37

Biochimica et Biophysica Acta, 432 (1976) 37--48 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98565

INHIBITION OF B A C I L L US SUBTILIS DNA POLYMERASE III BY ARYLHYDRAZINOPYRIMIDINES NOVEL PROPERTIES OF 2-THIOURACIL DERIVATIVES

GEORGE E. WRIGHT and NEAL C. BROWN

Department of Pharmacology, University of Massachusetts Medical School, Worcester, Mass. 01605 (U.S.A.) (Received October 20th, 1975)

Summary 6-(p-Tolylhydrazino)-uracil, 6-(p-tolylhydrazino)-isocytosine and 6-(p-tolylhydrazino)-2-thiouracil were synthesized and compared with respect to their chemical properties, their activity as inhibitors of DNA polymerase III of Bacillus subtilis, and their capacity to induce the formation of a complex between polymerase III and template DNA. As expected from earlier studies of analogous hydroxyphenylhydrazino compounds, the effects of the uracil derivative were reversed specifically by dGTP and those of the isocytosine derivative were reversed specifically by dATP. In contrast, reversal of the effects of the thiouracil derivative required both dGTP and dATP. The unique capacity of the 2-thiouracil analog to mimic either purine deoxyribonucleotide appears to reside in its ability to undergo tautomerism between the 2-thione and 2-thiol forms, which can pair with, respectively, template cytosine and thymine.

Introduction

Certain 6-(arylazo)-pyrimidines, when added to cultures of Gram-positive bacteria, i.e. Bacillus subtilis, selectively inhibit the replicative synthesis of DNA [ 1,2]. The mechanism of action of these compounds involves their metabolic conversion to an active, hydrazino form, which selectively inhibits DNA polymerase III [3--5], a replication-specific enzyme [6]. The molecular mechanism of inhibitor action on B. subtilis DNA polymerase III has been studied extensively, using the 6-(p-hydroxyphenylhydrazino) analogs of uracil and isocytosine (2-amino-4-pyrimidone) as model compounds [ 5,7,8]. The action of these inhibitors is derived in part from their capacities to mimic and compete with the two purine substrates of DNA polymerase III,

38 Q

0II

Uracil

Isocytosine

x

6 IcYT°s'NE I

.N. I

THYMINE

6 I

c.. Fig. 1. P r o p o s e d m e c h a n i s m o f p y r i m i d i n e - a r y l h y d r a z i n o p y r i m i d i n e cytosine. Right, arylhydrazinoisocytosine and thymine.

pairing. Left, arylhydrazinouracil and

dATP in the case of the isocytosine derivative and dGTP in the case of the uracil analog [9]. The basis for the capacity of the isocytosine and uracil derivatives to mimic dATP and dGTP is directly related to their ability to hydrogen bond, respectively, with thymine and cytosine [4] in the manner depicted in Fig. 1. The ability of the arylhydrazinopyrimidines to mimic and compete with purine deoxyribonucleotides is only one c o m p o n e n t of their inhibitory mechanism. A second and critical component, which is the actual cause of inhibition, involves the sequestration of the polymerase in a relatively stable, protein-drugtemplate-primer complex [ 5,7,8]. We have prepared and examined the activity of several series of arylhydrazinopyrimidines [10] in an a t t e m p t to test the inhibition model and to probe the structure and mechanism of B. subtilis DNA polymerase III. This article describes in particular the synthesis and the inhibitory and structural properties of a 2-thiouracil derivative, 6-(p-tolylhydrazino)-2-thio-4-pyrimidone. We report that the 2-thiouracil derivative, unlike its uracil analog, mimics not only dGTP, b u t also dATP. We propose that this dual capacity of 2-thio c o m p o u n d s derives from an unusual facility to form the 2-thione and 2-thiol tautomers, which can pair with, respectively, cytosine and thymine. Methods Synthesis and characterization o f the tolylhydrazinopyrimidines. 6-(p-Tolylhydrazino)-uracil, -isocytosine, and -2-thiouracil were prepared from the corresponding 6-aminopyrimidine and p-tolylhydrazine hydrochloride as described previously [ 10]. 6-(p-Tolylhydrazino)-2-methylmercapto-4-pyrimidone was obtained by stirring the 2-thiouracil derivative with methyl iodide in I M NaOH, and isolated b y precipitation with acetic acid. All c o m p o u n d s were purified by the standard m e t h o d [ 1 0 ] , and characterized by elemental analysis, nuclear magnetic resonance (see below) and mass spectral data. Full details will be published elsewhere. Nuclear magnetic resonance. NMR spectra were determined with a PerkinElmer R12B instrument (60 MHz) at a probe temperature of 35°C unless otherwise noted. Internal tetramethylsilane served as the lock signal. Perdeuteriodi-

39 methylsulfoxide and perdeuteriodimethylformamide were obtained from Stohler Isotope Chemicals and dried over Call2 before use. Studies of pairing of inhibitors with cytosine and thymine were done in dilute (5 mM) solutions of inhibitor in perdeuteriodimethylsulfoxide with the aid of a Nicolet TT-7 Fourier Transform accessory. Determination of b o u n d chemical shifts (AB) and association constants has been described [4]. Variable temperature studies were performed with the Perkin-Elmer variable temperature accessory; temperatures were considered accurate to + I°C. DNA polymerase III: preparation and assay conditions. DNA polymerase III was prepared as described previously [7] ; the Sephadex G-200 fraction, VII, was used in all experiments. The standard assay for polymerase was performed at 30°C for 5 min in 0.05 ml of an incubation mixture containing 20% glycerol, 50 mM Tris • HC1 (pH 7.6), 10 mM MgC12, 30 mM NaC1, 1.2 mM DNAase Iactivated, calf t h y m u s DNA (Worthington; cf. ref. 7 for details of preparation), 5 mM dithiothreitol, 0.1 mg per ml of bovine serum albumin (Armour, crystallized), dATP, dCTP, dGTP and [3H] TPP (100--200 cpm/pmol), 10 pM each, and 0 . 0 0 5 - 0 . 1 unit of enzyme. Preparation of the sample for the determination of acid-insoluble radioactivity is described elsewhere [4]. One unit of polymerase III is defined as that amount of enzyme which catalyses under standard conditions (30°C, 5 min) the incorporation of 1 nmol of [3H]TMP into an acid-insoluble form. Purineless assay conditions refer to the above procedure performed in the absence of dATP and dGTP. Miscellaneous. The DNAase I-activated calf thymus DNA used in the gel filtration assay of complex formation was sized by preparative filtration on Biogel A5M (Bio-Rad Laboratories) as described previously [7]. Arylhydrazinopyrmidines tend to oxidize slowly in basic solution to the corresponding azo forms. To avoid this problem the following procedure was followed. A 10 or 20 mM stock solution o f inhibitor was prepared in 50 mM NaOH and mixed at 30°C with an equal volume of fresh 100 mM Na2S204. The colorless solution of hydrazinopyrimidine or control diluent (25 mM NaOH/ 50 mM dithionite) was diluted immediately into assay or column media. Results

Potency o f the p-tolylhydrazinopyrimidines as inhibitors o f polymerase Standard conditions. Inhibition of DNA polymerase III as a function of concentration of three p-tolylhydrazino derivatives is summarized by part A of Fig. 2. The potencies of the isocytosine and uracil c o m p o u n d s were essentially identical with those which had been reported previously for the corresponding p - h y d r o x y p h e n y l derivatives [7]. The 2-thiouracil derivative was clearly a much weaker inhibitor than either o f its uracil and isocytosine analogs, and in these standard conditions, yielded only 8--10% inhibition at a concentration of 300 #M. Purineless conditions. The polymerase assay used in this study e m p l o y s a DNAase-treated DNA which is rich in short regions of unpaired template (5--50 nucleotide residues). Accordingly, this polymer, in sufficient amounts, allows polymerase III to catalyse the incorporation o f considerable amounts of [3H]TMP in the absence of one or more of the other three deoXyribonucleotides,

40 i.e. in the purineless condition. The purineless assay has two important advantages for the examination of arylhydrazinopyrimidine action. First, it is devoid of competitor nucleotide, and, therefore, provides a very sensitive m e t h o d for examining the activity of weak inhibitors like the 2-thiouracil derivative. Second, it permits convenient, direct determination of Ki, the concentration of arylhydrazinopyrimidine required to achieve 50% inhibition. The sensitivity of the purineless assay is demonstrated by the experiments summarized in Fig. 2B. The inhibitory effect of the thiouracil derivative at 0--200 pM was clearly expressed, revealing a K i of approx. 50 pM. The sensitivity of the purineless reaction is also apparent in the case of the uracil and isocytosine derivatives, each of which had a Ki value of approx. 1 pM. The latter values are essentially identical with those determined previously for the corresponding p - h y d r o x y p h e n y l h y d r a z i n o derivatives [7].

Reversal of inhibition by purine deoxyribonucleotides dGTP and dATP, both of which have Km values of approx. 0.8 pM in the DNA polymerase III system [7] reverse the inhibitory effects of, respectively, 6-(p-hydroxyphenylhydrazino)-uracil and 6-(p-hydroxyphenylhydrazino)-isocytosine [4,5]. The experiment shown in Fig. 3 indicates that the response of p-tolylhydrazino derivatives of uracil and isocytosine to purines was conven tional; dGTP (cf. part A) reversed specifically and completely the inhibitory effect of the uracil analog, and dATP (cf. part B) reversed specifically and completely the effect of the isocytosine analog. In the case of the ~-thiouracil analog the response to purine deoxyribonucleotide was somewhat anomalous; dATP at a concentration as high as 0.6 mM had no apparent competitive effect; dGTP (part B) antagonized the effect of the thiouracil derivative, b u t only partially, even at a concentration as high as 0.6 mM. Although dATP alone did not antagonise the action of thiouracil signifi-

A

I B

I00~

~

J

--

1

t- S0-

~

-

J o

60-

o u ~

40-

.o

2O

2o05

I00 200 300 INHIBITOR CONCENTRATION {pM)

IO

I5

0 IOO 150 200 INHIBITOR CON,.CENTRATION (pM)

Fig. 2. Comparison of the potencies of p-tolylhydrazino derivatives. 0.1 units of polymerase III was incubated in standard conditions (A), or in purineless conditions (B) in the presence of different concentrations of inhibitor: 4 uracil derivative; o, isocytosine derivative; X, 2-thiouracil derivative. In part B the upper scale on the abscissa refers to the concentration (0--2/~M) of the uracil or the isocytosine derivative; the lower scale (0--200/~M) applies only to the concentration of the thiouracll derivative. Control samples in experiment A incorporated 1 0 0 p m o l of [3H] T M P ; the control samples in B (minus d A T P and d G T P ) incorporated 19 p m o l of [3H] T M P .

41

cantly in purineless conditions, it clearly complemented the partial effect of dGTP; the complementation, which was suggested from the results of the assay of inhibitor potency in standard conditions (Fig. 2A), is demonstrated by the experiment in part C of Fig. 3 in which both purines were added simultaneously. The effect of the 2-thiouracil derivative at 160 ttM was reversed completely when each nucleotide was present at a concentration of 30 pM. The curves generated in comparable experiments with the isocytosine and uracil analogs are included in Fig. 3C for comparison.

Activity o f the 2-thiouracil analog as an inducer o f complex formation Since reversal of the inhibitory action of the 2-thiouracil derivative required both purine nucleotides, we suspected that it might act via an anomalous or

I00-

A.

J

80u

60.

o

40. o

~

201

_ _

- -

v--X

O-

DeoY,yr [bonu¢ieolideConcentrotion(pM} IOO.

B

I00.

/j 1 80.

80 i

C.

60'

60. X ~ x ~ X

40.

40. 20! J

~0

I

I

• I

o

I

I

~

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I

; I

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I.O 3.0 10 30 tO0 3 0 0 ~ Deoxyribonucleotide concentration(~M)

O-

I

!

I.

I

I

I

I

I

I0 0 I0 30 I00 SO0600 Deoxyribmtucleotlde concentrotion(pM)

Fig. 3. R e v e r s a l o f t h e i n h i b i t o r y e f f e c t o f t h e p - t o l y l h y d r a z i n o d e r i v a t i v e s b y p u r i n e d e o x y r i b o n u c l e o tides. T h e p u r i n e l e s s c o n d i t i o n s d e s c r i b e d i n B w e r e e m p l o y e d , a n d i n c u b a t i o n w a s p e r f o r m e d i n t h e a b s e n c e o r t h e p r e s e n c e o f t h e u r a c i l a n a l o g ( 1 6 #M, A), t h e i s o c y t o s i n e a n a l o g ( 1 6 # M , o), o r t h e 2 - t h i o u r a c i l a n a l o g ( 1 6 0 # M , X). T h e p u r i n e d e o x y r i b o n u c l e o t i d e c o n c e n t r a t i o n w a s v a r i e d as i n d i c a t e d . A, a d d i t i o n o f d A T P ; B, a d d i t i o n o f d G T P ; C, a d d i t i o n o f b o t h d A T P a n d d G T P . In p a r t C t h e a b c i s s a v a l u e refers to the concentration of each purine nucleotide and not the total nucleotide concentration.

42

I6IA 121

IO#M

E (3.

25)~M

. .40yM

12 u~

z o n~ 0 On~ 0 0 Z

16.B

--

12

0_ ~-

8

o

"1-

0

I0 15 5 FRACTION NUMBER

2O

1

6

I0

6 I0 6 I0 6 FRACTION NUMBER

I0

Fig. 4. I n d u c t i o n o f a p o l y m e r a s e • D N A c o m p l e x b y t h e 2 - t h i o u r a c i l d e r i v a t i v e . A c t i v a t e d , s i z e d calf t h y m u s D N A ( 0 . 0 7 5 p m o l ) a n d p o l y m e r a s e I I I ( 0 . 3 0 u n i t ) w e r e i n c u b a t e d f o r 1 m i n at 3 0 ° C in 0 . 1 2 5 m l o f c o l u m n b u f f e r ( 2 0 % g l y c e r o l , 5 m M d i t h i o t h r e i t o l , 0 . 1 m g p e r ml b o v i n e s e r u m a l b u m i n , 30 m M NaC1, 10 m M m a g n e s i u m a c e t a t e a n d 50 m M Tris • H C l ( p H 7 . 6 ) ) c o n t a i n i n g t h e 2 - t h i o u r a c i l a n a l o g ( 3 0 0 p M ) or c o n t r o l d i l u e n t . 0.1 m l o f e a c h m i x t u r e w a s f i l t e r e d at 4 ° C t h r o u g h a 2.0 m l c o l u m n (0.7 X 5.0 c m ) o f Bio-Gel A5M, equilibrated w i t h b u f f e r devoid of D N A and e n z y m e , but, otherwise, of a c o m p o s i t i o n i d e n t i c a l w i t h t h a t o f t h e i n p u t m i x t u r e . 0 . 1 - m l f r a c t i o n s w e r e c o l l e c t e d , a n d 25-pl p o r t i o n s w e r e a s s a y e d f o r p o l y m e r a s e a c t i v i t y ( i ) or t e m p l a t e a c t i v i t y (o). d A T P a n d d G T P w e r e i n c l u d e d in a s s a y m i x t u r e s at a c o n c e n t r a t i o n o f 0 . 7 5 m M e a c h t o allow t h e e x p r e s s i o n o f e n z y m e a c t i v i t y i n t h e p r e s e n c e o f i n h i b i t o r . In the assay for t e m p l a t e activity, D N A was deleted f r o m the standard assay m i x t u r e and replaced by polym e r a s e I I I ( 0 . 0 5 u n i t p e r 50 pl a s s a y ) . T o a s s a y t h e p o l y m e r a s e c o n t e n t o f c o l u m n f r a c t i o n s t h e s t a n d a r d 50 pl a s s a y m i x t u r e w a s s u p p l e m e n t e d w i t h 0.1 p m o l of a c t i v a t e d D N A . R e c o v e r y o f e n z y m e w a s 8 6 % in b o t h c o l u m n s . A, c o n t r o l ; B, Plus i n h i b i t o r . Fig. 5. C o m p l e x f o r m a t i o n as a f u n c t i o n o f i n h i b i t o r c o n c e n t r a t i o n . E l u t i o n p r o f i l e s o f f r a c t i o n s c o n t a i n i n g c o m p l e x . O n e u n i t o f p o l y m e r a s e I n a n d 0 . 0 7 5 p m o l o f a c t i v a t e d , s i z e d calf t h y m u s D N A w e r e i n c u b a t e d in 0 . 1 2 5 m l o f c o l u m n b u f f e r c o n t a i n i n g t h e i n d i c a t e d c o n c e n t r a t i o n s o f t h e 2 - t h i o u r a c i l d e r i v a t i v e ; 0.1 m l o f e a c h i n c u b a t i o n m i x t u r e w a s f i l t e r e d as d e s c r i b e d in Fig. 4 t h r o u g h a c o l u m n e q u i l i b r a t e d w i t h c o l u m n b u f f e r c o n t a i n i n g i n h i b i t o r at t h e s a m e c o n c e n t r a t i o n . A 25-~zl p o r t i o n o f e a c h c o l u m n f r a c t i o n w a s a s s a y e d f o r p o l y m e r a s e I I I c o n t e n t as d e s c r i b e d in Fig. 4.

trivial mechanism which did not involve induction of the formation of a drugspecific DNA polymerase III • DNA complex. Therefore, we examined the complex-inducing capacity of the 2-thiouracil derivative directly with a gel filtration technique employed previously [7] with the p-hydroxyphenylhydrazino analogs. Fig. 4 depicts the patterns of elution of template and polymerase activity from columns run in the absence and presence of 200 pM inhibitor. The profiles clearly demonstrate that the 2-thio derivative, typical of other active arylhydrazinopyrimidines, promoted the sequestration of the polymerase in a DNA • protein • drug complex.

Relationship o f complex formation to the concentration and Ki of the 2-thiouracil derivative If the 2-thiouracil derivative induced a DNA • enzyme complex by the normal mechanism involving the formation of a drug • DNA • polymerase complex, its

43 Ki should be essentially identical with Kcx, the concentration required for halfmaximal complex formation [7]. Fig. 5, which describes the formation of complex as a function of drug concentration, indicates that the value of Kcx was essentially identical with that of Ki. Part A shows elution profiles obtained at inhibitor concentrations between 0 and 300 pM; in this range, the amount of enzyme coeluting with DNA increased directly with drug concentration, reaching a maximum at 200 pM. The data of part A were used to construct a double reciprocal plot of the total enzyme activity in the position of complex as a function of drug concentration; the result, which is not shown, indicated a linear relationship, which, upon analysis yielded Kcx of approx. 50 pM.

Effect of purine deoxyribonucleotides on the induction of complex formation Table I compares the effect of addition of dATP and dGTP, alone or in combination, on the induction of complex by the uracil, isocytosine, and 2-thiouracil derivatives; the results essentially mirrored those obtained for experiments examining the effect of dATP and dGTP on the drug-induced inhibition of polymerase (cf. Fig. 3). The complex-inducing capacity of the isocytosine derivative at 10 pM was specifically and completely reversed by 0.5 mM dATP and that of the uracil derivative at 10 pM was reversed in an analogous manner by 0.5 mM dGTP. The capacity of the 2-thiouracil derivative (200 pM) to induce complex formation was only partially reversed by 0.5 mM dGTP, and even at a concentration of 1.5 mM, dGTP produced no additional destruction of complex (results not shown), dATP, alone, at 0.5 mM had no discernible effect on complex formation; however, at the same concentration it complemented the partial effect of dGTP, destroying completely the dGTP-resistant complex. Ki of the 2-thiouracil derivative in the presence of a high concentration of a single purine deoxyribonucleotide The results of the competition experiments with the purine nucleotides strongly suggested that the 2-thiouracil derivative was distributed, under assay conditions, into two inhibitory forms, a major form which mimicked dGTP, and a minor form which mimicked dATP. We attempted to estimate the conTABLE I EFFECT OF dATP AND dGTP HYDRAZIN OPYRIMIDINES Derivative used

ON T H E I N D U C T I O N

OF COMPLEX

FORMATION

BY p - T O L Y L -

m U n i t s o f e n z y m e in p o s i t i o n o f c o m p l e x No addition

+0.5 mM dGTP

+0.5 mM dATP

+0.5 mM dGTP +0.5 mM dATP

None 10/IM uracil 10 ~uM i s o c y t o s i n e 2 0 0 ~ M 2-thiou~acil

14 144 138 155

II 16 142 96

13 152 13 157

11 --10

T h e c o n d i t i o n s f o r t h e f o r m a t i o n a n d assay o f t h e p o l y m e r a s e • D N A c o m p l e x w e r e t h o s e d e s c r i b e d in Fig. 5; d A T P a n d dGTP0 w h e n p r e s e n t ° w e r e i n c l u d e d in b o t h t h e i n c u b a t i o n m i x t u r e a n d t h e a g a r o s e c o l u m n t h r o u g h w h i c h it w a s f i l t e r e d .

44

>- I00.

_> t.~ 80. '~ .._1

I1~11.

o

'

'

'

~

~6o'

'

2~)o

'

'sbo

INHIBITOR (pM) Fig. 6. P o t e n c y of the 2 - t h i o u r a c i l d e r i v a t i v e in the p r e s e n c e of high c o n c e n t r a t i o n s of d A T P or d G T P . 0.1 u n i t of e n z y m e was a s s a y e d in t h e p r e s e n c e o f the i n d i c a t e d c o n c e n t r a t i o n o f i n h i b i t o r in purineless c o n d i t i o n s (0); in t h e P r e s e n c e o f 0.S m M d G T P (m); o r in t h e p r e s e n c e o f 0.8 m M d A T P (4).

tribution of each of these forms as a function of the total concentration of inhibitor; our approach was simply to determine the apparent Ki of one form b y selectively destroying the inhibitory effect of the other with a large excess (1000 times Km) of the appropriate purine deoxyribonucleoside triphosphate. The relevant experiments are summarized in Fig. 6. In the presence of 0.8 mM dATP the inhibitor curve was essentially the same as that obtained in the purineless condition {apparent Ki = 50/~M), suggesting that in these conditions the dGTP-specific form contributed the bulk of the inhibitory effect of the 2thiouracil derivative. In the presence of 0.8 mM dGTP, the inhibition curve was clearly displaced, indicating an apparent Ki value of 200 pM, approximately four times the the concentration of drug required for 50% inhibition in the purineless conditions.

NMR analysis Structure. The NMR spectra of the isocytosine and uracil inhibitors, the 2thiouracil inhibitor, and its S-methyl analog are summarized in Table II. The spectra of the uracil and isocytosine inhibitors were similar to those of the corresponding p - h y d r o x y p h e n y l derivatives [4] and indicated that they exist in the 2,4-diketo and 2-amino-4-keto forms as anticipated. The spectrum of the 2thiouracil c o m p o u n d showed the presence of only one form, which was determined to be the 2-thione-4-keto tautomer by comparison with the spectra of the uracil inhibitor and 6-amino-2-thiouracil (results n o t shown); the preference for the 2-thioketone was not considered unusual, since simple ~-thio azaheterocycles have been shown to exist exclusively as thiolactams [11,12]. At the probe temperature of 35°C in perdeuteriodimethylsulfoxide, ring NH resonances were clearly discernible in the uracil series of inhibitors [ 10] b u t absent in the 2-thiouracil derivatives (Table II, G.E.W., unpublished observations). When the probe temperature was lowered to --40°C (in perdeuteriodimethylformamide) a broad resonance of the 2-thiouracil inhibitor was observed at

45 T A B L E II NMR CHEMICAL SHIFTS OF p-TOLYLHYDRAZINOPYRIMIDINES

,

M M 1'\'-" / 2'

3'

Derivative

(1), 3 - H

6-NH

I'-NH

2',6'-H

3',5'-H

5-H

CH 3

Uracil (X: = O) Isocytosine (X: N H 2) 2-Thiottracfl (X: = S) 2-Methylmercapto (X: - S C H 3 )

10.2

8.12

7.63

7.02

6.65

4.49

2.15

n.o.

8.09

7.45

7.02

6.65

4.69

2.21

n.o.

8.08

7.80

7.04

6.67

4.88

2.18

11.7 (3-H)

8.71

"/.65

7.03

6.66

5.08

2.20

Spectra w e r e d e t e r m i n e d at p r o b e temperature in p e r d e u t e r i o d i m e t h y l s u l f o x i d e ; p p m (6) w i t h r e s p e c t t o i n t e r n a l t e t r a m e t h y l s i l a n e , n . o . , n o t o b s e r v e d .

Other

NH 2 : 6.36

SCH3:2.44

c h e m i c a l shifts are in

12.15 ppm. This resonance, which integrated for one proton, was tentatively assigned to the 3-NH on the basis of preliminary studies of the temperature dependence of chemical shifts and on the position (11.7 ppm) of the 3-NH resonance of 2-methylmercapto-6-(p-tolylhydrazino)-4-pyrimidone. We were unable to find, even at --50°C, evidence of the 1-NH resonance of 6-(p-tolylhydrazino)-2-thiouracil. At 35°C, the hydrazino 6-NH resonance of this comp o u n d had a linewidth (8--10 Hz) considerably larger than that of the corresponding uracil inhibitor (2--3 Hz). Reduction of the temperature to --50°C did n o t appreciably affect this anomalous broadening. The latter observations, which were made only in the case of the 2-thiouracil series of inhibitors, apparently reflect an exchange phenomenon. Inhibitor-base pairing. NMR analysis of the pairing reactions of the uracil and isocytosine inhibitors with the appropriate pyrimidine base (cytosine and thymine, respectively) confirmed the pairing specificity reported previously for the p-(hydroxyphenyl) c o m p o u n d s [4]. The 2-thiouracil inhibitor b o u n d exclusively to cytosine, although the interaction appeared somewhat weaker than that between the uracil derivative and cytosine. The results of several experiments indicated a b o u n d chemical shift of the 6-NH resonance of 1.9 ppm and association constants of 8.3 1 • moF 1 for uracil: cytosine and 2.9 1 • moF 1 for 2-thiouracil : cytosine pairing. The replacement of an N - H . . . O b y the weaker N-H . . . S hydrogen bond [13--15] would be expected to lead directly to a weaker hydrogen-bonded complex. Furthermore, the resultant increase in hydrogen bond distance ( N - H . . . O, 2.9 A [16] ; N - H . . . S, 3.3--3.5 A [13] ) also may weaken the other two hydrogen bonds of the complex. When thymine was added to a solution of the 2-thiouracil inhibitor no shifts indicative of base-

46 pairing were observed. This result was not surprising since our previous studies indicated that the binding between isocytosine inhibitors and thymine in perdeuteriodimethylsulfoxide is much weaker than that between uracil inhibitors and cytosine. Thus the even weaker binding of a 2-thiol tautomer and thymine would probably be undetectable under these conditions. Discussion

The mechanism o f action o f 6-(arylhydrazino)-2-thiopyrimidines: conventinal and unique aspects The results of the experiments employing assay in purineless conditions (cf. Fig. 2B) and those examining complex formation (cf. Figs. 4 and 5) indicate that 6-(p-tolylhydrazino)-2-thiouracil, like its more p o t e n t uracil and isocytosine analogs, inhibits DNA polymerase III conventionally, b y promoting the sequestration of enzyme in a relatively stable complex with primer template DNA. The unique aspect of the action of the 2-thiouracil analog, which is clearly indicated by the results of the competition experiments exploiting dATP and dGTP (cf. Fig. 3 and Table I), is its capacity to mimic n o t one b u t both purine deoxyribonucleotides. Tautomerism: the apparent basis for the dual, dATP-dGTP specificity The surprising capacity of the 2-thiouracil analog to mimic dATP could be explained by one or more of the following conditions: ( 1 ) c o n t a m i n a t i o n of the drug preparation with a non-thiol, dATP-specific contaminant; (2) ionization, which may alter the normal m o d e of interaction between drug and template, and ( 3 ) t a u t o m e r i s m , to produce a mixture of the 2-thioketone and 2thiol, which could pair with, respectively, cytosine and thymine; the proposed mechanism of tautomerization and the specific pairing reactions are depicted in Fig. 7. The evidence strongly favors tautomerism as the basis for dual specificity. Clearly, the conditions of synthesis of the 2-thiouracil derivative, the purity, source and chemistry of the starting materials, and the extensive purifi-

O II

O II

"

•

•

HN I N - -

o

0

I CYTOSINE ]

2-THIOKETONE

~

•

H

!

O~\sN"~ + 0 I THYMINE I

2-THIOL

Fig, 7. P r o p o s e d m e c h a n i s m o f interaction o f 6-(arylhydrazino)-2-thiouracil t a u t o m e r s w i t h t e m p l a t e pyrimidines.

47 cation methods (cf. refs. 4 and 10 for details) make the presence of a unique contaminant unlikely. Furthermore, the capacity to mimic dATP does n o t reside solely with any particular preparation used here; identical effects were observed for three separate samples of the p-tolylhydrazino analog and also for purified samples of the p-bromophenylhydrazino and m-fluorophenylhydrazino analogs (results n o t shown). Although ionization u n d o u b t e d l y produces a mixture of N-1 and N-3 monoanions in aqueous media [18], we do n o t believe that ions are involved at all in the mechanism of drug action for two reasons: first, an N-l-derived anion would be inactive as an inhibitor and, second, blockade of N-3 ionization by methylation, at least in the uracil series, yields inhibitors which are fully p o t e n t (unpublished results, G.E.W. and N.C.B.). Experimentally, we have been unable to demonstrate directly by NMR the presence of a thiol tautomer of the 2-thiouracil inhibitor; nevertheless, three characteristics of its NMR spectra, all of which suggest rapid proton exchange, are consistent with its existence. First, ring NH resonances of this c o m p o u n d were absent at probe temperature (interestingly, ring NH resonance also was absent in the NMR spectrum of 2',3'-O-isopropylidene-4-thiouridine [17]); second, only one ring NH was observed at --40°C, and; third, the hydrazino 6-NH resonance, in contrast to that of the uracil and isocytosine inhibitors and a "fixed t a u t o m e r " analog of the 2-thiouracil inhibitor, was unusually broad and resistant to sharpening at reduced temperature. We can only estimate the proportion of the 2-thiol tautomer which exists in assay conditions; the estimate is based on the results of the experiment of Fig. 6, which attempts to determine the apparent Ki of each tautomer by destroying, selectively, the inhibitory effect of the other with an excess of the appropriate purine nucleotide. If it is assumed that the two tautomers do n o t differ significantly in their potency as polymerase III inhibitors, the ratio of their apparent Ki values (approx. 50 /~M for the dGTP-specific thioketone and 200 ~zM for the dATP-specific thiol) indicate that 20--25% of the inhibitor could exist as the 2-thiol tautomer. Clearly, the validity of the latter estimate relies on the assumption that the 2-thioketone and the 2-thiol forms are equip o t e n t as enzyme inhibitors; there is no obvious reason, based on the chemistry of these tautomers, to expect a difference in their respective capacities to p r o m o t e complex formation or to pair with the appropriate template pyrimidine. The 2-thiouracil analogs as indicators o f the significance o f base-pairing as a determinant o f inhibitor potency In three different classes of inhibitor analogs (p-tolylhydrazino, p-bromophenylhydrazino and m-fluorolahenylhydrazino) the replacement of the 2-keto function of uracil b y sulfur decreases inhibitor potency by a factor of at least 50. The actual basis for the loss of potency is n o t yet clear; the weakness is only partly reflected by a change in the strength of the pairing reaction with cytosine, at least in perdeuteriodimethylsulfoxide, in which the association constant was a b o u t one-third of that observed for the uracil derivative (see results above). We suspect that a major basis for the loss of potency occurring with thio substitution resides in a change in the distance between the thiol/ thioketone moieties of the tautomers and the 4-moieties of their respective

48 pyrimidine partners. Clearly, the distances between these latter moieties and the relatively bulky 2-thiol/2-thioketone groups would be greater than those between analogous groups in the arylhydrazinoisocytosine : t h y m i n e and arylhydrazinouracil : cytosine pairs; these altered relationships may induce a tilt in the orientation of the inhibitor on its template partner and, thus, affect considerably its binding to enzyme. The detection of tilting or any other change of configuration of the inhibitor • template complex induced by 2-thio substitution will require more than speculation based on the chemistry of sulfur; one means of detection, the X-ray crystallographic analysis of inhibitor : pyrimidine pairs, has been initiated.

Acknowledgments The authors wish to thank Mr. Joseph D'Ambrosio and Mr. Joseph Gambino for their technical assistance. This work was supported by grants GM21747 (G.E.W.) and CA 15915 (N.C.B.) from the National Institutes of Health.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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