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.     

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.     

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.     

Strong binding in the DNA minor groove by an aromatic diamidine with a shape that does not match the curvature of the groove

A combination of biophysical techniques has been used to characterize the interaction of an antitrypanosomal agent, CGP 40215A, with DNA. The results from a broad array of methods (DNase I footprinting, surface plasmon resonance, X-ray crystallography, and molecular dynamics) indicate that this compound binds to the minor groove of AT DNA sequences. Despite its unusual linear shape that is not complementary to that of the DNA groove, a high binding affinity was observed in comparison with other similar but more curved diamidine compounds. The amidine groups at both ends of the ligand and the -NH groups on the linker are involved in extensive and dynamic H-bonds to the DNA bases. Complementary and consistent results were obtained from both the X-ray and molecular dynamics studies; both of these methods reveal direct and water-mediated H-bonds between the ligand and the DNA.     

Heterocyclic diamidine interactions at AT base pairs in the DNA minor groove: effects of heterocycle differences, DNA AT sequence and length

Given the increasing significance of diamidines as DNA-targeted therapeutics and biotechnology reagents, it is important to establish the variations in thermodynamic quantities that characterize the interactions of closely related compounds to different sequence AT binding sites. In this study, an array of methods including biosensor-surface plasmon resonance (SPR), isothermal titration microcalorimetry (ITC), circular dichroism (CD), thermal melting (Tm) and molecular modeling have been used to characterize the binding of dicationic diamidines related to DB75 (amidine-phenyl-furan-phenyl-amidine) with alternating and nonalternating AT sequences. Conversion of the central furan of DB75 to other similar groups, such as thiophene or selenophene, can yield compounds with increased affinity and sequence binding selectivity for the minor groove. Calorimetric measurements revealed that the thermodynamic parameters (Delta G, Delta H, Delta S) that drive diamidine binding to alternating and nonalternating oligomers can be quite different and depend on both DNA sequence and length. Small changes in a compound can have major effects on DNA interactions. By choosing an appropriate central group it is possible to "tune" the shape of the molecule to match DNA for enhanced affinity and sequence recognition.     

Amidine- and Amidoxime-Substituted Heterocycles: Synthesis,Antiproliferative Evaluations and DNA Binding

The novel 1,2,3-triazolyl-appended N- and O-heterocycles containing amidine 4–11 and amidoxime 12–22 moiety were prepared and evaluated for their antiproliferative activities in vitro. Among the series of amidine-substituted heterocycles, aromatic diamidine 5 and coumarine amidine 11 had the most potent growth-inhibitory effect on cervical carcinoma (HeLa), hepatocellular carcinoma (HepG2) and colorectal adenocarcinoma (SW620), with IC₅₀ values in the nM range. Although compound 5 was toxic to non-tumor HFF cells, compound 11 showed certain selectivity. From the amidoxime series, quinoline amidoximes 18 and 20 showed antiproliferative effects on lung adenocarcinoma (A549), HeLa and SW620 cells emphasizing compound 20 that exhibited no cytostatic effect on normal HFF fibroblasts. Results of CD titrations and thermal melting experiments indicated that compounds 5 and 10 most likely bind inside the minor groove of AT-DNA and intercalate into AU-RNA. Compounds 6, 9 and 11 bind to AT-DNA with mixed binding mode, most probably minor groove binding accompanied with aggregate binding along the DNA backbone.     

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.     

DNA小沟结合二脒:水分子的识别作用

采用分子动力学模拟了DB921-DNA复合物, 通过7 ns的模拟研究表明: DB921一端的氨基氮原子与一个水分子形成氢键, 同时, 水分子又与DNA的5位A碱基的氮原子形成一个氢键. 水分子在DB921与DNA小沟结合中起了桥连的作用, 使得直线型的芳香二脒化合物DB921通过水桥与DNA小沟结合, 水分子诱导DB921分子与DNA的小沟域构型相适应, 与DNA小沟域的AATTC碱基有较强的结合作用. 在分子水平上提供了DB921与双螺旋DNA相互作用的结构及复合物的动态变化情况, 指出水分子在DNA小沟结合二脒化合物中的识别作用, 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.     

Synthesis and DNA-binding study of A thiazole-containing analog of netropsin

In order to study the influence of the nature of the heterocyclic rings of Netropsin (Nt) on the binding to specific nucleotide sequences in the minor groove of DNA, a thiazole-containing analogue (Thia-Nt) was designed. Optimized syntheses of the key compound 2-aminothiazole-4-carboxylic acid and Thia-Nt were reported together with the results of a preliminary study of Thia-Nt-DNA binding.     

Theoretical study of binding affinity for diamidine with DNA

Diamidine molecules, which have been recognized as the powerful gene drug candidates over the past decades, can bind in the DNA minor groove, inhibit the duplication of morbid sequences, and fight against a number of human and animal diseases. In this paper, on the basis of the binding models of a series of diamidines with DNA, the important influencing factors for the binding affinity of diamidines with DNA were systematically analyzed. The obtained results demonstrated that the curvature, length, distal group, and heteroaromatic ring of diamidine are four important factors, which could influence their binding affinities. Specifically, the better the curvature of the diamidine fits DNA minor groove, the higher the binding affinity is; increasing the molecular length within a certain range can make the binding affinity higher; changing the distal group of diamidine from amidino to imidazole or pyrimidine is favorable for improving the corresponding binding affinity; and the introduction of central heteroaromatic rings of diamidine molecules influences their binding affinities. One diamidine (named as DB103d) with ideal DNA binding affinity validates the four important factors proposed in the present work. The results obtained in this work might be helpful for the design of new efficient diamidine-based drug candidates.

     

Structural characterization of a rigidified threading bisintercalator

NMR spectroscopy was used to explore the sequence-specific interaction of DNA with a new threading bisintercalator (C1) consisting of two intercalating 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) units connected by a rigid, tricyclic spiro linker. A structural model of C1 complexed to d(CGGTACCG)(2) was calculated using distance constraints obtained from solution NMR data. The model was also supported by the results from residual dipolar coupling (RDC) measurements obtained using Pf1-phage as a cosolvent. According to the model, the central cyclohexane ring of the linker connecting the two NDI units lies flat in the minor groove of DNA. Linker length, hydrogen bonding, steric, and hydrophobic factors all appear to contribute to the observed sequence specificity of binding. These results serve to illustrate the versatility of threading polyintercalation given that, in a previous study, a ligand consisiting of two NDI units joined by a flexible peptide linker was shown to bind sequence specifically within the major groove of this same sequence of DNA.     

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.     

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.     

Synthesis, biological evaluation, and modeling of dimeric PPI analogues as novel DNA minor groove binders

A series of symmetrical dimeric proton pump inhibitor (PPI) analogues, designed as novel type DNA minor groove binders, was synthesized and evaluated for anti-tumor activity. Some of these new compounds showed IC(50) values below 10 microM in an in vitro anti-tumor test. A molecular modeling study was performed to confirm the sequence selectivity of these compounds towards AT base pairs in DNA. Two effective compounds were selected and docked into the minor groove of DNA. The snug binding may be responsible for their cytotoxic and anti-tumor effects.     

A synthetic miniprotein that binds specific DNA sequences by contacting both the major and the minor groove

Attachment of a slightly modified basic region of a bZIP protein (GCN4) to a distamycin-related tripyrrole provides a bivalent system capable of binding with high affinity to specific DNA sequences. Appropriate adjustment of the linker between the two units has led to a hybrid that binds a 9 base-pair-long DNA site (TTTTATGAC) with low nanomolar affinity at 4 degrees C. Circular dichroism and gel retardation studies indicate that the binding occurs by simultaneous insertion of the bZIP basic region into the DNA major groove and the tripyrrole moiety into the minor groove of the flanking sequence. Analysis of hybrids bearing alternative linkers revealed that tight, specific binding is strongly dependent on the length and nature of the connecting unit.     

Fluorescence‐Labeled Bis‐benzamidines as Fluorogenic DNA Minor‐Groove Binders: Photophysics and Binding Dynamics

In recent decades there has been great interest in the design of highly sensitive sequence‐specific DNA binders. The eligibility of the binder depends on the magnitude of the fluorescence increase upon binding, related to its photophysics, and on its affinity and specificity, which is, in turn, determined by the dynamics of the binding process. Therefore, progress in the design of DNA binders requires both thorough photophysical studies and precise determination of the association and dissociation rate constants involved. We have studied two bis‐benzamidine (BBA) derivatives labeled by linkers of various lengths with the dye Oregon Green (OG). These fluorogenic binders show a dramatic fluorescence enhancement upon binding to the minor groove of double‐stranded (ds) DNA, as well as significant improvement in their sequence specificity versus the parent BBA, although with decreased affinity constants. Detailed photophysical analysis shows that static and dynamic quenching of the OG fluorescence by BBA through photoinduced electron transfer is suppressed upon insertion of BBA into the minor groove of DNA. Fluorescence correlation spectroscopy yields precise dynamic rate constants that prove that the association process of these fluorogenic binders to dsDNA is very similar to that of BBA alone and that their lower affinity is mainly a consequence of their weaker attachment to the minor groove and the resultant faster dissociation process. The conclusions of this study will allow us to go one step further in the design of new DNA binders with tunable fluorescence and binding properties.     

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。