Binding Of Daunomycin Research Articles

Mechanism of Daunomycin Intercalation into DNA from Enhanced Sampling Simulations.

Daunomycin is a widely used anticancer drug, yet the mechanism underlying how it binds to DNA remains contested. 469 all-atom trajectories of daunomycin binding to the DNA oligonucleotide d(GCG CAC GTG CGC) were collected using weighted ensemble (WE)-enhanced sampling. Mechanistic insights were revealed through analysis of the ensemble of trajectories. Initially, the binding process involves a ubiquitous hydrogen bond between the DNA backbone and the NH3+ group on daunomycin. During the binding process, most trajectories exhibited similar structural changes to DNA, including DNA base pair rise, bending, and minor groove width changes. Variability within the ensemble of binding trajectories illuminates differences in the orientation of daunomycin as it initially intercalates; around 10% of trajectories needed minimal rearrangement from intercalation to reaching the fully bound configuration, whereas most needed an additional 1-5 ns to rearrange. The results here emphasize the utility of generating an ensemble of trajectories to discern biomolecular binding mechanisms.

In silicostudy of the binding of daunomycin and phenylalanine transfer RNA: probe molecular recognition for structure-based drug design

Structure-based design of drugs targeting RNAs relies on a systematic study of the molecular-level recognition mechanismviacomputational modelling and simulations.

Binding of anticancer drug daunomycin to parallel G-quadruplex DNA [d-(TTGGGGT)]4 leads to thermal stabilization: A multispectroscopic investigation

Guanine rich DNA sequences form four stranded G-quadruplex structures found in telomeric DNA and oncogene promoters. Small molecules binding and stabilizing the G-quadruplex disrupt telomere maintenance and gene regulation, thereby limiting the proliferation of cancer cells. The anti-cancer drug daunomycin binds to both G-quadruplex DNA and duplex DNA. We have undertaken a study of interaction of daunomycin to [d-(TTGGGGT)]4 comprising telomeric DNA sequence from Tetrahymena thermophilia to understand the mechanisms of its action. Absorbance, fluorescence and circular dicroism spectra show significant change on interaction with no change in wavelength maxima. The daunomycin dimers present in free state in solution are disrupted on binding. Presence of all sequential short inter proton distance contacts in nuclear magnetic resonance spectra confirm external binding. The observed inter molecular nuclear Overhauser enhancements, changes in chemical shift and molecular docking studies establish well defined binding of daunomycin at two different sites of G-quadruplex DNA. Thermal stabilization of [d-(TTGGGGT)]4 by 10–15 °C upon daunomycin binding is expected to reduce access of telomerase to its functional site at telomeres. The present studies on mode of action pave the way for alternate derivatives/analogues by chemical modification of anthracyclines to arrive at a more potent telomerase inhibitor.

Molecular Recognition of Parallel G-quadruplex [d-(TTGGGGT)]₄ Containing Tetrahymena Telomeric DNA Sequence by Anticancer Drug Daunomycin: NMR-Based Structure and Thermal Stability.

The anticancer drug daunomycin exerts its influence by multiple strategies of action to interfere with gene functioning. Besides inhibiting DNA/RNA synthesis and topoisomerase-II, it affects the functional pathway of telomere maintenance by the telomerase enzyme. We present evidence of the binding of daunomycin to parallel-stranded tetramolecular [d-(TTGGGGT)]4 guanine (G)-quadruplex DNA comprising telomeric DNA from Tetrahymena thermophilia by surface plasmon resonance and Diffusion Ordered SpectroscopY (DOSY). Circular Dichroism (CD) spectra show the disruption of daunomycin dimers, suggesting the end-stacking and groove-binding of the daunomycin monomer. Proton and phosphorus-31 Nuclear Magnetic Resonance (NMR) spectroscopy show a sequence-specific interaction and a clear proof of absence of intercalation of the daunomycin chromophore between base quartets or stacking between G-quadruplexes. Restrained molecular dynamics simulations using observed short interproton distance contacts depict interaction at the molecular level. The interactions involving ring A and daunosamine protons, the stacking of an aromatic ring of daunomycin with a terminal G6 quartet by displacing the T7 base, and external groove-binding close to the T1–T2 bases lead to the thermal stabilization of 15 °C, which is likely to inhibit the association of telomerase with telomeres. The findings have implications in the structure-based designing of anthracycline drugs as potent telomerase inhibitors.

Binding of anticancer drug daunomycin to a TGGGGT G-quadruplex DNA probed by all-atom molecular dynamics simulations: additional pure groove binding mode and implications on designing more selective G-quadruplex ligands.

DNA G-quadruplex structures are emerging cancer-specific targets for chemotherapeutics. Ligands that bind to and stabilize DNA G-quadruplexes have the potential to be anti-cancer drugs. Lack of binding selectivity to DNA G-quadruplex over DNA duplex remains a major challenge when attempting to develop G-quadruplex ligands into successful anti-cancer drugs. Thorough understanding of the binding nature of existing non-selective ligands that bind to both DNA quadruplex and DNA duplex will help to address this challenge. Daunomycin and doxorubicin, two commonly used anticancer drugs, are examples of non-selective DNA ligands. In this study, we extended our early all-atom binding simulation studies between doxorubicin and a DNA duplex (d(CGATCG)2) to probe the binding between daunomycin and a parallel DNA quadruplex (d(TGGGGT)4) and DNA duplex. In addition to the end stacking mode, which mimics the mode in the crystal structure, a pure groove binding mode was observed in our free binding simulations. The dynamic and energetic properties of these two binding modes are thoroughly examined, and a detailed comparison is made between DNA quadruplex binding modes and DNA duplex binding modes. Implications on the design of more selective DNA quadruplex ligands are also discussed. Graphical abstract Top stacking and groov binding modes from the MD simulations.

Binding of the Anti-Cancer Drug Daunomycin to DNA Probed by Second Harmonic Generation

Second harmonic generation (SHG) was used to selectively probe DNA-drug interactions without the need for chemical labels or invasive detection methods. In particular, the binding constant of the anticancer drug daunomycin to a recognition triplet sequence in a 33-mer of double stranded DNA was determined. The SHG method, which is interface selective, probed the binding of daunomycin to DNA that was tethered to the surface of colloidal microparticles suspended in aqueous solution. Probing biomolecule coated colloids is expected to yield larger SH signals and provides experimental flexibility as compared to experiments performed at planar interfaces. The change in SHG intensity as daunomycin was added to the microparticle solution was fit to a Langmuir binding model, which yielded an equilibrium constant of 2.3 (±0.7) × 10(5) M(-1); the corresponding Gibbs free energy change at 20 °C is -7.2 ± 0.2 kcal/mol. Control experiments established that daunomycin preferentially binds to DNA at the recognition sequence. The equilibrium was found to be unaffected by the presence of free DNA in solution, and hyper-Rayleigh scattering from bulk molecules did not change with increasing daunomycin concentration. The extracted equilibrium constants are in agreement with the range of reported values found in the literature.

Multistep Drug Intercalation: Molecular Dynamics and Free Energy Studies of the Binding of Daunomycin to DNA

Atomic-scale molecular dynamics and free energy calculations in explicit aqueous solvent are used to study the complex mechanism by which a molecule can intercalate between successive base pairs of the DNA double helix. We have analyzed the intercalation pathway for the anticancer drug daunomycin using two different methods: metadynamics and umbrella sampling. The resulting free energy pathways are found to be consistent with one another and point, within an equilibrium free energy context, to a three-step process. Daunomycin initially binds in the minor groove of DNA. An activated step then leads to rotation of the drug, coupled with DNA deformation that opens a wedge between the base pairs, bends DNA toward the major groove, and forms a metastable intermediate that resembles structures seen within the interfaces between DNA and minor-groove-binding proteins. Finally, crossing a small free energy barrier leads to further rotation of daunomycin and full intercalation of the drug, reestablishing stacking with the flanking base pairs and straightening the double helix.

Evidence for the genotoxic effect of daunomycin in multipotent hematopoietic cells of mouse bone marrow: Chromatin proteins analysis

Severe bone marrow suppression and anemia are the main side effects of using chemotherapeutic agents like daunomycin. In this study the effect of daunomycin on viability and chromosomal proteins of multipotent hematopoietic cells (MHCs) of mouse bone marrow was investigated. The cells were separated from mature cells according to their adherence, incubated in the absence and presence of various concentrations of daunomycin and viability was determined by trypan blue exclusion and MTT assay. The histones and high mobility group (HMG) proteins were extracted by acid and salt, respectively and analyzed on SDS-PAGE and immunoblot. The results revealed that daunomycin exhibited time and dose dependent cytotoxicity on the cells and upon increasing the concentrations of drug, the extractability of histone and HMG proteins decreased. Agarose gel electrophoresis showed aggregation of DNA in the presence of drug. Flow cytometry analysis suggested that there are differences in type and amount of histones H2A and H2B of these cells in comparison with thymus histones. Moreover histone H3 was not subjected to trimethylation at lysine 9 and daunomycin decreased H3K9 dimethylation and H3K9 acetylation in these cells in a dose-dependent manner. From these results it is concluded that the binding of daunomycin to chromatin precedes chromatin of multipotent hematopoietic cells of bone marrow into compaction/aggregation and beside DNA, histones and HMG proteins also play an important role in this process.

In vitro induction of G bands in human chromosomes.

When added to cells in culture, daunomycin produces a marked decrease in mitotic index, an inhibition of the cellular synthesis of DNA, RNA and protein probably due to a binding of daunomycin to DNA. When added in low concentration to lymphoid cells in G2-phase, it also induces banding in the metaphase chromosomes. The bands are found to be G bands. The possible mechanism behind this in vitro induction is discussed.

Studies on drug–DNA complexes, adriamycin–d‐(TGATCA)2 and 4′‐epiadriamycin–d‐(CGATCG)2, by phosphorus‐31 nuclear magnetic resonance spectroscopy

The complexes of adriamycin-d-(TGATCA)(2) and 4'-epiadriamycin-d-(CGATCG)(2) are studied by one- and two-dimensional (31)P nuclear magnetic resonance spectroscopy (NMR) at 500 MHz in the temperature range 275-328 K and as a function of drug to DNA ratio (0.0-2.0). The binding of drug to DNA is clearly evident in (31)P-(31)P exchange NOESY spectra that shows two sets of resonances in slow chemical exchange. The phosphate resonances at the intercalating steps, T1pG2/C1pG2 and C5pA6/C5pG6, shift downfield up to 1.7 ppm and that at the adjacent step shift downfield up to 0.7 ppm, whereas the central phosphate A3pT4 is relatively unaffected. The variations of chemical shift with drug to DNA ratio and temperature as well as linewidths are different in each of the two complexes. These observations reflect change in population of B(I)/B(II) conformation, stretching of backbone torsional angle zeta, and distortions in O-P-O bond angles that occur on binding of drug to DNA. To the best of our knowledge, there are no solution studies on 4'-epiadriamycin, a better tolerated drug, and binding of daunomycin or its analogue to d-(TGATCA)(2) hexamer sequence. The studies report the use of (31)P NMR as a tool to differentiate various complexes. The specific differences may well be the reasons that are responsible for different antitumor action of these drugs due to different binding ability and distortions in DNA.

Is ATP binding responsible for initiating drug translocation by the multidrug transporter ABCG2?

ABCG2 confers resistance to cancer cells by mediating the ATP-dependent outward efflux of chemotherapeutic compounds. Recent studies have indicated that the protein contains a number of interconnected drug binding sites. The present investigation examines the coupling of drug binding to ATP hydrolysis. Initial drug binding to the protein requires a high-affinity interaction with the drug binding site, followed by transition and reorientation to the low-affinity state to enable dissociation at the extracellular face. [3H]Daunomycin binding to the ABCG2 R482G isoform was examined in the nucleotide-bound and post-hydrolytic conformations. Binding of [3H]daunomycin was displaced by ATP analogues, indicating transition to a low-affinity conformation prior to hydrolysis. The low-affinity state was observed to be retained immediately post-hydrolysis. Therefore, the dissociation of phosphate and/or ADP is likely to be responsible for resetting of the transporter. The data indicate that, like ABCB1 and ABCC1, the 'power stroke' for translocation in ABCG2 R482G is the binding of nucleotide.

Affinity of anticancer drug, daunomycin, to core histones in solution: comparison of free and cross?linked proteins

The interaction of anthracycline anticancer drugs with chromatin, nucleosomes and histone H1 has been extensively studied. In the present study, for the first time, we have investigated the binding of anthracycline antibiotic, daunomycin, to free and cross-linked thymus core histones (CL-core) in solution and in the absence of DNA. Fluorescence, UV/Vis spectroscopy and equilibrium dialysis techniques were used. The UV spectroscopy results show that daunomycin induces hypochromicity in the absorption spectra of the core histones. Fluorescence emission intensity is decreased upon daunomycin binding and the process is concentration dependent. The equilibrium dialysis shows that the binding is positive cooperative with the binding sites as Scatchard plot and Hill Coefficient confirm it. The results suggest that daunomycin shows much higher affinity to core histones free in solution than to CL-core, implying that the binding is most likely due to the accessibility of these proteins to the environment. It is suggested that daunomycin binds strongly to open state of histones, such as in tumor cells, rather than to their compact structure seen in normal chromatin.

Multiple drugbinding sites on the R482G isoform of the ABCG2 transporter

Drug-resistant cancer cells frequently display efflux pumps such as P-glycoprotein (P-gp), the multidrug resistance associated protein (MRP1) or the transporter ABCG2. These transporters are each capable of mediating the active efflux of numerous anticancer drugs and display relatively distinct substrate preferences. The last, most recently discovered member, ABCG2, plays a major role in resistance in several types of cancer and the precise pharmacology of this multidrug transporter remain unresolved as does the nature of substrate binding. Plasma membranes from insect cells expressing ABCG2 were used to characterise binding of [3H]daunomycin to the multidrug transporter. The kinetics of association and dissociation for this substrate and several other compounds were also determined in this experimental system. The dissociation constant for [3H]daunomycin binding was 564 +/- 57 nM and a Hill slope of 1.4 suggested cooperative binding. Doxorubicin, prazosin and daunomycin completely displaced the binding of radioligand, while mitoxantrone and Hoechst 33342 produced only a partial displacement. Analysis of the dissociation rates revealed that [3H]daunomycin and doxorubicin bind to multiple sites on the transporter. Both kinetic and equilibrium data support the presence of at least two symmetric drug binding sites on ABCG2, which is distinct from the asymmetry observed for P-gp. The data provide the first molecular details underlying the mechanism by which this transporter is capable of interacting with multiple substrates.

Binding of Antitumor Antibiotic Daunomycin to Histones in Chromatin and in Solution

Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.

DAUNOMYCIN BINDING TO DEOXYPOLYNUCLEOTIDES WITH ALTERNATING SEQUENCES: COMPLETE THERMODYNAMIC PROFILES OF HETEROGENEOUS BINDING SITES

ABSTRACT Complete thermodynamic binding profiles for the interaction of the anticancer drug, daunomycin with natural DNA and synthetic deoxypolynucleotides were described. Fluorescence titration method was used to estimate the equilibrium binding constants. Binding isotherms were found to be surprisingly complex in some cases, presumably because there were heterogeneous sites even in simple deoxypolynucleotides of repeating sequence. Some polynucleotides consisting of alternating sequence contain at least two different binding sites for daunomycin. The binding affinity of the primary binding sites of alternating and non-alternating sequences was found to differ by two orders of magnitude. An isothermal microtitration calorimeter was used to directly measure the binding enthalpy at 25°C with a high sensitivity. The binding enthalpy of poly[d(A-T)] was found to be −5.5 Kcal/mol, which was much lower than any other polynucleotides, while the binding constant of the high affinity sites, was similar. In this report, the complete thermodynamic profiles of daunomycin binding to deoxypolynucleotides were reliably shown for the first time.

Structural and Thermodynamic Analysis of Daunomycin Binding with Desoxyhexanucleotides with Different Base Sequences by NMR Spectroscopy

Complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary desoxyhexanucleotides with different base sequences in the chain, 5′-d(CGTACG) and 5′-d(CGCGCG), is studied in aqueous solution. Homonuclear 2D-1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H–31P NMR spectroscopy (HMBC) are used for complete assignment of the nonexchangeable proton resonances and the phosphorus signals and for qualitative determination of preferable DAU binding sites, respectively. Daunomycin is preferably intercalated into the first d(CG)-site of each hexanucleotide, and the aminosaccharide ring of DAU lies in the minor groove of the hexamer duplex, partly overlapping the third base pair. Quantitative analysis of DAU complexation with desoxyhexanucleotides was done by analyzing the concentration and temperature dependences of the DAU proton chemical shifts; the equilibrium reaction constants and the thermodynamic parameters of the formation of the 1:1, 1:2, 2:1, and 2:2 DAU complexes with hexamers, as well as the limiting values of the DAU proton chemical shifts were determined in aqueous solution. The antibiotic preferably binds with the triplet sections of the nucleotide sequence containing two neighboring CG-pairs of nitrogen bases flanked by the AT-pair in 5′-d(CGTACG) compared to the triplets consisting of a sequence of three CG-pairs in the 5′-d(CGCGCG) hexamer. The specific binding of daunomycin depends on the character of the hydrophobic interactions of the aminosaccharide ring of the antibiotic in the minor groove of the DNA double helix. Calculations have been carried out to determine the most probable spatial structures of the 1:2 DAU–desoxyhexanucleotide complexes; the results agree well with X-ray diffraction data.

Daunomycin-induced Unfolding and Aggregation of Chromatin

Using equilibrium dialysis and sedimentation velocity analysis, we have characterized the binding of the anti-tumor drug daunomycin to chicken erythrocyte chromatin before and after depletion of linker histones and to its constitutive DNA under several ionic strengths (5, 25, and 75 mM NaCl). The equilibrium dialysis experiments reveal that the drug binds cooperatively to both the chromatin fractions and to the DNA counterpart within the range of ionic strength used in this study. A significant decrease in the binding affinity was observed at 75 mM NaCl. At any given salt concentration, daunomycin exhibits higher binding affinity for DNA than for linker histone-depleted chromatin or chromatin (in decreasing order). Binding of daunomycin to DNA does not significantly affect the sedimentation coefficient of the molecule. This is in contrast to binding to chromatin and to its linker histone-depleted counterpart. In these instances, preferential binding of the drug to the linker DNA regions induces an unfolding of the chromatin fiber that is followed by aggregation, presumably because of histone-DNA interfiber interactions.

Contrasting Hydration Changes for Ethidium and Daunomycin Binding to DNA

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTContrasting Hydration Changes for Ethidium and Daunomycin Binding to DNAXiaogang Qu and Jonathan B. ChairesView Author Information Department of Biochemistry University of Mississippi Medical Center 2500 North State Street Jackson, Mississippi 39216-4505 Cite this: J. Am. Chem. Soc. 1999, 121, 11, 2649–2650Publication Date (Web):March 3, 1999Publication History Received21 December 1998Published online3 March 1999Published inissue 1 March 1999https://doi.org/10.1021/ja984366bCopyright © 1999 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views288Altmetric-Citations32LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (30 KB) Get e-AlertsSUBJECTS:Carbohydrates,Genetics,Hydration,Molecules,Stress Get e-Alerts

Deciphering the fluorescence signature of daunomycin and doxorubicin

The fluorescence characteristics of daunomycin (DNM), doxorubicin (DXR), and other anthracycline drugs are often used to monitor localization of the drug within lipid bilayers and liposomal delivery systems and to assess interaction of the drug with DNA and other macromolecules. However, the binding of DNM and DXR to proteins and membrane systems has been observed to exhibit variable effects on the anthracycline's fluorescence. We have delineated the spectroscopic response of DXR and DNM to their surroundings in several systems, including solvents of differing dielectric constant, aqueous solutions of varying pH or fluorophore concentration, and the reverse micellar system of AOT/heptane/water with a range of doxorubicin concentrations. We have observed that the ratio of fluorescence intensities at the two principal λ max values shows a parabolic dependence on solvent dielectric constant, i.e. inverted solvatochromism. This behavior has been overlooked by previous investigators and, together with the appearance of a long-wavelength band near 630 nm in solvents of low dielectric strength (also previously not reported), is key to understanding the partitioning of anthracyclines in membrane systems as well as resolving the conflicting interpretation of data in the literature. © 1998 Elsevier Science B.V. All rights reserved.

Binding of daunomycin to diaminopurine- and/or inosine-substituted DNA.

The binding of the anticancer drug daunomycin to double-helical DNA has been investigated by DNase I footprinting and fluorescence titration, using a series of polymerase chain reaction (PCR) synthesized DNA fragments that contained systematic base substitutions to alter the disposition of functional groups within the minor groove. The 160 bp tyrT DNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substituted for adenine, and (iii) both inosine and diaminopurine were substituted for guanosine and adenine, respectively, were studied. These fragments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experiments confirmed that daunomycin binds preferentially to 5'(A/T)GC and 5'(A/T)CG triplets in the normal fragment. Substitution of inosine for guanosine, with the concomitant loss of the N-2 in the minor groove, weakened binding affinity but did not dramatically alter the sequence preference associated with daunomycin binding. Complete reversal of the location of the N-2 group by the double substitution, however, completely altered the sequence preference of daunomycin and shifted its binding from the canonical triplets to ones with a 5'IDD motif. These results have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating binding to preferred sites. In a parallel study, both macroscopic and microscopic binding to the normal tyrT fragment were investigated, experiments made possible by using PCR to prepare large quantities of the long, defined DNA sequence. The results of these experiments underscored the complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different binding sites. A class of high-affinity sites, most probably corresponding to the 5'(A/T)GC and 5'(A/T)CG triplets, was identified and characterized in macroscopic binding isotherms.