cause the accumulation of tension and lead to DNA entanglements in the forms of supercoiled DNA, catenanes, or knots, which are toxic for cells if left unresolved. Based on differences in their catalytic mechanisms, topoisomerases can be classified into two types: members of the type Ⅰ family cut one strand of a double-stranded DNA, whereas type Ⅱ enzymes cut both strands to allow the passage of a second DNA duplex through the DNA double-stranded break (DSB). Type II family can be further divided into IIA and IIB subclasses based on sequence and structural differences. Mammalian cells possess two functionally distinct type ⅡA topoisomerase (Top2) isoforms, Top2α and Top2β. The homodimeric architecture of Top2 features the presence of three dissociable dimer interfaces (or gates) termed N-gate, DNA-gate, and C-gate. According to the widely accepted “two-gate mechanism”, the opening of N-gate allows entry and cleavage of the G-segment DNA by the active site tyrosine to form the so-called cleavage complex, the T-segment DNA can then pass through the cleaved G-segment and depart via the C-gate, which achieves a change in DNA topology. Since Top2 is abundantly expressed in cancer cells and indispensable for cellular functions, it has long been regarded as an anticancer drug target. Small molecules capable of disrupting Top2 function can be classified as poisons and catalytic inhibitors according to their mechanisms of actions. Poisons are known to induce the formation of toxic DSB by stabilizing the Top2 cleavage complex, which effectively causes death of cancer cells. In contrast, catalytic inhibitors interfere with Top2 function by preventing DNA binding or blocking the ATP-binding site without producing DSB, therefore, they are in general safer but less effective. Though both types of Top2-targeting drugs have been applied clinically for over 40 years, serious side effects such as the emergence of secondary cancer and drug resistance call for the development of new drugs. Toward this goal, the team led by Professor Marco De Vivo has developed a set of new anticancer agents by employing a pharmacophore hybridization strategy, which combines the E-ring of etoposide, a clinically active Top2 poison, with the thiobarbituric core of merbarone, a well-known catalytic inhibitor of Top2. The resulting compound, ARN16267, was found to be a promising Top2 blocker, capable of inducing pronounced accumulation of Top2-mediated DSBs. Also, by introducing different functional groups at the meta position of the six-membered E-ring, three derivatized compounds (termed 3f, 3g, 3i) were found to exhibit improved inhibitory activity. The aim of this study is to elucidate the structural bases underlying the Top2-poisoning/inhibiting activities of these compounds. Given our lab’s experience in performing structural analysis of poison-stabilized Top2 cleavage complexes, we utilized the DNA binding and cleavage core (DBCC) of human Top2β (hTop2β) for this study. Compared to full-length human Top2β, the DBCC region is much easier to work with and can be purified and crystallized more efficiently. Hence, the proposed mechanistic dissection will be conducted by characterizing the structures of hTop2β DBCC in complexes with these novel hybrid compounds, in hoping to facilitate the development of new drugs. To this end, we have produced highly purified hTop2β DBCC and conducted cleavage assay for validating protein activity and drugs’ Top2-poisoning/inhibiting activity. Also, we performed thermal shift assay to confirm the interaction between hTop2β DBCC and drugs with and without the presence of DNA. Furthermore, using the vapor diffusion method, we performed co-crystallization of hTop2β DBCC-drug binary complexes, with partial data sets been collected to 7.86 Å and 6.93 Å resolution. Besides, by post-crystallization drug replacement, we obtained crystals of the hTop2β DBCC-DNA-3f complex, which belong to the space group P3 and diffract to ~10 Å resolution. Crystallization conditions are currently being optimized to improve the size and diffraction quality of these crystals.