前情回顧：細(xì)胞工廠搬運(yùn)工——驅(qū)動(dòng)蛋白（Kinesin）活性檢測(cè)試劑盒

真核細(xì)胞驅(qū)動(dòng)蛋白馬達(dá)蛋白利用 ATP 水解的能量沿細(xì)胞骨架微管網(wǎng)絡(luò)移動(dòng)貨物，如染色體和囊泡 。它們?cè)诩?xì)胞內(nèi)運(yùn)輸?shù)姆椒矫婷娑及l(fā)揮著重要作用，并廣泛參與各種生理過(guò)程，包括胚胎發(fā)育、軸突運(yùn)輸和細(xì)胞分裂。許多驅(qū)動(dòng)蛋白在細(xì)胞分裂中起著重要作用，以及驅(qū)動(dòng)蛋白的過(guò)度表達(dá)與癌癥（如視網(wǎng)膜母細(xì)胞瘤）有關(guān)的事實(shí)，使它們成為開(kāi)發(fā)抗有絲分裂藥物非常有潛力的靶標(biāo)。許多驅(qū)動(dòng)蛋白僅在細(xì)胞分裂期間表達(dá)的證據(jù)也表明，它們可能優(yōu)于當(dāng)前的抗有絲分裂藥物靶標(biāo)，例如普遍表達(dá)的Tubulin。

驅(qū)動(dòng)蛋白的一般結(jié)構(gòu)：

驅(qū)動(dòng)蛋白分類和結(jié)構(gòu)多樣性

迄今為止，已經(jīng)在從酵母（釀酒酵母 含有 6 種驅(qū)動(dòng)蛋白）和真菌到人類（目前有13種驅(qū)動(dòng)蛋白）等物種中鑒定了大約 150 種驅(qū)動(dòng)蛋白。分類基于馬達(dá)蛋白域的同源性。目前一致認(rèn)為至少有九類驅(qū)動(dòng)蛋白，驅(qū)動(dòng)蛋白分類如表1所示，驅(qū)動(dòng)蛋白命名（在表 1 中以粗體顯示）基于驅(qū)動(dòng)蛋白內(nèi)馬達(dá)蛋白結(jié)構(gòu)域的位置，因此 N-蛋白質(zhì)在蛋白質(zhì)的氨基末端包含一個(gè)運(yùn)動(dòng)結(jié)構(gòu)域，而 C- M-馬達(dá)蛋白分別位于羧基末端和蛋白質(zhì)中間。

考慮到不同類驅(qū)動(dòng)蛋白的馬達(dá)之間相對(duì)較高的百分比差異，很可能識(shí)別類特異性的驅(qū)動(dòng)蛋白藥物，這一結(jié)構(gòu)證據(jù)得到了生化數(shù)據(jù)的支持，這些數(shù)據(jù)表明不同類別的果蠅驅(qū)動(dòng)蛋白表現(xiàn)出對(duì)各種 ATP 類似物的不同的利用效率。實(shí)驗(yàn)表明，即使在同一的物種內(nèi)，不同驅(qū)動(dòng)蛋白之間也存在明顯的結(jié)構(gòu)差異，可用于選擇性藥物開(kāi)發(fā)。

表 1：驅(qū)動(dòng)蛋白超家族蛋白的分類及功能

用于 HTS 分析的驅(qū)動(dòng)蛋白靶標(biāo)的選擇

從上表我們可以看到，驅(qū)動(dòng)蛋白的細(xì)胞功能主要可分為兩大類：膜囊泡和細(xì)胞器的運(yùn)輸和定位，以及有絲分裂紡錘體的形態(tài)發(fā)生和染色體運(yùn)動(dòng) [12]，九類驅(qū)動(dòng)蛋白中有四類專門參與細(xì)胞分裂（表 1 的黃色/綠色陰影區(qū)域），兩類（C- 和 M-）同時(shí)包含囊泡轉(zhuǎn)運(yùn)蛋白和有絲分裂馬達(dá)蛋白（表 1 的淺黃色陰影區(qū)域），三個(gè)類別（NI、NIII 和 N-IV）僅在囊泡運(yùn)輸中起作用（表 1 的無(wú)陰影區(qū)域）。

我們從每個(gè)驅(qū)動(dòng)蛋白類中選擇了一個(gè)人類同源蛋白[6,17,21,22,26,28,29,30,39]，從Aspergillus(一種人類病原體)中選擇了兩個(gè)真菌驅(qū)動(dòng)蛋白[34]， 一個(gè)人類病原體作為 HTS 檢測(cè)的基礎(chǔ)（這些顯示在表 1 中）。通過(guò)用化合物庫(kù)篩選每種蛋白質(zhì)，可以生成一個(gè)初級(jí)化合物庫(kù)，該化合物庫(kù)應(yīng)該允許識(shí)別選擇性靶向細(xì)胞分裂特定類別的人類驅(qū)動(dòng)蛋白（表 1 的深色陰影區(qū)域）的化合物，同時(shí)對(duì)囊泡轉(zhuǎn)運(yùn)驅(qū)動(dòng)蛋白沒(méi)有影響。這種化合物很有可能是治療人類疾病如癌癥的有效的抗有絲分裂劑。同樣，可以將與曲霉驅(qū)動(dòng)蛋白特異性反應(yīng)的化合物用作潛在的抗真菌藥物。

驅(qū)動(dòng)蛋白為什么能作為抗有絲分裂藥物篩選靶點(diǎn)？

在開(kāi)始一項(xiàng)昂貴的藥物開(kāi)發(fā)計(jì)劃之前，必須嚴(yán)格評(píng)估候選蛋白的適用性，以滿足有效藥物靶標(biāo)的標(biāo)準(zhǔn)。驅(qū)動(dòng)蛋白將成為抗有絲分裂藥物開(kāi)發(fā)的絕佳靶點(diǎn)的證據(jù)來(lái)自一系列實(shí)驗(yàn)方法：包括突變分析 [40]、抗體抑制實(shí)驗(yàn) [7,32] 和驅(qū)動(dòng)蛋白活性的反義抑制 [19]。這些證據(jù)總結(jié)在表 1 中，可以看出特定有絲分裂特異性驅(qū)動(dòng)蛋白的功能性抑制導(dǎo)致細(xì)胞分裂的抑制。

在低等真核生物中，突變分析有助于闡明有絲分裂驅(qū)動(dòng)蛋白的作用。單個(gè)驅(qū)動(dòng)蛋白的突變通常會(huì)導(dǎo)致有絲分裂阻滯和致死表型。我們選擇作為抗真菌靶點(diǎn)的 AnBimC 蛋白就是這種情況[34]。然而，在某些情況下，兩個(gè)高度同源的基因執(zhí)行相同的功能從而導(dǎo)致功能冗余[41]，在這些情況下，必須同事敲除兩個(gè)基因才能產(chǎn)生有絲分裂表型變化。

重要的是，所有數(shù)據(jù)表明，針對(duì)有絲分裂驅(qū)動(dòng)蛋白的抗體會(huì)特異性影響有絲分裂過(guò)程，但對(duì)驅(qū)動(dòng)蛋白囊泡轉(zhuǎn)運(yùn)功能沒(méi)有影響 [42]。事實(shí)上，對(duì)特定驅(qū)動(dòng)蛋白的抑制通常會(huì)誘導(dǎo)一種非常具有細(xì)胞周期特異性的表型。例如，抑制 M 類驅(qū)動(dòng)蛋白 MCAK 中的運(yùn)動(dòng)區(qū)域?qū)忓N體組裝沒(méi)有影響，但確實(shí)抑制了染色體運(yùn)動(dòng) [32]。該數(shù)據(jù)表明驅(qū)動(dòng)蛋白馬達(dá)的特定抑制劑將只會(huì)特異性地抑制有絲分裂過(guò)程而不影響其他關(guān)鍵細(xì)胞功能。

驅(qū)動(dòng)蛋白靶標(biāo)相對(duì)于當(dāng)前抗有絲分裂靶標(biāo)的優(yōu)勢(shì)：

微管的紡錘體蛋白Tubulin是目前抗有絲分裂藥物如紫杉醇和長(zhǎng)春堿的主要靶點(diǎn) [43,44]。人們普遍認(rèn)為，這些藥物通過(guò)在有絲分裂期間直接抑制微管動(dòng)力學(xué)發(fā)揮作用 [45,46]。由于有絲分裂細(xì)胞中的微管動(dòng)力學(xué)比靜止細(xì)胞大得多，因此對(duì)分裂細(xì)胞的特異性是有利的。藥物處理導(dǎo)致有絲分裂阻滯，在此期間細(xì)胞進(jìn)入凋亡途徑并死亡 [47]。但由于其作用機(jī)制的性質(zhì)，所有抗有絲分裂劑，包括紫杉醇和長(zhǎng)春堿，都會(huì)在某種程度上對(duì)正常有絲分裂細(xì)胞產(chǎn)生不利影響，例如存在于胸腺、睪丸、小腸、結(jié)腸和胎盤中的細(xì)胞 [48] .

然而，有絲分裂驅(qū)動(dòng)蛋白作為潛在的抗有絲分裂藥物靶點(diǎn)的一個(gè)主要優(yōu)點(diǎn)，是它們的表達(dá)通常受到嚴(yán)格調(diào)節(jié)，從而與有絲分裂事件一致。例如，HsMCAK 僅在增殖細(xì)胞中表達(dá)，其表達(dá)已被證明在轉(zhuǎn)錄水平受到嚴(yán)格調(diào)控 [6]；HsEg5 僅在有絲分裂期間與微管相關(guān)，在體內(nèi)間期與 MT 不相關(guān) [7]；染色質(zhì)驅(qū)動(dòng)蛋白僅在增殖細(xì)胞中表達(dá)，在那里它們是壽命較短的蛋白質(zhì)，可能受細(xì)胞周期蛋白降解機(jī)制的調(diào)節(jié) [51]；研究表明，CENP-E在核破裂后立即與著絲點(diǎn)結(jié)合，并一直保持完全結(jié)合，直到B后期，當(dāng)它重新定位到B后期紡錘體，隨后通過(guò)類似周期素的途徑降解 [52]。有絲分裂驅(qū)動(dòng)蛋白表達(dá)的嚴(yán)格調(diào)控預(yù)示著這些將是高度特異性的抗有絲分裂靶點(diǎn)，具有最小的劑量限制副作用。

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