Studies on Bleomycin-DNA and Bleomycin-Iron Interactions

Abstract

The bleomycins, a group of antitumor antibiotics (Figure 1), cause the degradation of DNA by a process requiring iron(II) and dioxygen (1,2). DNA degradation appears to involve two steps: association of the drug with the nucleic acid and degradation of the DNA. As part of studies directed toward achieving an understanding of how the bleomycins degrade DNA, we have examined various properties of the drug using a variety of chemical and physico- chemical techniques, including NMR and Mössbauer spectroscopy. We have studied both the interaction of the antibiotic with its target (DNA) as well as its association with its metal ion cofactor. This work has been performed on the intact drug and its derivatives as well as on synthetic models of the parent drug. This paper reviews and updates the recent work from this laboratory on the bleomycins.     

Deglyco-bleomycin. Degradation of DNA and formation of a structurally unique Fe(II) . CO complex

In analogy with bleomycin, deglyco-bleomycin B2 has been found to form a stable, diamagnetic complex with Fe(II) and CO. Although the stoichiometry of this complex appeared to be the same as that formed with bleomycin, the geometry of the deglyco-bleomycin complex was fundamentally different, especially as regards orientation of the beta-aminoalanine moiety. In the presence of Fe(II) and O2, deglyco-bleomycin A2 and deglyco-bleomycin B2 were found to release [3H]thymine from radiolabeled PM-2 DNA; when employed at limiting concentrations, deglyco-bleomycin A2 and B2 gave about half as much [3H]thymine release as the respective bleomycins. In view of the spectral evidence (Burger, R. M., Horwitz, S. B., Peisach, J., and Wittenberg, J. B. (1979) J. Biol. Chem. 254, 12299-12302) that Fe(II) . bleomycin . CO has the same geometry as the complex formed by initial association of bleomycin, Fe(II), and O2, the accumulated data suggest strongly that all metal complexes of bleomycin (derivatives) capable of DNA degradation need not have the same geometry.     

DNA折纸纳米载体在药物递送中的应用

DNA纳米技术是基于沃森克里克碱基配对原则产生可编程核酸结构的技术。因其具有高精度的工程设计、前所未有的可编程性和内在的生物相容性等特点，运用该技术合成的纳米结构不仅可以与小分子、核酸、蛋白质、病毒和癌细胞相互作用，还可以作为纳米载体，递送不同的治疗药物。DNA折纸作为一种有效的、多功能的方法来构建二维和三维可编程的纳米结构，是DNA纳米技术发展的一个里程碑。由于其高度可控的几何形状、空间寻址性、易于化学修饰，DNA折纸在许多领域具有巨大的应用潜力。本文通过介绍DNA折纸的起源、基本原理和目前进展，归纳总结了运用DNA折纸进行药物装载和释放的方式，并基于此技术，展望了今后的发展趋势以及所面临的机遇和挑战。     

MDM4 enhances p53 stability by promoting an active conformation of the protein upon DNA damage

Stabilization of p53 protein is an important step in the activation of its function. p53 levels are regulated by ubiquitin-dependent and -independent degradation pathways. MDM4 (MDMX) is an important regulator of p53, able to both stimulate and antagonize p53 degradation. Both of these activities have been attributed to the ability of MDM4 to potentiate or antagonize the function of MDM2, the main ubiquitin ligase of p53, depending on their relative levels. Here, we have investigated the stabilizing function of endogenous MDM4 using genetic models of knockout MEFs and RNA interference in human non-transformed cell lines. Our data demonstrate that MDM4 is able to stabilize p53, protecting it from proteasome-mediated degradation in a MDM2- and ubiquitin-independent manner. Upon DNA damage, MDM4 is associated to p53 independently of MDM2 and promotes a conformational change of the protein toward an active form. This correlates with a decreased association of p53 to the proteasome and increased protein levels. The association between MDM4 and p53 is evidenced in the cytoplasmic compartment, supporting the role of cytoplasmic stabilization of p53 during its activation. This work demonstrates that the ability of MDM4 to enhance p53 stability is actually a specific property of MDM4 accomplished upon DNA damage. In addition, these data support the hypothesis of distinct functions of MDM4 under different growth conditions.     

DNA strand scission by activated bleomycin group antibiotics

The bleomycins (BLMs) are a structurally related group of antitumor antibiotics used clinically for the treatment of certain malignancies. The mechanism of action of the BLM is believed to involve DNA strand scission, a process that requires O2 and an appropriate metal ion; the therapeutically relevant metal is probably iron or copper. DNA strand scission by activated Fe X BLM involves oxygenation C-4' of deoxyribose and leads to two sets of products. One set results from scission of the C-3'--C-4' bond of deoxyribose, with concomitant cleavage of the DNA chain. The other set of products consists of free bases and an alkali-labile lesion, the latter of which leads to DNA chain cleavage on subsequent treatment with base. The structures of all of these degradation products have now been established by direct comparison with authentic synthetic samples. Also studied was the activation of BLM with (mono)oxygen surrogates such as iodosobenzene. The chemistry of the activated BLM so formed was remarkably similar to that of activated cytochrome P-450 and structurally related metalloporphyrins, which suggests a mechanistic analogy between the two. Remarkably, both Fe X BLM and Cu X BLM were also shown to be activated by NADPH cytochrome P-450 reductase in a transformation that was dependent on metal ion, O2 and NADPH.     

Bleomycin pulmonary toxicity: production of fibrosis by bithiazole-terminal amine and terminal amine moieties of bleomycin A2

We have previously demonstrated that endotracheal administration of the terminal amines of several bleomycins, when administered as the free amines, produce pulmonary fibrosis of severity comparable to the intact drug. In the present report, bleomycin A2 and its sulfonium ion-containing terminal substituents, with and without the bithiazole rings, were administered to mice endotracheally. The incidence and severity of epithelial metaplasia was greater with the intact drug in comparison to the terminal substituents. In contrast, the terminal substituents and intact drug produced similar degrees of fibrosis. These results underscore the importance of the variable bleomycin terminal substituents in the pathogenesis of pulmonary fibrosis.     

The interaction with DNA of unfused aromatic systems containing terminal piperazino substituents. Intercalation and groove-binding

A number of unfused tricyclic aromatic intercalators have shown excellent activity as amplifiers of the anticancer activity of the bleomycins and the 4',6-diphenylpyrimidines, 2a and 2b, with terminal basic functions (4-methylpiperazino groups) have been synthesized to test the structural requirements for amplifier-DNA interactions. The terminal piperazine rings are bulky, have limited flexibility, and are twisted out of the phenyl ring plane in both 2a and 2b. With 2a the pyrimidine is unsubstituted at position 5 and the conformation predicted by molecular mechanics calculations has a 25-30 degrees twist between the phenyl and pyrimidine ring planes. With 2b the 5-position is substituted with a methyl group and this causes a larger twist angle (50-60 degrees) between the phenyl and pyrimidine planes. These conformational variations lead to markedly different DNA interactions for 2a and 2b. Absorption, CD and NMR spectral, viscometric, flow dichroism and kinetics results indicate that 2a binds strongly to DNA by intercalation while 2b binds more weakly in a groove complex. The general structure and conformation of 2a, a slightly twisted, unfused-aromatic system with terminal piperazino groups is more similar to groove-binding agents such as Hoechst 33258 than to intercalators. The fact that 2a forms a strong intercalation complex with DNA is unusual but in agreement with studies on other amplifiers of anticancer drug action. Molecular modeling studies provide a second unusual feature of the 2a intercalation complex. While most well-characterized intercalators bind with their bulky and/or cationic substitutents in the DNA minor groove, the cationic piperazino groups of 2a are too large to bind in the minor groove in an intercalation complex but can form strong interactions with DNA in the major groove. The tricyclic aromatic ring system of 2a stacks well with adjacent base-pairs in the major-groove complex and the piperazino groups have good electrostatic and van der Waals interactions with the DNA backbone.     

Structural and energetic insights into sequence-specific interaction in DNA–drug recognition: development of affinity predictor and analysis of binding selectivity

Although the molecular mechanism and thermodynamic profile of a wide variety of chemical agents have been examined intensively in the past decades in terms of specific recognition of their protein receptors, to date the physicochemical nature of DNA–drug recognition and association still remains largely unexplored. The present study focused on understanding the structural basis, energetic landscape, and biological implications underlying the binding of small-molecule ligands to their cognate or non-cognate DNA receptors. First, a new method to capture the structural features of DNA–drug complex architecture was proposed and then used to correlate the extracted features with binding affinity of the complexes. By employing this method, a statistical regression-based predictor was developed to quantitatively evaluate the interaction potency of drug compounds with DNA in a fast and reliable manner. Subsequently, we use the predictor to examine the binding behavior of a number of structure-available, affinity-known DNA–drug complexes as well as a large pool of randomly generated DNA decoys in complex with the same drugs. It was found that (1) as compared with protein–DNA recognition, small-molecule agents have relatively low specificity in selecting their cognate DNA targets from the background of numerous random decoys; (2) the abundance of A–T base pairs in the DNA core motif exhibits a significant positive correlation with the affinity of drug ligand binding to the DNA receptor; and (3) high affinity seems not to be closely related to high selectivity for a DNA-targeting drug, although high-affinity drug entities have a greater possibility of being ranked computationally as top binders. We hope that this work will provide a preliminary insight into the molecular origin of sequence-specific interactions in DNA–drug recognition.

Histone H1 subtype preferences of DFF40 and possible nuclear localization of DFF40/45 in normal and trichostatin A-treated NB4 leukemic cells

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.     

Bleomycin A2 and B2 purification by flash chromatography for chemical and biochemical studies.

The clinically used formulation of the anticancer antibiotic, Blenoxane, is a mixture of bleomycin congeners. A new approach to separating the major A2 and B2 congeners has been developed utilizing the flash chromatography technique. A 5-6-inch column of fine mesh silica gel with a solvent system of 1% ammonium formate:methanol (2:3) was used. Low air pressure was applied to the column to increase the flow rate such that separation was complete in approximately 20 min. Reverse phase size exclusion gravity chromatography with Sephadex G-15 column bedding was an effective, rapid procedure for removal of the 1% ammonium formate, the lowest percentage practical for separating the bleomycins. This separation approach does not damage the antibiotics, as demonstrated by NMR spectroscopy, thin layer chromatography, and DNA cleaving activity. Although not as useful for detection of trace amounts of the drug in biological systems as some of the known HPLC methods, this method is excellent for separating large quantities of the drug (8-32 mg) in order to obtain congeners pure enough for synthetic, biochemical, and biophysical studies.     

Amplification of bleomycin-induced DNA cleavage by pyrrole triamide.

We investigated the amplification of bleomycin-induced DNA cleavage by synthetic pyrrole triamide (PyPyPy) using 32P-labeled DNA fragments obtained from human genes. Peplomycin, a kind of bleomycins, plus Fe(II) caused DNA cleavage at the 5'-GC-3' and 5'-GT-3' sequences (damaged bases are underlined). The addition of PyPyPy enhanced the cleavage at cytosine and thymine residues 3' to consecutive guanines, particularly at the 5'-GGGGC-3' and 5'-GGGGT-3' sequences. These results suggest that PyPyPy binds to DNA to induce its conformational change, resulting in alteration of the site specificity and amplification of DNA cleavage. The present study on amplifiers of antitumor drugs would show a novel approach to the establishment of more effective chemotherapy.     

Unraveling the interaction between doxorubicin and DNA origami nanostructures for customizable chemotherapeutic drug release

Doxorubicin (DOX) is a common drug in cancer chemotherapy, and its high DNA-binding affinity can be harnessed in preparing DOX-loaded DNA nanostructures for targeted delivery and therapeutics. Although DOX has been widely studied, the existing literature of DOX-loaded DNA-carriers remains limited and incoherent. Here, based on an in-depth spectroscopic analysis, we characterize and optimize the DOX loading into different 2D and 3D scaffolded DNA origami nanostructures (DONs). In our experimental conditions, all DONs show similar DOX binding capacities (one DOX molecule per two to three base pairs), and the binding equilibrium is reached within seconds, remarkably faster than previously acknowledged. To characterize drug release profiles, DON degradation and DOX release from the complexes upon DNase I digestion was studied. For the employed DONs, the relative doses (DOX molecules released per unit time) may vary by two orders of magnitude depending on the DON superstructure. In addition, we identify DOX aggregation mechanisms and spectral changes linked to pH, magnesium, and DOX concentration. These features have been largely ignored in experimenting with DNA nanostructures, but are probably the major sources of the incoherence of the experimental results so far. Therefore, we believe this work can act as a guide to tailoring the release profiles and developing better drug delivery systems based on DNA-carriers.     

Bleomycins: new methods will allow reinvestigation of old issues

The bleomycins (BLMs) are used clinically in combination chemotherapy, their clinical usefulness being limited by the accompanying pulmonary toxicity. Much has been learned about the structure and function of BLMs in vitro. However, the mechanism of their cytoxicity in vivo, including their target(s), metal cofactor(s) effecting nucleic acid cleavage and its (their) oxidation state, concentrations of BLM in the nucleus of the cell, BLM metabolism, hot spots for double-strand DNA cleavage, and their repair, have remained elusive. New methods offer new opportunities to revisit and solve old problems, which could ultimately lead to development of a more effective therapeutic.     

Three-dimensional modeling of cytomegalovirus DNA polymerase and preliminary analysis of drug resistance

Cytomegalovirus (CMV) is the leading cause of congenital infection and a frequent opportunistic agent in immunocompromised hosts such as transplant recipients and AIDS patients. CMV DNA polymerase, a member of the polymerase B family, is the primary target of all available antivirals (ganciclovir, cidofovir, and foscarnet) and certain variations of this enzyme could lead to drug resistance. However, understanding the drug resistance mechanisms at the atomic level is hampered by the lack of its three-dimensional (3D) structure. In the present work, 3D models of two different conformations (closed and open) for CMV DNA polymerase have been built based on the crystal structures of bacteriophage RB69 DNA polymerase (a member of the polymerase B family) by using the 3D-Jury Meta server and the program MODELLER. Most of the variations on CMV DNA polymerase pertinent to ganciclovir/cidofovir and foscarnet resistance can be explained well based on the open and closed conformation models, respectively. These results constitute a first step towards facilitating our understanding of drug resistance mechanisms for CMV and the interpretation of novel viral mutations.     

Cdc13 telomere capping decreases Mec1 association but does not affect Tel1 association with DNA ends

Chromosome ends, known as telomeres, have to be distinguished from DNA breaks that activate DNA damage checkpoint. Two large protein kinases, ataxia-teleangiectasia mutated (ATM) and ATM-Rad3-related (ATR), control not only checkpoint activation but also telomere length. In budding yeast, Mec1 and Tel1 correspond to ATR and ATM, respectively. Here, we show that Cdc13-dependent telomere capping attenuates Mec1 association with DNA ends. The telomeric TG repeat sequence inhibits DNA degradation and decreases Mec1 accumulation at the DNA end. The TG-mediated degradation block requires binding of multiple Cdc13 proteins. The Mre11-Rad50-Xrs2 complex and Exo1 contribute to DNA degradation at DNA ends. Although the TG sequence impedes Exo1 association with DNA ends, it allows Mre11 association. Moreover, the TG sequence does not affect Tel1 association with the DNA end. Our results suggest that the Cdc13 telomere cap coordinates Mec1 and Tel1 accumulation rather than simply covering the DNA ends at telomeres.     

Effect of deoxyribonucleic acid replication inhibitors on bacterial recombination.

Two inhibitors of replicative deoxyribonucleic acid (DNA) synthesis, nalidixic acid (NAL) and 6-(p-hydroxyphenylazo)-uracil (HPUra), showed different effects on genetic recombination and DNA repair in Bacillus subtilis. Previous work (Pedrini et al., 1972) showed that NAL does not interfere with the transformation process of B. subtilis. The results reported in this work demonstrated that the drug was also without effect on the transfection by SPP1 or SPO-1 phage DNA (a process that requires a recombination event). The drug was also ineffective on the host cell reactivation of ultraviolet-irradiated SPP1 phage, as well as on transfection with ultraviolet-irradiated DNA of the same phage. HPUra instead markedly reduced the transformation process, as well as transfection, by SPO-1 DNA, but it did not affect the host cell reactivation of SPO-1 phage. In conclusion, whereas the NAL target seems to be specific for replicative DNA synthesis, the HPUra target (i.e., the DNA polymerase III of B. subtilis) seems to be involved also in recombination, but not in the excision repair process. The mutations conferring NAL and HPUra resistance used in this work were mapped by PBS-1 transduction.     

Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.