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New oligonucleotide derivatives as unreactive substrate analogues and potential inhibitors of human apurinic / apyrimidinic endonuclease APE 1

Human apurinic/apyrimidinic endonuclease APE1 is one of the key enzymes of the base excision DNA repair system. The main biological function of APE1 is the hydrolysis of the phosphodiester bond on the 50-side of an apurinic/apyrimidinic site (AP-site) to give the 50-phosphate and 30-hydroxyl group. It has long been known that AP-sites have mutagenic and cytotoxic effects and their accumulation in DNA is a potential hazard to the cell lifecycle. The structural and biochemical studies of APE1 are complicated by its high catalytic activity towards the AP-site and its cyclic or acyclic analogues. This work has focussed on the design, synthesis and analysis of oligonucleotide derivatives as potentially unreactive APE1 substrates. We have shown that the replacement of oxygen atoms in the phosphate group on the 50-side from the AP-site analogue tetrahydrofuran (F) considerably decreases the rate of enzymatic hydrolysis of modified oligonucleotides. We have calculated that a N30–P50 phosphoramidate linkage is hydrolysed about 30 times slower than the native phosphodiester bond while phosphorothioate or primary phosphoramidate linkages are cleaved more than three orders of magnitude slower. The value of IC50 of the oligonucleotide duplex containing a primary phosphoramidate linkage is 2.5 10 7 M, which is in accordance with the APE1 association constant of DNA duplexes containing AP-sites. Thus, it is demonstrated that oligonucleotide duplexes with chemical modifications could be used as unreactive substrates and potential competitive inhibitors of APE1.


Introduction
Keeping DNA intact is the key to safeguarding genetic information and smooth running of all vital processes in the cell. In a mammalian genome of billions of nucleotides some 100 000 lesions per day may occur. [1][2][3][4] Genotoxicity may result from a number of endogenous factors such as side effects of normal cell metabolites, and exogenous factors such as UV irradiation, ionising radiation and a variety of mutagenic chemical compounds. 5,6 It is thought that a majority of nucleobase lesions are removed via the base excision repair (BER) pathway. [7][8][9] This process is initiated by DNA glycosylases, which find lesions amongst all the undamaged bases and catalyse break-up of N-glycosidic bonds. 10,11 After the action of DNA glycosylases the resulting apurinic/apyrimidinic site (AP-site) is targeted by apurinic/apyrimidinic endonuclease that cleaves the 5 0 -phosphate bond. The nick contains a hydroxyl group on the 3 0 -end and a 2 0 -deoxyribonucleoside-5 0 -phosphate on the 5 0 -end. Later stages of repair are performed by DNA polymerases and DNA ligases, which restore the initial DNA sequence. 12,13 Disruptions in the work of DNA repair enzymes cause severe consequences such as premature aging, cancer and other diseases. [14][15][16][17] It was shown that cells or animals in which some DNA glycosylase genes are knocked out become more sensitive to DNA-damaging factors. [18][19][20][21] At the same time exclusion of the second player of the BER pathway AP-endonuclease results in cell death, which reveals a critical role APE1 plays in the restoration of the native DNA structure. 22 Thus, the level of enzymatic activity of human AP-endonuclease APE1 has a considerable influence on the process of the removal of DNA lesions. APE1 is an extremely active enzyme, 23,24 which is able to process many synthetic analogues of the AP-site such as tetrahydrofuran (F-site) or a,o-alkanediols. [24][25][26] Analysis of crystal structures of free APE1 [27][28][29] and its complexes with DNA [30][31][32] shows that currently there is still some uncertainty in our understanding of the multiple catalytic functions of the enzyme. Moreover, the stoichiometry of the divalent cations and their catalytic functions are still not clear. [27][28][29]33,34 Therefore, the important aspects of the APE1-mediated catalytic mechanism remain unresolved due to the lack of convenient unreactive analogues of APE1 substrates.
The objective of this study was to prepare modified oligonucleotide derivatives and ascertain their utility in vitro as unreactive substrate analogues of human apurinic/apyrimidinic endonuclease APE1. Some of these derivatives may also have potential as inhibitors of APE1 in vivo. APE1 inhibition is expected to enhance genotoxicity of DNA lesions and accelerate cell death, which is the goal of radiation therapy and chemotherapy of cancer. On-going development of effective systems for cell delivery of DNA and RNA derivatives 35 allows us to believe that those oligonucleotide inhibitors of APE1 may find their use in near future as effective and stable in vivo therapeutics.

Results and discussion
Rational design of oligonucleotide derivatives as potential APE1 inhibitors Information on the mechanism of the APE1-catalysed reaction 25,28,30,36,37 prompted us to hypothesise that if we substitute the NH group for the 3 0 -bridging oxygen atom in the phosphate group of the nucleotide on the 5 0 -side from the AP-site to form an N3 0 -P5 0 phosphoramidate, or sulphur S, an amino group NH 2 or a 1,1,3,3-tetramethylguanidine (Tmg) group for any of the non-bridging oxygen atoms in the same phosphate group (Fig. 1), we disrupt its interactions with Mg 2+ cations and amino acid residues in the active centre of the enzyme, which will likely decrease the rate of the catalytic reaction. Thus, in this work we set out to synthesise oligonucleotides incorporating those substitutions ( Fig. 2 and Table 1). As APE1 exhibits also 3 0 -5 0 -exonuclease activity albeit less pronounced compared to its endonuclease activity, the 3 0 -terminal internucleotide phosphate group was chemically modified by the replacement of its oxygen atom by either a sulphur or a Tmg group to make the phosphate resistant to the exonuclease activity of the enzyme ( Fig. 2 and Table 1).
Instead of the natural AP-site, the oligonucleotides incorporated its chemically stable analogue (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran (F-site). It is known that such a substitution scarcely affects the activity of APE1. [24][25][26]38,39 Chemical synthesis of oligonucleotides with modified phosphate groups An oligonucleotide with a N3 0 -P5 0 phosphoramidate group was prepared using a dimer phosphoramidite I according to the approach described in ref. 40. Chemical synthesis of the dimer phosphoramidite I was accomplished as shown in Fig. 3. A known 5 0 -protected (2R,3S)-2-(hydroxymethyl)-3-hydroxy-tetrahydrofuran II 41 was converted to 3 0 -protected compound III. After its 5 0 -phosphitylation and hydrolysis of the transient phosphoramidite H-phosphonate diester IV was obtained that was in turn reacted with protected 3 0 -deoxy-3 0 -aminocytidine V. 40 After the selective removal of the levulinyl group and 3 0 -phosphitylation the desired dimer phosphoramidite I was obtained that was subsequently used in the synthesis of an oligonucleotide containing the AP-site mimic F and the N3 0 -P5 0 phosphoramidate internucleotide linkage on the 5 0 -side of the F-site. To confirm that the P-N bond would be stable to the conditions of oligonucleotide synthesis, the protected dimer VI was unblocked by applying standard procedures of oligonucleotide deprotection, i.e. conc. aq. ammonia, 3 h at 55 1C, then 80% acetic acid, 30 min. The product was analysed by mass-spectroscopy. Its ESI-MS spectrum contained the only peak with m/z 405.10 that corresponded to the molecular ion [M À H] À of the fully unprotected dinucleotide VI.
Dimer phosphoramidite I was used in automated oligonucleotide synthesis together with regular nucleoside phosphoramidites to obtain the model oligonucleotides ( Fig. 2 and Table 1).   Table 1. Table 1 Chemical modifications of phosphate groups and abbreviations of oligonucleotides

Abbreviation
Chemical modification of the phosphate group Analysis of the rate of hydrolysis by APE1 of oligonucleotides with modified phosphate groups incorporated into DNA substrate duplexes The efficiency of enzymatic hydrolysis by APE1 of doublestranded (ds) DNA substrates containing oligonucleotides with modified phosphate groups was ascertained by the separation of hydrolysis products by PAGE. Observed rate constants of chain scission for duplexes with substitutions only in the phosphate group on the 5 0 -side of the F-site were found to decrease in the order of Fig. 4 and Table 2). Interestingly, the N3 0 -P5 0 phosphoramidate linkage 1b in the DNA duplex F 30N /G O is hydrolysed only about 30 times slower than the native phosphodiester group 1a in the unmodified DNA duplex F O /G O . At the same time, oligonucleotides with phosphorothioate group 1c or primary phosphoramidate group 1d are hydrolysed more than three orders of magnitude slower than the unmodified duplex. Therefore, oligonucleotides containing those modifications could be potentially promising candidates for binding to and inhibiting the activity of APE1. As could be seen from the above data, the endonuclease activity of APE1 is considerably retarded for DNA duplexes containing phosphate modifications on the 5 0 -side of the F-site.
However, analysis of the products of chain scission of those duplexes has revealed that some 3 0 -5 0 -exonuclease reaction that removes the 3 0 -terminal nucleotide has also occurred. To suppress the removal of the 3 0 -terminal nucleotide from the model oligonucleotides, their 3 0 -terminal internucleotide phosphate group was modified ( Fig. 2 and Table 1). The tetramethyl phosphoryl guanidine group (Tmg) was employed as a nuclease-resistant phosphate group isostere. 42 It was observed that such a modification blocks 3 0 -5 0 -exonuclease activity of APE1 for more than 12 h. The group was also tested as a protecting group for the phosphate group on the 5 0 -side of the F-site 1e (Fig. 1). The data obtained by PAGE analysis of reaction products have shown that the duplex containing the dinucleotide 1e was resistant to the endonuclease action of APE1 for at least 3 h ( Fig. 5 and Table 2).
Comparison of cleavage efficacy ( Fig. 6) of the modified oligonucleotide derivatives has shown that the modifications reduce enzymatic activity of APE1 in the order

Binding of DNA duplexes to APE1
To ascertain the efficacy of APE1 binding to modified DNA duplexes, the kinetics of enzyme-substrate complex formation was studied by the ''stopped-flow'' method. The kinetic curve of the APE1 reaction with duplex F O /G O goes through characteristic phases (Fig. 7). The data for the APE1 interaction with a Obtained by fluorescence titration. duplexes incorporating the F-site that were published in ref. 38 and 39 prompted us to conclude that the initial (up until 0.1 seconds) decrease in fluorescence intensity could be attributed to the process of the formation of an enzyme-substrate complex that is responsible for the catalytic hydrolysis of the internucleotide phosphodiester bond. After that initial period within 0.1-10 seconds the complex dissociates with the concomitant release of the products of the reaction, which results in the increase in fluorescence intensity. As it is shown in Fig. 7, the interaction of APE1 with duplexes F N /G O and F NB /G B until 0.1 s was accompanied by only the decrease in fluorescence intensity that correlates with the formation of an enzyme-substrate complex. At the same time the interaction of APE1 with duplex F BB /G B did not result in any change in fluorescence intensity. This result may suggest that the presence of a bulky Tmg group on the 5 0 -side of the F-site has prevented the formation of an enzyme-substrate complex.
To estimate the dissociation constant of the enzyme/inhibitor complex, APE1 was titrated with F 30N /G O , F N /G O and F S /G O duplexes. The change in APE1 protein fluorescence is shown in Fig. 8.
Quenching of the Trp fluorescence was registered for all these DNA duplexes. The calculated dissociation constants K d are listed in Table 2. It is worthy of note that values of K d for F N /G O and F S /G O duplexes are approximately the same considering the standard deviation interval. It is also noteworthy that the F 30N /G O duplex is partially cleaved during the titration time. Probably, due to this fact the value of K d for the F 30N /G O duplex is 2-3 times lower in comparison with F S /G O or F N /G O duplexes, respectively.  (Fig. 9C), which is in accordance with the value of APE1 association constants with F-containing DNA duplexes. 38,39 Thus, the replacement of one of the non-bridging oxygen atoms in the phosphate group on the 5 0 -side from the F-site by the amino group to form a primary phosphoramidate did not significantly affect the affinity of APE1 for the DNA substrate. At the same time, the catalytic activity of APE1 has decreased considerably.

Assessment of inhibition efficacy
As a control, we have studied the kinetics of the interaction of APE1 with a DNA duplex 5 0 -d(CTCTCGCCTTCC)-3 0 /3 0d(GAGAGCGGAAGG)-5 0 (G/C), which did not contain any of the modifications. As seen from Fig. 9B and C, duplex G/C slightly retards enzymatic activity of APE1 resulting in IC 50 = 8.0 Â 10 À6 M. Thus, inhibitory activity of the duplex F N /G O is related to specific interactions of APE1 with the F-site.
It should be noted that IC 50 for most of the reported smallmolecule inhibitors of APE1 is in the 1.0-10.0 Â 10 À6 M range. 43,44 One of the potent inhibitors of APE1 identified in the library of pharmacologically active compounds (LOPAC) was aurintricarboxylic acid (ATA), displaying an IC 50 value of 5.5 Â 10 À8 M. 45 The developments in oligonucleotide synthesis provided an   opportunity for their use as therapeutic agents. 46,47 Therefore, the compounds reported in the present work could be used not only for biochemical and structural studies of AP endonucleases but also could have potential for subsequent application for therapy.

Experimental
The following chemicals were used in this work: N,N,N 0 ,N 0 -tetraisopropyl-2-cyanoethylphosphordiamidite, N,N-diisopropylethylamine (DIEA), 1H-tetrazole, levulinic acid and acetylacetone were from Aldrich (USA), 1-methylimidazole, N,N 0 -dicyclohexylcarbodiimide (DCC) and hydrazine hydrate were from Merck (FRG), the remainder were from ''Reakhim'' (Russia). All the reagents and solvents were used as purchased unless stated otherwise. Thin layer chromatography (TLC) was carried out on Kieselgel 60 F 254 plates (Merck, FRG). The spots were visualized under UV light (254 nm) or by spraying with 0.25% cysteine solution in 30% H 2 SO 4 and heating. Flash column chromatography was on Kieselgel 55-100 mm (Merck, FRG). NMR spectra were recorded on a Brücker AM-400 spectrometer (FRG). Chemical shifts d are reported in ppm against tetramethylsilane as an internal standard for 1 H and 13 C NMR spectra, and 85% H 3 PO 4 as an external standard for 31 P NMR spectra. 13

Oligonucleotide synthesis
Automated synthesis of oligonucleotides was carried out on an ASM-800 DNA/RNA synthesiser (''Biosset'', Russia) using dimer phosphoramidite I together with standard commercial 2-cyanoethyl deoxynucleoside phosphoramidites and CPG solid supports from Glen Research (USA). Dimer phosphoramidite I was used as a 0.1 M solution in anhydrous acetonitrile. The volumes of I and activator 1H-tetrazole solutions were increased from 20 to 80 ml, and the reaction time was extended from 1 till 10 min. Phosphorothioate groups were introduced using sulfurizing reagent II (3-((dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-3thione) from Glen Research, USA (0.1 M in pyridine-acetonitrile 3 : 2 v/v) according to the manufacturer's protocol. Oligonucleotides with internucleotide tetramethyl phosphoryl guanidine group (1e) were obtained as described in ref. 42. Oligonucleotides with an internucleotide primary phosphoramidate group (1d) were prepared as described in ref. 49 Native and modified oligonucleotides were isolated by reverse-phased HPLC on an Agilent 1200 HPLC system (USA) using a Zorbax SB-C18 5 mm column 4.6 Â 150 mm in a linear gradient of acetonitrile 0-50% in 20 mM triethylammonium acetate, pH 7.0 in 30 min, a flow rate of 2 ml min À1 . Fractions containing the desired product were pooled, concentrated in vacuo, dissolved in 0.1 ml of deionised water and precipitated by 2% LiClO 4 in acetone. After centrifugation at 14 500 rpm for 2 min, washing with acetone and drying on air for 20 min, oligonucleotide pellets were dissolved in deionised water and stored at À20 1C. Oligonucleotide homogeneity was assessed by 20% denaturing PAGE in 8 M urea, 0.1 M Tris-borate buffer, pH 8.3. The bands were visualised by staining with Stains-All (Sigma, USA).
Molecular masses of oligonucleotides were checked by LC-MS/MS ESI MS on an Agilent G6410A mass spectrometer (USA) in a negative ion mode. The samples were prepared by dissolving oligonucleotides in 20 mM triethylammonium acetate in 60% aq. acetonitrile until 0.1 mM concentration. The volume of the sample was 10 ml. Analysis was carried out using 80% aq. acetonitrile as an eluent, a flow rate of 0.1 ml min À1 and standard device settings. Molecular masses were calculated using experimental m/z values, obtained for each sample. The results are given in Table 3 and Fig. 8.

Enzymes and DNA duplexes
DNA duplexes that were used as substrates are given in Fig. 2. Oligonucleotides were purified by ion exchange HPLC on a Hamilton PRP-X500 12-30 mm 3.9 Â 300 mm column followed by reverse-phased HPLC on a Nucleoprep 100-20 C18 10 Â 250 mm column (Macherey-Nagel, FRG). Oligonucleotide homogeneity was checked by 20% denaturing PAGE.
APE1 enzyme was isolated from the E. coli Rosetta2 cell line transformed by a plasmid pET11a carrying human APE1 gene. 50 To The extent of cleavage was determined by radioautography and digital processing by Gel-pro Analyzer 4.0 software (Media Cybernetics, USA). The degree of cleavage was calculated as a ratio of the area of product peaks to the sum of the areas of the product and starting material peaks. All experiments were repeated in triplicate. The presented data points reflect the average value obtained from a set of repeats.
The fraction product was fitted by the single exponential curve using Origin software (Originlab Corp.) (eqn (1)).
where A is the amplitude, k obs is the observed rate constant, and t is the reaction time (s).

Stopped-flow fluorescence measurements
Fluorescent kinetic curves were recorded on a stopped-flow spectrometer SX.18MV (Applied Photophysics, UK). For Trp fluorescence, excitation wavelength was 290 nm. The change in Trp fluorescence emission was observed on wavelengths above 320 nm (Schott filter WG 320

Fluorescence titration of APE1 with oligodeoxyribonucleotide inhibitors
Dissociation constant K d of the complex of APE1 with the inhibitors was determined by fluorescence titration. Each point on the fluorescence titration curves was obtained by the measurement of the fluorescence intensity of the reaction mixture (100 ml) containing APE1 (1.0 Â 10 À6 M) and an oligonucleotide inhibitor at the required concentration in a buffer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 0.1 mM EDTA, 5 mM MgCl 2 , 1 mM DTT, 9% glycerol). The mixtures were incubated at 25 1C for 2 min. Fluorescence spectra were measured at an excitation wavelength of 290 nm using a Varian Cary Eclipse spectrofluorimeter (Agilent Technologies, USA). Values of fluorescence intensity at the emission maximum (335 nm) were used to calculate the dissociation constants. The values of K d were determined by non-linear regression analysis of data according to eqn (2), using program Origin software (Originlab Corp.).
where, F 0 , F and F complex are fluorescence intensities of APE1 without added inhibitor, at any given concentration of the inhibitor, and at the saturating concentration of the inhibitor, respectively.
[APE1] 0 and [dsDNA] 0 are the total concentrations of APE1 and the inhibitor.

Conclusions
This work has focussed on the design, synthesis and analysis of oligodeoxyribonucleotide derivatives as unreactive substrate analogues of a key enzyme of the DNA repair, AP-endonuclease APE1. It was found that either S-or N-substitution of the oxygen atoms in the phosphate group on the 5 0 -side of the tetrahydrofuran analogue of the AP-site (F-site) leads to a significant decrease in the APE1 activity. The kinetic analysis demonstrated that these modified oligonucleotide duplexes could be used in vitro as specific uncleavable DNA substrate analogues of APE1 for biochemical or structural studies of AP-endonuclease mechanisms. Besides, the compounds in our opinion show promise for future cell and animal studies.  Pyridine  TEA  Triethylamine  Tet  1H-Tetrazole  Tmg 1,1,3,3-Tetramethylguanidine AP-site Apurinic/apyrimidinic site F ( 2 R,3S)-2-(Hydroxymethyl)-3-hydroxytetrahydrofuran