Molecular dynamics simulations and binding free energy analysis of DNA minor groove complexes of curcumin

Curcumin is a natural phytochemical that exhibits a wide range of pharmacological properties, including antitumor and anticancer activities. The similarity in the shape of curcumin to DNA minor groove binding drugs is the motivation for exploring its binding affinity in the minor grooves of DNA sequences. Interactions of curcumin with DNA have not been extensively examined, while its pharmacological activities have been studied and documented in depth. Curcumin was docked with two DNA duplexes, d(GTATATAC)₂ and d(CGCGATATCGCG)₂, and molecular dynamics simulations of the complexes were performed in explicit solvent to determine the stability of the binding. In all systems, the curcumin is positioned in the minor groove in the A·T region, and was stably bound throughout the simulation, causing only minor modifications to the structural parameters of DNA. Water molecules were found to contribute to the stability of the binding of the ligand. Free energy analyses of the complexes were performed with MM-PBSA, and the binding affinities that were calculated are comparable to the values reported for other similar nucleic acid–ligand systems, indicating that curcumin is a suitable natural molecule for the development of minor groove binding drugs.     

Binding of Cu(II) complexes of oxicam NSAIDs to alternating AT and homopolymeric AT sequences: differential response to variation in backbone structure

Besides their principal functions as painkillers and anti-inflammatory agents, drugs belonging to the nonsteroidal anti-inflammatory drug (NSAID) group also have anticancer properties. Cu(II) complexes of these drugs enhance the anticancer effect. How they exert this effect is not clear. As a possible molecular mechanism, our group has already shown that the Cu(II) complexes of two oxicam NSAIDs with anticancer properties, viz. piroxicam and meloxicam, can directly bind to the DNA backbone. AT stretches are abundant in the eukaryotic genome. These stretches are more accessible to binding of different ligands, resulting in expression of different functions. AT stretches containing both alternating base pairs and homopolymeric bases in individual strands show subtle differences in backbone structures. It is therefore of interest to see how the Cu(II)–NSAID complexes respond to such differences in backbone structure. Binding studies of these complexes with alternating polydA–dT and homopolymeric polydA–polydT have been conducted using UV–vis absorption titration studies, UV melting studies and circular dichroism spectroscopy. Competitive binding with the standard intercalator ethidium bromide and the minor groove binder 4′,6-diamidino-2-phenylindole was monitored using fluorescence to identify the possible binding mode. Our results show that Cu(II)–NSAID complexes are highly sensitive to the subtle differences in backbone structures of polydA–dT and polydA–polydT and respond to them by exhibiting different binding properties, such as binding constants, effect on duplex stability and binding modes. Both complexes have a similar binding mode with polydA–dT, which is intercalative, but for polydA–polydT, the results point to a mixed mode of binding.     

Design,synthesis and biological evaluation of a novel series of anthrapyrazoles linked with netropsin-like oligopyrrole carboxamides as anticancer agents

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIα-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIα decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA–topoisomerase IIα covalent complex indicating that they acted as topoisomerase IIα poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.     

Molecular dynamics simulation studies for DNA sequence recognition by reactive metabolites of anticancer compounds

The discovery of novel anticancer molecules 5F‐203 (NSC703786) and 5‐aminoflavone (5‐AMF, NSC686288) has addressed the issues of toxicity and reduced efficacy by targeting over expressed Cytochrome P450 1A1 (CYP1A1) in cancer cells. CYP1A1 metabolizes these compounds into their reactive metabolites, which are proven to mediate their anticancer effect through DNA adduct formation. However, the drug metabolite–DNA binding has not been explored so far. Hence, understanding the binding characteristics and molecular recognition for drug metabolites with DNA is of practical and fundamental interest. The present study is aimed to model binding preference shown by reactive metabolites of 5F‐203 and 5‐AMF with DNA in forming DNA adducts. To perform this, three different DNA crystal structures covering sequence diversity were selected, and 12 DNA‐reactive metabolite complexes were generated. Molecular dynamics simulations for all complexes were performed using AMBER 11 software after development of protocol for DNA‐reactive metabolite system. Furthermore, the MM‐PBSA/GBSA energy calculation, per‐nucleotide energy decomposition, and Molecular Electrostatic Surface Potential analysis were performed. The results obtained from present study clearly indicate that minor groove in DNA is preferable for binding of reactive metabolites of anticancer compounds. The binding preferences shown by reactive metabolites were also governed by specific nucleotide sequence and distribution of electrostatic charges in major and minor groove of DNA structure. Overall, our study provides useful insights into the initial step of mechanism of reactive metabolite binding to the DNA and the guidelines for designing of sequence specific DNA interacting anticancer agents. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural insights into the effect of hydration and ions on A-tract DNA: a molecular dynamics study

DNA structure is known to be sensitive to hydration and ionic environment. To explore the dynamics, hydration, and ion binding features of A-tract sequences, a 7-ns Molecular dynamics (MD) study has been performed on the dodecamer d(CGCAAATTTGCG)(2). The results suggest that the intrusion of Na(+) ion into the minor groove is a rare event and the structure of this dodecamer is not very sensitive to the location of the sodium ions. The prolonged MD simulation successfully leads to the formation of sequence dependent hydration patterns in the minor groove, often called spine of hydration near the A-rich region and ribbon of hydration near the GC regions. Such sequence dependent differences in the hydration patterns have been seen earlier in the high resolution crystal structure of the Drew-Dickerson sequence, but not reported for the medium resolution structures (2.0 approximately 3.0 A). Several water molecules are also seen in the major groove of the MD simulated structure, though they are not highly ordered over the extended MD. The characteristic narrowing of the minor groove in the A-tract region is seen to precede the formation of the spine of hydration. Finally, the occurrence of cross-strand C2-H2.O2 hydrogen bonds in the minor groove of A-tract sequences is confirmed. These are found to occur even before the narrowing of the minor groove, indicating that such interactions are an intrinsic feature of A-tract sequences.     

Drug-DNA sequence-dependent interactions analysed by electric linear dichroism.

The interactions between 20 drugs and a variety of synthetic DNA polymers and natural DNAs were studied by electric linear dichroism (ELD). All compounds tested, including several clinically used antitumour agents, are thought to exert their biological activities mainly by virtue of their abilities to bind to DNA. The selected drugs include intercalating agents with fused and unfused aromatic structures and several groove binders. To examine the role of base composition and base sequence in the binding of these drugs to DNA, ELD experiments were carried out with natural DNAs of widely differing base composition as well as with polynucleotides containing defined alternating and non-alternating repeating sequences, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT),poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC). Among intercalating agents, actinomycin D was found to be by far the most GC-selective. GC selectivity was also observed with an amsacrine-4-carboxamide derivative and to a lesser extent with methylene blue. In contrast, the binding of amsacrine and 9-aminoacridine was practically unaffected by varying the GC content of the DNAs. Ethidium bromide, proflavine, mitoxantrone, daunomycin and an ellipticine derivative were found to bind best to alternating purine-pyrimidine sequences regardless of their nature. ELD measurements provided evidence for non-specific intercalation of amiloride. A significant AT selectivity was observed with hycanthone and lucanthone. The triphenyl methane dye methyl green was found to exhibit positive and negative dichroism signals at AT and GC sites, respectively, showing that the mode of binding of a drug can change markedly with the DNA base composition. Among minor groove binders, the N-methylpyrrole carboxamide-containing antibiotics netropsin and distamycin bound to DNA with very pronounced AT specificity, as expected. More interestingly the dye Hoechst 33258, berenil and a thiazole-containing lexitropsin elicited negative reduced dichroism in the presence of GC-rich DNA which is totally inconsistent with a groove binding process. We postulate that these three drugs share with the trypanocide 4',6-diamidino-2-phenylindole (DAPI) the property of intercalating at GC-rich sites and binding to the minor groove of DNA at other sites. Replacement of guanines by inosines (i.e., removal of the protruding exocyclic C-2 amino group of guanine) restored minor groove binding of DAPI, Hoechst 33258 and berenil. Thus there are several cases where the mode of binding to DNA is directly dependent on the base composition of the polymer. Consequently the ELD technique appears uniquely valuable as a means of investigating the possibility of sequence-dependent recognition of DNA by drugs.     

Molecular dynamics simulations of A. T-rich oligomers: sequence-specific binding of Na+ in the minor groove of B-DNA

Molecular dynamics simulations have been employed to probe the sequence-specific binding of sodium ions to the minor groove of B-DNA of three A. T-rich oligomers having identical compositions but different orders of the base pairs: C(AT)(4)G, CA(4)T(4)G, and CT(4)A(4)G. Recent experimental investigations, either in crystals or in solution, have shown that monovalent cations bind to DNA in a sequence-specific mode, preferentially in the narrow minor groove regions of uninterrupted sequences of four or more adenines (A-tracts), replacing a water molecule of the ordered hydration structure, the hydration spine. Following this evidence, it has been hypothesized that in A-tracts these events may be responsible for structural peculiarities such as a narrow minor groove and a curvature of the helix axis. The present simulations confirm a sequence specificity of the binding of sodium ions: Na(+) intrusions in the first layer of hydration of the minor groove, with long residence times, up to approximately 3 ns, are observed only in the minor groove of A-tracts but not in the alternating sequence. The effects of these intrusions on the structure of DNA depend on the ion coordination: when the ion replaces a water molecule of the spine, the minor groove becomes narrower. Ion intrusions may also disrupt the hydration spine modifying the oligomer structure to a large extent. However, in no case intrusions were observed to locally bend the axis toward the minor groove. The simulations also show that ions may reside for long time periods in the second layer of hydration, particularly in the wider regions of the groove, often leading to an opening of the groove.     

Variation in the inhibition of restriction enzyme cleavage of lambda phage DNA produced by two covalent binding antitumor agents: Anthramycin and mitomycin C

DNA-drug complexes containing various levels of covalently bound mitomycin C (MC) or anthramycin were subjected to the actions of a number of restriction enzymes. While MC presented only a partial block to the actions of a number of these enzymes, anthramycin, at high binding ratios, blocked enzymatic activity very well. The contrast seen in the restriction cleavage of these DNA-drug complexes may be related to the different points of attachment in DNA (minor groove vs. major groove) for these drugs. Although similarities in electrophoretic band patterns exist for both drug complexes, certain differences are indicative of preferences in binding sequences that these drugs may have for DNA. The results show that these sequences do not necessarily lie immediately within the restriction cut sites but may effect the cutting of these sites from a distance. The results also further support anthramycin's potential usage as a selective/reversible blocking agent for recombinant research.     

Synthesis, DNA binding, topoisomerases inhibition and cytotoxic properties of 4-arylcarboxamidopyrrolo-2-carboxyanilides

Three 4-arylcarboxamidopyrrolo-2-carboxyanilides bearing different substituents on the pyrrole nitrogen were synthesized and evaluated for their capacities to bind to specific sequences within the minor groove of DNA and to inhibit human topoisomerases I and II in vitro. The cytotoxicity of the drugs correlates with their DNA binding affinities. The two drugs bearing a N-methyl or N-benzyl pyrrole stabilize topoisomerase I-DNA complexes.     

An unexpected major groove binding of netropsin and distamycin A to tRNA(phe)

Crystalline complexes of yeast tRNA(phe) and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 O(6), U52 O(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.     

Thermodynamics of drug-DNA interactions

Many anticancer, antibiotic, and antiviral drugs exert their primary biological effects by reversibly interacting with nucleic acids. Therefore, these biomolecules represent a major target in drug development strategies designed to produce next generation therapeutics for diseases such as cancer. In order to improve the clinical efficacy of existing drugs and also to design new ones it is necessary to understand the molecular basis of drug-DNA interactions in structural, thermodynamic, and kinetic detail. The past decade has witnessed an increase in the number of rigorous biophysical studies of drug-DNA systems and considerable knowledge has been gained in the energetics of these binding reactions. This is, in part, due to the increased availability of high-sensitivity calorimetric techniques, which have allowed the thermodynamics of drug-DNA interactions to be probed directly and accurately. The focus of this article is to review thermodynamic approaches to examining drug-DNA recognition. Specifically, an overview of a recently developed method of analysis that dissects the binding free energy of these reactions into five component terms is presented. The results of applying this analysis to the DNA binding interactions of both minor groove drugs and intercalators are discussed. The solvent water plays a key role in nucleic acid structure and consequently in the binding of ligands to these biomolecules. Any rational approach to DNA-targeted drug design requires an understanding of how water participates in recognition and binding events. Recent studies examining hydration changes that accompany DNA binding by intercalators will be reviewed. Finally some aspects of cooperativity in drug-DNA interactions are described and the importance of considering cooperative effects when examining these reactions is highlighted.     

Experimental DNA-binding and computer modelling studies on an analogue of the anti-tumor drug amsacrine

The DNA-binding properties of the anti-cancer drug amsacrine and a 9-aminoacridine analogue substituted at the 4 position with a 4-methanesulphonanilido-group, have been examined by means of unwinding, melting and equilibrium binding experiments. These find that the latter compound is at least as effective as a DNA-binder and intercalator as amsacrine itself. Molecular modelling and energetic calculations have confirmed this, and have produced plausible intercalation geometries. These show that there are subtle differences in the low-energy minor groove arrangements adopted by the substituents of the two drugs. Speculation is advanced that these differences may be relevant to the marked differences in cytotoxicity shown by the two compounds.     

The protein HU can displace the LexA repressor from its DNA-binding sites

The major bacterial histone-like protein HU is a small, basic, dimeric protein composed of two closely related subunits. HU is involved in several processes in the bacterial cell such as the initiation of replication, transposition, gene inversion and cell division. It has been suggested that HU could introduce structural changes to the DNA which would facilitate or inhibit the binding of regulatory proteins to their specific sites. In this study we investigated the effect of HU on the binding of LexA protein, the regulator of SOS functions, to three of its specific binding sites. We show that HU can displace LexA from its binding sites on the operators of the lexA, recA and sfiA genes. The lexA operator was the most sensitive while the higher affinity sfiA operator was the least sensitive. Since HU, like its homologue IHF, probably binds DNA in the minor groove we tested the effect of distamycin, a drug which binds to the minor groove, on LexA binding. Like HU, this drug disrupted LexA–operator complexes. These results suggest that distortion of the minor groove of the lexA operators excludes the binding of the repressor to the major groove.     

An Unexpected Major Groove Binding of Netropsin and Distamycin A to tRNAphe

Abstract

Crystalline complexes of yeast tRNA^phe and the oligopeptide antibiotics netropsin and distamycin A were prepared by diffusing drugs into crystals of tRNA. X-ray structure analyses of these complexes reveal a single common binding site for both drugs which is located in the major or deep groove of the tRNA T-stem. The netropsin-tRNA complex is stabilized by specific hydrogen bonds between the amide groups of the drug and the tRNA bases G51 0(6), U52 0(4) and G53 N(7) on one strand, and is further stabilized by electrostatic interactions between the positively charges guanidino side chain of the drug and the tRNA phosphate P53 on the same strand and the positively charged amidino propyl side chain and the phosphates P61, P62 and P63 on the opposite strand of the double helix. These results are in contrast to the implicated minor groove binding of these drugs to non-guanine sequences in DNA. The binding to the GUG sequence in tRNA implies that major groove binding to certain DNA sequences is possible.     

Structural comparison of anticancer drug-DNA complexes: adriamycin and daunomycin

The anticancer drugs adriamycin and daunomycin have each been crystallized with the DNA sequence d(CGATCG) and the three-dimensional structures of the complexes solved at 1.7- and 1.5-A resolution, respectively. These antitumor drugs have significantly different clinical properties, yet they differ chemically by only the additional hydroxyl at C14 of adriamycin. In these complexes the chromophore is intercalated at the CpG steps at either end of the DNA helix with the amino sugar extended into the minor groove. Solution of the structure of daunomycin bound to d(CGATCG) has made it possible to compare it with the previously reported structure of daunomycin bound to d(CGTACG). Although the two daunomycin complexes are similar, there is an interesting sequence dependence of the binding of the amino sugar to the A-T base pair outside the intercalation site. The complex of daunomycin with d(CGATCG) has tighter binding than the complex with d(CGTACG), leading us to infer a sequence preference in the binding of this anthracycline drug. The structures of daunomycin and adriamycin with d(CGATCG) are very similar. However, there are additional solvent interactions with the adriamycin C14 hydroxyl linking it to the DNA. Surprisingly, under the influence of the altered solvation, there is considerable difference in the conformation of spermine in these two complexes. The observed changes in the overall structures of the ternary complexes amplify the small chemical differences between these two antibiotics and provide a possible explanation for the significantly different clinical activities of these important drugs.     

Inhibition of Zn(II) Binding Type IA Topoisomerases by Organomercury Compounds and Hg(II)

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.     

DNA minor groove binding agents interfere with topoisomerase II mediated lesions induced by epipodophyllotoxin derivative VM-26 and acridine derivative m-AMSA in nuclei from L1210 cells

This study demonstrated that agents capable of interacting with the minor groove in nuclear DNA interfere with topoisomerase II mediated effects of antitumor drugs such as VM-26 and m-AMSA. Distamycin, Hoechst 33258, and DAPI were used as agents capable of AT-specific binding in the minor groove of DNA while producing no profound long-range distortion of DNA structure. In intact nuclei from L1210 cells, these minor groove binders inhibited the induction of topoisomerase II mediated DNA damage (DNA-protein cross-links and DNA double-strand breaks) by VM-26 and m-AMSA. The inhibitory effects of distamycin reflected prevention of formation of new lesions but not reversal of preexisting damage. The minor groove binders did not differentiate between lesions induced by an intercalator, m-AMSA, or by a DNA-nonbinding drug, VM-26. All three groove binders inhibited DNA breaks more strongly than DNA-protein cross-links. The inhibitory potency correlated with the size of minor groove binders and the size of their DNA-binding sites: distamycin (5 bp) greater than Hoechst 33258 (4 bp) greater than DAPI (3 bp). The results showed that DNA minor groove binders are a new type of modulators of the action of topoisomerase II targeted drugs.