人類胸腺嘧啶核酸激酶與抑制物YMU1之複合體結構解析

Abstract

在快速分裂的癌細胞中，DNA複製的速度顯著高於正常細胞，使得癌細胞對於DNA的損傷非常敏感，因此DNA修復機制的運作與效率尤為重要。先前研究指出，DNA修復的效率與修復過程所需之dNTPs的濃度高低密切相關。dNTPs的生合成是以核苷酸新合成路徑 (de novo pathway)或是回收路徑 (salvage pathway)所產生的NDPs為原料，再經由ribonucleotide reductase (RNR)催化之還原反應將NDP還原成dNDP，這個步驟在dNTPs的合成扮演關鍵的角色。而大部分的dNDPs包含dADP、dGDP、dCDP和dUDP都可以經由RNR催化NDPs的還原而生成，然而，只有dTDP的合成是經由TMPK (thymidylate kinase)所催化，此酵素以dTMP為其受質，利用ATP做為磷酸根的供給者，藉由對dTMP的磷酸化以合成dTDP，有鑒於hTMPK對於dTTP合成不可或缺的重要性，因此，抑制TMPK或許可作為治療癌症的手段之一 。 目前許多化療藥物仍被廣泛的用於癌症治療，如doxorubicin可以造成DNA雙股斷裂，使癌細胞生長停止或是誘發細胞凋亡。然而這些化療藥物的副作用也不可忽視，病人可能會出現噁心、嘔吐等症狀，亦可能產生心臟毒性。研究發現，當以siRNA技術使癌細胞的TMPK表達下降，會增強doxorubicin的毒殺效果，因此發展針對TMPK的專一性抑制物或許有助於降低doxorubicin的IC50，進而減少不必要的副作用。這個概念在近期使用TMPK抑制物YMU1進行動物實驗後得到進一步的支持：YMU1自身並無顯著的細胞毒性，但與低劑量的doxorubicin合併使用則可以使癌細胞凋亡。因此YMU1應可做為發展抗癌藥物的先導化合物。 為了瞭解YMU1如何與TMPK交互作用並抑制其活性，我們著手TMPK與YMU1複合體的結構解析。首先利用大腸桿菌表現大量的人類TMPK蛋白，並且利用液相層析法純化蛋白，最後再以共結晶的方式培養複合體的晶體，另一方面，我們同樣以共結晶的方式，取得TMPK與dTMP複合體之晶體，之後或許可以將晶體浸泡於含有YMU1的溶液中，取得TMPK與YMU1的複合體晶體。此結構亦可能加深我們對TMPK催化反應機制的認識。

In fast-proliferating cancer cells, DNA replication usually occurs at a higher frequency compared to normal cells. Therefore, cancer cells are in general more sensitive to DNA-damaging agents and rely more heavily on the DNA repair mechanisms. Previous studies have established that the efficiency of DNA repair, which involves synthesis of new DNA at the damage site, is tightly linked to the cellular concentration of dNTPs. Several enzymes are known to regulate the level of dNTPs pools by participating in the de novo and salvage pathways. Ribonucleotide reductase (RNR)-mediated reduction reactions play a crucial role in dNTPs biosynthesis by producing dNDPs as precursors. While the majority of dNDPs, including dADP, dGDP, dCDP and dUDP are generated from NDPs by RNR, the synthesis of dTDP is catalyzed by thymidylate kinase (TMPK). Using dTMP as the substrate and ATP as the phosphate donor, TMPK produces dTDP for the subsequent biosynthesis of dTTPs. It has been suggested that the inhibition of TMPK may be exploitable in cancer therapy. The drug doxorubicin, which is widely used in anticancer chemotherapy, is highly effective in killing cancer cells by inducing DNA double-strand break (DSB). However, patients receiving doxorubicin may develop nausea, vomiting and irreversible myocardial toxicity. Because TMPK knockdown has been shown to sensitize cancer cells toward doxorubicin treatment, thus the use of TMPK inhibitor may suppress the undesired side effects by reducing the IC50 of doxorubicin. This concept is supported by the finding that YMU1, a specific inhibitor of human TMPK with no obvious cytotoxicity, may be used in conjunction with low dose of doxorubicin to induce cancer cell-specific apoptosis. Therefore, it appears that YMU1 is a promising lead compound for drug development. To understand how YMU1 interacts with and inhibits the activity of TMPK, we have initiated structural analysis on the TMPK-YMU1 complex. First, large amount of human TMPK was obtained using the Escherichia coli expression system, and liquid chromatography was performed for protein purification. Co-crystallization of human TMPK with YMU1 has been performed using highly purified protein. As an alternative approach, we have crystallized TMPK in complexes with dTMP. These crystals will be exposed to various soaking buffers that contain YMU1 to produce the inhibitor-bound crystals for structural determination.