DNA与二脒DB293复合物的分子动力学模拟

采用分子动力学模拟了DNA小沟与芳香二脒化合物DB293结合形成的复合物,通过5ns的模拟研究表明,DB293分子可紧密结合在DNA的AATT小沟区域,和双螺旋d[CGCGAATTCGCG]2形成稳定的复合物。DB293苯并咪唑的氮原子N2能够与DNA胸腺嘧啶碱基T7的O2原子和T19的O2原子形成两个较强的氢键,同时,其末端氨基的N3原子和T20的O2原子形成一个较弱的氢键。本文在分子水平上提供了DB293直接与双螺旋DNA相互作用的结构及复合物的动态变化情况,为设计出更高活性的芳香二脒类DNA小沟结合剂提供一定的理论依据。     

DNA小沟结合药物DB818的分子动力学模拟和结合自由能分析

采用分子动力学模拟了DNA小沟结合芳香二脒药物DB818形成的复合物. 通过5 ns的模拟研究表明: DB818药物分子可紧密结合在DNA的AATTC小沟区域, 和双螺旋d[CGCGAATTCGCG]2形成稳定的复合物. 由于噻吩硫原子的弱电负性, 使DB818能够以更大的伸展程度与DNA的小沟结合, 形成更强的结合力. DB818苯并咪唑的氮原子能够与DNA 7位和19位T碱基上的氧原子形成两个稳定的氢键, 同时, DB818末端氨基氮原子分别与DNA 的20位T碱基的氧原子和9位C碱基的氧原子形成两个氢键. 另外, 运用MM_PBSA方法计算了DB293-DNA和DB818-DNA复合物的结合自由能, 计算结合能与实验值能较好的吻合, 通过比较其结合自由能, 从热力学能量角度说明了DB818有较大的熵值与较小的焓值贡献, 从而与DNA小沟结合的结合力比DB293强. 本文在分子水平上提供了DB818直接与双螺旋DNA相互作用的结构及复合物的动态变化情况, 为设计出更高生物活性的DNA小沟结合剂提供一定的理论依据.     

诺氟沙星-DNA复合物的分子动力学模拟

采用分子模建的方法构建了诺氟沙星-DNA复合物的初始结构, 通过2 ns的分子动力学(MD)模拟研究表明: 诺氟沙星能够和双螺旋d[ATATCGATAT]₂形成稳定的复合物, 药物分子可紧密结合在DNA的小沟区域, 并且能够与DNA的鸟嘌呤碱基形成两个稳定的氢键. 在分子水平上提供了诺氟沙星直接与双螺旋DNA相互作用的结构及复合物的动态变化情况.     

核酸碱基与甘氨酸二肽间氢键作用的最佳位点

优化得到了碱基腺嘌呤、胸腺嘧啶、尿嘧啶、鸟嘌呤及胞嘧啶与甘氨酸二肽分子形成的28个氢键复合物的稳定结构并计算了结合能, 探讨了五种碱基与甘氨酸二肽分子间氢键作用的最佳位点. 本文研究发现: 每种碱基均可以通过不同位点与二肽分子形成氢键复合物, 腺嘌呤、胸腺嘧啶、尿嘧啶、鸟嘌呤及胞嘧啶分别最倾向使用A3、T1、U1、G3及C1位点与甘氨酸二肽分子形成氢键复合物; 碱基分子某位点的质子化反应焓变越负所形成的氢键复合物越稳定, 去质子化反应焓变越小所形成的氢键复合物越稳定; 由氢键复合物的结合能计算得到的稳定性次序与由碱基分子质子化和去质子化反应焓变推得的稳定性次序一致.     

米托蒽醌与B-DNA相互作用的分子模拟

针对嵌插型抗癌药物米托蒽醌(mitoxantrone,MTX)同B-DNA间作用模式的争议,采用分子模拟方法研究了米托蒽醌分子与B-DNA分子的相互作用.结果表明:米托蒽醌分子插入到B-DNA中有大小沟选择性及碱基对特异性,更倾向从小沟方向插入到DNA分子中;对5'-CG碱基对有特异性识别.通过详细能量项的分析,揭示了米托蒽醌插入DNA分子的驱动力及对碱基的特异性识别作用主要是空间相互作用特别是静电相互作用.在最佳作用位点复合物的构象分析则表明蒽醌环只有一部分插入碱基对中,侧链在小沟中延磷酸基骨架以3'-5'方向伸展,并通过静电作用进一步增强米托蒽醌与B-DNA的结合.     

7,8-二氢-8-氧鸟嘌呤碱基对的量子化学研究

遗传信息的完整性不断受氧化基因的威胁,7,8-二氢-8-氧鸟嘌呤（8-oxo-G）是氧化DNA损伤最常见的产物. 氧化碱基会引起基因突变、癌变及衰老等. 应用量子化学方法分析得出：鸟嘌呤（G）被氧化为8-oxo-G后,其电荷分布、氢键的供体和受体位点的数目和位置随之改变,N7和O6原子所带的电荷变得更负,使得它们作为氢键供体的能力增强. 从而G被误认为其他碱基,与正常碱基形成多种氢键复合物. 可将8-oxo-G划分为3个作用位点与正常碱基相互作用. 与正常的单体相比,碱基对中形成氢键的受体原子上所带电荷平均变负0.05e,占原电荷的8%; 供体H原子所带电荷平均变正0.02e,占原电荷的4%. 1位点与正常碱基作用形成的氢键复合物更稳定,2位点和3位点性质相似,水溶剂使碱基对的结合能力减弱,其中与C作用形成氢键复合物的结合能减弱程度最大,且使碱基对结合能力的次序改变. 在8-oxo-G导致的GC→TA突变中,亲核反应位点从G所在链转到A（C）所在链,影响酶对碱基的识别,从而产生基因突变.     

溴化乙锭对B-DNA碱基对特异性识别的分子模拟研究

溴化乙锭是一种常用的DNA荧光探针, 其作用机制是通过插入作用与DNA分子形成稳定的复合物. 分子模拟显示, 溴化乙锭插入碱基对过程中有大小沟选择性, 对结合能的统计分析发现, 溴化乙锭分子更倾向从小沟方向插入到DNA分子中. 由溴化乙锭从小沟方向插入不同碱基对的结合能考察发现, 溴化乙锭对DNA碱基对有特异性识别, 并且与CA碱基对结合能最强. 对溴化乙锭插入DNA分子的驱动力和序列特异性识别的作用力分析, 揭示溴化乙锭插入DNA分子的驱动力和碱基对的特异性识别均以静电作用为主.     

HIV-1病毒DNA与整合酶结合后的构象变化

用分子动力学(MD)模拟方法优化了HIV-1病毒DNA与整合酶(IN)二聚体(IN2)复合物模型结构, 并分析了HIV-1病毒DNA结合IN2后的构象变化. 结果表明, 按照HIV-1病毒DNA与IN2结合能力的强度, 病毒DNA可分为五个区域: 非结合区、强结合区1、弱结合区、强结合区2和反应区, 并用结合自由能计算验证了该分区的合理性. 与未结合IN2的病毒DNA相比, 复合物模型中病毒DNA除了非结合区碱基外, 其它四个区域的碱基构象变化较大. 复合物模型中病毒DNA主链较大程度地偏离标准B型DNA以及结合部位的小沟变宽都是识别IN的结构基础. 模拟结果与实验数据吻合较好, 为基于HIV-1 IN的药物分子设计提供了一定的结构信息.     

醇及脱氧核糖与水分子间三体作用强度的快速准确计算

快速准确预测醇及脱氧核糖分子与水形成的氢键复合物的三体作用强度, 对准确模拟水环境下蛋白质和DNA的结构和功能至关重要. 基于对多体极化作用的理解, 在可极化偶极-偶极作用模型(PBFF)基础上, 将体系中的极性化学键视为化学键偶极, 通过模拟键偶极的极化计算了醇及脱氧核糖与水分子形成的氢键复合物的三体作用能. 通过拟合甲醇与水氢键复合物的三体作用能随分子间距离变化的能量曲线确定了所需的参数. 将模型和所确定的参数应用于计算更多的甲醇、 乙醇及脱氧核糖与水氢键复合物的三体作用能, 检验了模型的准确性和参数的可转移性. 计算结果表明, 可极化偶极-偶极作用模型及所确定的参数能够较好地预测具有不同结构的氢键复合物的三体作用强度, 其精度可与MP2方法的计算精度相当.     

精氨酸侧链和核酸碱基间离子氢键作用强度分析

采用MP2/6-31+G(d,p)方法优化得到了22个由精氨酸侧链与碱基尿嘧啶、 胸腺嘧啶、 胞嘧啶、 鸟嘌呤及腺嘌呤形成的氢键复合物的气相稳定结构, 使用包含BSSE校正的MP2/aug-cc-pVTZ方法计算得到了复合物的气相结合能, 通过MP2/6-31+G(d,p)方法和PCM模型优化得到了复合物的水相稳定结构, 采用MP2/aug-cc-pVTZ方法和PCM模型计算得到了复合物的水相结合能. 研究发现, 精氨酸侧链与碱基间的离子氢键作用强度与单体间电荷转移量、 氢键临界点电子密度及二阶作用稳定化能密切相关. 与中性氢键相比, 离子氢键作用具有更显著的共价作用成分. 研究还发现, 精氨酸侧链和碱基间形成的氢键复合物的稳定性次序可以通过氢键受体碱基分子上氧原子和氮原子的质子化反应焓变进行预测, 质子化反应焓变越负, 形成的氢键复合物越稳定.     

Water-mediated binding of agents that target the DNA minor groove

Small molecule complexes with DNA that incorporate linking water molecules are rare, and the DB921-DNA complex has provided a unique and well-defined system for analysis of water-mediated binding in the context of a DNA complex. DB921 has a benzimidazole-biphenyl system with terminal amidines that results in a linear conformation that does not possess the appropriate radius of curvature to match the minor groove shape and represents a new paradigm that does not fit the classical model of minor groove interactions. To better understand the role of the bound water molecule observed in the X-ray crystal structure of the DB921 complex, synthetic modifications have been made in the DB921 structure, and the interactions of the new compounds with DNA AT sites have been evaluated with an array of methods, including DNase I footprinting, biosensor-surface plasmon resonance, isothermal titration microcalorimetry, and circular dichroism. The interaction of a key compound, which has the amidine at the phenyl shifted from the para position in DB921 to the meta position, has also been examined by X-ray crystallography. The detailed structural, thermodynamic, and kinetic results provide valuable new information for incorporation of water molecules in the design of new lead scaffolds for targeting DNA in chemical biology and therapeutic applications.     

DNA sequence dependent monomer-dimer binding modulation of asymmetric benzimidazole derivatives

A number of studies indicate that DNA sequences such as AATT and TTAA have significantly different physical and interaction properties. To probe these interaction differences in detail and determine the influence of charge, we have synthesized three bisbenzimidazole derivatives, a diamidine, DB185, and monoamidines, DB183 and DB210, that are related to the well-known minor groove agent, Hoechst 33258. Footprinting studies with several natural and designed DNA fragments indicate that the synthetic compounds bind at AT sequences in the minor groove and interact more weakly at sites with TpA steps relative to sites without such steps. Circular dichroism spectroscopy also indicates that the compounds bind in the DNA minor groove. Surprisingly, Tm studies as a function of ratio indicate that the monoamidines bind to TTAA sequences as dimers, whereas the diamidine binds as a monomer. Biosensor-surface plasmon resonance (SPR) studies allowed us to quantitate the interaction differences in more detail. SPR results clearly show that the monoamidine compounds bind to the TTAA sequence in a cooperative 2:1 complex but bind as monomers to AATT. The dication binds to both sequences in monomer complexes but the binding to AATT is significantly stronger than binding to TTAA. Molecular dynamics simulations indicate that the AATT sequence has a narrow time-average minor groove width that is a very good receptor site for the bisbenzimidazole compounds. The groove in TTAA sequences is wider and the width must be reduced to form a favorable monomer complex. The monocations thus form cooperative dimers that stack in an antiparallel orientation and closely fit the structure of the TTAA minor groove. The amidine groups in the dimer are oriented in the 5' direction of the strand to which they are closest. Charge repulsion in the dication apparently keeps it from forming the dimer. It instead reduces the TTAA groove width, in an induced fit process, sufficiently to form a minor groove complex. The dimer-binding mode of DB183 and DB210 is a new DNA recognition motif and offers novel design concepts for selective targeting of DNA sequences with a wider minor groove, including those with TpA steps.     

Oxidative damage to DNA: counterion-assisted addition of water to ionized DNA

Oxidative damage to DNA, implicated in mutagenesis, aging, and cancer, follows electron loss that generates a radical cation that migrates to a guanine, where it may react with water to form 8-oxo-7,8-dihydroguanine (8-OxoG). Molecular dynamics and ab initio quantum simulations on a B-DNA tetradecamer reveal activated reaction pathways that depend on the local counterion arrangement. The lowest activation barrier, 0.73 eV, is found for a reaction that starts from a configuration where a Na(+) resides in the major groove near the N7 atoms of adjacent guanines, and evolves through a transition state where a bond between a water oxygen atom and a carbon atom forms concurrently with displacement of a proton toward a neighboring water molecule. Subsequently, a bonded complex of a hydronium ion and the nearest backbone phosphate group forms. This counterion-assisted proton shuttle mechanism is supported by experiments exploiting selective substitution of backbone phosphates by methylphosphonates.     

Design of DNA minor groove binding diamidines that recognize GC base pair sequences: a dimeric-hinge interaction motif

The classical model of DNA minor groove binding compounds is that they should have a crescent shape that closely fits the helical twist of the groove. Several compounds with relatively linear shape and large dihedral twist, however, have been found recently to bind strongly to the minor groove. These observations raise the question of how far the curvature requirement could be relaxed. As an initial step in experimental analysis of this question, a linear triphenyl diamidine, DB1111, and a series of nitrogen tricyclic analogues were prepared. The goal with the heterocycles is to design GC binding selectivity into heterocyclic compounds that can get into cells and exert biological effects. The compounds have a zero radius of curvature from amidine carbon to amidine carbon but a significant dihedral twist across the tricyclic and amidine-ring junctions. They would not be expected to bind well to the DNA minor groove by shape-matching criteria. Detailed DNase I footprinting studies of the sequence specificity of this set of diamidines indicated that a pyrimidine heterocyclic derivative, DB1242, binds specifically to a GC-rich sequence, -GCTCG-. It binds to the GC sequence more strongly than to the usual AT recognition sequences for curved minor groove agents. Other similar derivatives did not exhibit the GC specificity. Biosensor-surface plasmon resonance and isothermal titration calorimetry experiments indicate that DB1242 binds to the GC sequence as a highly cooperative stacked dimer. Circular dichroism results indicate that the compound binds in the minor groove. Molecular modeling studies support a minor groove complex and provide an inter-compound and compound-DNA hydrogen-bonding rational for the unusual GC binding specificity and the requirement for a pyrimidine heterocycle. This compound represents a new direction in the development of DNA sequence-specific agents, and it is the first non-polyamide, synthetic compound to specifically recognize a DNA sequence with a majority of GC base pairs.     

Structural and dynamic variations in DNA hexamers containing T-T and F-F single and tandem internal mispairs

Molecular dynamics simulations of double-helical DNA oligomers have been performed to investigate differences in the structure, dynamics, and hydration of F-F and T-T mispairs. Hexamers containing F-F pairs were found to be more dynamic, especially in the region of the mispair itself. This dynamic variability derives from greater flexibility of F-F pairs. The T-T mispairs, on the other hand, were found to be comparatively tightly bound as wobble pairs. The major and minor groove edges of the T-T pairs were observed to be solvated at exposed carbonyl positions by at least one water molecule, while F-F pairs lacked solvating waters. Stacking interactions were nearly identical for T-T and F-F pairs, leading to similar average structures, even though F stacking was more dynamically variable. Solvation differences between F-F and T-T therefore support the steric exclusion model for nucleotide incorporation in DNA replication. Large differences in the orientation of minor groove functional groups, in addition to differences in solvation, further rationalize why F bases present during DNA extension events induce stalls. Two novel nucleotides are proposed to further elucidate minor groove interactions of DNA with polymerase molecules.Electronic Supplementary Material This Material consists of equilibration protocol, plots of center-of-mass stacking, water radial distribution functions, helical parameter dynamics, and dynamics data for a control AT sequence. Supplementary material is available in the online version of this article at Contribution to the Jacopo Tomasi Honorary Issue     

Induced fit conformational changes of a "reversed amidine" heterocycle: optimized interactions in a DNA minor groove complex

To better understand the molecular basis for recognition of the DNA minor groove by heterocyclic cations, a series of "reversed amidine" substituted heterocycles has been prepared. Amidine derivatives for targeting the minor groove have the amidine carbon linked to a central heterocyclic system, whereas in the reverse orientation, an amidine nitrogen provides the link. The reverse system has a larger dihedral angle as well as a modified spatial relationship with the groove relative to amidines. Because of the large dihedral, the reversed amidines should have reduced binding to DNA relative to similar amidines. Such a reduction is observed in footprinting, circular dichroism (CD), biosensor-surface plasmon resonance (SPR), and isothermal titration calorimetric (ITC) experiments with DB613, which has a central phenyl-furan-phenyl heterocyclic system. The reduction is not seen when a pyrrole (DB884) is substituted for the furan. Analysis of a number of derivatives defines the pyrrole and a terminal phenyl substituent on the reversed amidine groups as critical components in the strong binding of DB884. ITC and SPR comparisons showed that the better binding of DB884 was due to a more favorable binding enthalpy and that it had exceptionally slow dissociation from DNA. Crystallographic analysis of DB884 bound to an AATT site shows that the compound was bound in the minor groove in a 1:1 complex as suggested by CD solution studies. Surprisingly, unlike the amidine derivative, the pyrrole -NH of DB884 formed an H-bond with a central T of the AATT site and this accounts for the enthalpy-driven strong binding. The structural results and molecular modeling studies provide an explanation for the differences in binding affinities for related amidine and reversed amidine analogues.     

The inefficiency of incisions of ecteinascidin 743-DNA adducts by the UvrABC nuclease and the unique structural feature of the DNA adducts can be used to explain the repair-dependent toxicities of this antitumor agent.

BACKGROUND: Ecteinascidin 743 (Et 743), a natural product derived from a marine tunicate, is a potent antitumor agent presently in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine via a unique mechanism involving catalytic activation. The sequence selectivity of Et 743 is governed by different patterns of hydrogen-bonding to DNA, which results in differential reversibility of the covalent adducts. As determined by nuclear magnetic resonance spectroscopy, the preferred sequences 5'-PuGC and 5'-PyGG are stabilized by a hydrogen-bonding network, while the non-preferred sequences 5'-NG(A/T) are much less stabilized due to the lack of a key hydrogen bond to the GC base pair on the 3'-side of the alkylated guanine. RESULTS: Mammalian cell lines (XPB, XPD, XPF, XPG, and ERCC1) deficient in the nucleotide excision repair (NER) gene products show resistance to Et 743. The recognition and subsequent incision of Et 743-DNA adducts by the bacterial multisubunit endonuclease UvrABC were used to evaluate DNA repair-mediated toxicity as a rationale for the resistance of NER-defective cell lines and the antitumor activity of Et 743. The Et 743-DNA adducts are indeed recognized and incised by the UvrABC repair proteins; however, the pattern of incision indicated that the non-preferred, and less stable, sequences (i.e. 5'-NG(A/T)) modified with Et 743 are generally incised at a much higher efficiency than the preferred, more stable sequences (i.e. 5'-PuGC or 5'-PyGG). In addition, within the same Et 743 recognition sequence, the level of incision varies, indicating that flanking regions also contribute to the differential incision frequency. CONCLUSIONS: The inefficient repair incision by the UvrABC nuclease of Et 743-DNA adducts provides a basis for rationalizing the observed repair-dependent cytotoxicities of these DNA adducts, if other associated structural properties of Et 743-DNA adducts are taken into account. In particular, the wedge-shaped Et 743, which forces open the minor groove of DNA, introducing a major groove bend, and the extrahelical protrusion of the C-subunit of Et 743 provide unique characteristics alongside the hydrogen-bonding stabilization of a covalent DNA adduct, which we propose traps an intermediate in NER processing of Et 743-DNA adducts. This trapped intermediate protein-Et 743-DNA adduct complex can be considered analogous to a poisoned topoisomerase I- or topoisomerase II-DNA complex. In the absence of an intact NER nuclease complex, this toxic lesion is unable to form, and the Et 743-DNA adducts, although not repaired by the NER pathway, are less toxic to cells. Conversely, elevated levels of either of these nucleases should lead to enhanced Et 743 toxicity.