Evaluation of cross-links in covalent complexes of 1–nitroacridines and bis-1-nitroacridines with DNA

A C T A U N I V E R S I T A T I S L O D Z I E N S I S FOLIA BIOCHIMICA ET BIOPHYSICA 6, 1988

K a z i m i e rz S tu dzia n, D a n u ta S er af in -K u ns t , D o r ot a W i l m a ń sk a M a r e k G n ia z d o w sk i

E V A L U A T I O N OF C RO S S - LI N K S IN C O V AL E N T C OM P LE X ES OF 1 - N I TR O A C R I D IN E S AN D B I S -1 - N I T R O A C R I D I N E S W I T H DNA*

An anticancer drug nitracrine (Ledakrin, C-283) and its other

bio-logically active 1-nitroacridine analogues in the presence of dithio-

threitol form with DNA irreversible complexes. The complex formation

may be demonstrated by the decrease of transcriptional template acti-vity of DNA previously incubated with a drug and sulfhydryl compounds. The aim of the present experiments is to assay the content of cross-links in the 1-nitroacridines and bis-l-nitroacridines complexes with

DNA. The complexes were analyzed by means of column chromatography on

hydroxylapatite following thermal denaturation. The comparison of the

data concerning the biological effects of different analogues of ni -tracrine with the observations on thiol-dependent inhibition of RNA synthesis in vitro and the formation of covalent bifunctional

comple-xes both in vitro and in the cell indicates that these phenomena are

manifestations of the same property of the drugs.

It has be e n p r e v i o u s l y sh ow n [1, 3, 4, 9] that an an t ica nc er d ru g n i t r a c ri n e (Ledakrin, C-283) a nd s eve r al of its l -ni tro- 9- - a m i n oa l k y l a c ri d i n e d e r i v a t i v e s in the p r es e n c e of d it h i o t h r ei t o l e x h i b it e d a h i g h i nc re as e of the t em pl a te t o x ic i t y in D NA -d ep en- de nt R NA s yn t he s is in v i t r o system. Th e e n h a n ce m e n t of the i n h i -b i ti o n is d ue to a c o v a l e n t -b i n di n g of the d ru g to DNA. T hi s p h e -no m e n o n is o b se r v e d for the ni t ra c r i n e an a lo g ue s wh i c h ex hi bi t c y t o st a t i c an d c y t ot o x i c a c ti v i t y w h i l e it do es not occur for a c r i di n es f ro m this g r o u p w i t h low b io l o gi c a l a c t iv i ty [4, 9].

* This work was supported by the Polish National Cancer Program, grant No.

2304.

K. Studzian, D. Serafin-Kunst, D. Wilmańska

» ... .... ■ ■ --

---T he a i m of t he p r e se n t e x p e r i m e n t s is to a s s a y the c o n t en t of c r o s s - l i n k s in the n i t r o a c r i d i n e - D N A c o m p le x e s fo r me d in vitro.

MATERIALS AND METHODS

M o n o m e r i c a c r i d i n e s (see Fig. 1 for th e ir form ulae ) w e r e gif ts of Prof. A. L e d d c h o w s k i (T ec h ni c al U n i v e r s i t y of G dans k, Po land),

a)

©

©

)3-© /—\ /—N)3-©

Fig. 1. Formulae of acridines studied

a) nitracrine (Ledakrin, C-283), R * -(0 1 1 2 ) 2 ” (CH^^S C-846, R - -(0 1 1 2 )2

- NH 2C H( CH3 )2 ; C-849, R - - ( C H ^ N H . , « ^ C-857, R = - ( C H ^ O H ; C-921, R

« -(CH2 )2COOH; C-1006, R » -(CH2 )3NH 2 (CH2 )5CH 3 ; b) A-339 and A-340

Rys. 1. Wzory badanych akrydyn

a) nitrakryna (Ledakrin, C-283), R ■ C-846, R

B i s - l - n i t ro a c r i d i ne s (Fig. 1) w er e k i nd l y d o n a t e d by Dr J.-B. Le P ec q (In stit ut G us tav e -R o us s y, Vi lle ju if , France). E sc h er i ch i a co li D N A - d e p en d e t R NA p o l y m er a s e wa s is o la t ed as p r e v i ou s l y d e s -c ri b e d [11]. Ot h er m a t e ri a l s u s ed w er e the sam e as b ef o re [2, 4, 9, 11]. Th e c o m p l ex e s w e r e for me d by i n cu b at i on of ca lf t hy mus D NA (400 yg/ml) w i t h 1 -n i tr o a c r i d in e s (0.2 mmo le /1 ) or bi s- l- n lt r o- a cr i d in e s (0. 09-0 .1 mm ole /1 ) in the p re se n ce of d i t h io t hr e it o l (2 mm ole/ 1) for 1 h at 0.1 m o l e / 1 of KC 1 an d t h en p u r if i e d b y e x -t r a c -ti o n w i -t h i s ob u -ta n ol a nd d i a ly s is [2, 4, 9, 11]. T he te mpl at e a c t i vi t y of the c om p l ex e s w as a s s ay e d u si ng the a mo un t of the c o m p l e x e q u i v al e n t to 20 vig of D N A an d [14C] A TP as a lab el led s ub s t ra t e [4, 8, 9, 11] a nd e x p r e s se d as a p e r c e n t ag e of R NA s y n t h e s i z e d w i t h the c o n tr o l D N A (i.e., D NA w i t ho u t an inhibitor). T he c o m p l e x es of 8 m e t ho x y p s o r al e n (46 ymole/1) w i t h D N A w e re f o r -m e d by i r r a d i at i o n a nd p u r i f i e d as d e s c r i b e d [2, 11]. Th e he at d e n a t u r e d c o m pl e x e s we r e s u b je c te d to c h r o m a t o gr a p h y o n h y d r ox y l a p a t i t e c o l um n as d e s c r i b e d b e fo r e [2]. T he f ra ctio ns w er e e l u t e d w i t h l inea r g r a di e nt of 0. 1 0.5 m o l e / 1 of s od i um p h o -s ph at e b u ff e r p H 6.8 a nd a bs o r ba n c e of D N A w as m e a s u r e d at 260 n m [2] . D N A c o n c e n t r a t i o n on th e c o m pl e x e s we re e s t i m a t e d sp ec tr op ho- t o m e tr i c a l l y [2, 11]. RESULTS H i g h in h i b it i o n of R N A sy nth esis , 8.8, 7.6, 8.6 an d 10.4% of t he t e m p l at e a c t i v it y of c o n t ro l D N A (Tab. 1), w a s f ou nd w it h c o mp l e x es of n it rac ri ne , C-846, C- 84 9 a nd C - 10 0 6 w i t h D N A r e s p e -ct ivel y. C-8 57 a nd C- 9 21 e x h i b i t e d lower i n h i b it o r y e f fe c ts (32.8 a nd 18 .3% of t he c o nt r ol r es p ec tiv el y) . B o th b i s - l - n i t ro a c r i d i ne s s h o w ed a l so low er i n h i b i t o r y e f fe c t t ha n n i t r a c r i ne u se d e ith er at t w o f ol d low er or s im il a r c o n c e n t r a t i o n in t he d i t h io t hr e it o l- - de p e nd e n t r e a ct i o n w i t h D N A (Tab. 1). C o v a l e n t b i n d in g of a r e a c ti v e m o l e c u l e to D N A m a y be e ith er m o n o f u n c t i o n a l or b if un c ti o na l . In the latt er c ase a d r ug m a y fo rm i n te r s t r a n d cr os s- l in k s. T h e y m a y be c o n v e n i e n t l y d e t ec t ed af te r t h e r m al d e n a t u r a t i o n b y the t e n d e nc y of the c r o s s- l in k ed D N A to r ec o n s t i tu t e the d o u b l e - s t r a n d e d s tr u c tu r e u p o n s ub se qu ent

A

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Fig 2 Hydroksylapatite chromatography of denatured complexes of DNA with 1-

-nitro and bis-l-nitroacridine derivatives. Thermally denatured DNA (control

DNA) and denatured complexes of DNA with 8-methoxypsoralen (a), C-846 (b),

p

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cooling. T h e r m a l l y d e n a t u r e d ( pr e do m in a nt l y s in g le -st ra nd ed ) and d o u b l e - s t r a n d e d D NA ca n be e lu t e d fro m h yd r o x y l a pa t i t e c ol u mn at d if f e re n t p ho s p ha t e c on cen t ra t io n s. As a l re a dy sh ow n [2] a d e n a -tu re d n i t r a c r i ne - D N A c o m p l ex c o n t ai n s a small (15%, see Tab. 1) but d i s t i nc t f r a ct i on of r en a t u r at e d DNA.

A p r o f i l e of d e n a t u r e d c o nt r ol D NA p re s en t s t yp i ca l l y a m a in p ea k w h i c h is e l ut e d at the p h o s p h at e c o nc e n t r a t io n s of 0 .14 -0. 15 m ol e / 1 an d fo l lo w ed b y a tai l w h i c h do es not f rom a d i s cr e te pea k (Fig. 2a, s ol id line). D e n a t u re d c o mp l ex e s of D NA w i t h 8-m etox y- pso rale n. an e ff i c ie n t c r o s s- l i n k i n g agent, w er e u se d as p os it iv e c on t r ol s in o ur e x p e r i m en t s [2]. T wo peaks, the fi rs t c o r r e s p o n -d in g to s i n g l e - s t r a nd e d D N A and the s ec on d one c o n t a i ni n g about 75% of the u l t ra v i o l e t a b s o rb i n g m a t e ri a l w er e e lu t e d fr om the c o lu m n (Fig. 2a, d a s he d line). C om p le x es of C-8 46 (Fig. 2b), C -1 00 6 (Fig. 2d) an d A-3 39 (Fig. 2e) w i t h D N A g iv e two d i s c r e -te peaks. A sm al l er shi ft of the u lt r a v i ol e t a b s o r bi n g m at er i al to the r e gi o n of d o u b l e - s t r a n d e d D N A w as o b s e r v ed in the e lu ti o n p r of i le of the c o m p l ex e s of C-8 57 (Fig. 2c), C- 84 9 an d C-921 (unshown) w h i l e the c o m p le x of A- 34 0 w a s e l u t e d as a s in gle p eak (Fig. 2f). T he i nc r ea s e of the a mo un t of D N A in the s ec on d p eak wa s e s t i m a t e d by s ub s t r a ct i o n of the re l at i ve a bs o r b an c e of c o n -trol D N A e l ut e d in this r eg i on f ro m the o p ti c al d e n si t y of c o r -r e s p o nd i n g f -ra c ti o ns of the c om p le x e s [2]. T h e r es u lts of s e v e -ral d e t e r m i n a t i o n s are r e c o rd e d in Tab. 1. T he c o n te n t of rena tu- red D NA in c o mp l e x es of n it rac ri ne , C-846, C -1 00 6 a nd A- 33 9 was on the a v er a ge 15, 20.5 a nd 21.3% w h i l e the ot he r co mp le x es s ho we d low er (C-849 a n d C-857) if a ny (C-921, A-339) in crea se in t he r e n a t u r a t i o n a b i l i t y (Tab. 1).

C-857 (c), C-1006 (d), A-339 (e) and A-340 (f) were subjected to

chromato-graphy on hydroxylapatite column. Ultraviolet absorpion of each fraction is

expressed as a percentage of the total absorbance recovered in the effluent

( ... ) - sodium phosphate concentration (mole/1); ( ) - absorbance of

DNA; (--- ) - of 8-methoxypsoralen complex (see Fig. 2a); ( ) "

absor-bance of nitroacridines complexes (Figs. 2b-2f)

Rys. 2. Profile zdenaturowanych kompleksów DNA z 1-nitro i bis-1-nitroakrydy-

nami rozdzielanymi na kolumnie hydroksyapatytowej. DNA - kontrolny i DNA-

-metoksypsoralen (a), DNA - C-846 (b), DNA - C-857 (c), DNA />

DNA - A-339 (e) i DNA - A-340 (f) po denaturacji poddano analizie

chromatogra-ficznej. Absorpcję (260 nm) każdej frakcji wyrażono jako procent całkowitej

absorbancji materiału wypływającego z kolumny

( ... ) - gradient buforu fosforanowego (mol/litr); (“ J- ; ) ' absorpcja DNA

(---- ) . kompleks DNA z 8-metoksypsoralenem (patrz rys. 2a); ( ) -

T a b l e 1

Relative transcriptional template activity and the content of cross-linked fractions in purified complexes of DNA with 1-nitroacridines

Względna aktywność matrycowa i zawartość frakcji usieciowanej oczyszczonych kompleksów 1-nitroakrydyn z DNA

Drug Template activity (%) Cross-linked fraction (%)

nitracrine 8.8a 15a

C-846 7.6 ± 1.4 (3) 20.5 ± 7.2 (3) C-849 8.6 ± 2.6 (3) 7.7 ± 3.8 (2) C-857 32.8 ± 12.8 (3) ,6.8 ± 1.6 (5) C-921 18.3 ± 3.7 (3) 0 (3) C-1006 10.4 t 5.4 (3) 21.8 ± 7.1 (7) A-339 24.5 ± 1.5 (2) 19.0 ± 8.8 (3) A-340 60.0 ± 8.0 (2) 0 b a See ref. [2] .

k No ultraviolet absorbing material followed the main peak, see Fig. 2f.

N o t e : Increase of the amount of DNA eluted in the region corresponding to double-stranded DNA (the cross-linked fraction) was estimated by subtraction

of the absorbance (in relative units) of control DNA eluted in this region

from the optical density of corresponding fractions of the complexes. Nitra-

crine-DNA complex formed at the drug concentration of 0.1 mmole/1 had a

tem-plate activity of about 13%. The number of independent experiments is

indica-ted in the parentheses. The averages of independent experiments ± range v a

-lues (for n = 2) or standard deviation values (for n > 2) are shown.

DISCUSSION

H i g h b i o l o g i c a l a c t i v i t y of n i t ra c r i n e an d th e m o n o m e r i c 1-ni- t r o - a c r i d i n e s a s s a y e d h e r e w a s d e m o n s t r a t e d b o t h o n c e ll g r o w t h in v i t r o a nd e x p e r i m e n t a l t um o ur b e a r i n g m i c e (see [1, 3, 5, 7] an d the l i t e r a t u r e c i t e d t he rei n). F o r e x a m pl e d r u g c o n c e n t r a t i o n s c a -u s i n g 50% i n h i b i t i o n of H eL a c e l l g r o w t h ( ED 5 Q ) for all of the 1- - n i t r o- a c r i d i ne s , e x c e p t for C- 921 , w e r e w i t h i n the ra n ge 2-15 y m o l e / 1 , w h i l e E D 5Q for the l a tt e r w as a bo u t 4 m m o l e / 1 [7]. T he y s h o w e d i n t e r s t r a n d D N A c r o s s - l i n k f or ma ti on , w h i c h w a s c o r r e l a t e d w i t h t h ei r c y t o t o x i c a n d a n t i t u m o u r a c t i v i t y [7]. U n l i k e oth er a c r i d i n e s the b i o l o g i c a l l y a c t i v e 1 - n i tr o - a c r i d i n e s f o rm w i t h D NA in t he p r e s e n c e of t h io l c o m p o u n d s i r r e v e r s i bl e p r o b a b l y c o v al e nt

co mp lexe s. As no a n a l y t i c a l l y u se f ul c h an ge s in o pti c al p r o -pe rt i es of the c o m p l e xe s h av e b ee n f ou nd t he d e c r e a s e of the t e m -pl a te a c t i v i t y of D NA w i t h E, co li RN A p o l y me r a s e in s ta nd ar d c on d i t i on s is u se d as a m e a s u r e of the d ru g bi nding. H ig h er i n h i -b i ti o n of R NA s yn t he s is w a s f ou n d w i t h the l - n i t ro - 9 -a m in o al k yl d e r i v a t iv e s b e a ri n g the a dd i t i o na l a mi n o a l ky l g ro up s in the side ch a in (n itracrine, C-846, C-849, and C-1006, see Fig. 1 and Tab. 1). Th r e e of th em (ex cept C-849) fo rm c o mp l e x e s of cons id er abl y, i n cr e a s ed c o n te n t of fa st r e n a t u r a ti n g f r a ct i on of D NA due to the i n te r s t r an d c o v al e nt bi ndin g. It is g e n e r a l l y a s s u me d that the 1 -n it r o a n d the a d d i t i o na l a m in o a lk y l g r ou p s are t wo r ea ct iv e s i -tes in the m o l e c u l e r e s p o n si b l e for b io l o gi c a l a c t iv i t y of 1-ni- t r o a c r i di n e s [1, 3, 5, 7]. C-85 7 a nd C-9 21 h a vi n g h y d ro x yl and c a r bo c yl g ro u ps in the si de c h a i n (Fig. 1) e x h ib i t lower i n h i b i -t or y e f f e c -t a nd c r o s s - l i n k i n g p o -t e n c y (Tab. 1).

In a ny c as e h o w e v e r b i f u nc t i o n a l b i n d i n g of 1- ni t ro a cr i di n es le ad i ng to c r o s s - l i n k f o r m a ti o n in D N A see ms to be a r e la t iv e ly rare ev e nt w h e n c o m p a r e d w i t h the p s o r a l e n d e r iv a t i v e (Fig. 2a, d a s h e d line). Th e t ot a l b i n d i n g of 8 - m e t h o x y p s o r a l e n d e t e r mi n e d w i t h the t r i r i a t e d p r e p a r a t i o n w as un de r th e se c o nd i ti o ns abo ut five m o l e c u l e s p er 10 D N A nu cl eot id es . A n o b s e r v a t i o n m a de w i t h n i tr a c r i n e tha t a lo g/ lo g pl ot of the re la t iv e t emp la te ac ti v it y ve r su s th e nu m be r of ^ C - l a b e l l e d d ru g m o le c u l e s b o u n d per 1 0 3 DNA n u c l e ot i d e s p r es e n t s a li nea r d e p en d e n c e [8] a ll ow s to f in d the a p p r o x i ma t e d e n s i t y of C-846, C- 84 9 a nd C -1 00 6 b in di n gs to be ab o ut 8-15 m o l e c u l e s p er 1 0 3 n u cle ot id es . T he lev el of the a c t i -v i t y of D N A c o m p le x e s w i t h C-85 7 a nd C-92 1 is e q u i v al e n t to abou t t e n f o l d low er bi ndin g. R e c e n t l y s e ve r al d im e rs of n it r a c ri n e ha ve b e e n s y n th e s i z e d (L e P e c q et al. [6] u n p u b l i s h e d e x p e r i -me nts, see al s o [10]). Th e tw o b i s- l - n i t r o a c r i d i n e s a ss a y e d in

thi s pa p e r e x hi b it b i o lo g i c a l ef fe c ts in se ve r al tes ts in vi vo an d in v i t r o a l t ho u g h th ei r a c t i v i t y is lo we r t han tha t of the pa r en t c o mp o u n d (L e P e c q et al. [6] u n p ub l i s h e d results). S i m i l a r y to t he ir m o n o m e r s the d im e rs b i nd i r r e v e r s ib l y to D NA in the p re s e n c e of d i t h i o t h r e i t o l , d e cr e a s i n g the te m pl a te ac ti v it y (Tab. 1). T he lev el of b i n d i n g a p p r ox i m a t e d as ab ov e is abo ut te n fo l (A — 339) a nd t w e n t y f o l d (A-340) lower t ha n th at of n i t r a -crine. As the d im e rs ha ve a p p a r e n t l y a do u b l e nu m be r of rea cti ve si tes it ha s b e e n a ss u m e d th at t he y s ho u l d f or m c ro s s - li n k s in D N A u p o n bin din g. In d ee d d e n a t u r e d c o mp l e x e s of D N A w i t h A - 339 are r e s o lv e d on h y d r o x y l a p a t i t e c o l u m n in to tw o pe a ks (Fig. 2e).

R a th e r s u r p r i s i n g l y no c r o s s - l i n k s a re d e t e c t e d in c o m p l e x of A- -340 w h i c h is r e p r o d u c i b l y e l u t e d f rom the c o l u m n as a si ngl e a nd v e r y n a r r o w p e a k (Fig. 2f). T h e d i f f e r e n c e b e t w e e n the two li ga n ds d e p e n d s on th e s t r u c t u r e of th e linkers. C o n f o r m a t i on a l r i g i d i t y of th e d i e t h y l d i p i p e r i d i n e c ha i n of A- 34 0 (Fig. 1) m a y re d uc e th e a b i l i t y of b o t h r e a ct i v e si t es in the d r u g m o l e c u l e a c t i v a t e d b y d i t h i o t h r e i t o l to i n t er a ct s i m u l t a n e o u s l y w i t h DNA. No s u ch l i m i ta t i o n of m o b i l i t y m a y be a s c r i b e d to a fl e xi b le s p e r m i n e c h a i n of A -3 39 (Fig. 1). T h e c o m p a r i s o n of t he da ta c o n c e r n i n g th e b i o lo g i c a l e f f e c t s of d i f f e r e n t a n a lo g u e s of ni- t r a c r i n e w i t h t he o b s e r v a t i o n s o n t h i o l - d e p e n d e n t i n h i b i t i o n of R N A s y n t he s i s in vi tro , an d th e f o rm a t i o n of c o v a l e n t b i f u n c t i o -n al c o m p l e x e s b o t h i-n v i t r o a nd in th e c ell i nd i ca t e s tha t th ese p h e n o m e n a are m a n i f e s t a t i o n s of the sa me p r o p e r t y of th e drugs.

ACKNOWLEDGEMENTS W e t h a n k Dr J.-B. Le P e c q a n d Dr J. M a rk o v i t s for a ge ne r ou s g if t of b i s - l - n i t r o a c r i d i n e s a n d c o m m u n i c a t i o n of t he re su lt s p r io r to t he i r publication'. W e a p p r e c i a t e th e r e a di n g of the m a -n u sc r i p t b y Dr L. S z m i g i e r o a nd E, Sz a do wsk a, M.A. T he ca pa b le t e c h n i c a l a s s i s t a n c e of M is s M. A f f e l t o w i c z is ac kn ow l ed g ed . REFERENCES [ 1] D e n n y W. A., B a g u 1 e y B. C., C a i n B. F., W a

-r i n g M. J. (1983), Molecular aspects of anticancer drug action,

eds. S. Neidle, M. J. Waring, Macmillan, London, 1-34.

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-g i e r o L. (1981), Chem.-Biol. Interact., 34, 355-366.

[ 3 ] G n i a z d o w s k i M., F i l i p s k i J., C h o r ą ż y M.

(1979), Antibiotics. Mechanism of action of antieucariotic and antiviral

compounds, ed. F. E. Hahn, Springer-Verlag, Berlin, Vol. V/2, 275-297.

[ 4 ] G n i a z d o w s k i M . , S z m i g i e r o L., W i l m a ń

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Department of General Chemistry Institute of Physiology and Biochemistry Medical Academy of Łódź

Kazimierz Studzian, Danuta Serafin-Kunst, Dorota Wilmańska Marek Gniazdowski

BADANIE WIĄZAŃ POPRZECZNYCH W NIEODWRACALNYCH KOMPLEKSACH NITROAKRYDYN

I BIS-NITROAKRYDYN Z DNA

Lek przećiwnowotworowy Ledakrin (nitrakryna, C-283) i jego aktywne

biolo-giczne pochodne 1-nitro w obecności ditiotreitolu tworzą z DNA nieodwracalne

kompleksy. Powstawanie kompleksów można wykazać poprzez badanie obniżenia

akty-wności transkrypcyjnej DNA uprzednio inkubowanego z lekiem w obecności związków

sulfhydrylowych. Celem pracy było zbadanie zawartości frakcji usieclowanej w

kompleksach 1-nitroakrydyn oraz bis-l-nitroakrydyn z DNA. Kompleksy po denatura- cji termicznej analizowano metodą chromatografii na kolumnie hydroksyapatytowej.

Porównanie danych odnoszących się do efektu biologicznego różnych analogów nitrakryny ze stopniem hamowania syntezy RNA in vitro oraz tworzeniem przez nie dwufunkcyjnych wiązań z DNA, wskazuje, że zjawiska te zależą od tych samych