Antibiotic Drugs Aminoglycosides Cleave DNA at Abasic Sites

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Antibiotic Drugs Aminoglycosides Cleave DNA at Abasic Sites: Shedding New Light on their Toxicity? Maralise Perigolo de Oliveira, Jean-Francois Constant, Marine Peuchmaur, Ivan Pitta, and Jean-Luc Decout Chem. Res. Toxicol., Just Accepted Manuscript • Publication Date (Web): 15 Oct 2013 Downloaded from http://pubs.acs.org on October 20, 2013

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Chemical Research in Toxicology

Antibiotic Drugs Aminoglycosides Cleave DNA at Abasic Sites: Shedding New Light on their Toxicity? Maralise Perigolo de Oliveira,† Jean-François Constant,§* Marine Peuchmaur,† Ivan Pitta,‡ Jean-Luc Décout†* †

Université de Grenoble I/CNRS, UMR 5063, Département de Pharmacochimie Moléculaire,

ICMG FR 2607, 470 rue de la Chimie, BP 53, F-38041 Grenoble, France §

Université de Grenoble I/CNRS, UMR 5250, Département de Chimie Moléculaire, ICMG FR

2607, 570 rue de la Chimie, BP 53, F-38041 Grenoble, France ‡

Universidade Federal de Pernambuco, Instituto Nacional de Ciência e Tecnologia para

Inovação Farmacêutica, Laboratório de Planejamento e Síntese de Fármacos, Recife, Brazil.

Keywords: aminoglycosides, DNA cleavage, abasic sites, neomycin B, paromomycin, neamine, ribostamycin, kanamycin A, tobramycin, gentamicin, geneticin, streptomycin, spectinomycin, apramycin, 4’-neamine derivative, 5-neamine derivatives, 4’,5-neamine derivative, adenine.

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ABSTRACT

Abasic sites are probably the most common lesions in DNA resulting from the hydrolytic cleavage of glycosidic bonds that can occur spontaneously and through DNA alkylation by anticancer agents, by radiotherapy and during the repair processes of damaged nucleic bases. If not repaired, the abasic site can be mutagenic or lethal. Thus, compounds able to specifically bind and react at abasic sites have attracted much attention for therapeutic and diagnostic purposes. Here, we report on the efficient cleavage activity of characteristic antibiotic drugs of the major aminoglycosides (AG) family at abasic sites introduced either by depurination in a plasmidic DNA or site specifically in a synthetic oligonucleotide. Among the antibiotic AG drugs selected for this study, neomycin B is the most efficient (a 0.1 µM concentration induces 50% cleavage of an abasic site containing DNA). This cleavage activity could be related to the aminoglycoside toxicity but also find medicinal applications through potentiation of cancer radiotherapy and chemotherapy with alkylating drugs. In the search for antibiotic and antiviral agents, we have previously modified the small aminoglycoside neamine corresponding to ring I and II of neomycin B constituted of four rings and the cleavage activity at abasic sites of four neamine derivatives previously synthesized are also reported. One of them appeared to be much more active than the parent compound neamine with cleavage efficiency close to that of neomycin.


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TABLE OF CONTENTS GRAPHIC

6'

4'

HO HO 3'

NH2 5'

O

2'

NH2

HO

O H2N

OH

3

2

NH2

O O

NH2

OH

1' H2N

O

O

5

6

OH 1

OH

Neomycin B



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INTRODUCTION Ribosomal RNA (rRNA) is the main target of clinically used major antibiotics such as the natural drugs aminoglycosides (AG), for examples, neomycin B 1, paromomycin 2, kanamycin A 5, tobramycin 6, gentamicin C 7 and geneticin 8 (Figure 1).1 These antibacterial agents are potent antibiotics active against both Gram (+) and Gram (-) pathogens. AG are pseudo-oligosaccharides, which carry amine and/or guanidine functions positively charged at pH 7. AG act through binding to rRNA causing mRNA decoding errors, mRNA and tRNA translocation blockage, ribosome recycling inhibition and in fine protein synthesis alteration.112

R

6' 4'

NH2

5' O

HO O Ring I HO HO 1' H2N 1 R 3 Ring II 3' 2' NH2 2 H2N 2 O R NH2 HO O O NH2 6 1 5 OH HO O NH2 OH OH O 3: neamine, R = NH2 Ring III 1 2 OH O 5: kanamycin A, R = R = OH: O O 1 2 NH2 H2N 6: tobramycin, R = H, R = NH2 4: ribostamycin, R = NH2 OH Ring IV

HO NH

O OH O

HO

OH

OH

HN

O O

OH

O 9: streptomycin

R

NH2 O HO

H2N NH2 O

1

2

O

HO

7: gentamicin C 3

C1: R , R = H, R = CH(CH3)NHCH3 1

2

CH3 NH

OH

3

C1a: R , R = H, R = CH2NH2 1

O

OH

OH NH

OH

HO

O

HN H2N

HO NH

NH

O

1

C2: R1, R2 = H, R3 = CH(CH3)NH2

1: neomycin B, R = NH2 2: paromomycin, R = OH

H2N

R3

R2

O

OH

H2N HO

3

O HN

OH H N

O

2

8: geneticin, R , R = OH, R = CH(OH)CH3

OH 10: spectinomycin

OH

O O 11: apramycin

O H2N

H2N O HO

NH2 OH

Figure 1. Structure of the aminoglycosides used in this study: antibiotic aminoglycosides drugs 1, 2, 5-11 and of neamine 3 and ribostamycin 4 corresponding to parts of neomycin B 1.

The broad antimicrobial spectrum of AG (Figure 1), their rapid bactericidal action even if the bacterial inoculum is large, and their ability to act synergistically with other drugs, such as penicillins and vancomycin, have made them especially useful in the treatment of serious infections with a widespread clinical use despite reduction of their clinical efficacy due to the


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appearance of resistant bacteria.8-14 Unfortunately, side toxicities, mainly nephrotoxicity and ototoxicity have been observed.15-22 AG have long been one of the most common causes of drug-induced nephrotoxicity.15, 16

Unless administrated with careful serum monitoring and optimized administration, AG can

induce acute nephrotoxicity in 5–25% of treated patients, and even more in populations at risk.17 AG such as gentamicin C 7 accumulated by epithelial cells are mainly localized in endosomal and lysosomal vacuoles18, 19 but are also localized with the Golgi complex.18 A recent study demonstrated the key role of lysosomal iron and early reactive oxygen species (ROS) production in gentamicin induced lysosomal membrane permeation and apoptosis.20 AG antibiotics have long been known to have ototoxic effects.21, 22 These antibiotics, including neomycin C, tobramycin, kanamycins, gentamicin C, streptomycin (Figure 1) and amikacin, are associated with hearing loss and vestibular dysfunction due to hair cell loss. However, AG are used in the treatment of the Ménière’s disease of the inner ear characterized by recurring attacks of disabling vertigo, hearing loss and tinnitus.23 In regard to the emergence of resistances to AG and to their nephrotoxic and ototoxic effects, new strategies to counter resistance and new therapeutic approaches have been developed through chemical modifications of the first generation of antibiotic AG drugs.23-31 In this aim, we have prepared neamine derivatives, which are potent antibacterial agents targeting the bacterial membranes32,

33

, efficient vectors for gene transfection in vitro34 or

strong HIV TAR RNA binders.35-39 The potential toxicity of these modified neamine derivatives merits attention in regard to the toxicity of the parents AG. The non specific binding of antibiotic AG to nucleic acids and their ability to cleave this macromolecule can be also another source of toxicity.40-51 First, Tor et al. have reported that neomycin B hydrolyses the phosphate ester bonds in a di-ribonucleoside 5’-monophosphate (ApA).41 Hairpin ribozyme cleavage catalyzed by AG antibiotics and the polyamine spermine 5 ACS Paragon Plus Environment


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was then described42 and tRNAPhe was found to be susceptible to aminoglycoside mediated cleavage.43 This highly specific tRNAPhe cleavage induced by neomycin B has been related to the presence of the hyper modified guanine base-wybutine and requires its previous depurination to form an abasic site.43 We have also demonstrated that the neamine moiety attached to a peptide nucleic acid (PNA) sequence designed to target the HIV-1 TAR RNA, was able to induce selective RNA strand cleavage.37,

38

More recently, a number of

aminoglycoside antibiotics, especially neomycin B, were demonstrated to promote strand cleavage of RNA oligonucleotides (minimized HIV-1 TAR element and prokaryotic ribosomal A-site), by binding and causing sufficient distortion to the RNA backbone to render it more susceptible to intramolecular transesterification.44 The majority of AG can chelate transition cations such as copper (II) ions by vicinal amino and hydroxyl groups and the oxidative cleavage of RNA by copper ions AG antibiotics complexes was also described45-48 and might be related partly to the AG toxicity.48 Hydrolytic and oxidative cleavage of doublestrand DNA by copper ions-AG complexes were also reported.49- 51 Since aliphatic diamines and polyamines have been shown to catalyze the cleavage of DNA at abasic sites,52 which are probably the most common damages in DNA,53 we were interested in examining whether AG used as antibiotic drugs may catalyze a similar cleavage. Most of the aminoglycosides studied here carry protonated and unprotonated amino groups at physiological pH54-56 and could also catalyze such a cleavage. For example, the antibiotic AG neomycin B 1 (Figure 1) carries six amino groups with pKa values, ranging from 6.9 to 9.6, that could cooperate for cleavage via acido-basic catalysis.54 An abasic site results from the loss of a nucleic base that can occur spontaneously53 through a variety of processes, either chemically by protonation or alkylation of a purine base (Scheme 1)57 or through modification by physical agents such as radiation.58 This baseless lesion appears to be among the most common damages induced by chemotherapeutic DNA 6 ACS Paragon Plus Environment

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alkylating agents.59, 60 Abasic sites are also produced enzymatically in the course of the repair of modified or abnormal nucleic bases.61

ODNA R O P O O

N O O

O P O ODNA

ODNA

ODNA

O N

NH

O P O

O P O

O

O

O N

NH2

R= H, alkyl

O

O H

O O P O ODNA

H

ODNA

ODNA

O P O

O

H3N

O

O

O P O ODNA

+ guanine or 7-alkylguanine

O P O

O

O H

NH2

O P O H3N ODNA

NH2

OH O

H

O

O

O P O ODNA δ-elimination

β-elimination

+

O OH

OH

OH

ODNA

O P O

O O

Scheme 1. Abasic sites lesions are generated by hydrolysis of the N-glycosidic bond facilitated either by protonation or alkylation of nucleobases. Aldehydic form and α,β hemiacetals of AP sites are shown in tautomeric equilibrium.62 The aldehydic form is not stable and can undergo β-elimination of the oligonucleotide fragment 3′ to the abasic site yielding a 5′-phosphorylated 3′-fragment and a 3′-phosphorylated 5′-fragment carrying a reactive α,β-unsaturated aldehyde group which can lead through a second δ-elimination to reactive carbonyl species potentially toxic.

If not repaired, the abasic site, which is a non-informative lesion, may promote misincorporation of nucleotides 63 and can be cytotoxic and mutagenic.64-67 Molecules, which recognize and cleave DNA specifically at abasic sites in vitro, such as a tryptophan containing tripeptide

Lys-Trp-Lys,68,

69

9-aminoellipticine70 and 9-

aminocarbazole71 have been reported. Conjugates composed of three parts, an acridine intercalator linked by a diamino chain to a nucleobase diaminopurine (DTAc) or adenine (ATAc) (Figure 2), have also been shown to exhibit cleavage efficiency at apurinic sites in plasmidic DNA at nanomolar concentrations.72-73



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Since abasic sites are formed in the cell with fairly high frequency either spontaneously or enzymatically as intermediates during the repair process of modified bases (base excision repair pathway (BER)),59 compounds that cleave at abasic sites are thus good candidates for designing drugs able to interfere with the repair process. For instance, an inhibitory effect might be useful to sensitize tumor cells to alkylating drugs. Indeed, a number of anticancer drugs, mostly of the alkylating family, have been reported to generate abasic sites and are thus susceptible to potentiation by abasic-site specific molecules.57,

74, 75

Sensitization of cancer

cells to bis-chloroethylnitrosurea (BCNU), a clinically useful anticancer drug, by DTAC and ATAC has also been observed.74 Therefore, compounds able to interfere with the abasic site repair are of great interest for diagnostic and therapeutic purposes in order to potentiate the cytotoxic effects of alkylating drugs or radiations.75

NH 2 N

N R

N

N (CH 2) 2 NH

DTAC: R = NH 2 ATAC: R = H

(CH 2 )3 NH (CH 2 )3 NH OCH 3

Cl

N

Figure 2. DTAC and ATAC structures. Here, we report on the study of the cleaving efficiency at abasic sites in DNA of eleven antibiotic AG drugs and of four synthesized derivatives. All compounds were tested on abasic lesions generated by controlled depurination of pBR322 plasmid DNA. The most efficient AG identified in this study, neomycin B, was also tested on an abasic lesion specifically introduced in a short synthetic oligonucleotide. It is important to mention that all the


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experiments described here were performed using naked DNA. Recent studies on abasic sites stability when present in nucleosome particles revealed a significant decrease of the abasic site half-life due to its reaction with neighbouring histone lysines.76 The cleavage activity observed could partly explain the AG toxicity and could also find applications in potentiating the therapeutic effects of anticancer treatments such as γ radiations and alkylating agents known to generate massive amounts of baseless sites.

MATERIAL AND METHODS Enzymes and reagents. AG were dissolved in water to prepare stock solutions at concentrations ranging from 10-3 to 10-5 M. Polynucleotide kinase was from fermentas and Endonuclease IV from England Biolabs.

32

P γ radiolabeled ATP (3000 Ci/mmol) was

purchased from Perkin Elmer. Preparation of DNA Containing Apurinic Sites (AP-pBR322 DNA). A sample of pBR322 DNA was dissolved in 25 mM sterilized acetate buffer (pH 4.9) at the concentration of 0.05 mg/mL and heated at 70 °C for 20 min.53, apurinic sites per DNA molecules.53,

72

72

This treatment introduced approximately 2

DNA was precipitated by ethanol (70% v/v) in the

presence of sodium acetate (0.3 M final concentrations). After rinsing with 70% ethanol, the DNA sample was dried and resuspended in 10 mM phosphate buffer containing various KCl concentrations. Incision of AP-pBR322 DNA. A sample of AP-pBR322 DNA (0.125 µg in a final volume of 10 µl) was incubated at 37 °C for 20 min in the presence of varying concentrations of AG and KCl in 10 mM phosphate buffer. The abasic sites formation was controlled by treating with DTAC at a 0.5 µM concentration known to induce 100% cleavage at the lesion site.72 The reaction was stopped with 2 µl of loading buffer containing 0.1% bromophenol blue, 0.1% xylene cyanol and 30% of glycerol. 9 ACS Paragon Plus Environment


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Quantitation of AP-pBR322 strand breakage. The nuclease activity of the different compounds was estimated by measuring their ability to induce single strand breaks in depurinated pBR322 plasmid DNA. The nicked (form II) and supercoiled (form I) DNA were separated by agarose gel electrophoresis (0.8%) using TBE (45 mM Tris-borate, 1 mM EDTA pH 8.0) as a migrating buffer and a constant voltage of 150 V for 1.5 h. The DNA was stained by ethidium bromide incorporated in the agarose gel (1 µg/mL). The intercalated ethidium bromide fluorecence is strongly enhanced and DNA can be quantitated by scanning the gel with a Typhoon 9410 Scanner Control, which measures the fluorescence in arbitrary units (λexc= 532 nm; λem= 620 nm). The analysis of the relative amounts of the two forms of the plasmid was performed using the Image Quant Tools software. The experiments were done in triplicate and the nicked form fraction F was calculated according to the following equations where we define: (1) for the reference (depurinated plasmid in absence of aminoglycoside), the fluorescence intensity of the nicked form INr and supercoiled form ISr and (2) for an experiment i (in the presence of an aminoglycoside), the fluorescence intensity of the nicked form INi and supercoiled form ISi. Thus, the nicked form fraction in the starting plasmid is FNr= INr/( INr + ISr). The fluorescence intensity measured corresponding to the total amount of DNA in the experiment i is INi + ISi and the fluorescence intensity of nicked plasmid coming from the starting plasmid in the experiment i was xi= FNr (INi + ISi). The fluorescence intensity of nicked plasmid due to the cleavage in the experiment i is INi xi= INi – [FNr (INi + ISi)]. Thus, the nicked form fraction due to the cleavage by an aminoglycoside in the experiment i was calculated as FNi= (INi – xi) / (INi + ISi - xi)= [(INi – (ISi INr / ISr)] / (INi +ISi). No correction relative to the difference in the fluorescence characteristics of the BET-nicked DNA and BET-supercoiled DNA complexes was applied.


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Study of the AP site breakage by neomycin B in a synthetic undecamer oligonucleotide. Oligonucleotide preparation. The oligonucleotides were purchased from Eurogentec (Belgium) and were purified by HPLC and ion exchange chromatography. The abasic site lesion (AP site) was incorporated in the DNA strand d(CGCTCXCACGC) as a silylated precursor of the hemiacetal form. The silyl protecting group was removed by a treatment in 25 mL of acetic acid 80% for 30 min at room temperature. Water was then added (25 mL) and the mixture incubated for an additional 4 h. The reaction was quenched with 1mL of phosphate buffer 2M, pH 7. The sample was then desalted on a C18-column by washing with water and eluting with a mixture of water/acetonitrile (60/40). After evaporation to dryness, the sample was resuspended in water and the oligonucleotide content was quantified by measuring the UV absorbance at 260 nm (molar extinction coefficient: 83000 cm-1.L.mol-1). Reaction at the abasic site. The 5' 32P labelled AP site containing oligonucleotide was either used as a single strand or annealed with its complementary strand d(GCGAGGGTGCG). In a typical experiment, the DNA fragment at a 2.5 micromolar final concentration is incubated in presence of neomycin B (50 µM) for 15 min, 30 min and 1 h in phosphate buffer 10 mM, pH 7.0. The chemical AP-lyase DTAC was used at 1 µM concentration for 30 min at 37 °C in the same buffer. For the E coli endonuclease IV reaction, the abasic duplex was incubated for 30 min in presence of 10-3 units of enzyme in the assay buffer provided by the furnisher.

RESULTS Nine natural AG used as antibiotic drugs of the two main classes (neomycin- and kanamycin-classes), were first selected for this study (Figure 1). These classes differ from each other by their substituents at position 5 or 6 in the 2-deoxystreptamine ring I that defines the neomycin-class (neomycin B 1, paromomycin 2) and the kanamycin-class (kanamycin A 11 ACS Paragon Plus Environment


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5, tobramycin 6, gentamicin C 7, geneticin 8), respectively (Figure 1). Two small aminoglycosides (neamine 3 and ribostamycin 4) corresponding to fragments of neomycin B 1 were also studied in order to determine some structure-activity relationships. Neamine 3 corresponds to rings I and II of the neomycin B core and ribostamycin 4 corresponds to rings I, II and III of this core (Figure 1). We also investigated three other natural AG interesting for their structure and their particular antibiotic use. Streptomycin 9, mainly used for the treatment of tuberculosis, differs from the other natural AG by the presence of only one hindered secondary amine function and the presence of the two guanidine groups fully protonated at physiological pH.77, 78 Spectinomycin 10 is constituted by three fused rings and is mainly used in the therapy of acute gonorrheal urethritis and proctitis due to Neisseria gonorrhoeae infection.78,

79

Apramycin 11, a structurally unique antibiotic that contains a

bicyclic sugar moiety, is only used for the treatment of bacterial infections in animals and stands out among AG for its mechanism of action based on translocation blockage and its ability to also bind to the eukaryotic decoding site.80, 81

Cleavage at AP sites in pBR322 plasmid DNA, a first selection of the most efficient antibiotic AG drugs The cleavage activity at abasic site of a series of eleven AG was evaluated on a depurinated supercoiled plasmid DNA. The cleavage activity can be analyzed by measuring the ability of AG to induce single strand breaks at abasic sites in pBR322 plasmid DNA. These single strand breaks convert the circular covalently closed form (supercoiled or form I) into the nicked circular form (relaxed or form II). These two conformations of the plasmid were separated by agarose gel electrophoresis and quantified after ethidium bromide staining. The cleavage percentage was calculated as shown in the experimental section. Depurination of pBR322 plasmid DNA was effected under controlled conditions (sodium acetate buffer, 25 12 ACS Paragon Plus Environment

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mM, pH 4.9, 70ºC, 20 min) to produce an average of approximately 2 apurinic sites per DNA molecule.72 Because trace amounts of copper can be found in buffers and DNA samples and because AG copper (II) complexes have been reported to behave as artificial nucleases, through a hydrolytic mechanism,45-51 we made a control experiment in order to verify the absence of cleavage of non modified pBR322 plasmid DNA under the conditions used for the experiments. In this study, the non depurinated DNA was incubated at 37 ºC, pH 7.2, 100 mM KCl concentrations, for 20 minutes in the presence of the aminoglycosides antibiotics 1-11 at 5.10-4, 5.10-5 and 5.10-6 M. In these conditions, AG appeared to be unable to cleave non depurinated plasmid. Nine out of the eleven AG tested (compounds 1-8 and 11, Table 1) showed significant cleavage activity at apurinic sites at 5.10-4 and 5.10-5 M concentrations and six others were active at 5.10-6 M (compounds 1, 3-7). For the AG antibiotics exhibiting a strong cleaving activity at abasic sites the half maximal effective concentration (EC50) was determined at pH 7.2 in the presence of 100 mM KCl. The values obtained are reported in Table 2. Among the AG studied and used for their antibiotic properties, by far, the most efficient one was neomycin B 1 with a 0.1 µM EC50. Streptomycin 9 and spectinomycin 10 showed very weak cleavage activities (Table 1) and were not further studied. In the neomycin-class of AG, paromomycin 2 and ribostamycin 4 led to the lowest fractions of cleavage in comparison to neomycin B 1 and neamine 3. In the kanamycin-class of AG (AG 5-8), geneticin 8 showed the lowest activity. In regard to tobramycin 6 and gentamicin C 7, a weaker cleaving activity was found at 5 and 50 µM for kanamycin A 5. Tobramycin, gentamicin and geneticin were kept for the next EC50 determination experiments in regard to their use as antibiotic drugs and, also geneticin, in regard to its interest in the treatment of genetic diseases. Indeed, geneticin 8 (G418) has been found to allow, via binding to rRNA, the read-through by the translation 13 ACS Paragon Plus Environment


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complex of disease-causing non-sense mutations and, therefore, the synthesis of full-length active proteins.82-85 Such a read-through activity of geneticin and analogues was evaluated for the treatment of genetic diseases such as Cystic Fibrosis. The weaker cleaving activity of paromomycin and kanamycin A in comparison to tobramycin, gentamicin and neomycin B, respectively, could be related to their lower numbers of amine functions (Figure 1). The steric hindrance in spectinomycin 10 and the presence of two secondary amine functions probably could explain its weak activity. Spectinomycin 10 and apramycin 11 showed significant cleaving effects at 50 and 500 µM as geneticin 9 and paromomycin 2. In regard to their specific uses in antibiotherapy, 10 and 11 were not kept for EC50 determination.

Table 1. Fractions of apurinic site cleavage after 20 min incubation at pH 7.2, 37ºC, 100 mM KCl by aminoglycosides antibiotics at 5, 50 and 500 µM concentrations. AG, 1: neomycin B, 2: paromomycin, 3: neamine, 4: ribostamycin, 5: kanamycin A, 6: tobramycin, 7: gentamicin, 8: geneticin, 9: streptomycin, 10: spectinomycin, 11: apramycin.

Aminoglycosides 1

2

3

4

5

6

7

8

9

10

11

5 µM

0.73

0.11

0.48

0.28

0.26

0.58

0.56

0.08

0.05

0.08

0.06

50 µM

0.83

0.26

0.80

0.64

0.64

0.80

0.76

0.26

0.11

0.13

0.34

500 µM

0.90

0.58

0.85

0.76

0.80

0.84

0.80

0.58

0.17

0.18

0.63

Cleavage at AP sites in pBR322 plasmid DNA, quantification of the effects of the most efficient antibiotic AG (EC50) The most active aminoglycoside, neomycin B 1, showed an approximatively EC50 500 fold higher than paromomycin 2 which carries a hydroxyl group at 6’-position in ring II.


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Thus, replacement of this group by an amino group in neomycin B increases strongly the cleavage activity. At pH 7, due to the presence of several amino functions of various pKa values,54 AG carry protonated and unprotonated amines, which may play different roles: the protonated nitrogen constitutes an additional DNA-binding site involving eletrostatic interaction with the phosphates, while unprotonated amino group can cooperate for strand breakage through a β-elimination process. Moreover, in the neomycin-class compounds 1-4, neamine 3 (0.89 µM EC50) and ribostamycin 4 (2.2 µM EC50) appeared to be much less active than neomycin 1 (0.094 µM) suggesting the major role of ring IV of neomycin in the affinity for DNA and/or the cleaving activity. For the kanamycin-class (compounds 6-8), tobramycin 6 which carries six amino groups showed a slightly better cleavage capacity (0.63 µM EC50) than gentamicin C 7 (0.79 µM EC50) and strongly higher than geneticin 8 (32 µM EC50). One major difference between gentamicin C 7 which is a mixture of three AG (Figure 1) and geneticin 8 is the presence of an additional 6’-amino group in gentamicin. Such a group present in neomycin, neamine, ribostamycin, tobramycin and gentamicin appeared essential for a strong cleaving activity. It is protonated at pH 7.2 and plays probably an essential role in DNA binding through electrostatic interaction with phosphodiester anions. The artificial AP-lyase DTAC (Figure 2), which is one of the best reported artificial cleavers at abasic sites72 was used for comparison. Under similar conditions, DTAC induces 50% cleavage at a 5 nM concentration, which is several orders of magnitude more efficient than the well-known tripeptide Lys-Trp-Lys.73 DTAC is more than two orders of magnitude more efficient than neomycin 1 and three orders of magnitude more efficient than tobramycin 6 and gentamicin 7 as they induce 50% DNA cleavage at 0.094 µM, 0.63 µM and 0.74 µM concentrations, respectively (Table 2).



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Table 2. Aminoglycoside concentrations for which 50% of abasic cleavage (EC50) was determined in a pBR322 plasmid DNA containing an average of two abasic sites per molecule after 20 min incubation at 37 °C and pH 7.2 in the presence of 100 mM KCl.

Compounds EC50 µM, pH 7.2, 100 mM KCl Neomycin B 1 0.094 Paromomycin 2 42 Neamine 3 0.89 Ribostamycin 4 2.2 Tobramycin 6 0.63 0.79 Gentamicin C 7 Geneticin 8 32 0.15 12 1.1 13 0.71 14 0.79 15

Synthetic neamine derivatives and cleavage at abasic sites in depurinated pBR322 plasmid DNA The neamine derivatives 12-15 have been synthesized in our laboratory with the aim at developping new compounds possessing antibacterial or antiviral properties and ribonuclease like activity (Figure 3).35-38 We also determined the EC50 of these derivatives (Table 2). In compounds 12-14, one or two purinic nucleic bases, adenine, are attached by their amino group at the 4’- and/or 5-positions of the neamine core by a hexyl linking arm.35 The presence of such adenine rings could increase the affinity for apurinic DNA through replacement of the missing purines and intercalation. In compound 15,35 the presence of additional positive charge(s) in comparison to neamine 3 and to compounds 12-14 should increase the binding to DNA and the incorporated imidazole ring could also participate in the catalysis of cleavage at abasic sites. Among the neamine derivatives evaluated, compound 12 carrying one adenine ring attached at the 4’-position appeared to be the most active with an EC50 much lower than the


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one measured for the parent neamine 3 and slighly higher than the EC50 of neomycin B 1 (Figure 4). The other neamine derivatives 13-15 showed EC50 close to the one of neamine. In 12, attachment at the 4’-position of the adenine ring probably favours binding to apurinic DNA without disturbing the cleavage activity whereas in 13 and 15 the attachment at the 5and at the 4’,5- positions of one or two adenine rings, respectively, do not improve the cleaving properties in comparison to neamine probably due to steric effects. This result is interesting in the search for neamine derivatives able to interfere selectively at abasic site in the repair processes.

NH2 O

O HO

NH2 NH N

H 2N O HO

O

N H

NH2 H2N O O

NH2 OH

13

HO

NH

NH2 O

NH2 O HO NH2 H2N O O

N N

HO NH2 O

N

NH2 O

NH2 OH

12 N

N

HO HO

NH

H2N

NH

OH

N NH2

O

NH

OH 15

N

N HN

NH2

NH

14

NH O NH2

N

N

N N

N H

Figure 3. Synthetic aminoglycosides used in this study.



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Figure 4. Fraction of nicked plasmid DNA as a function of molar concentration of neomycin B 1, neamine 3 and compound 12. Fractions of cleavage of pBR322 plasmid DNA containing an average of two abasic sites per molecule were measured after 20 min incubation at 37 °C and pH 7.2 in the presence of 100 mM KCl.

Effect of the ionic strength on the cleaving activity Since AG have a polycationic character, their binding to DNA and their cleaving properties at abasic sites should be influenced by the ionic strength. We analyzed the abasic site cleavage activity of AG at three concentrations (5, 50 and 500 µM) at 37 °C and pH 7.2 in the presence of 50, 100, 150 and 200 mM KCl concentrations (Figure 5). As expected the cleavage activity of all the compounds was strongly dependent on the ionic strength. The lower is the ionic strength, the higher is the cleavage activity and vice-versa. The counterion effect of the potassium ions bound to DNA phosphates reduces the eletrostatic interaction of the protonated amino groups of AG with DNA and thus, modulates the cleavage activity. For


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the most active AG, neomycin B 1, the cleavage fractions decrease significantly at the lowest 5 µM concentration in AG and in the presence of KCl at concentrations higher than 100 mM.

Figure 5. Fraction of nicked plasmid DNA as a function of the ionic strength. Cleavage fractions of pBR322 plasmid DNA containing an average of two abasic sites per molecule were measured after 20 min incubation at 37ºC and pH 7.2 in the presence of AG tested at 5.10-6 M (blue bar), 5.10-5 M (red bar) and 5.10-4 M (green bar) and 50, 100, 150 and 200 mM KCl solutions.

Role of pH in the cleaving activity Neomycin B 1 appeared to be the most efficient AG for the cleavage of DNA at abasic sites. Neamine 3, which corresponds to rings I and II of the neomycin core, showed an activity similar to those of the most active natural AG drugs studied here, tobramycin 6 and gentamicin C 7. Thus, in order to confirm the role of the pKa of amine functions in the cleavage activity and confirm the necessity of the presence of unprotonated amino groups in 19 ACS Paragon Plus Environment


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our conditions (pH 7.2), we chose neamine 3 as a representative example. A pH effect has been reported previously in the study of the mechanism of HIV-1 TAR RNA cleavage catalyzed by the neamine core attached to a peptide nucleic acid (PNA) sequence directed against the viral RNA.38 The pKa values of the four amine functions of neamine have been determined at 25 °C by 1H NMR titration experiments: pKa1 6.44 ± 0.13 for N3 of ring I, pKa2 7.23 ± 0.09 for N2' of ring II, pKa3 7.77 ± 0.19 for N1 of ring I, and pKa4 8.08 ± 0.15 for N6' of ring II.56 We measured its cleavage activity as a function of the concentration in the presence of 100 mM of K+ ions (buffer and KCl) at three pH values 6.0, 7.2 and 7.5 (Figure 6). The choice of such a narrow pH range has been dictated by some experimental limitations. At low pH, the protonation of the amino groups should increase the global positive charge of the AG and favor its binding to DNA but this will be at the expand of the cleavage reaction catalysis performed by non protonated amino groups. On the other hand, higher pH will favor the presence of active unprotonated amino groups (accompanied by a decrease of the DNA binding) but will also lead to the alkaline degradation of the abasic sites. For these reasons, the highest pH used was 7.5 in order to limit the non-specific cleavage and the lowest pH was 6.0 in order to keep unprotonated amino groups available. The difference in the cleavage activity at the three pH used appeared to be minor with an EC50 close to 0.8-1 µM (Figure 6). The cleaving activity did not decrease significantly at pH 6 (EC50= 1 µM). Here, it is difficult to conclude from the results obtained in this limited pH range


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Figure 6. Fraction of nicked plasmid DNA as a function of pH and concentration of neamine 3: pH= 6.0 (blue bar), 7.2 (red bar) and 7.5 (green bar). Fractions of cleavage of pBR322 plasmid DNA containing an average of two abasic sites per molecule were measured after 20 min incubation at 37 °C in the presence of 100 mM of K+ ions (buffer and KCl).

Cleavage of neomycin B at an AP site generated in a short synthetic DNA duplex In order to analyze more accurately the cleavage mechanism of the most active compound neomycin B, the cleavage reaction was performed with a 5'-32P labelled synthetic undecamer containing an abasic site. In these experiments, the labelled oligonucleotide was hybridized with its complementary strand and incubated in presence of various neomycin 1 concentrations. The cleavage pattern was compared with those found using the artificial APlyase DTAC (Figure 2) and endonuclease IV from E. coli, which affords the 3'-OH fragment resulting from hydrolytic cleavage of the phosphodiester bond on the 5'-side of the abasic site. In presence of a fixed concentration of neomycin 1 at various incubation times (15 min to 1 hour), cleavage products similar to those produced by DTAC were formed (Figure 7). The fraction of cleavage increased with time, being almost quantitative after 1 hour. The endo IV cleavage product corresponding to the 3'-hydroxyl fragment migrates at a slightly higher 21 ACS Paragon Plus Environment


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position than the product observed with neomycin 1. These results clearly demonstrate that neomycin B behaves as a very efficient artificial AP-lyase.

Figure 7. Analysis by PAGE under denaturing conditions of the reaction products obtained from (32P)dCGCTCXCACGC (X: abasic site), (2.5 µM) annealed in buffer at pH 7.0 (10 mM sodium phosphate, 100 mM potassium chloride) with its complementary strand: lane 1, DNA alone incubated 1 h at 37 °C; lanes 2, 3 and 4 correspond to incubation with neomycin 1 (50 µM) for 15, 30 and 60 min at 37 °C respectively; lane 5, incubation with DTAC (Figure 2, 1 µM for 30 min at 37 °C); lane 6, cleavage catalysed by endonuclease IV from E. coli (10-3 units) for 30 min at 37°C.

DISCUSSION AND CONCLUSION

In the present work, we clearly demonstrate that a series of antibiotic AG drugs and synthetic AG are able to cleave efficiently DNA at abasic sites. Neomycin B 1 appeared to be the most efficient cleaving AG. Neamine 3 corresponding to rings I and II of the neomycin core retains a part of the cleaving activity of neomycin since the other most active natural 22 ACS Paragon Plus Environment

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antibiotic AG drugs identified, tobramycin 6 and gentamicin C 7, showed cleaving activities similar to that of neamine. As reported in the Literature with polyamines, for a high cleaving activity of AG through an acid-base catalysis, the concomitant presence of, at least, two well located and close amine functions should be necessary.52,

73

A first function of high pKa value is

protonated and binds to a phosphate group in a close vicinity of the abasic site and a second amine function of low pKa value and thus unprotonated under physiological pH abstracts acidic β- hydrogen atom of the aldehydic form of the abasic site leading to strand breakage via elimination of the 3'-phosphate group (Scheme 1). This assumption is confirmed by the low cleaving activity of streptomycin, which carries only one hindered secondary amine function and two guanidine groups fully protonated at physiological pH. Spectinomycin 10 carrying two hindered secondary amine functions appeared also to be weakly active. The much lower cleaving activity of paromomycin 2 in comparison to neomycin 1 and neamine 3 points out the major role of the protonable 6’-amine function present on ring II in neomycin54 and neamine55,

56

and replaced by a hydroxyl group in paromomycin 2. The corresponding

ammonium group is the less hindered of all AG and probably plays a major role in optimizing interaction with phosphate groups adjacent to the abasic site. The concomitant presence of this 6’-ammonium group in ring II and of an other amine function unprotonated at physiological pH (possibly the 3-amine function in ring I of neomycin with a pKa= 6.9 at 25 °C)54 explains probably the high cleaving activity at DNA abasic sites observed for the neomycin-class of AG drugs, neomycin B, kanamycins (EC50 not determined for kanamycin A), tobramycin and gentamicin C (Figure 2). The decrease of the cleaving activity with the ionic strength increase confirms the major role played by the ammonium groups in the binding to DNA.



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The cleavage of DNA at basic sites performed by antibiotic AG drugs could be partly related to their toxicity. It was shown that cytosolic gentamicin C 7 could either act directly on mitochondria by causing the release of intermembrane proteins, as shown for a variety of polycations including aminoglycosides,86 or indirectly through impairment of Bax proteosomal degradation87 upon drug binding to the β-9 proteasome subunit.88 This suggests that AG could interfere with DNA repair in mitochondria. The possibility for AG to interfere with DNA repair could also be involved in their antimicrobial activity upon exposure to DNA damaging agents such as UV radiation. The cleavage at basic sites could also have medicinal applications as AG may interfere with DNA repair and potentiate the anticancer effects of ionizing radiations and alkylating agents used in radiotherapy and chemotherapy, respectively. Regarding chemotherapy, AG nucleophilic functions should react with anticancer alkylating agents such as BCNU and, thus, the effects of differed delivery of each drug should be evaluated.

ASSOCIATED CONTENTS Supporting Information Available: Images of two typical agarose gels obtained by electrophoresis showing the cleavage of apurinic-pBR322 strand as a function of neomycin and neamine concentrations. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding authors: *Jean-François Constant: Phone +33 4 76 52 08 39, Fax: +33 4 76 52 08 05, E-mail: [email protected]; *Jean-Luc Decout: Phone +33 4 76 63 53 17, Fax +33 4 76 63 52 98, E-mail: [email protected]. Funding and acknowledgments: Maralise Perigolo de Oliveira gratefully thanks "Région Rhône-Alpes" for a MIRA grant. This work was supported by CAPES (Brazilian 24 ACS Paragon Plus Environment

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Coordination for Enhancement of Higher Education Personnel) and COFECUB (French Committee for the Evaluation of Academic and Scientific Cooperation with Brazil), which are also gratefully acknowledged.

ABBREVIATIONS AG, aminoglycoside; AP, apurinic.

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Antibiotic Drugs Aminoglycosides Cleave DNA at Abasic Sites

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