Binding Of Mitoxantrone Research Articles

Interactions of pixantrone with apurinic/apyrimidinic sites in DNA.

Pixantrone and mitoxantrone are structurally related anticancer drugs which have been shown to generate covalent conjugates at apurinic/apyrimidinic (AP) sites in DNA. Mitoxantrone binding to AP sites induces DNA strand cleavage and inhibits the endonuclease activity of human AP endonuclease 1 (APE1). Here, pixantrone was demonstrated to have similar properties, but relative to mitoxantrone, it was significantly less potent in both DNA incision and APE1 inhibition. Consistent with these observations, pixantrone had ~ 15-fold lower affinity for DNA containing an AP site analogue, tetrahydrofuran, as measured by a Thiazole Orange (ThO) displacement assay.

MOSR and NDHA Genes Comprising G-Quadruplex as Promising Therapeutic Targets against Mycobacterium tuberculosis: Molecular Recognition by Mitoxantrone Suppresses Replication and Gene Regulation.

Occurrence of non-canonical G-quadruplex (G4) DNA structures in the genome have been recognized as key factors in gene regulation and several other cellular processes. The mosR and ndhA genes involved in pathways of oxidation sensing regulation and ATP generation, respectively, make Mycobacterium tuberculosis (Mtb) bacteria responsible for oxidative stress inside host macrophage cells. Circular Dichroism spectra demonstrate stable hybrid G4 DNA conformations of mosR/ndhA DNA sequences. Real-time binding of mitoxantrone to G4 DNA with an affinity constant ~105-107 M-1, leads to hypochromism with a red shift of ~18 nm, followed by hyperchromism in the absorption spectra. The corresponding fluorescence is quenched with a red shift ~15 nm followed by an increase in intensity. A change in conformation of the G4 DNA accompanies the formation of multiple stoichiometric complexes with a dual binding mode. The external binding of mitoxantrone with a partial stacking with G-quartets and/or groove binding induces significant thermal stabilization, ~20-29 °C in ndhA/mosR G4 DNA. The interaction leads to a two/four-fold downregulation of transcriptomes of mosR/ndhA genes apart from the suppression of DNA replication by Taq polymerase enzyme, establishing the role of mitoxantrone in targeting G4 DNA, as an alternate strategy for effective anti-tuberculosis action in view of deadly multi-drug resistant tuberculosis disease causing bacterial strains t that arise from existing therapeutic treatments.

Exploring the binding of mitoxantrone to DNA using optical tweezers.

Mitoxantrone (MTX) is a synthetic anti-cancer drug that displays intercalation binding mode with DNA. It has been widely studied using conventional methods, but not using single molecule techniques which can provide molecular level details of the interactions. Here we explored the binding of MTX with DNA using optical tweezers. Our optical tweezers set-up uses two counter propagating lasers to trap and manipulate a single DNA molecule that is stretched to measure the force response as a function of extension. The DNA stretching was repeated in the presence of various nano-molar concentrations of MTX at different pulling rates. Stretching at much slower pulling rates exhibited closer to binding equilibrium, indicating MTX is a slow binder with the time scale of seconds compared to classical intercalators which have microsecond binding kinetics. This will provide more detail about the binding mechanism of MTX that can be used to better understand the binding of MTX and also develop similar drug designs.

Intercalation of anticancer drug mitoxantrone into DNA: Studied by spectral and surface plasmon resonance methods

Studying DNA interaction with different drugs is interesting and important in discovering the interaction mechanism and developing novel drugs. Mitoxantrone (MTX) is a potent antitumor drug used to treat certain types of cancer. In this work, we evaluated the binding strength of MTX to 16 base pair double strands (ds) DNA. For the first time, this work investigates the intercalation of anticancer drugs into DNA by spectral fluorescence spectroscopy and SPR (surface plasmon resonance) based methods. Furthermore, the thermodynamic and kinetic parameters, including KD, KA, entropy change (ΔS), enthalpy change (ΔH), and ΔG, were calculated using different concentrations of ds DNA at three different temperatures. The results indicated that MTX binding to DNA was an exothermic and spontaneous process. The negative value of both ΔH and ΔS calculated by SPR and fluorescence data analysis showed that hydrogen bonding was the major binding force for the complexation of MTX to DNA. In addition, fluorescence data analysis revealed that MTX could quench the DNA fluorescence via the static quenching mechanism. This study shows that the investigation of intercalation anticancer drugs into DNA can be studied by spectral methods and can be investigated using SPR based method, and obtained data from the SPR method is as reliable as the fluorescence method. The present work shows hydrogen bonding is the major binding force in intercalating MTX to DNA.

Mitoxantrone stacking does not define the active or inactive state of USP15 catalytic domain

Ubiquitin specific protease USP15 is a deubiquitinating enzyme reported to regulate several biological and cellular processes, including TGF-β signaling, regulation of immune response, neuro-inflammation and mRNA splicing. Here we study the USP15 D1D2 catalytic domain and present the crystal structure in its catalytically-competent conformation. We compare this apo-structure to a previous misaligned state in the same crystal lattice. In both structures, mitoxantrone, an FDA approved antineoplastic drug and a weak inhibitor of USP15 is bound, indicating that it is not responsible for inducing a switch in the conformation of active site cysteine in the USP15 D1D2 structure. Instead, mitoxantrone contributes to crystal packing, by forming a stack of 12 mitoxantrone molecules. We believe this reflects how mitoxantrone can be responsible for e.g. nuclear condensate partitioning.We conclude that USP15 can switch between active and inactive states in the absence of ubiquitin, and that this is independent of mitoxantrone binding. These insights can be important for future drug discovery targeting USP15.

NMR based structure reveals groove binding of mitoxantrone to two sites of [d-(TTAGGGT)]4 having human telomeric DNA sequence leading to thermal stabilization of G-quadruplex

Interaction of mitoxantrone (MTX) with G-quadruplex, leading to inhibition of telomerase enzyme and anticancer action, is not understood. Titrations of MTX with [d-(TTAGGGT)]4, comprising human telomere single repeat sequence, have been monitored by fluorescence and 1H/31P NMR spectroscopy. Binding induces chemical shift changes in GNH (~0.3ppm), T2/T7/A3 base protons and sequence specific shifts in 31P resonances. Absence of large downfield shifts in 31P signals and presence of all sequential NOEs show that classical intercalation of drug between base quartets does not occur. The upfield shift (~0.53ppm) in 2/3H, 1/4OH and minor shift in 6/7H aromatic protons of MTX in complex rule out end stacking as a possible mode of interaction. The 26 short inter molecular contacts of 1/4OH, 11NH, 6/7H and 12CH2 protons of MTX with T1, T2, G6, G7 protons in the structure of complex obtained by restrained Molecular Dynamics simulations show binding at grooves accompanied by 4 hydrogen bonds and partial stacking of MTX with base pairs. Interaction causes thermal stabilization, ΔTm=35°C, which may be attributed to telomerase inhibition. The present study is the first report on solution structure of mitoxantrone-[d-(TTAGGGT)]4 complex and is significant for structure based designing of anthraquinone derivatives as future drugs.

Thermodynamic parameters analysis of ethidium bromide and mitoxantrone binding with DNA by adsorption isotherms

Analysis of the adsorption isotherms by virtue of experimentally obtained data of anti-tumorous compound mitoxantrone and ethidium bromide binding with DNA has been carried out. The obtained data showed that throughout the comparatively simple linear isotherm, the more precise values of the binding constant K and number of nucleotides n, per binding site were received. Based on the values of K, those for the enthalpy changes at complex-formation of these ligands with DNA were obtained.

NMR structure of dual site binding of mitoxantrone dimer to opposite grooves of parallel stranded G-quadruplex [d-(TTGGGGT)

The formation of complex between anti-cancer drug mitoxantrone (MTX) and tetra-molecular parallel G-quadruplex DNA [d-(TTGGGGT)]4 has been studied by solution state one and two dimensional NMR spectroscopy. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. The Job's method of continuous variation and thermal melting studies independently ascertain binding stoichiometry of 4:1 in mitoxantrone:DNA complex. The existence of only four guanine NH peaks corresponding to the four G-quartets during the course of titration shows that C4 symmetry of G-quadruplex is intact upon binding of mitoxantrone. The specific inter molecular short distance contacts between protons of two mitoxantrone molecules of dimer, that is, ring A protons with ring C and side chain methylene protons, confirms the formation of mitoxantrone head-to-tail dimer. The observed 38 Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and G-quadruplex DNA indicate formation of a well-defined complex. The three dimensional structure of 4:1 mitoxantrone:[d-(TTGGGGT)]4 complex computed by using experimental distance restraints followed by restrained Molecular Dynamics (rMD) simulations envisages the critical knowledge of specific molecular interactions within ligand-G-quadruplex complex. The findings are of direct interest in development of anti-cancer therapeutic drug based on G-quadruplex stabilization, resulting in telomerase inhibition.

A 4:1 stoichiometric binding and stabilization of mitoxantrone-parallel stranded G-quadruplex complex established by spectroscopy techniques

Small molecule ligands which specifically bind and stabilize G-quadruplex structures in telomeric ends inhibit the activity of telomerase enzyme, an important marker for cancer. Understanding of the binding mode of ligand-G quadruplex complex is important for evaluating relative efficacy of anti-tumor drugs. The present study is focused on interaction of anti-tumor drug mitoxantrone (MTX) with tetra-molecular parallel stranded G-quadruplex sequence d-TTGGGGT using absorbance, fluorescence and circular dichroism spectroscopy techniques. Absorbance of mitoxantrone shows hypochromism up to MTX (D)/DNA quadruplex (N) ratio ~5, followed by hyperchromism up to D/N=0.21 accompanied by a red shift of 15nm. The fluorescence emission of MTX shows decrease up to D/N ~5 and then increases with red shift of 8nm. The two observed fluorescent lifetimes, 0.17ns (91%) and 0.44ns (9%), indicate dual binding mode. Absence of isobestic and isoemissive point indicates presence of multiple complexes. Circular Dichroism spectra showing positive induced band at 645nm and two exciton bands centered at 619 and 664nm suggest binding of mitoxantrone as a dimer. Proton NMR studies show intermolecular MTX-MTX short contacts confirming existence of stacked dimer of MTX. Thermal melting transitions of DNA saturate at D/N=4 with ΔTm=25°C. The results establish highly specific external groove binding of 4 molecules of mitoxantrone as two dimers at two distinct sites of DNA.

Theoretical study of functionalized single-walled carbon nanotube (5, 5) with Mitoxantrone drug

Objective(s): First principles calculations were performed to study four multiple sclerosis drugs namely, Ampyra, Fingolimod, Mitoxantrone and Eliprodil in gas and liquid phases using Density Functional Theory (DFT). Computational chemistry simulations were carried out to compare calculated quantum chemical parameters for Ampyra, Fingolimod, Mitoxantrone and Eliprodil. Materials and Methods: All calculations were performed using DMol3 code which is based on DFT. The Double Numerical basis set with Polarization functions (DNP) was used.Results: Mitoxantrone has highest HOMO energy, global softness, solvation energy and molecular mass and lowest LUMO energy, energy gap, global hardness and total energy in comparison to Ampyra, Fingolimod and Eliprodil in gas and solvent phases. Calculations were carried out to study the interaction of covalently binding Mitoxantrone to functionalized carbon nanotube. The Mitoxantrone local reactivity was studied through the Fukui indices in order to predict both the reactive centers and the possible sites of nucleophilic and electrophilic attacks. The Mitoxantrone binding energy is calculated to be 6.507 eV in gas phase and -9.943 eV in solvent phase that is a decrease in BE as the drug phase changes from gas to liquid.Conclusion: The simulation results show Mitoxantrone is quite a reactive drug. The quantum chemical parameters of pristine nanotube and f-SWNT-Mitoxantrone showed that reactivity of f-SWNT-Mitoxantrone increased in comparison to pristine nanotube in both phases.

Antitumor Drug Mitoxantrone Do Not Preclude Salt-driven B-Z Transition of Poly (dG- dC)

Despite of the existence in literature of large number of reports the binding modes of mitoxantrone (MTX) with DNA must be subject to refinement. Moreover there is no any report yet on interaction of MTX with Z-DNA. Here we present results of circular dichroism (CD) studies on MTX binding to salt driven left-handed conformation of poly (dG- dC). It was shown, that in contrast with classical intercalating ligands (ethidium bromide, acridine orange etc.) that bind with B-form DNA and obstacle B-Z transition at high concentrations of NaCl MTX interferes B-Z transition, but does not prevent the transitions process, thus suggest the semi intercalating (partial stacking) mode of MTX interaction with Z-form of DNA.

Intercalative interaction of the anticancer drug mitoxantrone with double stranded DNA: A calorimetric characterization of the energetics

The interaction of the anticancer agent mitoxantrone with DNA was investigated using microcalorimetry. The results show that the binding of mitoxantrone to DNA was predominantly enthalpy driven with a small but favorable entropic contribution. The equilibrium constant for the binding reaction (mitoxantrone+DNA=mitoxantrone−DNA complex) was calculated to be (5.05±0.06)·106M−1 at T=298.15K and the binding stoichiometry value show that ∼2 base pairs were spanned by each mitoxantrone molecule. The equilibrium constant decreased, the standard molar enthalpy change increased and the standard molar entropy change decreased with increasing temperature. However, the change in temperature had little effect on the standard molar Gibbs energy change. The negative value of the standard molar heat capacity change along with enthalpy–entropy compensation behavior suggests the involvement of dominant hydrophobic forces in the binding process. A small but favorable electrostatic component to the binding was revealed from salt dependent calorimetric data and the parsing of the standard molar Gibbs energy change. The binding increased the thermal stability of DNA and the value of the equilibrium constant calculated from the melting data was (6.26±0.17)·106M−1.

Chromium(VI) but Not Chromium(III) Species Decrease Mitoxantrone Affinity to DNA

Binding of mitoxantrone (MXT) to double-stranded DNA has been investigated as a model drug-DNA binding system to evaluate the effects of various forms of chromium on the binding properties. We have found that Cr(III), which binds strongly to DNA, does not affect the MXT affinity to DNA. In contrast, Cr(VI), in the form of chromate ions CrO(4)(2-), decreases the MXT affinity to DNA despite electrostatic repulsions with phosphate-deoxyribose chains of DNA. The MXT-DNA binding constant was found to decrease from (1.96 ± 0.005) × 10(5) to (0.77 ± 0.018) × 10(5) M(-1) for Cr(VI) concentration changing from 0 to 30 μM. The influence of Cr(VI) on MXT-DNA binding has been attributed to the oxidation of guanine residue, thus interrupting the intercalation of MXT into the DNA double helix at the preferential CpG intercalation site. This supposition is corroborated by the observed increase in the MXT binding site size from 2 bp (base pairs) to 4-6 bp in the presence of Cr(VI). The measurements of the MXT-DNA binding constant and the MXT binding site size on a DNA molecule have been carried out using spectroscopic, voltammetric, and nanogravimetric techniques, providing useful information on the mechanism of the interactions.

A Physiologically Based Pharmacokinetic Model of Mitoxantrone in Mice and Scale-up to Humans: a Semi-Mechanistic Model Incorporating DNA and Protein Binding

We conducted a pharmacokinetic (PK) study of mitoxantrone (Novantrone®), a clinically well-established anticancer agent, in mice and developed a mechanism-based PBPK (physiologically based pharmacokinetic) model to describe its disposition. Mitoxantrone concentrations in plasma and six organs (lung, heart, liver, kidney, spleen, and brain) were determined after a 5mg/kg i.v. dose. We evaluated three different PBPK models in order to characterize our experimental data: model 1 containing Kp values, model 2 incorporating a deep binding compartment, and model 3 incorporating binding of mitoxantrone to DNA and protein. Among the three models, only model 3 with DNA and protein binding captured all the experimental data well. The estimated binding affinity for DNA (K (DNA)) and protein (K (macro)) were 0.0013 and 1.44μM, respectively. Predicted plasma and tissue AUC values differed from observed values by <19 %, except for heart (60 %). Model 3 was further used to simulate plasma mitoxantrone concentrations in humans for a 12-mg/m(2) dose, using human physiological parameters. The simulated results generally agreed with the observed time course of mitoxantrone plasma concentrations in patients after a standard dose of 12mg/m(2). In summary, we reported for the first time a mechanism-based PBPK model of mitoxantrone incorporating macromolecule binding which may have clinical applicability in optimizing clinical therapy. Since mitoxantrone is a substrate of the efflux transporters ABCG2 and ABCB1, the incorporation of efflux transporters may also be necessary to characterize the data obtained in low-dose studies.

Thermodynamical study of interaction of histone H1 chromosomal protein and mitoxantrone anticancer drug

Using ultraviolet spectroscopy technique, we have investigated the interaction of anticancer drug, mitoxantrone with calf thymus histone H1 chromosomal protein in 100 mM phosphate buffer, pH 7.0, at temperatures 300 and 310 K. UV spectroscopy results show interactions between mitoxantrone and histone H1 with a positive cooperative binding process which was confirmed by Scatchard plot. According to the obtained results, it is concluded that histone H1 can be considered as a target for mitoxantrone binding at the chromatin level.

Calorimetric and spectroscopic studies on the interaction of anticancer drug mitoxantrone with human serum albumin

Binding of the anticancer drug mitoxantrone with the protein human serum albumin (HSA) has been studied by using isothermal titration calorimetry (ITC), in combination with fluorescence, UV–visible, and circular dichroism spectroscopy. The thermodynamic parameters of binding have been evaluated from ITC and spectroscopic results and compared. The ITC results demonstrate that the binding of mitoxantrone with HSA occurs according to two sets of binding sites on the protein as opposed to the fluorescence and UV–visible spectroscopic results. Blockage of one binding site on HSA for mitoxantrone in the presence of NaCl indicates strong involvement of electrostatic interactions in the binding of the drug with the protein. An insignificant temperature dependence of the association constant observed in fluorescence measurements suggests a very low enthalpy of binding which is in close agreement with the results obtained from ITC measurements. Fluorescence life time measurements suggest formation of a static complex between mitoxantrone and HSA. The discrepancies in the ITC and fluorescence results suggest that one of the binding sites on the protein for mitoxantrone does not contain tryptophan residue in its immediate vicinity. The calorimetric and spectroscopic results have provided quantitative information on the binding of mitoxantrone with HSA and suggest that the binding is dominated by electrostatic interactions.

Spectral Studies on the Molecular Interaction of Anticancer Drug Mitoxantrone with CTAB Micelles

The interaction of anticancer drug mitoxantrone with cationic surfactant cetyltrimethylammonium bromide (CTAB) has been investigated by absorption spectroscopy as a function of surfactant concentration ranging from the premicellar to postmicellar region at pH 7.4 and 10. Interaction of mitoxantrone with CTAB micelles induces a bathochromic shift of both absorption maxima and spectral data showed that the micellization reduces the dimerization process and mitoxantrone is bound into micelles in the monomeric form. Binding constant and partition coefficient were estimated using the red shifts of the absorption maxima in the presence of surfactant. From the resulting binding constants for mitoxantrone-surfactant interactions, it was concluded that the hydrophobic interactions have a great effect on the binding of mitoxantrone to CTAB micelles. Also, by comparing the partition coefficients obtained using pseudo-phase model, the hydrophobic interactions have a major role in the distribution of mitoxantrone between micelle-water phases. Gibbs free energy of binding and distribution of mitoxantrone between the bulk aqueous medium and surfactant micelles were calculated.

Mitoxantrone Induced Impediment of Histone Acetylation and Structural Flexibility of the Protein

Mitoxantrone (MTX), a choice of drug in cancer chemotherapeutic regime, is a potent and less toxic among anthracycline class of drugs. Here, we study the molecular interaction of MTX, with histone and its acetylation dynamics. Its binding with histone core protein was predicted with CD and UV-visible spectroscopic techniques. The MTX-protein complex resulted in the impediment of the histone acetyltransferase (HAT) activity in a dose dependent manner on MTX binding. Interestingly, the concentration dependent reduction in acetylated state of specific lysines K9/K14 was also observed on MTX treatment in vivo. The molecular distance r, between donor (histone H3) and acceptor (MTX) was estimated using Förster's theory of non-radiation energy transfer and the detailed binding phenomenon was expounded. MTX binding site near N-terminal lysines is characterized with an association constant of the order of 10(4). The positive thermodynamic values of both ∆H° and ∆S° were suggestive that the hydrophobic interactions dominate in MTX-protein binding. The binding site allocation predicted by computational modeling placed the drug molecule near N-terminal lysine K9 and K14 of histone H3, and corroborate with the thermodynamic interaction model. The study establishes that MTX-histone interaction affects protein acetylation state and also provided a mechanistic model for its binding. Hence, MTX interaction may affect chromatin structure and implicates its role in transcriptional regulation at epigenetic level.

Interaction of mitoxantrone, as an anticancer drug, with chromatin proteins, core histones and H1, in solution

In the present study, for the first time we have investigated the interaction of anticancer drug mitoxantrone with histone H1 and core histone proteins in solution using fluorescence, UV/Vis, CD spectroscopy and thermal denaturation techniques. The results showed that mitoxantrone reduced the absorbencies of H1 and core histone proteins at 210 nm (hypochromicity) and fluorescence emission intensity was decreased in a dose dependent. Binding of mitoxantrone changed secondary structures of the proteins as circular dichroism analysis confirmed it. Also, mitoxantrone increased the melting temperature of core histones at the final step of denaturation. The results suggest higher affinity of mitoxantrone to histone H1 compared to core histones providing histone proteins as a new target for mitoxantrone action at the chromatin level.

Inhibition of transcription factor assembly and structural stability on mitoxantrone binding with DNA

MTX (mitoxantrone) is perhaps the most promising drug used in the treatment of various malignancies. Comprehensive literature on the therapeutics has indicated it to be the least toxic in its class, although its mechanism of action is still not well defined. In the present study, we have evaluated the associated binding interactions of MTX with naked DNA. The mechanism of MTX binding with DNA was elucidated by steady-state fluorescence and a static-type quenching mechanism is suggested for this interaction. Thermodynamic parameters from van 't Hoff plots showed that the interaction of these drugs with DNA is an entropically driven phenomenon. The binding mode was expounded by attenuance measurements and competitive binding of a known intercalator. Sequence specificity of these drug-DNA complexes was analysed by FTIR (Fourier-transform infrared) spectroscopy and molecular modelling studies. CD spectroscopy and the plasmid nicking assay showed that the binding of this drug with DNA results in structural and conformational perturbations. EMSA (electrophoretic mobility-shift assay) results showed that these drug-DNA complexes prevent the binding of octamer TF (transcription factor) to DNA. In summary, the study implicates MTX-induced conformational instability and transcription inhibition on DNA binding.