ESR studies of structure and kinetics of radicals from hydroxyurea

FreeRadicalBiology & Medicine,Vol. 6, pp. 241-244, 1989

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Original Contribution ESR STUDIES OF STRUCTURE AND KINETICS OF RADICALS FROM HYDROXYUREA An Antitumor Drug Directed Against Ribonucleotide Reductase G. LASSMANN* and B. LIERMANN Central Institute of Molecular Biology, Academy of Sciences of GDR, GDR-1115 Berlin-Buch, Robert-R6ssle-Stral~e 10 (Received 10 December 1987; Revised 2 May 1988; Accepted 17 May 1988)

Abstract--Hydroxyurea (HU) is a clinically applied antineoplastic drug, which quenches tyrosine radicals in the active site of ribonucleotide reductase (RR) and inhibits DNA synthesis in proliferating cells. Under oxidizing conditions (Cu 2÷ or H202) long-lived.radicals from HU have been found by ESR. The structure of HU radicals was established to be: H2N--CO--N---O.The kinetics of formation and decay of HU radicals after reaction

I

H of HU with H202 is complex; it exhibits a lag-phase, a maximum, and a decay, all depending on the concentration of HU. Biological consequences of HU radicals for the inhibition of RR as well as their role in cytotoxic events during chemotherapy of cancer are discussed. Keywords--Hydroxyurea radicals, Kinetics, ESR, Antitumor drug, Ribonucleotide reductase

INTRODUCTION

MATERIAL AND METHODS

Hydroxyurea (HU) inhibits ribonucleotide reductase (RR), which supplies deoxyribonucleotides for DNA synthesis in proliferating cells of bacteria up to mammals, and is therefore used as an antitumor drug. ~The IDso value for inhibition of RR, however, is rather high ( 2 - 5 . 10-4M) so that high doses are necessary for treatment, which induce cytotoxic effects. L2 The inhibitory action of HU on RR is discussed either by scavenging of the catalytic essential tyrosine radical or by chelating the iron of the non-hem iron complex, which stabilizes the tyrosine radical. L3 In order to discriminate one effect from the other, a series of inhibitors of RR have been compared, first for the radical scavenging ability against stable model radicals, and second for the complex formation with Cu 2÷ by ESR. 4 In the case of HU, after reaction with Cu 2÷ in DMSO, a long-lived radical from HU was observed rather than formation of a Cu E÷-HU-complex. In the present paper, an ESR analysis of the structure of HU radicals as well as the kinetics of formation and decay after oxidation of HU is given.

Hydroxyurea was obtained from Serva (FRG) and has been recrystallized; H202 from Laborchemie Apolda (GDR); dimethylsulfoxide (DMSO) from Reachim (USSR) and D20 (99.7% D) from Isocommerz (GDR). An ESR spectrometer VARIAN E3 coupled on-line with a minicomputer KRS 4201 (Robotron, GDR) was used. Absolute spin concentrations were determined by double integration and comparison with a 3 0 / t M aqueous solution of the nitroxide spin probe TEMPO.

RESULTS

Structure of the hydroxyurea radical

Oxidation of hydroxyurea (HU) by Cu 2÷ in an H20DMSO mixture (Fig. 1A) and by H202 in aqueous solution (Fig. 1B) induces ESR spectra with 6 equal intense lines according to splittings from a nitrogen and a proton (triplet of doublets). An analogous experiment with H202 in a solution of D20 (Fig. 1C) ensured that the doublet originates from an exchangeable NH-proton. From the type of reaction and the long life of the

*Author to whom correspondence should be addressed. 241

242

G. LASSMANNand B. LIERMANN

radical (Fig. 2) an oxidation of --N--OH

A

to ~ l ~ l - - O

I

I

H

H

is assumed. The structure of the HU radical is:

H\

O

II

g -- 2.0060

.

H/N--C--N--Oj H

B

ar~ -" 8.0 gauss NH

a H -" 11.6 gauss

In D20 a radical is formed with ao u° = 1.8 gauss (aN = 7.9 gauss, g = 2.0060), which is theoretically a value of 6.5 times less that of a~H. The line width in D:O is 0.75 gauss (in H20, 2.0 gauss), indicating that the protons of the NH2-group contribute to an unresolved hyperfine splitting. Additional lines in Figure 1C having also a linewidth of 0.75 gauss but a splitting as in Figure IB (dotted sticks) can be explained to stem from a partially deuterated radical:

f ".W]

I

"'t' '//,,yls o

D2N--CO--iq--O.

I

H

Formation and decay of hydroxyurea radicals Under strongly oxidizing conditions with 3% H202 the kinetics of formation and decay of HU radicals exhibit a complicated shape. HU radicals are formed after a pronounced lag-phase, reach a maximum ([flU] = 30/tM and 110/aM for 100 mM and 200 mM initial concentration of HU, respectively), and decay thereafter (Fig. 2). This means that even at strong oxidation in the maximum of the kinetic curve only one HU radical per 2 0 0 0 - 3 0 0 0 initial HU molecules is visualized by ESR. The lag-phase and decay-time decrease and maximal radical concentration increases with increasing concentration of HU. Thus HU radicals persists about 45 min for 200 mM HU and about 75 min for 100 mM HU. In the case of oxidation of HU (11 mM) with C C + in aqueous DMSO ( > 6 0 % ) a low concentration of HU radicals (2.4/aM) was detected after 30 min, which persist longer so as after 1 day about 1 /aM was still observable. DISCUSSION

Hydroxyurea radicals and mechanisms of inhibition of ribonucleotide reductase Although radicals from hydroxyurea have already been detected, for example, after reaction with Ce(504)25,6 with very close hyperfine coupling constants as in the present case, the interest in HU radicals has recently been grown considerably because HU is used as a chemotherapeutic drug against cancer, and analogous other hydroxamic acid derivatives having

Fig. 1. ESR spectra of hydroxyurea (HU) radicals after oxidation A) of HU (11 raM) with Cu 2. (0.62 raM) in DMSO (75%). B) of HU (100 raM) with H202" in H20. C) of HU (100 raM) with H202" in D20. *Final concentration: 3% Spectrometer settings: A) 40 roW, ~.5 gauss, 6.2 x 105

B) 40 roW, 1 gauss, 2.5 x 10s C) 40 roW, 0.5 gauss, 6.2 x l& (Line positions and his coupling constants are indicated and explained in the text.) also the - - - C - - N H O H moiety are possible candidates for such drugs. 2'7 HU was the first clinically used inhibitor of the proliferation-linked enzyme RR. 8'9 It is well established that RR contains a stable tyrosine radical stabilized by an iron-complex in the active site and any destruction of this radical inactivates RR. 1.3 Therefore, mechanisms of inhibition of RR are discussed, first as quenching of tyrosine radicals by radical scavenger or, second, as destruction of the iron-complex, by means of chelating agents. In the case of HU, an one-electron transfer from the inhibitor HU to the tyrosine radical of RR forming an inactive M2 subunit of RR and a nitroxide radical from HU was postulated. 1 O OH

II II

+ H2N~C--N

I

H 0 OH + H 2 N - - C - - N - - O

I

H

Hydroxyurearadicals

7"1,

0

~

! 30

243

o

46

O

eo

*

75

t (m~l

I.

~mixing

Fig. 2. Kinetcs of formation and decay of hydroxyurea(HU) radicals after mixing of: A) 200 mM HU in 3% H202. B) 100 mM HU in 3% H202 (ESR amplitude I (Fig. 1) in dependenceon time.) Spectrometersettings for both: 40 mW, 2.5 gauss, 1.25 × 105.

Tyrosine radicals of active RR as well as their quenching by HU were detected by means of ESR in different cell systems.I-4 Nevertheless, HU radicals have not yet been visualized in this way. Therefore, data about structure, formation and decay-time of above postulated HU radicals as given here for a model system may be helpful for understanding the mechanisms of this antitumor drug. We found that a small part (in model systems maximal 0 . 0 2 - 0 . 0 5 % ) of initial HU concentration is oxidized to HU radicals, which occur after a definite induction period and persist for a definite rather long time. The presence of long-living HU radicals can be assumed also at inhibition of RR by HU as postulated in the equation above.~ Concerning the following typical conditions (0.5 mM HU corresponding to 50% inhibition of RR, and maximal 2/LM M2-subunit of RR ]3) for the initial concentration of reactants the expected concentration of HU radicals should be much lower than 1/tM (as found for 11 mM HU and Cu2+), and thus beyond the limit of ESR detection. Nevertheless, HU radicals should persist for many days at low concentrations. With regard to the mechanism of inhibition of RR by HU our results favour a radical-scavenging mechanism rather than a metal-chelating, since firstly, we found in redox-reactions just this radical which was predicted, t and secondly, we did not find a Cu2+-HU complex, whereas a complex is easily formed between Cu 2+ and another type of inhibitor of R R - - n a m e l y - thiosemicarbazone.4

Hydroxyurea radicals and cytotoxic response hydroxyurea The clinical usefulness of HU is limited, due to the high toxicity of the drug. One of the unwanted effects is the S-phase-specific cell death of normally proliferating cells, which can be effectively avoided by a combined application of HU with radical scavengers, such as, for example, propylgallate, 5 ot-tocopherol6-8 as well as enzymes (SOD, POD, catalase 6) protecting against radicals. Since ot-tocopherol is a potent inhibitor of lipid peroxidation and protects cell membranes against damage by HU, it is assumed that the main cytotoxic event of HU is lipid peroxidation of cell membranes by HU radicals during the S-phase.~S Teratogenic effects of HU, which can be inhibited by propylgallate 15 were discussed under involvement of the - - N H - - - O H - - m o i e t y of HU.]9 It is reasonable to assume that HU radicals are formed at chemotherapy of cancer on the one hand after inactivation of RR, and on the other hand additionally by cellular redox-systems, possibly in much higher concentrations than in the former case. Primarily, HU radicals seem to be responsible for cytotoxic damage of cell membranes by HU, which can be effectively diminished by ettocopherol. The provision of direct evidence for HU radicals in liquid state by ESR, their order of concentration as well as conditions of their formation and persistence time may be useful to minimize cytotoxic events leading to an improved clinical treatment with HU.

244

G. LASSMANNand B. LIERMANN

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