Bleomycins: new methods will allow reinvestigation of old issues

doi:10.1016/j.cbpa.2004.02.008

Current Opinion in Chemical Biology

Volume 8, Issue 2, April 2004, Pages 175-181

https://doi.org/10.1016/j.cbpa.2004.02.008 Get rights and content

Abstract

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.

Introduction

The bleomycins (BLMs, Figure 1) are natural products used clinically in combination chemotherapy in the treatment of germ cell tumors, lymphomas, Kaposi’s sarcoma, cervical cancers, and squamous cell carcinomas of the head and neck [1]. The BLM’s mode of cytotoxicity is thought to be related to their ability to cleave nucleic acids in the presence of their essential metal cofactor(s), O₂ and a reductant 2., 3., 4.. The focus of most investigations has centered on single-stranded (ss) and double-stranded (ds) DNA cleavage events. The difficulty of repairing ds-lesions within DNA has been postulated to be the major source of BLM’s cytoxicity [5]. Recently, RNA has also been implicated as a potential target contributing to BLM’s cytotoxicity [6^•]. The therapeutic efficacy of the BLMs has been hampered by the accompanying pulmonary toxicity that can result in lung fibrosis [1]. New methods to deliver the drug at lower doses and an understanding of the basis for pulmonary toxicity could greatly expand the usefulness of these compounds clinically.

Much has been learned about the structure and function of BLMs in vitro. BLMs can now be synthesized by solution and solid phase methods in a modular fashion, which has facilitated understanding of the functions of the domains of BLMs in nucleic acid recognition and cleavage 3., 7., 8., 9.. The basis of BLM’s sequence specificity and its specific binding to DNA is attributed to the H-bonds between the pyrimidine (3-N and 4-amino group) in the metal-binding domain and the guanine that is 5′ to the cleavage site (2-amino group and the 3-N) [10]. The bithiazole tail is responsible for multiple modes of DNA binding including partial intercalation and binding within the minor groove. The linker domain appears to be essential for the efficiency of ds-DNA cleavage 11., 12.. These recent studies have convincingly demonstrated that the whole of BLM is much greater than the sum of its parts, indicating that any tinkering within these three domains has dramatic effects on the ds-cleavage properties of the drug. Thus, if ds-DNA cleavage is the basis for BLM’s efficacy, manipulation within these three domains might not lead to a better drug. Despite these advances, the role of the sugars (the fourth domain) in BLM remains speculative due to the challenges of synthesizing glycosylated BLMs. Recently, the genes for the biosynthetic pathway for BLMs have been identified and sequenced [13^••]. The putative glycosyl transferases have been identified within the gene clusters that can attach these sugars, presumably to the BLM aglycone. New methods have been developed for the chemical [14] or chemoenzymatic 15., 16. synthesis of nucleoside diphosphate sugars, the substrates for glycosyl transferases. These new advances might facilitate elucidation of the role of the sugars, if promiscuity in sugar substitution is feasible.

Past studies have asked the appropriate questions concerning the active form of the drug and its cytotoxic mechanism in vivo [17]. The availability of new tools is allowing a reinvestigation of these old unresolved issues with the promise of more informative outcomes. These new synthetic and biosynthetic tools will assist in identifying the metal that is coordinated to BLM within the cell, the ligands involved in the coordination of the iron, and determination of the location and concentration of BLM in the cell. The ability to radiolabel BLM in a non-reactive position should allow the study of the uptake of BLM into cells and its subsequent metabolism. Ligation-mediated PCR methods for detection of DNA damage and elucidation of the mechanisms of ds-break repair should allow further investigations on BLM-mediated DNA damage in the nucleus of the cell and the repair of the resulting ds-lesions. A variety of BLM analogs with differing abilities to effect ds-DNA cleavage should establish if ds-cleavage events are correlated with cytotoxicity.

Section snippets

Structure and reactivity of metallo-BLMs

Both iron (Fe²⁺/Fe³⁺) and copper (Cu⁺/Cu²⁺) BLMs have been considered as the active form of BLM in vivo with the former being favored. The ligands and their chiral organization around the metal (iron or copper) have remained controversial due to lack of structural information. Co³⁺-OOH and Zn²⁺-BLMs (inactive forms of BLM), amenable to structural analysis via 2D NMR methods, have provided the structure models for iron and copper BLMs. In the past year, both heteronuclear single-quantum

Ds-DNA cleavage and models for cytotoxicity

A structural model for the mechanism by which a single molecule of BLM can cause ds-cleavage without dissociation from DNA has been proposed by Stubbe and Kozarich [10] (Figure 4a). A recent competing model has been proposed by the Hecht group based on their studies on the Dickerson dodecamer [30] (Figure 4b). The Stubbe/Kozarich model is based on the structure of BLM-Co³⁺-OOH and studies on ds-cleavage using oligonucleotide hairpin technology. The model requires a partial intercalative mode of

Conclusions

Studies in vitro have provided a detailed picture about the chemistry of BLM activation and cleavage of DNA. New tools available will allow a re-examination of BLM’s mode(s) of cytotoxicity in vivo guided by the wealth of knowledge from the in vitro studies. Recent discovery of the biosynthetic genes of BLM and the explosion of knowledge of the glycosyl transferases 37., 38., suggests that BLMs with modifications on the sugars will provide tools to study the role of BLM in vivo. This knowledge

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

•
of special interest
••
of outstanding interest

Acknowledgements

This research has been supported by the National Institute of Health (GM34454).

References (39)

S. Sleijfer
Bleomycin-induced pneumonitis
Chest
(2001)
L.F. Povirk
DNA damage and mutagenesis by radiomimetic DNA-cleaving agents: bleomycin, neocarzinostatin and other enediynes
Mutat Res
(1996)
A.T. Abraham et al.
RNA cleavage and inhibition of protein synthesis by bleomycin
Chem Biol
(2003)
V. Wittmann et al.
1H-tetrazole as catalyst in phosphomorpholidate coupling reactions: efficient synthesis of GDP-fucose, GDP-mannose, and UDP-galactose
J Org Chem
(1997)
M. Sugiyama et al.
The 1.6-Å crystal structure of the copper(II)-bound bleomycin complexed with the bleomycin-binding protein from bleomycin-producing Streptomyces verticillus
J Biol Chem
(2002)
D. Lukoyanov et al.
ENDOR determination of the distance between bleomycin-bound iron and $^{19} F$ of 2′-fluorocytidine in a DNA target sequence
J Am Chem Soc
(2001)
R.M. Burger
Cleavage of nucleic acids by bleomycin
Chem Rev
(1998)
D.L. Boger et al.
Bleomycin: synthetic and mechanistic studies
Angew Chem Int Ed Engl
(1999)
C.A. Claussen et al.
Nucleic acid recognition by metal complexes of bleomycin
Chem Rev
(1999)
K.L. Smith et al.
Deglycobleomycin: solid-phase synthesis and DNA cleavage by the resin-bound ligand
Org Lett
(2002)

S.M. Hecht

Bleomycin: new perspectives on the mechanism of action

J Nat Prod

(2000)

C.J. Thomas et al.

Solid-phase synthesis of bleomycin A(5) and three monosaccharide analogues: exploring the role of the carbohydrate moiety in RNA cleavage

J Am Chem Soc

(2002)

J. Stubbe et al.

Bleomycins: A structural model for specificity, binding, and double strand cleavage

Acc Chem Res

(1996)

D.L. Boger et al.

A systematic evaluation of the bleomycin A(2) l-threonine side chain: Its role in preorganization of a compact conformation implicated in sequence-selective DNA cleavage

J Am Chem Soc

(1998)

D.L. Boger et al.

Definition of the effect and role of the bleomycin A(2) valerate substituents: preorganization of a rigid, compact conformation implicated in sequence-selective DNA cleavage

J Am Chem Soc

(1998)

L. Du et al.

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase

Chem Biol

(2000)

W.A. Barton et al.

Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering

Proc Natl Acad Sci USA

(2002)

W.A. Barton et al.

Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization

Nat Struct Biol

(2001)

D.H. Petering et al.

The role of redox-active metals in the mechanism of action of bleomycin

Chem Biol Interact

(1990)

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Two distinct rotations of bithiazole DNA intercalation revealed by direct comparison of crystal structures of Co(III)•bleomycin A<inf>2</inf> and B<inf>2</inf> bound to duplex 5′-TAGTT sites
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Citation Excerpt :
Despite years of effort, however, our knowledge of several basic aspects of the BLM-DNA interaction remains incomplete.10,13 For example, while models14,15 to explain the DNA cleavage activities of this drug have been influenced by evidence suggesting alternative drug-DNA binding modes,16,17 details of these binding modes and possible accompanying structural reorganizations14 remain to be verified experimentally. Towards our understanding of bleomycin, we reported the first crystal structures of Co(III)•BLMB2 bound to a DNA oligonucleotide.18
Bleomycins constitute a family of anticancer natural products that bind DNA through intercalation of a C-terminal tail/bithiazole moiety and hydrogen-bonding interactions between the remainder of the drug and the minor groove. The clinical utility of the bleomycins is believed to result from single- and double-strand DNA cleavage mediated by the HOO-Fe(III) form of the drug. The bleomycins also serve as a model system to understand the nature of complex drug-DNA interactions that may guide future DNA-targeted drug discovery. In this study, the impact of the C-terminal tail on bleomycin-DNA interactions was investigated. Toward this goal, we determined two crystal structures of HOO-Co(III)•BLMA₂ “green” (a stable structural analogue of the active HOO-Fe(III) drug) bound to duplex DNA containing 5′-TAGTT, one in which the entire drug is bound (fully bound) and a second with only the C-terminal tail/bithiazole bound (partially bound). The structures reported here were captured by soaking HOO-Co(III)•BLMA₂ into preformed host–guest crystals including a preferred DNA-binding site. While the overall structure of DNA-bound BLMA₂ was found to be similar to those reported earlier at the same DNA site for BLMB₂, the intercalated bithiazole of BLMB₂ is “flipped” 180˚ relative to DNA-bound BLMA₂. This finding highlights an unidentified role for the C-terminal tail in directing the intercalation of the bithiazole. In addition, these analyses identified specific bond rotations within the C-terminal domain of the drug that may be relevant for its reorganization and ability to carry out a double-strand DNA cleavage event.
Loss of an ABC transporter in Arabidopsis thaliana confers hypersensitivity to the anti-cancer drug bleomycin
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Bleomycin (BLM) is used as an anti-cancer drug clinically. However, some cancer cells are resistant to BLM, which limits the usage of BLM in chemotherapy. But the underlying mechanism of such resistance is poorly understood. Here we show that the ATP binding cassette (ABC) transporter ABCC3 is required for the BLM-resistance in Arabidopsis. In a genetic screen for ddrm (DNA damage response mutants), we found that loss of ABCC3 confers the hypersensitivity to BLM. In contrast, overexpression of ABCC3 enhances the resistance to BLM. We further found that the expression of ABCC3 is induced by BLM, which is dependent on the protein kinase ATM and the transcription factor SOG1, two master regulators of DNA damage response. Our study revealed that the ABC transporter contributes to BLM-resistance, indicating that the combination of ABC transporter inhibitors and BLM may enhance the efficacy of BLM in cancer therapy.
Electrochemical detection of DNA damage induced by Bleomycin in the presence of metal ions
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Citation Excerpt :
The exact mechanism of the drug cannot be met and different theories about the mechanism of BLM action have been proposed. Based on previous researches [8–11] the antitumor activity of BLM is related to capability for single strain (ss) and double strain (ds) DNA cleavage that various reactions can lead to these degradations. Some reports suggest that in presence of some metal ions, BLM can forms a binary complex and reactive oxygen species (ROS) and eventually destroy DNA [10,11].
A simple and inexpensive electrochemical methodology was used to develop a new method to inspect the biochemical action of Bleomycin (BLM). Furthermore, the effect of some metal ions such as Fe(II), Fe(III), Cu(I), Cu(II), Zn(II), Mn(II) and Co(II) on the operation of BLM was investigated. To prove the role of ionic metals some chelators and reducers were used too. In this study, a pretreated pencil graphite electrode (PGE) modified with gold nanoparticles (AuNPs) and a synthesized zinc(II)-Schiff base complex (ZnSB) decorated with ds-DNA was used to identify DNA damage induced by BLM in the presence of metal ions. The change in the charge transfer resistance (R_ct) using EIS method as well as appearance of 8-Oxo guanine (8-OxoG) peak in DPV method after immersion of the DNA biosensor in BLM solution were used as indicators of DNA damage. Our results showed that the BLM alone cannot destroy DNA and in the other words the BLM has not any interaction with ds-DNA but BLM in the presence of some metal ions can destroy the ds-DNA. By comparing the performance of metal ions and investigation of their effect on action of BLM, the mechanism of DNA damage induced by BLM was presented.
Comparative study of human neuronal and glial cell sensitivity for in vitro neurogenotoxicity testing
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Just one exception was made in the case of the highest concentration chosen for BLM (50 μg/ml), which was selected despite the low viability of SH-SY5Y cells (41%) in order to make a proper comparison with A172 cells. BLM is a well-known clastogenic agent, capable of inducing a wide spectrum of mutagenic lesions in mammalian cells, including DNA base damage, abasic sites, and alkali-labile sites (Chen and Stubbe, 2004; Milic and Kopjar, 2004; Povirk and Austin, 1991), which eventually result in DNA single (SSBs) and double (DSBs) strand breaks. Consequently, it has been extensively used as DNA damage inductor and positive control in genotoxicity assays (Laffon et al., 2010; Mira et al., 2013; Valdiglesias et al., 2013a).
Cell cultures from neuronal and glial origin have proven to be powerful tools for elucidating cellular and molecular mechanisms of nervous system development and physiology, and as neurotoxicity models to evaluate in vitro the possible effects of chemicals. But cellular heterogeneity of nervous system is considerable and these cells have been shown to respond diversely to neurotoxic insults, leading to disparate results from different studies. To shed more light on suitability of cellular models of nervous origin for neurotoxicity screening, the objective of this study was to compare the sensitivity to genetic damage induction of two nervous cell lines. To this aim, neurons (SH-SY5Y) and glial (A172) cells were treated with differently-acting genotoxic agents (bleomycin, actinomycin-D, methyl methanesulfonate, mitomycin C, and griseofulvin). After discarding cytotoxicity, genotoxicity was evaluated by a battery of assays encompassing detection of different genetic lesions. Results obtained showed that glial cells are generally more resistant to genotoxic damage induced by clastogenic agents, but more sensitive to aneugenic effects. These results highlight the need of proper design of in vitro neurotoxicology studies, especially for neurogenotoxicity screening, emphasizing the importance of employing more than one nervous cell type for testing the potential toxicity of a particular exposure.
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A better understanding of the mechanism of action of these molecules would have the potential to lead to better bleomycin therapeutics with lower toxicity, and thus great benefit in medicine. Bleomycin can cause either single stranded or double-stranded cleavage of DNA [11]. When the DNA is cleaved in only a single strand, the second strand remains intact, and as such permits repair processes to operate.
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Recent advances in understanding the structural properties of mononuclear heme-peroxo intermediates are reviewed. The peroxo adducts of hemes are pivotal intermediates generated in the active site of heme enzymes, which catalyze dioxygen activation. The transient nature of the peroxo intermediates under physiological conditions makes isolation and spectroscopic characterizations difficult. Thus, our aim was to generate and capture peroxo intermediates using specifically designed porphyrin complexes at low temperatures, where the cryogenic spectroscopic analyses were performed. Our studies revealed the first reliable resonance Raman (RR) evidence for heme-peroxo intermediate models and provided important insight into the structural mechanism of side-on and end-on (hydro)peroxo-bound hemes.

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Bleomycins: new methods will allow reinvestigation of old issues

Abstract

Introduction

Section snippets

Structure and reactivity of metallo-BLMs

Ds-DNA cleavage and models for cytotoxicity

Conclusions

References and recommended reading

Acknowledgements

Chest

Mutat Res

Chem Biol

J Org Chem

J Biol Chem

J Am Chem Soc

Cleavage of nucleic acids by bleomycin

Chem Rev

Bleomycin: synthetic and mechanistic studies

Angew Chem Int Ed Engl

Nucleic acid recognition by metal complexes of bleomycin

Chem Rev

Deglycobleomycin: solid-phase synthesis and DNA cleavage by the resin-bound ligand

Org Lett

Bleomycin: new perspectives on the mechanism of action

J Nat Prod

Solid-phase synthesis of bleomycin A(5) and three monosaccharide analogues: exploring the role of the carbohydrate moiety in RNA cleavage

J Am Chem Soc

Bleomycins: A structural model for specificity, binding, and double strand cleavage

Acc Chem Res

A systematic evaluation of the bleomycin A(2) l-threonine side chain: Its role in preorganization of a compact conformation implicated in sequence-selective DNA cleavage

J Am Chem Soc

Definition of the effect and role of the bleomycin A(2) valerate substituents: preorganization of a rigid, compact conformation implicated in sequence-selective DNA cleavage

J Am Chem Soc

The biosynthetic gene cluster for the antitumor drug bleomycin from Streptomyces verticillus ATCC15003 supporting functional interactions between nonribosomal peptide synthetases and a polyketide synthase

Chem Biol

Expanding pyrimidine diphosphosugar libraries via structure-based nucleotidylyltransferase engineering

Proc Natl Acad Sci USA

Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization

Nat Struct Biol

The role of redox-active metals in the mechanism of action of bleomycin

Chem Biol Interact