铂类抗肿瘤药物与蛋白质的作用机理

铂类抗肿瘤药物在癌症化疗中被广泛应用,通常认为铂类药物通过与肿瘤细胞的DNA形成交联物,抑制DNA复制,造成DNA损伤,进而诱导细胞凋亡.然而,近年来的研究表明,铂类药物在进入细胞核与DNA结合之前,会与细胞内外的多种蛋白质发生复杂的相互作用.这些作用一方面会影响铂类药物的运输、代谢以及与DNA靶点的作用,从而对铂类药物的药效、耐药性产生影响;另一方面,铂类药物的作用会影响蛋白质的活性,与铂类药物的毒副作用以及蛋白质靶点所贡献的药效紧密相连.本文将从这两方面介绍近年来铂类药物与蛋白质相互作用研究取得的进展、小分子化合物对铂类药物与蛋白质作用的影响,并对铂类药物与非抗癌作用靶点蛋白的作用进行了介绍,以及研究这些相互作用的常用方法.     

细胞研究中的金属配合物-核酸相互作用

金属配合物与核酸相互作用的探讨对新型抗癌金属药物的设计、核酸结构的特异识别和核酸水解断裂的研究一直起着巨大的推动作用。不仅如此,配合物与核酸的相互作用已被引入细胞生物学研究中。本文利用一些典型的研究结果,对配合物与核酸相互作用应用于胞内DNA定位成像,对胞内信号转导和表观遗传的影响,以及金属配合物作为非病毒基因载体进行了总结。     

亚硝基脲对DNA损伤作用的研究进展

亚硝基脲是重要的抗癌烷化剂,应用于多种恶性肿瘤的临床治疗。亚硝基脲分解生成活泼亲电中间体而与生物大分子作用,导致DNA碱基烷化、互补碱基对横向交联等损伤,最终诱导癌细胞凋亡而发挥其抗癌作用。由于亚硝基脲在动物实验和临床应用中表现出的复杂生理活性,有关亚硝基脲对DNA损伤作用机制的研究,已成为该类药物设计开发以及癌症防治领域中的热点问题。本文对近年来国内外有关亚硝基脲对DNA损伤作用机制的研究进行了综述。     

顺铂,碳铂与鸟苷,L—蛋氨酸反应动力学比较研究

自发现顺铂具有抗癌活性以来,人们一直致力于它的抗癌机理研究。很多结果表明顺铂与蛋白质核苷酸(DNA)作用时主要与DNA中鸟嘌呤的N_7配位,通过形成链内交联作用造成DNA损伤。顺铂在显示抗癌活性的同时,也表现出较强的毒副作用,有文献认为毒性可能与形成Pt—S键抑制生物体内的巯基酶有关。     

用共振Rayleigh散射光谱研究盐酸氯丙嗪和盐酸异丙嗪与核酸相互作用

研究了结构相似的吩噻嗪类药物盐酸氯丙嗪(CPZ·HCl)和盐酸异丙嗪(PZ·HCl)与核酸 (NA) 的相互作用, 发现CPZ-NA体系可产生强烈的共振Rayleigh散射(RRS), 而PZ-NA反应较微弱. 采用量子化学AM1和密度泛函B3LYP方法计算了两个反应的能量关系, 讨论了CPZ与DNA分子的结合位点和作用方式, 获得一些有意义的结果. RRS法有望成为研究核酸的分子识别和基因药物的分子设计的有用手段. 此外, 用盐酸氯丙嗪作试剂, RRS法测定核酸具有很高的灵敏度和良好的选择性, 因此也为核酸的定量测定提供了一种新的灵敏、简便、快速的新方法.     

Schiff碱非铂抗癌络合物初步筛选的荧光法研究

依据药物与DNA发生相互作用能抑制DNA的复制、合成和增殖且这种作用在体内与在体外的一致性,用荧光探剂溴化乙锭(EB,EthBr)示踪药物/DNA作用而提出了荧光筛选新方法。该法具有可靠性强、灵敏度高、操作简便、耗费很小、周期较短等特点,适合于抗癌药物的初步筛选。以所合成的Schiff碱非铂抗癌络合物为例,考察了方法的可行性,并进一步确定了Schiff碱络合物与DNA的结合常数K_M,发现其大小与抗癌活性顺序相一致。这一结果为解释络合物抗癌作用的生化基础提供了初步依据,同时还说明络合有利于抗癌并以载体或整体起作用。     

Ru(phen)2dppx2+光谱探针法研究道诺霉素与脱氧核糖核酸的相互作用

以核酸分子“光开关”Ru(phen)2dppx2 为探针,分别采用紫外-可见吸收光谱法和荧光光谱法研究了抗癌药物道诺霉素(daunomycin,DNM)与小牛胸腺DNA之间的作用方式,结果表明:DNM是以插入的方式与DNA相互作用。Scatchard方程的研究表明,Ru(phen)2dppx2 与小牛胸腺DNA的结合比为1∶4~1∶5;表观结合常数为2.58×107L/mol。DNM加入后,表观结合常数大大降低,这也表明道诺霉素主要是以插入方式与DNA结合。作为研究核酸分子的荧光探针,与溴化乙锭相比,Ru(phen)2dppx2 具有灵敏度高、毒性低、稳定性好、选择性好、使用方便等优点。     

钯卟啉室温磷光探针研究更生霉素与DNA相互作用

在分子水平研究抗癌药物与DNA的作用方式,对了解抗癌药物的作用机理极为重要,同时也能为设计新型抗癌药物提供有价值的信息.更生霉素D(ACTD)是目前临床上应用最广泛的的抗癌药物之一,它对恶性淋巴瘤、肾母细胞瘤、绒毛膜上皮癌及恶性葡萄胎等癌症有良好的疗效[1],抗癌活性高.人们研究ACTD与DNA作用的手段有X射线衍射法、凝胶电泳法、吸收光谱、荧光光谱[2]及NMR谱[1]等技术.运用室温磷光光度法研究ACTD抗癌机理未见报道.本文首次借助钯卟啉室温磷光探针研究了在水溶液中ACTD与小牛胸腺DNA的作用机制,同时说明钯卟啉探针可从室温磷光的角度进行DNA及其相关研究.     

新型吡啶卟啉-蒽醌磺酰丙胺季铵盐的合成

设计合成一些能作为结构探针和化学治疗剂的核酸断裂剂为不同学科背景的研究者涉足分子生物学领域提出的挑战之一.天然生物酶在许多研究领域具有重要的作用,但由于它们自身特性的局限性,使它们的普及应用受到了限制.而今,合成具有高度序列选择性的核酸断裂剂成为人们研究的重点[1,2].卟啉和蒽醌类化合物均为有效的核酸定位断裂剂,利用它们各自特征来合成新型的核酸断裂剂,引起分子生物学,抗癌和抗病毒领域的研究者的极大关注.为深入探讨此类化合物在DNA断裂中的作用,我们合成了两种未见文献报道的卟啉-蒽醌季铵盐化合物(产率20%～25%)和两种卟啉-丙胺季铵盐化合物(产率10%～12%),对它们进行了UV,IR,1H NMR,MS及荧光性质的研究和表征.     

博莱霉素金属络合物的抗癌活性及结构研究进展

博莱霉素是一类具有选择性抗癌活性的抗生素。在对其抗癌机理的研究中,发现某些微量过渡金属离子的存在,可大大增强其抗癌活性,体外实验同时表明,博莱霉素作用底物DNA链的断链程度也显著增加。这一现象,不仅引起了分子生物学家的关注,更引起了生物     

EFFECT OF METAL IONS ON THE LUMINESCENCE OF ACRIDINE DYES BOUND TO DNA

Abstract— The luminescence of acridine dyes intercalated in DNA was studied as a function of the concurrent binding of metal ions to DNA, in an effort to deduce specific site interactions of the dyes. Two dyes, proflavine (PF) and acridine orange (AO), and two metal ions, silver and mercuric, were used. Both ions quench the fluorescence of the dyes in aqueous solution at room temperature. The metal ions have a different effect on the fluorescence of these dyes when they are intercalated between the base pairs of DNA. The fluorescence of AO is decreased when silver is bound, while the fluorescence of PF is enhanced. Since Ag⁺ initially binds to GC sites in DNA, which quench the PF fluorescence, it ostensibly 'turns off' the quenching by DNA at these sites, and this effect is greater than the quenching effect of the silver ion itself. Hg²⁺ ion initially binds to AT sites in DNA. Since both dyes fluoresce from AT sites, Hg²⁺ is expected to quench their fluorescence. This behavior is observed at low r (metal ion/base). At higher r values, however, where Hg²⁺ is expected to begin binding to GC sites, the fluorescence of PF is enhanced. These quenching turn-off effects are tentatively interpreted in terms of a change in the structure of the dye/DNA complex which occurs when a metal ion binds at the intercalation site. At 77 K. no fluorescence enhancement is observed when metal ions bind; Ag⁺ quenches the fluorescence and enhances the phosphorescence of both dyes. Qualitatively similar results are obtained with Hg²⁺.     

DNA-bound lipids: computer modeling of DNA interaction with stearic acid and unsaturated fatty acids

It was shown for the first time by computer experiments that fatty acids are strongly bound to DNA. This is consistent with the presence of free fatty acids in the specimens of DNA-bound lipids isolated from various cells. Binding of all fatty acids to the DNA minor groove is stronger than to the major groove, which is correlated with the presence of two pools of free fatty acids isolated from DNA specimens by biochemical methods. Since DNA polymerase is also bound to the DNA minor groove, fatty acids can play an important role in the regulation mechanism of DNA replication and signal transmission. The energy of interaction of fatty acids with DNA depends on both the number of double bonds and the geometric configuration of the fatty acid and the nucleotide composition of DNA. Dependence on the bond energy in the DNA—fatty acid complex on the nucleotide composition attests to the possibility of site-specific binding of lipids to DNA. On passing from a saturated fatty acid to unsaturated acids containing one, two, or three double trans-bonds, the bond energy of DNA with the fatty acid gradually decreases. The presence of one or three double cis-bonds results in weakening of the strength of the DNA—fatty acid complexes compared to those with the saturated acid. The strongest binding between DNA and fatty acid was found for the unsaturated acid with two double cis-bonds (linoleic). This can be explained by the fact that the bent (boomerang) shape of the molecule of this acid follows the curve of the DNA helix. The pattern of variation of the energy of DNA complexes with stearic, linoleic, oleic, and linolenic acids correlates with experimental data on the melting points of these complexes: the more stable the DNA—fatty acid complex, the lower the melting point of DNA.     

Interaction of zanamivir with DNA and RNA: Models for drug–DNA and drug–RNA bindings

Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5 mM) and various zanamivir/polynucleotide (P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV–visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir–DNA and RNA complexes in aqueous solution. Structural analysis showed major interaction of zanamivir with G-C (major groove) and A-T (minor groove) base pairs and minor perturbations of the backbone PO₂ group with overall binding constants of K_{zanamivir–DNA} = 1.30 × 10⁴ M⁻¹ and K_{zanamivir–RNA} = 1.38 × 10⁴ M⁻¹. The drug interaction induces a partial B to A-DNA transition, while RNA remains in A-conformation.     

DNA recognition by the anthracycline antibiotic respinomycin D: NMR structure of the intercalation complex with d(AGACGTCT)2

Respinomycin D is a member of the anthracycline family of antitumour antibiotics that interact with double stranded DNA through intercalation. The clinical agents daunomycin and doxorubicin are the most well-studied of this class but have a relatively simple molecular architecture in which the pendant daunosamine sugar resides in the DNA minor groove. Respinomycin D, which belongs to the nogalamycin group of anthracyclines, possesses additional sugar residues at either end of the aglycone chromophore that modulate the biological activity but whose role in molecular recognition is unknown. We report the NMR structure of the respinomycin D-d(AGACGTCT)2 complex in solution derived from NOE restraints and molecular dynamics simulations. We show that the drug threads through the DNA double helix forming stabilising interactions in both the major and minor groove, the latter through a different binding geometry to that previously reported. The bicycloaminoglucose sugar resides in the major groove and makes specific contacts with guanine at the 5'-CpG intercalation site, however, the disaccharide attached at the C4 position plays little part in drug binding and DNA recognition and is largely solvent exposed.     

Sequence-specific recognition and cleavage of DNA by anti-tumour antibiotics

Abstract

DNA cleavage by a bleomycin—iron complex occurs preferentially at guanine-pyrimidine (5′ → 3′) sequences, in particular at G-C sites. Metallobleomycin binds in the minor groove of B-DNA and the bithiazole moiety probably plays an important role as an anchor on DNA duplex. The 2-amino group of guanine adjacent to the 5′-side of the cleaved pyrimidine base is one key element of the specific 5′-GC recognition by the bleomycin-metal complex. Endiyne anti-tumour antibiotics such as esperamicin A₁ and dynemicin A also interact with the minor groove of DNA, and their strong DNA splitting activity is due to phenylene diradical formation from the enediyne core. A possible binding mode between these antibiotics and B-DNA has been proposed by computer-constructed model building.     

Screening of threading bis-intercalators binding to duplex DNA by electrospray ionization tandem mass spectrometry

The DNA binding of novel threading bis-intercalators V1, trans-D1, and cis-C1, which contain two naphthalene diimide (NDI) intercalation units connected by a scaffold, was evaluated using electrospray ionization mass spectrometry (ESI-MS) and DNAse footprinting techniques. ESI-MS experiments confirmed that V1, the ligand containing the -Gly3-Lys- peptide scaffold, binds to a DNA duplex containing the 5'-GGTACC-3' specific binding site identified in previous NMR-based studies. The ligand formed complexes with a ligand/DNA binding stoichiometry of 1:1, even when there was excess ligand in solution. Trans-D1 and cis-C1 are new ligands containing a rigid spiro-tricyclic scaffold in the trans- and cis- orientations, respectively. Preliminary DNAse footprinting experiments identified possible specific binding sites of 5'-CAGTGA-5' for trans-D1 and 5'-GGTACC-3' for cis-C1. ESI-MS experiments revealed that both ligands bound to DNA duplexes containing the respective specific binding sequences, with cis-C1 exhibiting the most extensive binding based on a higher fraction of bound DNA value. Cis-C1 formed complexes with a dominant 1:1 binding stoichiometry, whereas trans-D1 was able to form 2:1 complexes at ligand/DNA molar ratios >or=1 which is suggestive of nonspecific binding. Collisional activated dissociation (CAD) experiments indicate that DNA complexes containing V1, trans-D1, and cis-C1 have a unique fragmentation pathway, which was also observed for complexes containing the commercially available bis-intercalator echinomycin, as a result of similar binding interactions, marked by intercalation in addition to hydrogen bonding by the scaffold with the DNA major or minor groove.     

邻菲啰啉并1,2,4-三嗪钴(Ⅲ)配合物的合成及其对小牛胸腺DNA的荧光识别

在4个邻菲啰啉并1,2,4-三嗪新型配体分子(ARTP1ARTP4)合成的基础上,通过ARTP1ARTP4与Co³⁺配位,制备了4个钴(Ⅲ)配合物(Co-ARTP1Co-ARTP4)。 通过傅里叶变换红外光谱仪(FT-IR)、核磁共振波谱仪(NMR)和高分辨质谱(HRMS)仪和紫外-可见分光计(UV-Vis)等技术手段表征了ARTP1ARTP4和Co-ARTP1Co-ARTP4的结构和性能。 结果表明,配合物的紫外吸收峰中出现减色效应,最大吸收峰红移。 配合物以插入的方式与小牛胸腺DNA结合,结合常数K_b分别为4.78×10⁵、6.52×10⁵、5.97×10⁵和6.01×10⁵ L/mol,表明配合物可作为潜在的CT-DNA荧光探针,为探索DNA与有机小分子的作用提供了重要的参考。     

Alternative heterocycles for DNA recognition: the benzimidazole/imidazole pair

Boc-protected benzimidazole-pyrrole, benzimidazole-imidazole, and benzimidazole-methoxypyrrole amino acids were synthesized and incorporated into DNA binding polyamides, comprised of N-methyl pyrrole and N-methyl imidazole amino acids, by means of solid-phase synthesis on an oxime resin. These hairpin polyamides were designed to determine the DNA recognition profile of a side-by-side benzimidazole/imidazole pair for the designated six base pair recognition sequence. Equilibrium association constants of the polyamide-DNA complexes were determined at two of the six base pair positions of the recognition sequence by quantitative DNase I footprinting titrations on DNA fragments each containing matched and single base pair mismatched binding sites. The results indicate that the benzimidazole-heterocycle building blocks can replace pyrrole-pyrrole, pyrrole-imidazole, and pyrrole-hydroxypyrrole constructs while retaining relative site specifities and subnanomolar match site affinities. The benzimidazole-containing hairpin polyamides represent a novel class of DNA binding ligands featuring tunable target recognition sequences combined with the favorable properties of the benzimidazole type DNA minor groove binders.     

A FLUORESCENCE STUDY OF THE BINDING OF HOECHST 33258 and DAPI TO HALOGENATED DNAs

We have studied the time-resolved and the steady-state fluorescence of the DNA groove binders 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 with the double stranded DNAs poly(dA-dU) and poly(dI-dC) and their halogenated analogs, poly(dA-I5dU) and poly(dI-Br5dC). These studies were prompted by earlier observations that steady-state fluorescence of Hoechst 33258 is quenched on binding to halogenated DNAs (presumably due to an intermolecular heavy atom effect involving the halogen atom in the major groove), and recent studies which clearly point to a binding-site in the minor groove of DNA. Measurements of the time resolved fluorescence decay demonstrate that the fluorescence of Hoechst 33258 is quenched on binding to the halogenated DNAs, in agreement with previous observations. However, quenching studies carried out using the free halogenated bases IdUrd and BrdCyd in solution yielded bimolecular rate constants more than one order of magnitude larger than those expected for an intermolecular heavy atom effect. Moreover, the quenching of the Hoechst 33258 fluorescence was accompanied by an accelerated photochemical destruction of Hoechst 33258. We therefore conclude that the fluorescence quenching observed with halogenated DNAs is probably due to a photochemical reaction involving Hoechst 33258, rather than direct contact of Hoechst 33258 with the halogen substituents in the major groove of the DNA. The fluorescence decay measurements however, do provide clear evidence for at least two different modes of binding. Taking into account the alternating sequences used in this study and the possibility of two different conformations for bound dye, at least four different modes of binding are plausible. Our present data do not allow us to distinguish between these alternatives. The time-resolved fluorescence decays and fluorescence quantum yields of DAPI are not affected by the presence of the heavy atom substituents in the DNA major groove. Based on this observation and earlier reports that DAPI binds in one of the DNA grooves, we conclude that the high affinity sites for DAPI on DNA are located in the minor groove.     

Binding of naproxen and 1-anilino-8-naphtalenesulphonic acid to albumin: Characterization of their binding sites in bovine serum albumin

The binding of naproxen (NP) and 1-anilino-8-naphtalenesulphonic acid (ANS) to bovine serum albumin (BSA) has been studied by equilibrium dialysis and spectrophotofluorometry, respectively. The drug protein (D/P) ratio is found to determine the nature of binding sites for NP but not for ANS. At low D/P ratio, NP possesses a specific site in BSA and one of the tryptophan residues is part of it. Fluorometric investigation reveals non-displacement of ANS from its sites by the binding of NP to its specific site. At high D/P ratio, NP instead causes localized conformational changes in BSA. Furthermore, it is possible to infer the location of NP's specific site as loop 4 of BSA. NP and ANS binding sites have also been examined by employing Bromocresol Green (BCG) as a spectrophotometric probe. Competition studies are also able to establish distinct binding pattern for the two ligands in BSA. The displacement pattern points out the presence of independent sites for NP (specific site) and ANS (initially occupied site) in BSA in spite of their amphipathically similar nature.