1

Bleomycin

„the classic DNA-cleaving anti-cancer antibiotic“1

Team W:

Jan Hegen, Niklas Hummel,

Vanessa Kohl, Theresa Kissel

Module:

B.BME 23:

Studienprojekt zur Fachinformation "DaMocles"

Semester:

SS 2013

1 http://tuprints.ulb.tu-darmstadt.de/epda/pics/athene.gif

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Content

Basic Information

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Synthesis

4

Mode of Functioning

6

Bleomycin applications

9

References 12

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Basic Information

Bleomycin is an antibiotic and refers to the family/group of glycopetide antibiotics. It was

discoverd in 1962, where it was isolated from the actinobacterium Streptomyces verticillus.

Glycopetide antibiotics are a class of antibiotic drugs that solely affect Gram-positive bacertia

causing cell lysis. For pharmaceutical purposes water-soluble Bleomycin salts ( e.g. sulfates,

hydrochlorides) are used as cytostatics. They are utilized as an anti-cancer agents. The drug

is mainly used in the treatment of testicular cancer, Hodgkin’s lymphoma and cystic hygroma.

The antibiotic consists of two single compounds: to 55% to 70%

dimethylsulfoniumaminopropyl-derivate (bleomycin A2) and 25% - 32% out of the agmatin-

derivate (bleomycin B2). Before administration bleomycin needs to be dissolved in an

isotonic saline (solution) because it is applied intravenously. The antibiotic inhibits the

proliferation and the growth of the cells. In a certain extent it can also influence the RNA and

protein biosynthesis. Consequently bleomycin inhibits the replication and function in specific

phases (G2 and M-phase) of the cell cycle.

Bleomycin consists out of three different domains:

1. The DNA-binding domain consists of a bithiazole intercalator and a positively charged

guanidinium (B2) respectively a sulfonic group (A2)

2. The metal-binding domain contains a pyrimidine, a β-aminoalanine and a β-

hydroxyimidazole, which are involved in the formation of a stable bleomycin –metalcation-

complex. The named regions are in each case linked by a tripeptide.

3. The carbohydrate domain is not necessary for the DNA cleavage. The α-L-Glucose and

the α-D-Mannose disaccharide are responsible for the accumulation of the antibiotic in the

specific cancer cells.

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2 http://medlibrary.org/lib/images-rx/bleomycin-3/bleomycin-for-injection-usp1-figure-1-gbleomycin.jpg

DNA-binding domain (Bithiazol- domain)

metal-binding domain

carbohydrate domain

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Synthesis

There are various ways of synthesizing Bleomycin. Of special interest is the Bleomycin

aglycon biosynthesis in Streptomyces verticillus. The synthesis of the aglycone, which is

build of of two short peptide chains and one polyketide segment in between them, is

controlled and katalysized by the hybrid enzyme Bleomycin megasynthetase.

Bleomycin megasythetase is a combination out of a nonribosomal peptide synthetase

(NRPS) and a polyketide synthase (PKS). The hybrid of of these two enzymes works, based

on similarity of polypetide and polyketide synthesis, both are polycondensations. At the

beginning of the synthesis a serine, two asparagine, a Histidine and a Alanine are connceted

via a linear polycondensation to form the first oligopeptide. This step is highly selective. The

following step is the critical one, the oligoketide segmentis added. In this case it a Malate is

added first. After that, the resulting ß-ketothioester is reduced at the ß-keto group and

methylated in the ß-position. After transitioning the oligopeptide-oligopolyketide-hybrid to the

NRPS module, three more amino acids (ß-Ala, L-Cys, L-Cys) are continuously added. This

completes the aglycone.

By adding sugars at the Histidin and changing the C-terminus at the last Cystein Bleomycin

derivates can be synthesized.

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3 B.Shen et al. 2001

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The NRPS/PKS, NRPS/PKS Transition:

The figure beneath shows the transition from NRPS module to PKS module and thereafter

the transition from PKS module to NRPS module. For correct transitions from NRPS module

to PKS module the communication between the ketoacyl synthase (KS) and the peptidyl

carrier protein (PCP) is crucial. In normal PKS the KS catalyzes the transfer of the acyl-S-

acyl carrier Protein (ACP) from the upstream module to the active site cysteine of KS

(nucleophilic substitution) and the decarboxylative condensation between the acyl-S-KS and

malonyl-S-ACP. In the hybride enzyme bleomycin megasynthetase the KS transfers the

peptidyl-S-PCP from the upstream module to the active site cysteine of KS and then

catalyzes the decarboxylative condensation with malonyl-S-ACP. The KS-domain differs from

the KS-domain in normal PKS, while the PCP-domain does not really from other PCP-

domains. This means, that the KS-domain´s substrate selectivity changed, so it uses the

peptidyl-S-PCP. The ß-ketothioester is connected to the ACP of the PKS.

After the ß-keto group reduction and the methylation in ß-position the next important step

follows, the transition from PKS module to the downstream NRPS module. Usually the

Condensation(C)-domain catalyzes the nucleophilic substitution of peptidyl-S-PCP and acyl-

S-PCP by mediation of the C-N bond. The hybrid C-domain instead mediates the C-N bond

of the following L-Threonin, connected to the S-PCP, to substitute the acyl-S-ACP of the PKS

module. In this case the structure of PKS ACP is more similar to PCP. That means, the C-

domain can easily detect the ACP as a PCP and catalyze the reaction. After this step the

“peptide/polyketide” chain is connected to the next NRPS PCP-domain and the chain can

grow on with following NRPS modules.

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4 A, adenylation; ACP, acyl carrier protein ; AT, acyltransferase ; C, condensation ; KS, ketoacyl

synthase; PCP, peptidyl carrier protein 5 B.Shen et al. 2001

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Mode of functioning

The interaction of bleomycin with the DNA contains a two-stage process:

In a first step the antibiotic binds to the DNA. The flat aromatic system of the bithiazol domain

intercalates between two guanine bases particularly in G-T and G-C-rich sequences. The

interaction of the bithiazol domain with the DNA occurs sequence specific. Initially the amin-

substituent of the DNA-binding-domain binds a cobalt(II) ion and estabilshes a stable chelate

complex. The resulting Co(II)-amin-complex is now able to bind molecular oxygen. Reactive

hydroxylradicals are generated, which are capable of modifying guanine bases in an

oxidative manner and produce an alkali labile site. Guanin is known as the easiest oxidable

base, which is why the Co(II)-complex initiates a guanine specific DNA intercalation. The

supercoiled DNA is relaxed and the distance between the bases growth from 3.4Ӑ to 6.8Ӑ. At

a maximum every second intercalation place is attacked.

In the second step the DNA is cleaved oxidatively and afterwards fragmented. Therefore the

metal-binding-domain establishes a stable chelate complexes with Fe(II) cations.

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The produced bleomycin-Fe(III)-complex is now reduced by the NADPH-cytochrome-P-450-

reductase to a bleomycin-Fe(II)-complex. This complex is able to activate molecular oxygen.

A bleomycin-Fe(III)-complex and O2-▪ arises. The superoxide-anion-radicals produce in a so

called “Haber-Weiss-Reaction” hydroxylradicals (▪OH):

Bleomycin-Fe (III) Bleomycin-Fe (II)

Bleomycin-Fe (II) + O2 Bleomycin-Fe (III) + O2- ●

Bleomycin-Fe (II) + O2- ● Bleomycin-Fe (III) + H2O2

Bleomycin-Fe (II) +H2O2 Bleomycin-Fe (III) + ●OH + OH-

6 http://www.csj.jp/journals/bcsj/bc-cont/b98may_gif/ke70516con.gif

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For this reaction the bleomycin-Fe(II)-complex has to be regenerated. This is possible due to

sulhydryl- and mercaptan-groups (-SH,RSH) in cellular components. They are able to reduce

Fe(III) back to Fe(II). In a furhter step the hydroxylradicals or the bleomycin-Fe(II)-O2-

complex attacks the DNA. In both cases the desoxyribose of the DNA is deprotonated at the

4´ carbon atom by the abstraction of hydrogenradical (H▪). Afterwards oxygen adsorbs at the

analogous carbon atom and a radical chain reaction is activated. At the end the DNA is

fragmented whereby free bases and malondialdehyde are generated. In the absence of

NADPH or oxygen, no fragmentation or aldehyde synthesis takes place while in the absence

of bleomycin or iron the aldehyde synthesis only takes place at a slower pace.

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The formation of hydroxylradicals determines the effects of bleomycin, but nevertheless

explains the side effects and toxicity of the glycopetide antibiotic. In this mechanism, the

antibiotic is also attacked and damaged by the highly reactive hydroxide (free) radicals.

Given this fact, it can be explained, why the reaction only lasts for 20 minutes. Another

explanation could be the inhibition of the “pseudo-enzyme” caused by NADP+.

Bleomycin is able to evoke single- and double-strand breaks. The DNA is resolved into acid-

soluble fragments. The splitting of the DNA single-strand is followed by the reorganization.

The rotation about the bond between the two thiazol rings enables an intercalation with the

second DNA-strand. The fragmentation under the effect of bleomycin leads to a

destabilization and denaturation of the DNA, followed by conformational change of the

complex chromatin structure. Neither it is precisely understood how the bleomycin iron

complex is generated in the organism nor how it is reduced exactly.

7 I. Mahmutoglu ,1987

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Bleomycin is able to form complexes with different metal cations (for example iron). The

presence of Fe(II) activates the mechanism by forming reactive bleomycin Fe(II) complexes.

In the presence of copper (II), zinc (II) and cobalt metall cations the effect of bleomycin is

inhibited by inactive bleomycin metal complexes. These complexes do not lead to radical

formation and the subsequent fragmentation of the DNA

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Bleomycin applications

Bleomycin as an antibiotic

Bleomycin was used as an antibiotic for the creation of selective media or agars especially in

japan. The selection marker was the bleomycin hydrolase gene from streptomycis verticillus.

Because Bleomycin antibiotic effect is caused by random DNA double and single strand

breaks it can be used as antibiotic against eukarya, archea and bacteria.

Nowadays it is not in usage anymore due to its lounge toxicity.8

Bleomycin as an antitumor therapeuticum

Bleomycin is used to treat serveral forms of cancer such as cystic hygroma, different

lyphoma, squamous cell cancer and testicular cancer.9

Against cystic hygroma it can be used in non lounge toxic dosages (15 mg twice per week for

less than 13 weeks).10

Cystic hygroma is a disease of the lymphic system mostly appearing during the first three

years after birth. It causes the overproduction and secretion of lyphic liquid in the surrounding

tissiue. Because these hygroma moslty appear around head and neck the can be life

threatening. When they narrow the resperation system. A typical patient is shown below,

once before and once after treatment with bleomycin.

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8 Orford et al. 1995

9 Mosher et al. 1972

10 Williame Antholine et al. 1981

11 http://www.jcasonline.com/articles/2012/5/2/images/JCutanAesthetSurg_2012_5_2_133_99456_u1.j

pg

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The therapeutic succes in cases of csytic hygroma was achieved in 88 % of the treated

patients, therapeutic succes means curing or at leasing stopping of progression of the

disease.

For squamous cell cancer the therapeutic succes was shown to be around 66 % with a

dosage of 15 mg bleomycin per sqaure meter of affected tissue. The most common adverse

drug effect was the change of skin colour of the treated areas to grey.

For testicular cancer Bleomycin is not used anylonger due modern antitumor drugs that not

cause lounge fibroses in 10 % of the treated patient.

There is another application of bleomycin in cancer therapy. Bleomycin in imense dosis is

used on muribond patients with cancer in final states. In the case of those patient the lounge

toxicity of bleomycin does not matter because those patient would die anyways.12

The bleomycin animal model for human IPF

„IPF (Idiopathic Pulmonary Fibrosis) is a chronic progressive and ultimately fatal disease of

unknown etiology“ (Antje Moeller).

IPF affects 0,017 % of the population (classified in 2009) the survival ranges from two to four

years after diagnosis. Today only the symptomes, such as unproductive cough, rarely

dispnea, later lounge inflammatory and permanent dispnea, can be treated but the disease

itself can not be cured or even stopped in progression.

Bleomycin mostly effects the lounge because the lounge holds the lowest level of

bleomycinhydrolases, the detoxification enzyme for bleomycins, of all organs.

It was discovered that lounge fibrosis caused by bleomycin administration in animals strongly

resembled the human IPF.

This offered possibilities to create a bleomycin animal model for IPE. Eventually rodents

(mice, rats and hamsters) were selected because of high reproduction rates, small sizes and

the fact thar there were established laboratory strains.

The model has proven to have some advantages and some disadvantages.

The advantages are the idenfication of new target for either diagnosis or therapy of IPF.

Another advandtages is that model is easy handeld because bleomycin dosages for the

respective rhodents were found quickly and give highly reproductive output.

The disadvantages are that bleomycin induced lounge fibroses progresses over weeks and

not over years like human IPF. Also bleomycin simmulates an acute lounge injury but IPF is

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Friedlander et al. 1983

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not caused by any kind of knwon injury. So the transferability of drugs discovered againt

bleomycin induced lounge fibrosis in animals on human IPF is not easy.

One of the major results of this model was the identification of TGFβ (transformation growth

factor β) as one of the key factors in the progression of IPF.

Decorin an TGFβ-inhibitor was develloped and it showed a substantial reduction of the

fribrotic response to bleomycin in mice. Clinic studies on decorin against human IPF are

performed right now.

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References

Williame Antholine et al.; Interactions among iron(II)bleomycin,Lewis bases,and DNA;

Proc. NatL Acad. Sci. USA Vol. 78, No. 12, pp. 7517-7520, December 1981

Stefanie A. Kane, Hideaki Sasaki, and Sidney M. Hecht; Guanosine-Specific DNA

Damage by a Co(I1)Bithiazole Complex; Journal of the American Chemical Society, Volume

117, Number 36, September13, 1995

I. Mahmutoglu , M. E. Scheulen and H. Kappus; Oxygen radical formation and DNA

damage due to enzymatic reduction of bleomycin-Fe(III)*; Achives of Toxicology, Springer

Verlag, 1987

Marlon S. Mathews et al.; Photochemical internalization of bleomycin for glioma treatment;

Journal of Biomedical Optics, Volume 17, Issue 5, Research Papers: Therapeutic, May 03,

2012

Dindial Ramotar and Huijie Wang; Protective mechanisms against the antitumor agent

bleomycin: lessons from Saccharomyces cerevisiae; Current Genetics Lower Eukaryotes

and Organelles, Springer Verlag 2003

M. Friedlander et al.; Cervical carcinoma: a drug-responsive tumor--experience with

combined cisplatin, vinblastine, and bleomycin therapy.; Gynecol Oncol, 16(2):277-81, 1983

A. Moeller et al.; The bleomycin animal model: a useful tool to investigate treatment options

for idiopathic pulmonary fibrosis?;Int J Biochem Cell Biol. 2008 ; 40(3): 362–382

J. Orford et al.; Bleomycin Therapy for cystic Hygroma; Journal of Pediatric Surgery, Vol 30,

No 9 pp 1282 - 1287; 1995

B.Shen et al.; The biosynthetic gene cluster for the anticancer drug bleomycin from

Streptomyces verticillus ATCC15003 as a model for hybrid peptide-polyketide natural product

biosynthesis, Journal pf Industrial Microbiology & Biotechnology 2001

M. B. Mosher et al.; BLEOMYCIN THERAPY IN ADVANCED HODGKIN’S DISEASE AND

EPIDERMOID CANCERS; CANCER Vol 30; 1972

Internet:

- http://de.wikipedia.org/wiki/Bleomycin (01.06.2013)

- http://apps.who.int/phint/en/p/docf/ (01.06.2013)

- http://www.pnas.org/content/107/52/22419/F1.small.gif (01.06.2013)

- http://de.wikipedia.org/wiki/Polyketide (28.05.2013)

- http://medlibrary.org/lib/images-rx/bleomycin-3/bleomycin-for-injection-usp1-figure-1-

gbleomycin.jpg (25.05.2013)

- http://www.csj.jp/journals/bcsj/bc-cont/b98may_gif/ke70516con.gif (25.05.2013)