鮑偉偉

（溫州大學(xué)，浙江 溫州 310015）

Davies[1]于 1962年首次提出G-四聯(lián)體。G-四聯(lián)體是由富G序列在一定濃度的Na+、K+條件下所形成一種DNA二級結(jié)構(gòu)[2-4]。G-四聯(lián)體結(jié)構(gòu)具有多態(tài)性，主要是因為鏈的數(shù)量、鏈的結(jié)構(gòu)、鏈的取向以及環(huán)的結(jié)構(gòu)形態(tài)等存在著多樣性。G-四聯(lián)體的基本單元是4個鳥嘌呤通 過氫鍵圍繞形成平面得到G-四分體，其中的氫鍵包括Hoogste en (N1-O6)(N2-N7)和Watson-Crick，而 G-四分體之間通過π-π堆積得到G-四聯(lián)體。G-四聯(lián)體主要有平行結(jié)構(gòu)和反平行結(jié)構(gòu)，主要受到鳥嘌呤中的堿基和糖之間的syn和anti這2種糖苷鍵的影響。E Gavathiotis等對于平行的G-四聯(lián)體帶 有anti糖苷鍵的闡述非常明白[5]。其中最影響拓?fù)浣Y(jié)構(gòu)的應(yīng)當(dāng)是一條鏈中帶有對角環(huán)、側(cè)邊環(huán)以及螺旋槳環(huán)等多種環(huán)形結(jié)構(gòu)的G-四聯(lián)體，因為這樣形成的結(jié)構(gòu)中存在著4種形態(tài)的溝槽，比如對角環(huán)和對角環(huán)所形成的簡單環(huán)，但存在螺旋槳環(huán)就會很復(fù)雜。

在20世紀(jì)80年代初期，只有生物物理學(xué)家對其感興趣，而到80年代末期時，位于端粒末端的富G序列 能夠形成G-四聯(lián)體結(jié)構(gòu)的這個事實[6-7]以及在1991年時Zahl er[8]與其同伴證實了經(jīng)K+穩(wěn)定G-四聯(lián)體結(jié)構(gòu)具有限制端粒酶活性的功能后，G-四聯(lián)體就成為了抑制端粒酶的靶點[9-10]。隨著對G-四聯(lián)體研究的不斷深入，大量事實證實G-四聯(lián)體確實存在于活的生物體中[11-14]并且發(fā)揮著重要的生物性作用[15]。在真核細(xì)胞的基因組中，有很多關(guān)鍵的富G區(qū)域有望形成G-四聯(lián)體，像端粒[16]、免疫球蛋白開關(guān)區(qū)域[17]、致癌基因啟動子區(qū)域[18]以及核糖體DNA[19]。G-四聯(lián)體與小分子配體結(jié)合時能夠發(fā)揮重要作用，如限制端粒酶的活性[20]，干擾端粒的活性，影響端粒的維持[21-22]，進(jìn)一步影響細(xì)胞的生長與增殖[23]，甚至影響癌細(xì)胞的發(fā) 生與發(fā)展。

本文綜述了藥物及其衍生物作為G-四聯(lián)體的配體以及顯 示其優(yōu)越的抗腫瘤活性。

1 蒽醌類化合物

蒽醌類化合物廣泛存在于蓼科、豆科等眾多的植物中。早期因其對腫瘤細(xì)胞具有毒性而作為DNA嵌入劑。分子模擬研究表明蒽醌類化合物可能可以通過嵌插 模式與G-四聯(lián)體相互作用[24]，Sun等[25]于1997首先提出G-四聯(lián)體的配體BSU-1051，且抑制端粒酶活性的50%的有效濃度（teIIC50）為23μM。隨后大量報道經(jīng)取代反應(yīng)得到蒽醌類衍生物，其中包括1,4-, 1,5-, 1,8-, 2,6-,和2,7-等，其中的側(cè)鏈變化趨勢是氨基到特異性氨基酸官能團，并且考察了側(cè)鏈的大小和長度之間的關(guān)系以及活性[26-30]。

近年來，研究者發(fā)現(xiàn)經(jīng)肽基取代的蒽醌類衍生物對G-四聯(lián)體表現(xiàn)出極為卓越的選擇性。他們通過加入疏水性殘基苯丙氨酸到賴氨酸側(cè)鏈的2,6 或者 2,7上，表現(xiàn)出的選擇性要遠(yuǎn)遠(yuǎn)大于雙鏈DNA，并且賴氨酸被苯丙氨酸所取代的蒽醌類衍生物能夠在更低的濃度下使得細(xì)胞凋亡[31]。

2 喹叨啉類衍生物

喹叨啉于1977年第一次從南非的一種 植株中提取出來[32]，它具有許多優(yōu)良的性質(zhì)，包括抗菌、抗瘧疾、抗炎癥等。SYUIQ-5是一種廣為研究的喹叨啉類衍生物，一系列的抗癌研究表明SYUIQ-5具有抑制端粒酶的活性，可縮短端粒的長度，誘導(dǎo)細(xì)胞的衰老和生長的停止。另外，這種化合物能夠和c-myc啟動子區(qū)域上的G-四聯(lián)體相互結(jié)合并且在細(xì)胞的增殖和衰老的過程中占據(jù)重要作用[33]。

雙取代以及11號位上取代的喹叨啉衍生物都表現(xiàn)為使得G-四聯(lián)體更為穩(wěn)定，以及端粒酶抑制作用，但是它們對G-四聯(lián)體的選擇性與雙鏈DNA相比卻顯得差強人意。不過11位取代的喹叨啉衍生物表現(xiàn)出了更強的端粒酶活性，其中喹叨啉telIC50＞138μM；11位取代衍生物telIC50= 0.44～12.3μM[34]。另外5-N-甲基化喹叨啉因為正電荷中心與G-四聯(lián)體的負(fù)離子通道之間的相互作用，提高了其與反平行G-四聯(lián)體結(jié)構(gòu)之間的選擇性以及穩(wěn)定性[35]。

Jixun Dai等[36]通過了解溶液中的喹叨啉與G-四聯(lián)體（2∶1）復(fù)合物的結(jié)構(gòu)，表明藥物能夠誘導(dǎo)側(cè)翼序列形成一個新的綁定位點，并且強調(diào)了堆垛相互作用以及靜電相互作用間的重要性。他們首先強調(diào)了藥物的形狀以及2個側(cè)翼堿基在決定藥物綁定特異性方面的 重要作用。

3 小檗堿類衍生物

小檗堿是從中藥黃連中提取出來的一種藥物，在中 醫(yī)中主要是用來醫(yī)治濕熱癥。在早期，小檗堿因為其本身是一種抗生素使得其不是一種良好的抗腫瘤藥品，后來因獲得了一種混合小檗堿的新藥，使其成為 了抗腫瘤藥物[37]，引起了廣泛關(guān)注。小檗堿能夠抑制各種腫瘤細(xì)胞的生長[38-39]，其中包括乳腺癌細(xì)胞[40]、胰腺癌細(xì)胞[41]以及胃癌細(xì)胞[42-43]。后來，Tsuruo和其同伴發(fā)現(xiàn)小檗堿能夠抑制端粒酶的活性并且限制端粒的延長，同時也報道能夠和G-四聯(lián)體相互作用。Zhang等[44]發(fā)現(xiàn)9-取代小檗堿衍生物對G-四聯(lián)體結(jié)構(gòu)具有超強的相互結(jié)合能力并且能夠抑制端粒酶的活性。為了開發(fā)更具活性的端粒酶抑制劑，研究者開發(fā)了一系列的9-N-取代小檗堿衍生物，研究證實這些衍生物都能夠誘導(dǎo)并且穩(wěn)定c-myc上啟動子區(qū)域平行G-四聯(lián)體，還能夠抑制c-myc表達(dá)[45]。

4 卟啉類衍生物及其相關(guān)的類似物

眾所周知的卟啉是紅細(xì)胞中的色素— —亞鐵血紅素?；衔颰MPyP4是最具代 表性的陽離子卟啉類化合物。Hurley課題組對此進(jìn)行了深入而廣泛的研究[46-56]。TMPyP4對G-四聯(lián)體表現(xiàn)出較強的相互作用（ΔT1/2= 17℃），能夠有效抑制端粒酶的活性（IC50=6μM）。但問題是其對于平行以及反平行的G-四聯(lián)體缺乏明顯的選擇性[57-58]，甚至它對所有的核酸都具有選擇性(包括單鏈、雙鏈、三鏈以及四鏈)[59]。Parkinson等[60]報道了端粒G-四聯(lián)體與TMPyP4復(fù)合物的X單晶衍射結(jié)構(gòu)，表明TMPyP4不直接與G-四分體相互結(jié)合。

盡管TMPyP4表現(xiàn)出來的選擇性不盡人意， 但是研究者們對其的熱情卻不見消退，如Gerald等通過ITC、CD以及ESI-MS等方法來考察卟啉經(jīng)N-甲基-4-吡啶基取代的取代數(shù)目(如 5-N-甲基-4-吡啶基卟啉)、有效性以及取代后的結(jié)合能的大小，KTMPyP4，KP(5,15)＞ KP(5,10,15)＞ KP(5,10)，KP(5)，其中認(rèn)為P(5,15)和G-四聯(lián)體采用的是嵌插模式，而P(5,10,15)和G-四聯(lián)體采用的是底端堆垛模式[61]。

5 展望

自然界本身存在的藥物對新藥物的發(fā)現(xiàn)以及發(fā)展是一筆巨大寶貴的財富。其多態(tài) 性和結(jié)構(gòu)的復(fù)雜性，使其成為抗腫瘤藥物選更具選擇性，通過對G-四聯(lián)體的結(jié)構(gòu)特征以及功能的深入了解，我們期望在未來尋找到更多更好更具針對性的配體。

[1] Gellert M, Lipsett M N, Davies D R.Helix formation by guanylic acid[J].Proc Natl Acad Sci U S A, 1962, 48(12): 2013.

[2] Ambrus A, Chen D, Dai J, et al.Human tel omeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution[J].Nucleic Acids Res., 2006, 34(9): 2723-2735.

[3] Lim K W, Ng V C M, Martín-Pintado N, et a l.Structure of the human telomere in Na+ solution: an antiparallel (2+2) G-quadruplex scaffold reveals additional diversity[J].Nucleic Acids Res., 2013, 41(22): 10556-10562.

[4] Wang C, Jia G, Li Y, et al.Na+/K+switch of enantioselectivity in G-quadruplex DNA-based catalysis[J].Chemical Communications, 2013, 49(95): 11161-11163.

[5] Gavathiotis E, Searle M S.Structure of th e parallel-stranded DNA quadruplexd (TTAGGGT) 4 containing the human telomeric repeat: evidence for A-tetrad formation from NMR and molecular dynamics simulations[J].Organic & Biomolecular Chemistry, 2003(10): 1650-1656.

[6] Oka Y, Thomas C.The cohering telomeres of Oxytricha[J].Nucleic Acids Res., 1987, 15(21): 8877-8898.

[7] Williamson J R, Raghuraman M, Cech T R.Mo novalent cation-induced structure of telomeric DNA: the G-quartet model[J].Cell, 1989, 59(5): 871-880.

[8] Zahler A M, Williamson J R, Cech T R, et a l.Inhibition of telomerase by G-quartet DMA structures[J].Nature, 1991, 350(6320): 718-720.

[9] Sen D, Gilbert W.Formation of parallel fo ur-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis[J].1988, 334: 364-366.

[10] Kang C, Zhang X, Ratliff R, et al.Crysta l structure of four-stranded Oxytricha telomeric DNA[J].Nature, 1992, 356(6365): 126-131.

[11] Verma A, Yadav V K, Basundra R, et al.Ev idence of genome-wide G4 DNA-mediated gene expression in human cancer cells[J].Nucleic Acids Res., 2009, 37(13): 4194-4204.

[12] Paeschke K, Simonsson T, Postberg J, et a l.Telomere endbinding proteins control the formation of G-quadruplex DNA structures in vivo[J].Nature Structural & Molecular Biology, 2005, 12(10): 847-854.

[13] Lipps H J, Rhodes D.G-quadruplex structu res: evidence and function[J].Trends in Cell Biology, 2009, 19(8): 414-422.

[14] Tauchi T, Shin-Ya K, Sashida G, et al.Te lomerase inhibition with a novel G-quadruplex-interactive agent, telomestatin: in vitro and in vivo studies in acute leukemia[J].Oncogene, 2006, 25(42): 5719-5725.

[15] Cairns D, Anderson R J, Perry P J, et al.Design of telomerase inhibitors for the treatment of cancer[J].Current Pharmaceutical Design, 2002, 8(27): 2491-2504.

[16] Parkinson G N, Lee M P H, Neidle S.Cryst al structure of parallel quadruplexes from human telomeric DNA[J].Nature, 2002, 417(6891): 876-880.

[17] Rezler E M, Seenisamy J, Bashyam S, et al.Telomestatin and diseleno sapphyrin bind selectively to two different forms of the human telomeric G-quadruplex structure[J].J Am Chem Soc., 2005, 127(26): 9439-9447.

[18] Balasubramanian S, Hurley L H, Neidle S.Targeting G-quadruplexes in gene promoters: a novel anticancer strategy[J].Nature Reviews Drug Discovery, 2011, 10(4): 261-275.

[19] Han H, Bennett R J, Hurley L H.Inhibitio n of unwinding of G-quadruplex structures by Sgs1 helicase in the presence of N, N'-bis [2-(1-piperidino) ethyl]-3, 4, 9, 10-perylenetetracarboxylic diimide, a G-quadruplexinteractive ligand[J].Biochemistry, 2000, 39(31): 9311-9316.

[20] Cuesta J, Read M A, Neidle S.The design of G-quadruplex ligands as telomerase inhibitors[J].Mini reviews in medicinal chemistry, 2003, 3(1): 11-21.

[21] Deng R, Li W, Guan Z, et al.Acetylcholin esterase expression mediated by c-Jun-NH2-terminal kinase pathway during anticancer drug-induced apoptosis[J].Oncogene, 2006, 25(53): 7070-7077.

[22] Tauchi T, Shin-Ya K, Sashida G, et al.Ac tivity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells: involvement of ATM-dependent DNA damage response pathways[J].Oncogene, 2003, 22(34): 5338-5347.

[23] Riou J, Guittat L, Mailliet P, et al.Cel l senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands[J].Proceedings of the National Academy of Sciences, 2002, 99(5): 2672-2677.

[24] Tanious F A, Jenkins T C, Neidle S, et al.Substituent position dictates the intercalative DNA-binding mode for anthracene-9,10-dione antitumor drugs[J].Biochemistry, 1992, 31(46): 11632-11640.

[25] Sun D, Thompson B, Cathers B E, et al.In hibition of human telomerase by a G-quadruplex-interactive compound[J].J Med Chem., 1997, 40(14): 2113-2116.

[26] Perry P J, Gowan S M, Reszka A P, et al.1, 4-and 2, 6-disubstituted amidoanthracene-9, 10-dione derivatives as inhibitors of human telomerase[J].J Med Chem., 1998, 41(17): 3253-3260.

[27] Perry P J, Reszka A P, Wood A A, et al.H uman telomerase inhibition by regioisomeric disubstituted amidoanthracene-9, 10-diones[J].J Med Chem., 1998, 41(24): 4873-4884.

[28] Huang H-S, Chen I-B, Huang K-F, et al.Sy nthesis and human telomerase inhibition of a series of regioisomeric disubstituted amidoanthraquinones[J].Chemical & pharmaceutical bulletin, 2007, 55(2): 284-292.

[29] Huang H-S, Huang K-F, Li C-L, et al.Synt hesis, human telomerase inhibition and anti-proliferative studies of a series of 2,7-bis-substituted amido-anthraquinone derivatives[J].Bioorg Med Chem., 2008, 16(14): 6976-6986.

[30] Zagotto G, Sissi C, Lucatello L, et al.A minoacyl Anthraquinone Conjugates as Telomerase Inhibitors: Synthesis, Biophysical and Biological Evaluation[J].J Med Chem., 2008, 51(18): 5566-5574.

[31] Zagotto G, Ricci A, Vasquez E, et al.Tunin g G-quadruplex vs double-stranded DNA recognition in regioisomeric lysylpeptidyl-anthraquinone conjugates[J].Bioconjug Chem., 2011, 22(10): 2126-2135.

[32] Dwuma‐Badu D, Ayim J S, Fiagbe N I, et al.Constituents of West African medicinal plants XX: Quindoline from Cryptolepis sanguinolenta[J].Journal of pharmaceutical sciences, 1978, 67(3): 433-434.

[33] Ou T-M, Lin J, Lu Y-J, et al.Inhibition of cell proliferation by quindoline derivative (SYUIQ-05) through its preferential interaction with c-myc promoter G-quadruplex[J].J Med Chem., 2011, 54(16): 5671-5679.

[34] Caprio V, Guyen B, Opoku-Boahen Y, et al.A novel inhibitor of human telomerase derived from 10-indolo [3, 2-] quinoline[J].Bioorg Med Chem Lett., 2000, 10(18): 2063-2066.

[35] Lu Y-J, Ou T-M, Tan J-H, et al.5-N-Methylat ed quindoline derivatives as telomeric G-quadruplex stabilizing ligands: effects of 5-N positive charge on quadruplex binding affinity and cell proliferation[J].J Med Chem., 2008, 51(20): 6381-6392.

[36] Dai J, Carver M, Hurley L H, et al.Solution Structure of a 2∶1 Quindoline-c-MYC G-Quadruplex: Insights into G-Quadruplex-Interactive Small Molecule Drug Design[J].J Am Chem Soc., 2011, 133(44): 17673-17680.

[37] Hoshi A, Ikekawa T, Ikeda Y, et al.Antitumo r activity of berberrubine derivatives[J].Gann= Gan, 1976, 67(2): 321.

[38] Sun Y, Xun K, Wang Y, et al.A systematic re view of the anticancer properties of berberine, a natural product from Chinese herbs[J].Anti-cancer drugs, 2009, 20(9): 757-769.

[39] Tang J, Feng Y, Tsao S, et al.Berberine and Coptidis rhizoma as novel antineoplastic agents: a review of traditional use and biomedical investigations[J].Journal of ethnopharmacology, 2009, 126(1): 5-17.

[40] Kim J, Yu J-H, Ko E, et al.The alkaloid Ber berine inhibits the growth of Anoikis-resistant MCF-7 and MDA-MB-231 breast cancer cell lines by inducing cell cycle arrest[J].Phytomedicine, 2010, 17(6): 436-440.

[41] Pinto-Garcia L, Efferth T, Torres A, et al.Berberine inhibits cell growth and mediates caspase-independent cell death in human pancreatic cancer cells[J].Planta medica, 2010, 76(11): 1155-1161.

[42] Auyeung K K-W, Ko J K-S.Coptis chinensis in hibits hepatocellular carcinoma cell growth through nonsteroidal anti-inflammatory drug-activated gene activation[J].International journal of molecular medicine, 2009, 24(4): 571-577.

[43] Tang F, Wang D, Duan C, et al.Berberine inh ibits metastasis of nasopharyngeal carcinoma 5-8F cells by targeting Rho kinase-mediated Ezrin phosphorylation at threonine 567[J].Journal of Biological Chemistry, 2009, 284(40): 27456-27466.

[44] Zhang W-J, Ou T-M, Lu Y-J, et al.9-Substitu ted berberine derivatives as G-quadruplex stabilizing ligands in telomeric DNA[J].Bioorg Med Chem., 2007, 15(16): 5493-5501.

[45] Ma Y, Ou T-M, Hou J-Q, et al.9-N-Substitute d berberine derivatives: Stabilization of G-quadruplex DNA and downregulation of oncogene c-myc[J].Bioorg Med Chem., 2008, 16(16): 7582-7591.

[46] Sun D, Guo K, Rusche J J, et al.Facilitatio n of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents[J].Nucleic Acids Res., 2005, 33(18): 6070-6080.

[47] Siddiqui-Jain A, Grand C L, Bearss D J, et a l.Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription[J].Proceedings of the National Academy of Sciences, 2002, 99(18): 11593-11598.

[48] Dexheimer T S, Sun D, Hurley L H.Deconvolut ing the structural and drug-recognition complexity of the G-quadruplex-forming region upstream of the bcl-2 P1 promoter[J].J Am Chem Soc., 2006, 128(16): 5404-5415.

[49] Guo K, Pourpak A, Beetz-Rogers K, et al.For mation of Pseudosymmetrical G-Quadruplex and i-Motif Structures in the Proximal Promoter Region of the RET Oncogene[J].J Am Chem Soc., 2007, 129(33): 10220-10228.

[50] Han F X, Wheelhouse R T, Hurley L H.Interac tions of TMPyP4 and TMPyP2 with Quadruplex DNA.Structural Basis for the Differential Effects on Telomerase Inhibition[J].J Am Chem Soc., 1999, 121(15): 3561-3570.

[51] Izbicka E, Wheelhouse R T, Raymond E, et al.Effects of cationic porphyrins as G-quadruplex interactive agents in human tumor cells[J].Cancer Research, 1999, 59(3): 639-644.

[52] Shi D-F, Wheelhouse R T, Sun D, et al.Quadr uplexinteractive agents as telomerase inhibitors: synthesis of porphyrins and structure-activity relationship for the inhibition of telomerase[J].J Med Chem., 2001, 44(26): 4509-4523.

[53] Kim M-Y, Gleason-Guzman M, Izbicka E, et al.The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures[J].Cancer Research, 2003, 63(12): 3247-3256.

[54] Yang D, Hurley L H.Structure of the biologi cally relevant G-quadruplex in the c-MYC promoter[J].Nucleosides, Nucleotides, and Nucleic Acids, 2006, 25(8): 951-968.

[55] Liu W, Sun D, Hurley L H.Binding of G-quadr uplexinteractive agents to distinct G-quadruplexes induces different biological effects in MiaPaCa cells[J].Nucleosides, Nucleotides, and Nucleic Acids, 2005, 24(10-12): 1801-1815.

[56] Freyer M W, Buscaglia R, Kaplan K, et al.Bi ophysical Studies of the c-MYC NHE III1 Promoter: Model Quadruplex Interactions with a Cationic Porphyrin[J].Biophysical Journal, 2007, 92(6): 2007-2015.

[57] Wheelhouse R T, Sun D, Han H, et al.Cationi c porphyrins as telomerase inhibitors: the interaction of tetra-(N-methyl-4-pyridyl) porphine with quadruplex DNA[J].J Am Chem Soc., 1998, 120(13): 3261-3262.

[58] Anantha N V, Azam M, Sheardy R D.Porphyrin binding to quadruplexed T4G4[J].Biochemistry, 1998, 37(9): 2709-2714.

[59] Hershman S G, Chen Q, Lee J Y, et al.Genomi c distribution and functional analyses of potential G-quadruplex-forming sequences in Saccharomyces cerevisiae[J].Nucleic Acids Res., 2008, 36(1): 144-156.

[60] Parkinson G N, Ghosh R, Neidle S.Structural basis for binding of porphyrin to human telomeres[J].Biochemistry, 2007, 46(9): 2390-2397.

[61] Rowland G B, Barnett K, Dupont J I, et al.T he effect of pyridyl substituents on the thermodynamics of porphyrin binding to G-quadruplex DNA[J].Bioorg Med Chem., 2013, 21(23): 7515-7522.