J o u r n a l o f P h o t o c h e m i s t r y a n d Photobiology, B: Biology, 6 (1990) 207-220
207
M O L E C U L A R M E C H A N I C S A N D D Y N A M I C S OF D N A - F U R O C O U M A R I N C O M P L E X E S : E F F E C T OF T H E A R O M A T I Z A T I O N OF T H E PYRONE RING ON THE I N T E R C A L A T I O N GEOMETRY* JEAN-PHILIPPE DEMARET and SIMONE BRUNIE L a b o r a t o i r e d e B i o c h i m i e (CNRS UA 240), E c o l e P o l y t e c h n i q u e , 91128 P a l a i s e a u C d d e x (France) JEAN-PIERRE BALLINI L.P.C.B. (CNRS UA 198), I n s t i t u t C u r i e et Universit~ P a r i s VI, r u e P i e r r e et M a r i e C u r i e 11, 75231 P a r i s C d d e x 05 ( F r a n c e ) JEAN CADET L a b o r a t o i r e d e Chimie, D.R.F., Centre d ' E t u d e s Nucldaires, 38041 Grenoble C d d e x (France) PAUL VIGNY* L.P.C.B. (CNRS UA 198), I n s t i t u t C u r i e et Universitd P a r i s VI, r u e P i e r r e et M a r i e C u r i e 11, 75231 P a r i s C d d e x 05 (France) (Received November 24, 1989; accepted December 18, 1989)
Keywords. Angelicin, DNA, energy minimization, psoralen, model building, molecular dynamics, molecular mechanics, naphthofuran, non-covalent intercalation.
Summary Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide. *Paper presented at the Meeting on Photomedicine organized by the French Society for Photobiology, Paris, November, 1989. *Author to whom correspondence should be addressed.
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208 1. I n t r o d u c t i o n
Furocoumarins, and psoralens in particular, are intercalating DNA-binding drugs, which have been widely studied for their use in the photochemotherapy of skin diseases [1] and as molecular probes in molecular biology [2]. Their photobiological activity on UVA irradiation relies on their photochemical reactivity with pyrimidines. This reactivity generally implies the formation of two types of monoadduct and interstrand cross-links [3]. Since the photoformation of DNA interstrand cross-links is, at least partly, responsible for the mutagenicity and carcinogenicity of psoralens, monofunctional psoralens have been synthesized in recent years either by linking a voluminous group to the 3,4 double bond (3-carbethoxypsoralen (3-CPs) [4]) or by fusing a pyridine ring to the pyrone ring (methylpyridopsoralen (MePyPs) [5 ]). An alternative way to avoid the formation of cross-links is to use angular furocoumarins or angelicins (for a review, see ref. 6). The chemical structures of the various photoadducts formed within DNA by psoralens and angelicins are well understood [7-9]. It is now clear that the geometrical arrangement of the non-covalent psoralen-DNA intercalation and the psoralen excited state properties are the determining parameters for the subsequent photoreactions. We have recently shown that molecular mechanics (energy minimizations using empirical energy functions) can be used to study the structure of psoralen-DNA complexes [10]. In this paper, we focus on the structural effects related to the replacement of the pyrone ring (and the pyridine ring) by aromatic rings in order to estimate the role played by the oxygen heteroatoms of the pyrone ring and the related nitrogen atom of the pyridine ring in the overall positioning of the psoralen derivatives within the intercalation cavity. Molecular mechanics calculations were performed on the intercalation complexes of the duodecanucleotide d(CGCGATATCGCG)2 with psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen (MePyPs), 7-methylpyrido[4,3-c]psoralen (2N-MePyPs) (Fig. 1, compounds a, c, e and f respectively) and their aromatized derivatives (compounds b, d, g and h respectively). Molecular dynamics simulations were also performed with MePyPs (compound e), 2NMePyPs (compound f) and the related aromatized derivative (compound g) in order to compare the time-averaged structures with the energy-minimized structures and to check the validity of the molecular mechanics approach with the more sophisticated molecular dynamics approach.
2. M a t e r i a l s
and methods
Molecular mechanics and dynamics calculations were performed on a MicroVAX II minicomputer and a STAR ST-100 array processor (connected to a VAX 11/780 minicomputer), using CHARMm version 21 [11]. The STRANGE rigid body energy minimization routine [10] was used in the molecular mechanics calculations. Approximations were made to simulate
209 4
s
(a)
(b) 4
0
0
5' 4'
(c)
CH 3
(d)
CH 3
(e)
(f)
(g)
(h)
Fig. 1. Chemical structures of the furocoumarin derivatives: a and b, psoralen and its aromatized derivative; c and d, angelicin and its aromatized derivative; e and f, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen; g and h, their aromatized derivatives. the effect of counter-ions, solvent a n d shielding, since the calculations w e r e p e r f o r m e d for the v a c u u m state; the p h o s p h a t e g r o u p c h a r g e s were r e d u c e d so as to give a slightly negative c h a r g e on e a c h n u c l e o t i d e ( - 0 . 3 2 e ) [12] a n d a d i s t a n c e - d e p e n d e n t dielectric w a s c h o s e n [11, 13]. The p a r a m e t e r set u s e d [ 14] w a s tailored to a specific cut-off. Van der W a a l s ' e n e r g y t e r m s were shifted to yield a zero value at 9.5/~, a n d electrostatic a n d h y d r o g e n b o n d e n e r g y t e r m s w e r e z e r o e d b y a sigmoidal cut-off f u n c t i o n in the intervals 9 . 5 - 1 0 . 5 /~ a n d 4 . 0 - 7 . 0 /~ respectively. The additional p a r a m e t e r s for the f u r o c o u m a r i n s a n d related a r o m a t i z e d derivatives w e r e o b t a i n e d f r o m ref. 10; partial a t o m i c c h a r g e s w e r e o b t a i n e d f r o m CNDO calculations. E a c h p s o r a l e n or a r o m a t i z e d derivative w a s e n e r g y minimized u s i n g the a d a p t e d base N e w t o n - R a p h s o n (ABNER) s u b r o u t i n e of CHARMm with t h e s e p a r a m -
210 eters: this g a v e r.m.s, d i s p l a c e m e n t s b e t w e e n the initial a n d final s t r u c t u r e s in the 0.1 /~ range. I n t e r a c t i v e m o d e l building a n d m o l e c u l a r d y n a m i c s t r a j e c t o r y visualization w e r e p e r f o r m e d on an E v a n s & S u t h e r l a n d P S 3 9 0 interactive g r a p h i c s s y s t e m u s i n g the p r o g r a m s FRODO [15] a n d HYDRA (written b y R. H u b b a r d ) .
2.1. M o l e c u l a r s t r u c t u r e s The following s t a r t i n g s t r u c t u r e s w e r e used: (i) a c a n o n i c a l B - f o r m d(CGCGATATCGCG)2 d u o d e c a n u c l e o t i d e [ 16] with the following b a s e n u m bering convention A1 . 5'-C 3' - G B12 .
. G C .
. C G
. G C
.
A T
T A .
. A T .
T A
. C G .
A12 G C
C G
G-3' C - 5' B1
(ii) the refined c r y s t a l l o g r a p h i c s t r u c t u r e of M e P y P s [17]; (iii) the o t h e r derivatives w e r e built u s i n g s t a n d a r d s t e r e o c h e m i c a l d a t a [ 18].
2.2. M o d e l b u i l d i n g F o u r s t e p s w e r e n e c e s s a r y to build the n o n - c o v a l e n t c o m p l e x e s . (1) E n e r g y m i n i m i z a t i o n of the o l i g o n u c l e o t i d e (ABNER a l g o r i t h m ) until the r.m.s, e n e r g y g r a d i e n t w a s less t h a n 0.05 kcal m o l - 1 / ~ - 1 a n d the e n e r g y v a r i a t i o n b e t w e e n two m i n i m i z a t i o n cycles w a s less t h a n 10 -3 kcal mo1-1. (2) G r a p h i c m o d e l l i n g of an i n t e r c a l a t i o n site b e t w e e n the sixth a n d s e v e n t h b a s e p a i r s [19]: (i) the C 3 ' , O 3 ' b o n d s of the sixth t h y m i n e w e r e cut on b o t h strands; (ii) the t w o m o i e t i e s w e r e s e p a r a t e d b y 3.6 .~ a n d t h e n a d e t o r s i o n of 28 ° w a s m a d e a r o u n d the helicoidal s y m m e t r y axis [20]; (iii) the s u g a r s of the i n t e r c a l a t i o n site (i.e. the sixth a n d s e v e n t h n u c l e o t i d e s of e a c h strand) w e r e r e p u c k e r e d in a C 3 ' - e n d o ( 3 ' - 5 ' ) C 2 ' - e n d o c o n f i g u r a t i o n a n d the two s t r a n d s w e r e rebuilt b y altering the p h o s p h o - d i e s t e r c h a i n t o r s i o n angles; (iv) the c o n s t r a i n t s i n d u c e d in the two chains w e r e r e m o v e d with cycles of e n e r g y minimization; this m i n i m i z a t i o n m a i n l y a f f e c t e d the O 1 P a n d O 2 P p h o s p h a t e g r o u p a t o m s of the s e v e n t h n u c l e o t i d e of e a c h strand. (3) G r a p h i c a l i n t r o d u c t i o n of the d r u g into the i n t e r c a l a t i o n site. Of the two p o s s i b l e p o s i t i o n s of the f u r o c o u m a r i n s a n d the r e l a t e d a r o m a t i z e d d e r i v a t i v e s in the i n t e r c a l a t i o n site (in fact, four p o s i t i o n s r e d u c e d to t w o due to the s y m m e t r y of the o l i g o n u c l e o t i d e s e q u e n c e ) , w e c h o s e the o n e t h a t gives rise to the s t e r e o c o n f i g u r a t i o n s f o r m e d within DNA: c i s - s y n furanside a n d c i s - s y n p y r o n e - s i d e c o n f o r m a t i o n s for the linear c o m p o u n d s [ 7 - 9 ] ; c i s - s y n furan-side a n d c i s - a n t i p y r o n e - s i d e c o n f o r m a t i o n s f o r the a n g u l a r c o m p o u n d s [21]. T h e m o l e c u l e s w e r e t h e n fitted into the o l i g o n u c l e o t i d e such that good psoralen-base stacking interactions were obtained and bad c o n t a c t s with b a s e s w e r e avoided. (4) E n e r g y m i n i m i z a t i o n o f the c o m p l e x e s with b o t h CHARMm a n d STRANGE, until the t o t a l r.m.s, e n e r g y g r a d i e n t w a s less t h a n 0.05 kcal tool-1 /~-1 a n d the e n e r g y v a r i a t i o n b e t w e e n t w o m i n i m i z a t i o n cycles w a s
211 less than 1 0 - a kcal mol-1. Constraints were appfied to the dihedral angles of the intercalation site sugars (i.e. the sugars of the sixth and seventh nucleosides of each strand) in order to maintain the C 3 ' - e n d o ( 3 ' - 5 ' ) C2 ' - e n d o configuration during the initial refinement cycles; these were subsequently removed. About 1600 cycles of ABNER minimization (16 h of MicroVAX II CPU time) were necessary to reach the convergence criterion for each complex. Approximately 45 rain of MicroVAX II CPU time were spent in STRANGE for the rigid body minimization cycles.
2.3. M o l e c u l a r d y n a m i c s Since the current STAR implementation of CHARMm does not allow the manipulation of the complexes as segments of an infinite polymer, by defining the suitable translational and rotational symmetries, we applied small constraints (1.0 kcal tool -a) on the first and last base pairs of the oligonucleotide in order to model their interactions with the adjacent base pairs. This technique has no effect on the dynamics of the intercalation site which is considered in the current study and the r.m.s, fluctuations of the six central base pairs are similar in amplitude to those c o m p u t e d in completely unconstrained dynamics. The p r o c e d u r e for molecular dynamics was as follows: (i) a 15 ps thermalization from 0 to 300 K in 2 K steps, a given t em perat ure being obtained by assigning velocities from a gaussian distribution with a variance corresponding to this temperature; (ii) a 20 ps equilibration during which the velocities were randomly reassigned every 0.4 ps in order to provide a h o m o g e n e o u s velocity distribution; (iii) a 30 ps equilibration during which the velocities were rescaled ff the t e m p e r a t u r e fell outside a ± 10 K window, the temp er atu r e being checked every 0.4 ps; (iv) 50 ps " p r o d u c t i v e " dynamics (75 ps for the methylpyridopsoralen); (v) a time-averaged structure was c o m p u t e d over the "productive dynamics" and energy minimized. Newtonian equations of motion were integrated using the Verlet algorithm [22]. A 0.25 fs integration step was chosen as no constraint was applied on internal coordinates; the SHAKE algorithm [23, 24] was not used.
3. R e s u l t s
Figure 2 shows the calculated energy-minimized geometries of various furocoumarin derivatives intercalated between the two central alternating AT base pairs of the duodecanucleotide, in a projection plane orthogonal to the helix axis. The molecules are shown without the hydrogen atoms; the left thymine and the right adenine (full lines) are above the drug and the left adenine and the right thymine (broken lines) are below the drug. The photoreactive double bonds are shown in bold broken lines. The main geometrical features of the complexes are a complete intercalation of the furocoumarins and a parallelism between the drugs and the adjacent base pairs. The related parameters are r e p o r t e d in Table 1 which gives the distances
212
(a)
i~]" '~~'(
(b)
/ (c)
(d)
"" i"...... "//"/ -" "-x
(e)
,,/""
(0 ,..
//" ....
.,..
..'
(g)
(h) Fig. 2. Calculated intercalation geometries of the drugs between the two central AT base pairs, in a projection plane orthogonal to the helix axis (the left thymine and the right adenine (full lines) are above the drug, whereas the left adenine and the right thymine (broken lines) are below the drug): a and b, psoralen and its aromatized derivative; c and d, angelicin and its aromatized derivative; e and f, 7-methylpyrido[3,4-c ]psoralen and 7-methylpyrido[4,3 -c ]psoralen; g and h, their aromatized derivatives. between the two carbon atoms of the furocoumarin photoreactive double b o n d s (furan a n d p y r o n e ) a n d the c o r r e s p o n d i n g c a r b o n a t o m s of the t h y m i n e s o f s t r a n d s A a n d B. T h e t a b l e a l s o c o n t a i n s t h e c a l c u l a t e d v a l u e s o f t w o o p p o s i t e a n g l e s o f t h e v i r t u a l C4 r i n g s w h i c h p r e f i g u r e t h e c y c l o b u t a n e s w h i c h c a n b e f o r m e d i n s o m e c a s e s b y UVA i r r a d i a t i o n o f s u c h c o m p l e x e s . As c a n b e s e e n , t h e m i n i m u m d i s t a n c e is i n t h e r e g i o n o f 3 . 3 /~, w h i c h is a b o u t the s u m of the v a n der W a a l s ' radii of the c a r b o n atoms. M a n y angles lie i n t h e 9 0 ° r e g i o n ; t h e s e v a l u e s a r e c l o s e t o t h o s e r e p o r t e d f o r t h e
213 TABLE 1 Distances and angles between the reactive double bonds of the intercalated drug and the adjacent thymine in the complexes deduced from molecular mechanics studies
Compound
4'-5 (/~)
5'-6 (/~)
5-4'-5' (deg)
5'-6-5 (deg)
3-6 (/~)
4-5 (/~)
4-3-6 (deg)
4-5-6 (deg)
a b e d e f g h
3.40 3.62 4.75 4.49 3.25 3.32 3.57 3.32
3.79 3.72 4.50 4.32 3.36 3.38 3.62 3.44
111.5 80.2 40.6 44.7 92.2 86.4 81.9 98.3
92.7 76.1 53.0 53.0 87.8 85.2 80.4 93.4
4.62
4.48
133.1
127.1
4.30
4.94
41.4
67.8
4.18 3.89
4.07 3.86
59.1 81.6
66.3 83.7
4'-5, 5'-6, 3-6 and 4-5 represent the distances drug(C4')-thy(C5), drug(C5')-thy(C6), drug(C3)-thy(C6) and drug(C4)-thy(C5) respectively. 5 - 4 ' - 5 ' , 5'-6--5, 4 - 3 - 6 and 4 - 5 - 6 represent the angles thy(C5)-drug(C4')-drug(C5'), drug(C5')-thy(C6)-thy(C5), drug(C4)--drug(C3)-thy(C6) and drug(C4)-thy(C5)-thy(C6) respectively. In the case of angelicin, 3-6, 4-5, 4 - 3 - 6 and 4 - 5 - 6 should be replaced with 3-5, 4-6, 4 - 3 - 5 and 4--6-5 respectively. TABLE 2 Energy values (kcal mol- ) deduced from molecular mechanics studies of the DNA-furocoumarin complexes
Compound
Etot
Eoligo
Edmg
Elnter
Einter-VdW
Elnter-elec
a b c d e f
-460.0 -449.0 -461.6 -449.9 -465.1 -449.0 -450.9 -450.1
-430.6 -429.8 -431.6 -430.9 -430.7 -429.1 -430.1 -429.9
-0.2 7.7 0.3 8.2 3.1 13.8 13.5 13.5
-29.2 -26.9 -30.3 -27.2 -37.5 -33.7 -34.3 -33.7
-30.1 -27.7 -29.5 -27.2 -36.5 -36.7 -35.3 -34.7
0.9 0.6 -0.7 0.0 -1.1 3.0 1.0 1.0
g
h
Eo~go, potential energy of the unwound d(CGCGATATCGCG)2 oligonucleotide; Edm~, potential energy of the intercalated drug; Einter, external interaction energy between oligonucleotide and intercalated drug; Etot, Eol~o+Edrug +Elnter; Elnter-eLeoelectrostatic contribution to the interaction energy; Einter-VdW,van der Waals' contribution to the interaction energy. c y c l o b u t a n e r i n g o f t h e 8 - M O P - t h y m i n e f u r a n - s i d e p h o t o a d d u c t [ 2 5 ]. H o w e v e r , v a l u e s c a n r e a c h a s h i g h a s 4 . 7 /~ a n d 1 3 3 ° r e s p e c t i v e l y . Various potential energy terms can be deduced from the molecular m e c h a n i c s c a l c u l a t i o n s ( T a b l e 2). T h e s e a r e e i t h e r r e l a t e d t o t h e c o m p l e x (Etot a n d Einter) o r t o e a c h o f its m o i e t i e s (Eongo a n d Ea~g). T h e v a n d e r Waals' and electrostatic contributions to the interaction energy are also given. O f t h e e n e r g y t e r m s , t h e m o s t i n t e r e s t i n g is t h e i n t e r a c t i o n e n e r g y (Eint~r) w h i c h is i n d e p e n d e n t o f t h e z e r o e n e r g y r e f e r e n c e l e v e l a n d c a n b e r e l a t e d t o t h e e x p e r i m e n t a l l y m e a s u r a b l e i n t e r c a l a t i o n affinities. M o r e g e n e r a l l y , it
214 s h o u l d b e r e c a l l e d t h a t all t h e e n e r g y t e r m s d e d u c e d f r o m m o l e c u l a r m e c h a n i c s c a n o n l y be u s e d f o r c o m p a r i s o n s i n c e t h e y do n o t h a v e a b s o l u t e m e a n i n g . T h e s e l i m i t a t i o n s h a v e b e e n d i s c u s s e d in d e t a i l in a p r e v i o u s p a p e r [10]. F i g u r e 3 s h o w s t w o e x a m p l e s of t h e e v o l u t i o n of t h e d i s t a n c e b e t w e e n t h e p h o t o r e a c t i v e d o u b l e b o n d s at t h e f u r a n s i d e d u r i n g a 50 p s d y n a m i c
(a)
v
.8
~4
3 0
i
i
i
,
10
20
30
40
50
t(ps)
(b) A
.8 ~5 a.-,_
.~4
3 0
I
I
I
I
10
20
30
40
50
t (ps)
Fig. 3. Molecular dynamics: evolution of the distance between the 5,6 double bond of the sixth thymine of strand A and the 4',5' double bond of 7-methylpyrido[4,3-c]psoralen (a) and its aromatized derivative (b).
215 trajectory. These simulations do not show marked deviations from m ean values, thus indicating that the main geometrical features are preserved during the dynamics.
4. D i s c u s s i o n
In a recent paper, it has been shown that molecular mechanics calculations can provide a useful tool for understanding the structural aspects of photoreactions of psoralens with nucleic acids on UVA irradiation [10]. An interesting correlation has be e n observed between the geometrical arrangements of the non-covalently intercalated psoralens within DNA and the stereochemical structures of the phot oadduct s that are formed [7-9]. It has been p r o p o s e d to combine molecular mechanics with quantum chemistry in the design of new psoralen derivatives. Another interesting observation is the importance of the role played by the furan moiety which tends to maximize its stacking interactions with the adjacent base pairs. In order to understand more clearly the respective roles of the furan and prone moieties in the determination of the intercalation geometry, we have undertaken a comparative molecular mechanics study of various furocoumarins with their related aromatized derivatives where the pyrone rings, or in the case of pyridopsoralens the pyridine rings, are replaced by purely polycyclic aromatic moieties. In order to check the validity of our molecular mechanics approach, we also p e rf o r med molecular dynamics simulations. 4.1. P s o r a l e n vs. i t s a r o m a t i z e d
derivative
Psoralen itself was not studied in our previous paper [10]. It appears to have a significantly different behaviour from 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP). Compared with 8-MOP, its intercalation geometry appears to be less favourable for the formation of a furan-side adduct and very unfavourable for the formation of a pyrone-side adduct (Fig. 2(a) and Table 1). Although the furan ring tends to maximize its stacking interactions with the adjacent bases and is pushed towards strand A of the oligonucleotide, a couple, which is probably induced by the charges on the furan and pyrone rings, rotates the psoralen around an axis perpendicular to its plane. Such a movement, which pushes the 4',5' and 3,4 reactive bonds away from the 5,6 bonds of the adjacent thymines, is not possible for steric reasons in 8-MOP and for electrostatic reasons in 3-CPs and pyridopsoralens. In the case of the psoralen aromatized derivative ( c o m p o u n d b), the distance between the 4',5' bond and the 5,6 bond of the adjacent thymine is greater than for psoralen, while the angular values are closer to 90 ° (Table 1). This yields a more favourable intercalation geom et ry at the furan side (Fig. 2(b)). Due to the suppression of the charge densities on the pyrone ring, the couple is no longer present, and c o m p o u n d b maintains a rather
216 symmetrical position between the two strands. Furthermore, the furan ring is no longer pushed towards strand A of the oligonucleotide. Thus the displacement of the psoralen ring towards strand A appears to be induced not only by the maximization of the stacking between the furan ring and the adjacent bases, but also by electrostatic repulsive interactions between the pyrone ring and the adjacent bases. The suppression of the oxygen atoms of the pyrone moiety, as shown in c o m p o u n d b, therefore leads to a slightly more favourable geometrical arrangement for a further furan-side photoreaction than is the case with psoralen itself. 4.2. A n g e l i c i n vs. its a r o m a t i z e d d e r i v a t i v e The intercalation geometries of angular furocoumarins have been recognized for some time to be very different from those of psoralens [26]. We only report here the intercalation configurations which lead to a c i s - s y n furan-side m o n o a d d u c t or a cis-anti pyrone-side monoadduct. The other intercalation geometry which would lead to a ci s-syn pyrone-side adduct has never been experimentally observed, an aspect which could be related to a higher interaction energy [27]. In the first intercalation geometry, the present calculations show that angelicin is not very favourable for the formation of cis--syn furan-side or cis-anti pyrone-side monoadducts. This appears to be due to conflicting driving forces: the maximization of the furan ring stacking and the minimization of the van der Waals' interaction energy seem to have opposite effects in angelicins, whereas in psoralens both conditions can be fulfilled at the same time and yield an intercalation geom e t r y which is more favourable to furanside photoreaction. The geometrical parameters are slightly better for the angelicin aromatized derivative ( c o m p o u n d d) due to the suppression of the electrostatic perturbation induced by the cis-anti orientation at the pyrone side. It should be noted that these results do not indicate the impossibility of photoreaction in the angelicin-DNA complexes. Indeed, although the average structure is very unfavourable to photoreaction, molecular dynamics simulations have shown that relative positions of the furocoumarin and thymine moieties, which are favourable to photoreaction, may be reached [27]. 4.3. P y r i d o p s o r a l e n s vs. t hei r a r o m a t i z e d d e r i v a t i v e s Pyridopsoralens belong to the class of monofunctional derivatives in which one of the two reactive sites has been chemically blocked [5]. In this case, a decrease in reactivity is attempted by fusing the reactive pyrone bond with a pyridine ring. MePyPs ( c o m p o u n d e) has been demonstrated to be a monofunctional derivative which photobinds to DNA via its 4',5' furan bond [28]; the major photoadducts are two furan-side diastereoisomers formed with thymidine [29, 30]; additional effects have been noted recently [31]. As discussed previously [10], the present results show that MePyPs
217
is deeply intercalated in DNA (Fig. 2(e))*. The intercalation geometry is very favourable for the formation of a furan-side adduct as can be judged from the short distances between the reactive bonds and the near-90 ° angles between the atoms of these bonds. However, the formation of a pyrone-side adduct is much less favourable since the distances between the photoreactive double bonds are about 1/~ greater. These geometrical arrangements probably act with purely electronic factors (engagement of the 3,4 pyrone double bond) to abolish the photoreactivity of the pyrone site. 2N-MePyPs (compound f) differs from MePyPs only in the shift of the position of the nitrogen atom of the pyridine ring. From a physicochemical point of view, 2N-MePyPs shows a 2.5 times lower DNA photobinding capacity than MePyPs [32]. The intercalation geometry of 2N-MePyPs (Fig. 2(f)) is very similar to that of MePyPs, although slightly less favourable for the formation of a furan-side adduct. However, a striking difference between the two pyridopsoralens lies in their rather dissimilar interaction energy values, with a difference of around 10% ( - 3 6 . 5 kcal mo1-1 for MePyPs and - 3 3 . 7 kcal mo1-1 for 2N-MePyPs). This effect relies almost exclusively on the difference in charge densities between the two pyridopsoralens, as deduced from CNDO calculations. The aromatized derivative of MePyPs (compound g) appears to be intercalated in a similar fashion to 2N-MePyPs. However, it remains in a symmetrical position between the two strands, and is less pushed towards strand A. The distances and angles between its 4',5' double bond and the reactive double bond of the adjacent thymine are very favourable for subsequent photoreaction. This molecule has an interaction energy quite similar to that of 2N-MePyPs, with slightly less favourable van der Waals' interactions (less stacking between the furan ring and the adjacent bases), but a better electrostatic interaction energy (no unfavourable interaction between the pyrone nitrogen and the adjacent negative groups). A comparison between the dynamic behaviour of 2N-MePyPs and its aromatized derivative (compound h) is also of interest. As can be seen in Fig. 3, rather small fluctuations are observed around the average distances between reactive bonds over 50 ps. The amplitude of the fluctuations lies in the region of _ 0.5/~ for both the original pyridopsoralen and its aromatized derivative. This reinforces the idea that the contributions of the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not predominant in the precise positioning of the psoralen derivatives within the AT intercalation cavity. This does not exclude the fact that more specific effects may occur, related to the presence or positioning of the heteroatoms within the molecule. For example, a significant deviation from the average distances between reactive bonds is observed in the dynamics of 8-MOP and MePyPs. These aspects will be discussed in a forthcoming paper. *The nucleic acid force field has b e e n improved in CHARMra 21. Since our previous calculations were performed with CHARMm 19, w e had to minimize these complexes again to provide comparable energy values. It should be noted that, although the absolute values are different, the differences in interaction energy between the various molecules remain unchanged.
218
5. C o n c l u s i o n s T h e m o l e c u l a r m e c h a n i c s a n d d y n a m i c s c a l c u l a t i o n s w e r e p e r f o r m e d in an a t t e m p t to d e t e r m i n e the i m p o r t a n c e o f the o x y g e n h e t e r o a t o m s o f the p y r o n e ring o f the f u r o c o u m a r i n d e r i v a t i v e s in the g e o m e t r i c a l p o s i t i o n i n g of the p h o t o s e n s i t i z e r s within t h e a l t e r n a t i n g AT b a s e p a i r s of B - f o r m DNA. The i n v e s t i g a t i o n s are a c o n t i n u a t i o n o f a p r e v i o u s s t u d y o f s e v e r a l p s o r a l e n s w h i c h s e e m e d to indicate t h a t the i n t e r a c t i o n s b e t w e e n the f u r a n m o i e t y of the p s o r a l e n s a n d the a d j a c e n t b a s e p a i r s w e r e d e t e r m i n i n g f a c t o r s in the i n t e r c a l a t i o n g e o m e t r y . T h e p r e s e n t c o m p a r i s o n o f s e v e r a l linear a n d a n g u l a r f u r o c o u m a r i n s with r e l a t e d c o m p o u n d s in w h i c h the p y r o n e ring h a s b e e n a r o m a t i z e d e m p h a s i z e s a s o m e w h a t similar b e h a v i o u r , b o t h f r o m a m o l e c u l a r m e c h a n i c s a n d m o l e c u l a r d y n a m i c s p o i n t o f view. It r e i n f o r c e s t h e i m p o r t a n c e o f the s t a c k i n g i n t e r a c t i o n s of the f u r a n m o i e t y with the a d j a c e n t b a s e p a i r s in the p o s i t i o n i n g within t h e o l i g o n u c l e o t i d e . This d o e s n o t c o m p l e t e l y eliminate the role o f the i n t e r a c t i o n o f the p y r o n e m o i e t y with the a d j a c e n t b a s e pairs. A careful e x a m i n a t i o n o f the e n e r g y - m i n i m i z e d g e o m e t r i e s s h o w s t h a t the t e n d e n c y of f u r o c o u m a r i n s to m a x i m i z e t h e s t a c k i n g o f the f u r a n ring is m o d u l a t e d b y the i n t e r a c t i o n s l o c a t e d on the p y r o n e side. A n o t h e r c o n c l u s i o n of t h e p r e s e n t s t u d y is t h a t m o l e c u l a r m e c h a n i c s c a n be u s e d to p r e d i c t a n i n t e r c a l a t i o n g e o m e t r y f o r a r o m a t i z e d d e r i v a t i v e s o f f u r o c o u m a r i n s w h i c h m i g h t b e f a v o u r a b l e for t h e f o r m a t i o n ' o f f u r a n - s i d e m o n o a d d u c t s within DNA. E x p e r i m e n t a l w o r k o n the p h o t o r e a c t i o n s is currently being undertaken.
Acknowledgments W e are i n d e b t e d to Dr. J o c e l y n e Blais a n d Dr. Odilon Chalvet for t h e i r h e l p in the c a l c u l a t i o n o f t h e partial a t o m i c c h a r g e s a n d Dr. Mary L a p a d a t for critical r e a d i n g of t h e m a n u s c r i p t . W e also w i s h to t h a n k L.U.R.E., Universit~ de Paris Sud, Orsay, F r a n c e f o r the c o m p u t e r facilities. This w o r k w a s p a r t l y s u p p o r t e d b y the C e n t r e N a t i o n a l de la R e c h e r c h e Scientifique (UA 198 a n d 2 4 0 ) a n d the Minist~re de la R e c h e r c h e et de l ' E n s e i g n e m e n t Sup~rieur (grant 8704022).
References 1 J. A. Parrish, T. B. Fitzpatrick, L. Tanenbaum and M. A. Pathak, Photochemotherapy of psoriasis with oral methoxsalen and longwave ultraviolet light, N. Engl. J. Med., 2Pl (1974) 1207-1211. 2 G. D. Cimino, H. B. Gampen, S. T. Isaacs and J. E. Hearst, Psoralens as photoreactive probes of nucleic acid structure and functions: organic chemistry, photochemistry and biochemistry, A n n u . Rev. Biochem., 54 (1985) 1151-1193. 3 L. Musajo and G. Rodighiero, in A. C. Giese (ed.), Photophysiology, Vol. VII, Academic Press, New York, 1972, pp. 115-147.
219 4 P. Queval and E. Bisagni, NouveUe synth~se du psoral~ne et de compos6s apparent6s, Eur. J. Med. Chem., 9 (1974) 3 3 5 - 3 4 0 . 5 J. Moron, C. H. Nguyen and E. Bisagni, Synthesis of 5H-furo[3',2':6,7][1]benzo-pyrano[3,4c]pyridin-5-ones and 8H-pyrano[3',2':5,6]benzofuro[3,2-c]pyridin-8-ones (pyridopsoralens), J. Chem. Soc., Perkin Trans. 1, 1 (1983) 2 2 5 - 2 2 9 . 6 F. Dall'Acqua, D. Vedaldi, S. Caflieri, A. Gultto, F. Bordin and G. Rodighiero, Chemical basis of the photosensitizing activity of angelicins, Natl. Cancer Inst. Monogr., 66 (1984) 55-61. 7 J. E. Hearst, S. T. Isaacs, D. Kanne, H. Rapoport and K. Straub, The reaction of psoralen with deoxyribonucleic acid, Q. Rev. Biophys., 17 (1984) 1--44. 8 P. Vigny, F. Gaboriau, L. Voituriez and J. Cadet, Chemical structure of p s o r a l e n - n u c l e i c acid photoadducts, Biochimie, 67 (1985) 3 1 7 - 3 2 5 . 9 P. Vigny, A. Moysan, J. Cadet and L. Voituriez, in R. H. Douglas, J. Moan and G. Ronto (eds.), Light in Biology and Medicine, Vol. 2, Plenum, New York, 1989, in the press. 10 J.-Ph. Demaret, S. Brunie, J.-P. Ballini and P. Vigny, Geometry of intercalation of psoralens in DNA approached by molecular mechanics, Photochem. Phetobiol., 50 (1989) 7-21. 11 B. R. Brooks, R. E. Bruccoleri, B. D. Olafson, D. J. States, S. Swaminathan and M. Karplus, CHARMM: a program for macromolecular energy, minimization and cynamic calculations, J. Comput. Chem., 4 (1983) 1 8 7 - 2 1 7 . 12 B. Tidor, K. K. Irikura, B. R. Brooks a n d M. Karplus, Dynamics of DNA oligomers, J. Biomolec. Struct. Dyn., 1 (1983) 2 3 1 - 2 5 2 . 13 B. R. Gelin and M. Karplus, Mechanism of tertiary structural change in hemoglobin, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 8 0 1 - 8 0 5 . 14 L. Nilsson and M. Karplus, Empirical energy functions for energy minimization and dynamics of nucleic acids, J. Comput. Chem., 7 (1986) 5 9 1 - 6 1 6 . 15 T. A. Jones, A graphics model building and refinement system for macromolecules, J. Appl. CrystaUogr., 11 (1978) 2 6 8 - 2 7 2 . 16 S. Arnott, P. J. Campbell-Smith and R. Chandrasekaran, Atomic coordinates and molecular conformations for DNA-DNA, RNA-RNA and DNA-RNA helices, in G. D. F a s m a n (ed.), CRC Handbook of Biochemistry and Molecular Biology, Vol. II, CRC Press, Cleveland, 1975, pp. 411--423. 17 J. Delettr~, M.-E. Delaitre, P. Vigny and J. Moron, Structure de la methyl-7-5Hfuro[3',2':6,7]chromeno[3,4-c]pyridinone-5, Acta CrystaUogr., C, 42 (1986) 1 8 5 1 - 1 8 5 3 . 18 J. A. Pople and D. L. Beveridge, in Approximate Molecular Orbital Theory, McGrawHill, New York, 1970, p. 111. 19 C. J. Alden and S. Arnott, Visualization of planar drug intercalators in B-DNA, Nucleic Acids Res., 2 (1975) 1 7 0 1 - 1 7 1 7 . 20 G. W i e s e h a h n and J. E. Hearst, DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis, Proc. Natl. Acad. Sci. U.S.A., 75 (1978) 2 7 0 3 - 2 7 0 7 . 21 S. Caffieri, V. Lucchini, P. Rodighiero, G. Miolo and F. Dall'Acqua, 3,4 and 4',5'-photocycloadducts between 4'-methylangelicin and thymine from DNA, Photochem. Photobiol., 48 (1988) 5 7 3 - 5 7 7 . 22 L. Verlet, Computer " e x p e r i m e n t s " on classical fuids. I. Thermodynamical properties of L e n n a r d - J o n e s molecules, Phys. Rev., 159 (1967) 98. 23 J. P. Ryckaert, G. Ciccoti a n d H. J. C. Berendsen, Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes, J. Comput. Phys., 23 (1977) 327. 24 W. F. van Gunsteren and H. J. C. Berendsen, Algorithms for macromolecular dynamics and constraint dynamics, Mol. Phys., 34 (1977) 1311. 25 S. Peckler, B. Graves, D. Kanne, H. Rapoport, J. E. Hearst and S. H. Kim, Structure of a p s o r a l e n - t h y m i n e m o n o a d d u c t formed in photoreaction with DNA, J. Mol. Biol., 162 (1982) 157-172.
220 26 G. Rodighiero, F. Dall'Acqua and D. Averbeck, New psoralen and angelicin derivatives, in F. P. Gasparro (ed.), Psoralen DNA Photobiology, Vol. I, CRC Press, Boca Raton, FL, 1988, pp. 3 7 - 1 1 4 . 27 J. P. Demaret, unpublished work, 1989. 28 J. Blais, P. Vigny, J. Moron and E. Bisagni, Spectroscopic properties and photoreactivity with DNA of new monofunctional psoralens, the pyridopsoralens, Photochem. Photobiol., 39 (1984) 1 4 5 - 1 5 6 . 29 A. Moysan, Caractdrisation et dosage des produits de photoaddition de psoral~nes dans I'ADN "in vitro' et dans I'ADN cellulaire, Doctoral Thesis, Universit~ Paris VI, 1987. 30 A. Moysan, unpublished results, 1987. 31 R. Costalat, J. Blais, J.-P. Ballini, A. Moysan, J. Cadet, O. Chalvet and P. Vigny, Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: a triplet-triplet energy transfer mechanism, Photochem. Photobiol., 47 (1989) in the press. 32 J. Blais, D. Averbeck, J. Moron, E. Bisagni and P. Vigny, Effect of molecular structure on the photophysical properties, the photoreactivity with DNA and the photobiological activity of monofunctional pyridopsoralens, Photochem. Photobiol., 45 (1987) 465--472.
207
M O L E C U L A R M E C H A N I C S A N D D Y N A M I C S OF D N A - F U R O C O U M A R I N C O M P L E X E S : E F F E C T OF T H E A R O M A T I Z A T I O N OF T H E PYRONE RING ON THE I N T E R C A L A T I O N GEOMETRY* JEAN-PHILIPPE DEMARET and SIMONE BRUNIE L a b o r a t o i r e d e B i o c h i m i e (CNRS UA 240), E c o l e P o l y t e c h n i q u e , 91128 P a l a i s e a u C d d e x (France) JEAN-PIERRE BALLINI L.P.C.B. (CNRS UA 198), I n s t i t u t C u r i e et Universit~ P a r i s VI, r u e P i e r r e et M a r i e C u r i e 11, 75231 P a r i s C d d e x 05 ( F r a n c e ) JEAN CADET L a b o r a t o i r e d e Chimie, D.R.F., Centre d ' E t u d e s Nucldaires, 38041 Grenoble C d d e x (France) PAUL VIGNY* L.P.C.B. (CNRS UA 198), I n s t i t u t C u r i e et Universitd P a r i s VI, r u e P i e r r e et M a r i e C u r i e 11, 75231 P a r i s C d d e x 05 (France) (Received November 24, 1989; accepted December 18, 1989)
Keywords. Angelicin, DNA, energy minimization, psoralen, model building, molecular dynamics, molecular mechanics, naphthofuran, non-covalent intercalation.
Summary Results of molecular mechanics and dynamics calculations on intercalation complexes of DNA with various furocoumarins (psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen) and their corresponding aromatized derivatives are presented. These calculations were undertaken with the aim to elucidate the roles of the pyrone and pyridine moieties in the interactions which tend to orient the furocoumarins and pyridopsoralens between DNA base pairs. It appears that the intercalation geometries are very similar for the furocoumarins and related aromatized compounds. Therefore the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not important in the orientation of the drug within the oligonucleotide. *Paper presented at the Meeting on Photomedicine organized by the French Society for Photobiology, Paris, November, 1989. *Author to whom correspondence should be addressed.
1011-1344/90/$3.50
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208 1. I n t r o d u c t i o n
Furocoumarins, and psoralens in particular, are intercalating DNA-binding drugs, which have been widely studied for their use in the photochemotherapy of skin diseases [1] and as molecular probes in molecular biology [2]. Their photobiological activity on UVA irradiation relies on their photochemical reactivity with pyrimidines. This reactivity generally implies the formation of two types of monoadduct and interstrand cross-links [3]. Since the photoformation of DNA interstrand cross-links is, at least partly, responsible for the mutagenicity and carcinogenicity of psoralens, monofunctional psoralens have been synthesized in recent years either by linking a voluminous group to the 3,4 double bond (3-carbethoxypsoralen (3-CPs) [4]) or by fusing a pyridine ring to the pyrone ring (methylpyridopsoralen (MePyPs) [5 ]). An alternative way to avoid the formation of cross-links is to use angular furocoumarins or angelicins (for a review, see ref. 6). The chemical structures of the various photoadducts formed within DNA by psoralens and angelicins are well understood [7-9]. It is now clear that the geometrical arrangement of the non-covalent psoralen-DNA intercalation and the psoralen excited state properties are the determining parameters for the subsequent photoreactions. We have recently shown that molecular mechanics (energy minimizations using empirical energy functions) can be used to study the structure of psoralen-DNA complexes [10]. In this paper, we focus on the structural effects related to the replacement of the pyrone ring (and the pyridine ring) by aromatic rings in order to estimate the role played by the oxygen heteroatoms of the pyrone ring and the related nitrogen atom of the pyridine ring in the overall positioning of the psoralen derivatives within the intercalation cavity. Molecular mechanics calculations were performed on the intercalation complexes of the duodecanucleotide d(CGCGATATCGCG)2 with psoralen, angelicin, 7-methylpyrido[3,4-c]psoralen (MePyPs), 7-methylpyrido[4,3-c]psoralen (2N-MePyPs) (Fig. 1, compounds a, c, e and f respectively) and their aromatized derivatives (compounds b, d, g and h respectively). Molecular dynamics simulations were also performed with MePyPs (compound e), 2NMePyPs (compound f) and the related aromatized derivative (compound g) in order to compare the time-averaged structures with the energy-minimized structures and to check the validity of the molecular mechanics approach with the more sophisticated molecular dynamics approach.
2. M a t e r i a l s
and methods
Molecular mechanics and dynamics calculations were performed on a MicroVAX II minicomputer and a STAR ST-100 array processor (connected to a VAX 11/780 minicomputer), using CHARMm version 21 [11]. The STRANGE rigid body energy minimization routine [10] was used in the molecular mechanics calculations. Approximations were made to simulate
209 4
s
(a)
(b) 4
0
0
5' 4'
(c)
CH 3
(d)
CH 3
(e)
(f)
(g)
(h)
Fig. 1. Chemical structures of the furocoumarin derivatives: a and b, psoralen and its aromatized derivative; c and d, angelicin and its aromatized derivative; e and f, 7-methylpyrido[3,4-c]psoralen and 7-methylpyrido[4,3-c]psoralen; g and h, their aromatized derivatives. the effect of counter-ions, solvent a n d shielding, since the calculations w e r e p e r f o r m e d for the v a c u u m state; the p h o s p h a t e g r o u p c h a r g e s were r e d u c e d so as to give a slightly negative c h a r g e on e a c h n u c l e o t i d e ( - 0 . 3 2 e ) [12] a n d a d i s t a n c e - d e p e n d e n t dielectric w a s c h o s e n [11, 13]. The p a r a m e t e r set u s e d [ 14] w a s tailored to a specific cut-off. Van der W a a l s ' e n e r g y t e r m s were shifted to yield a zero value at 9.5/~, a n d electrostatic a n d h y d r o g e n b o n d e n e r g y t e r m s w e r e z e r o e d b y a sigmoidal cut-off f u n c t i o n in the intervals 9 . 5 - 1 0 . 5 /~ a n d 4 . 0 - 7 . 0 /~ respectively. The additional p a r a m e t e r s for the f u r o c o u m a r i n s a n d related a r o m a t i z e d derivatives w e r e o b t a i n e d f r o m ref. 10; partial a t o m i c c h a r g e s w e r e o b t a i n e d f r o m CNDO calculations. E a c h p s o r a l e n or a r o m a t i z e d derivative w a s e n e r g y minimized u s i n g the a d a p t e d base N e w t o n - R a p h s o n (ABNER) s u b r o u t i n e of CHARMm with t h e s e p a r a m -
210 eters: this g a v e r.m.s, d i s p l a c e m e n t s b e t w e e n the initial a n d final s t r u c t u r e s in the 0.1 /~ range. I n t e r a c t i v e m o d e l building a n d m o l e c u l a r d y n a m i c s t r a j e c t o r y visualization w e r e p e r f o r m e d on an E v a n s & S u t h e r l a n d P S 3 9 0 interactive g r a p h i c s s y s t e m u s i n g the p r o g r a m s FRODO [15] a n d HYDRA (written b y R. H u b b a r d ) .
2.1. M o l e c u l a r s t r u c t u r e s The following s t a r t i n g s t r u c t u r e s w e r e used: (i) a c a n o n i c a l B - f o r m d(CGCGATATCGCG)2 d u o d e c a n u c l e o t i d e [ 16] with the following b a s e n u m bering convention A1 . 5'-C 3' - G B12 .
. G C .
. C G
. G C
.
A T
T A .
. A T .
T A
. C G .
A12 G C
C G
G-3' C - 5' B1
(ii) the refined c r y s t a l l o g r a p h i c s t r u c t u r e of M e P y P s [17]; (iii) the o t h e r derivatives w e r e built u s i n g s t a n d a r d s t e r e o c h e m i c a l d a t a [ 18].
2.2. M o d e l b u i l d i n g F o u r s t e p s w e r e n e c e s s a r y to build the n o n - c o v a l e n t c o m p l e x e s . (1) E n e r g y m i n i m i z a t i o n of the o l i g o n u c l e o t i d e (ABNER a l g o r i t h m ) until the r.m.s, e n e r g y g r a d i e n t w a s less t h a n 0.05 kcal m o l - 1 / ~ - 1 a n d the e n e r g y v a r i a t i o n b e t w e e n two m i n i m i z a t i o n cycles w a s less t h a n 10 -3 kcal mo1-1. (2) G r a p h i c m o d e l l i n g of an i n t e r c a l a t i o n site b e t w e e n the sixth a n d s e v e n t h b a s e p a i r s [19]: (i) the C 3 ' , O 3 ' b o n d s of the sixth t h y m i n e w e r e cut on b o t h strands; (ii) the t w o m o i e t i e s w e r e s e p a r a t e d b y 3.6 .~ a n d t h e n a d e t o r s i o n of 28 ° w a s m a d e a r o u n d the helicoidal s y m m e t r y axis [20]; (iii) the s u g a r s of the i n t e r c a l a t i o n site (i.e. the sixth a n d s e v e n t h n u c l e o t i d e s of e a c h strand) w e r e r e p u c k e r e d in a C 3 ' - e n d o ( 3 ' - 5 ' ) C 2 ' - e n d o c o n f i g u r a t i o n a n d the two s t r a n d s w e r e rebuilt b y altering the p h o s p h o - d i e s t e r c h a i n t o r s i o n angles; (iv) the c o n s t r a i n t s i n d u c e d in the two chains w e r e r e m o v e d with cycles of e n e r g y minimization; this m i n i m i z a t i o n m a i n l y a f f e c t e d the O 1 P a n d O 2 P p h o s p h a t e g r o u p a t o m s of the s e v e n t h n u c l e o t i d e of e a c h strand. (3) G r a p h i c a l i n t r o d u c t i o n of the d r u g into the i n t e r c a l a t i o n site. Of the two p o s s i b l e p o s i t i o n s of the f u r o c o u m a r i n s a n d the r e l a t e d a r o m a t i z e d d e r i v a t i v e s in the i n t e r c a l a t i o n site (in fact, four p o s i t i o n s r e d u c e d to t w o due to the s y m m e t r y of the o l i g o n u c l e o t i d e s e q u e n c e ) , w e c h o s e the o n e t h a t gives rise to the s t e r e o c o n f i g u r a t i o n s f o r m e d within DNA: c i s - s y n furanside a n d c i s - s y n p y r o n e - s i d e c o n f o r m a t i o n s for the linear c o m p o u n d s [ 7 - 9 ] ; c i s - s y n furan-side a n d c i s - a n t i p y r o n e - s i d e c o n f o r m a t i o n s f o r the a n g u l a r c o m p o u n d s [21]. T h e m o l e c u l e s w e r e t h e n fitted into the o l i g o n u c l e o t i d e such that good psoralen-base stacking interactions were obtained and bad c o n t a c t s with b a s e s w e r e avoided. (4) E n e r g y m i n i m i z a t i o n o f the c o m p l e x e s with b o t h CHARMm a n d STRANGE, until the t o t a l r.m.s, e n e r g y g r a d i e n t w a s less t h a n 0.05 kcal tool-1 /~-1 a n d the e n e r g y v a r i a t i o n b e t w e e n t w o m i n i m i z a t i o n cycles w a s
211 less than 1 0 - a kcal mol-1. Constraints were appfied to the dihedral angles of the intercalation site sugars (i.e. the sugars of the sixth and seventh nucleosides of each strand) in order to maintain the C 3 ' - e n d o ( 3 ' - 5 ' ) C2 ' - e n d o configuration during the initial refinement cycles; these were subsequently removed. About 1600 cycles of ABNER minimization (16 h of MicroVAX II CPU time) were necessary to reach the convergence criterion for each complex. Approximately 45 rain of MicroVAX II CPU time were spent in STRANGE for the rigid body minimization cycles.
2.3. M o l e c u l a r d y n a m i c s Since the current STAR implementation of CHARMm does not allow the manipulation of the complexes as segments of an infinite polymer, by defining the suitable translational and rotational symmetries, we applied small constraints (1.0 kcal tool -a) on the first and last base pairs of the oligonucleotide in order to model their interactions with the adjacent base pairs. This technique has no effect on the dynamics of the intercalation site which is considered in the current study and the r.m.s, fluctuations of the six central base pairs are similar in amplitude to those c o m p u t e d in completely unconstrained dynamics. The p r o c e d u r e for molecular dynamics was as follows: (i) a 15 ps thermalization from 0 to 300 K in 2 K steps, a given t em perat ure being obtained by assigning velocities from a gaussian distribution with a variance corresponding to this temperature; (ii) a 20 ps equilibration during which the velocities were randomly reassigned every 0.4 ps in order to provide a h o m o g e n e o u s velocity distribution; (iii) a 30 ps equilibration during which the velocities were rescaled ff the t e m p e r a t u r e fell outside a ± 10 K window, the temp er atu r e being checked every 0.4 ps; (iv) 50 ps " p r o d u c t i v e " dynamics (75 ps for the methylpyridopsoralen); (v) a time-averaged structure was c o m p u t e d over the "productive dynamics" and energy minimized. Newtonian equations of motion were integrated using the Verlet algorithm [22]. A 0.25 fs integration step was chosen as no constraint was applied on internal coordinates; the SHAKE algorithm [23, 24] was not used.
3. R e s u l t s
Figure 2 shows the calculated energy-minimized geometries of various furocoumarin derivatives intercalated between the two central alternating AT base pairs of the duodecanucleotide, in a projection plane orthogonal to the helix axis. The molecules are shown without the hydrogen atoms; the left thymine and the right adenine (full lines) are above the drug and the left adenine and the right thymine (broken lines) are below the drug. The photoreactive double bonds are shown in bold broken lines. The main geometrical features of the complexes are a complete intercalation of the furocoumarins and a parallelism between the drugs and the adjacent base pairs. The related parameters are r e p o r t e d in Table 1 which gives the distances
212
(a)
i~]" '~~'(
(b)
/ (c)
(d)
"" i"...... "//"/ -" "-x
(e)
,,/""
(0 ,..
//" ....
.,..
..'
(g)
(h) Fig. 2. Calculated intercalation geometries of the drugs between the two central AT base pairs, in a projection plane orthogonal to the helix axis (the left thymine and the right adenine (full lines) are above the drug, whereas the left adenine and the right thymine (broken lines) are below the drug): a and b, psoralen and its aromatized derivative; c and d, angelicin and its aromatized derivative; e and f, 7-methylpyrido[3,4-c ]psoralen and 7-methylpyrido[4,3 -c ]psoralen; g and h, their aromatized derivatives. between the two carbon atoms of the furocoumarin photoreactive double b o n d s (furan a n d p y r o n e ) a n d the c o r r e s p o n d i n g c a r b o n a t o m s of the t h y m i n e s o f s t r a n d s A a n d B. T h e t a b l e a l s o c o n t a i n s t h e c a l c u l a t e d v a l u e s o f t w o o p p o s i t e a n g l e s o f t h e v i r t u a l C4 r i n g s w h i c h p r e f i g u r e t h e c y c l o b u t a n e s w h i c h c a n b e f o r m e d i n s o m e c a s e s b y UVA i r r a d i a t i o n o f s u c h c o m p l e x e s . As c a n b e s e e n , t h e m i n i m u m d i s t a n c e is i n t h e r e g i o n o f 3 . 3 /~, w h i c h is a b o u t the s u m of the v a n der W a a l s ' radii of the c a r b o n atoms. M a n y angles lie i n t h e 9 0 ° r e g i o n ; t h e s e v a l u e s a r e c l o s e t o t h o s e r e p o r t e d f o r t h e
213 TABLE 1 Distances and angles between the reactive double bonds of the intercalated drug and the adjacent thymine in the complexes deduced from molecular mechanics studies
Compound
4'-5 (/~)
5'-6 (/~)
5-4'-5' (deg)
5'-6-5 (deg)
3-6 (/~)
4-5 (/~)
4-3-6 (deg)
4-5-6 (deg)
a b e d e f g h
3.40 3.62 4.75 4.49 3.25 3.32 3.57 3.32
3.79 3.72 4.50 4.32 3.36 3.38 3.62 3.44
111.5 80.2 40.6 44.7 92.2 86.4 81.9 98.3
92.7 76.1 53.0 53.0 87.8 85.2 80.4 93.4
4.62
4.48
133.1
127.1
4.30
4.94
41.4
67.8
4.18 3.89
4.07 3.86
59.1 81.6
66.3 83.7
4'-5, 5'-6, 3-6 and 4-5 represent the distances drug(C4')-thy(C5), drug(C5')-thy(C6), drug(C3)-thy(C6) and drug(C4)-thy(C5) respectively. 5 - 4 ' - 5 ' , 5'-6--5, 4 - 3 - 6 and 4 - 5 - 6 represent the angles thy(C5)-drug(C4')-drug(C5'), drug(C5')-thy(C6)-thy(C5), drug(C4)--drug(C3)-thy(C6) and drug(C4)-thy(C5)-thy(C6) respectively. In the case of angelicin, 3-6, 4-5, 4 - 3 - 6 and 4 - 5 - 6 should be replaced with 3-5, 4-6, 4 - 3 - 5 and 4--6-5 respectively. TABLE 2 Energy values (kcal mol- ) deduced from molecular mechanics studies of the DNA-furocoumarin complexes
Compound
Etot
Eoligo
Edmg
Elnter
Einter-VdW
Elnter-elec
a b c d e f
-460.0 -449.0 -461.6 -449.9 -465.1 -449.0 -450.9 -450.1
-430.6 -429.8 -431.6 -430.9 -430.7 -429.1 -430.1 -429.9
-0.2 7.7 0.3 8.2 3.1 13.8 13.5 13.5
-29.2 -26.9 -30.3 -27.2 -37.5 -33.7 -34.3 -33.7
-30.1 -27.7 -29.5 -27.2 -36.5 -36.7 -35.3 -34.7
0.9 0.6 -0.7 0.0 -1.1 3.0 1.0 1.0
g
h
Eo~go, potential energy of the unwound d(CGCGATATCGCG)2 oligonucleotide; Edm~, potential energy of the intercalated drug; Einter, external interaction energy between oligonucleotide and intercalated drug; Etot, Eol~o+Edrug +Elnter; Elnter-eLeoelectrostatic contribution to the interaction energy; Einter-VdW,van der Waals' contribution to the interaction energy. c y c l o b u t a n e r i n g o f t h e 8 - M O P - t h y m i n e f u r a n - s i d e p h o t o a d d u c t [ 2 5 ]. H o w e v e r , v a l u e s c a n r e a c h a s h i g h a s 4 . 7 /~ a n d 1 3 3 ° r e s p e c t i v e l y . Various potential energy terms can be deduced from the molecular m e c h a n i c s c a l c u l a t i o n s ( T a b l e 2). T h e s e a r e e i t h e r r e l a t e d t o t h e c o m p l e x (Etot a n d Einter) o r t o e a c h o f its m o i e t i e s (Eongo a n d Ea~g). T h e v a n d e r Waals' and electrostatic contributions to the interaction energy are also given. O f t h e e n e r g y t e r m s , t h e m o s t i n t e r e s t i n g is t h e i n t e r a c t i o n e n e r g y (Eint~r) w h i c h is i n d e p e n d e n t o f t h e z e r o e n e r g y r e f e r e n c e l e v e l a n d c a n b e r e l a t e d t o t h e e x p e r i m e n t a l l y m e a s u r a b l e i n t e r c a l a t i o n affinities. M o r e g e n e r a l l y , it
214 s h o u l d b e r e c a l l e d t h a t all t h e e n e r g y t e r m s d e d u c e d f r o m m o l e c u l a r m e c h a n i c s c a n o n l y be u s e d f o r c o m p a r i s o n s i n c e t h e y do n o t h a v e a b s o l u t e m e a n i n g . T h e s e l i m i t a t i o n s h a v e b e e n d i s c u s s e d in d e t a i l in a p r e v i o u s p a p e r [10]. F i g u r e 3 s h o w s t w o e x a m p l e s of t h e e v o l u t i o n of t h e d i s t a n c e b e t w e e n t h e p h o t o r e a c t i v e d o u b l e b o n d s at t h e f u r a n s i d e d u r i n g a 50 p s d y n a m i c
(a)
v
.8
~4
3 0
i
i
i
,
10
20
30
40
50
t(ps)
(b) A
.8 ~5 a.-,_
.~4
3 0
I
I
I
I
10
20
30
40
50
t (ps)
Fig. 3. Molecular dynamics: evolution of the distance between the 5,6 double bond of the sixth thymine of strand A and the 4',5' double bond of 7-methylpyrido[4,3-c]psoralen (a) and its aromatized derivative (b).
215 trajectory. These simulations do not show marked deviations from m ean values, thus indicating that the main geometrical features are preserved during the dynamics.
4. D i s c u s s i o n
In a recent paper, it has been shown that molecular mechanics calculations can provide a useful tool for understanding the structural aspects of photoreactions of psoralens with nucleic acids on UVA irradiation [10]. An interesting correlation has be e n observed between the geometrical arrangements of the non-covalently intercalated psoralens within DNA and the stereochemical structures of the phot oadduct s that are formed [7-9]. It has been p r o p o s e d to combine molecular mechanics with quantum chemistry in the design of new psoralen derivatives. Another interesting observation is the importance of the role played by the furan moiety which tends to maximize its stacking interactions with the adjacent base pairs. In order to understand more clearly the respective roles of the furan and prone moieties in the determination of the intercalation geometry, we have undertaken a comparative molecular mechanics study of various furocoumarins with their related aromatized derivatives where the pyrone rings, or in the case of pyridopsoralens the pyridine rings, are replaced by purely polycyclic aromatic moieties. In order to check the validity of our molecular mechanics approach, we also p e rf o r med molecular dynamics simulations. 4.1. P s o r a l e n vs. i t s a r o m a t i z e d
derivative
Psoralen itself was not studied in our previous paper [10]. It appears to have a significantly different behaviour from 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP). Compared with 8-MOP, its intercalation geometry appears to be less favourable for the formation of a furan-side adduct and very unfavourable for the formation of a pyrone-side adduct (Fig. 2(a) and Table 1). Although the furan ring tends to maximize its stacking interactions with the adjacent bases and is pushed towards strand A of the oligonucleotide, a couple, which is probably induced by the charges on the furan and pyrone rings, rotates the psoralen around an axis perpendicular to its plane. Such a movement, which pushes the 4',5' and 3,4 reactive bonds away from the 5,6 bonds of the adjacent thymines, is not possible for steric reasons in 8-MOP and for electrostatic reasons in 3-CPs and pyridopsoralens. In the case of the psoralen aromatized derivative ( c o m p o u n d b), the distance between the 4',5' bond and the 5,6 bond of the adjacent thymine is greater than for psoralen, while the angular values are closer to 90 ° (Table 1). This yields a more favourable intercalation geom et ry at the furan side (Fig. 2(b)). Due to the suppression of the charge densities on the pyrone ring, the couple is no longer present, and c o m p o u n d b maintains a rather
216 symmetrical position between the two strands. Furthermore, the furan ring is no longer pushed towards strand A of the oligonucleotide. Thus the displacement of the psoralen ring towards strand A appears to be induced not only by the maximization of the stacking between the furan ring and the adjacent bases, but also by electrostatic repulsive interactions between the pyrone ring and the adjacent bases. The suppression of the oxygen atoms of the pyrone moiety, as shown in c o m p o u n d b, therefore leads to a slightly more favourable geometrical arrangement for a further furan-side photoreaction than is the case with psoralen itself. 4.2. A n g e l i c i n vs. its a r o m a t i z e d d e r i v a t i v e The intercalation geometries of angular furocoumarins have been recognized for some time to be very different from those of psoralens [26]. We only report here the intercalation configurations which lead to a c i s - s y n furan-side m o n o a d d u c t or a cis-anti pyrone-side monoadduct. The other intercalation geometry which would lead to a ci s-syn pyrone-side adduct has never been experimentally observed, an aspect which could be related to a higher interaction energy [27]. In the first intercalation geometry, the present calculations show that angelicin is not very favourable for the formation of cis--syn furan-side or cis-anti pyrone-side monoadducts. This appears to be due to conflicting driving forces: the maximization of the furan ring stacking and the minimization of the van der Waals' interaction energy seem to have opposite effects in angelicins, whereas in psoralens both conditions can be fulfilled at the same time and yield an intercalation geom e t r y which is more favourable to furanside photoreaction. The geometrical parameters are slightly better for the angelicin aromatized derivative ( c o m p o u n d d) due to the suppression of the electrostatic perturbation induced by the cis-anti orientation at the pyrone side. It should be noted that these results do not indicate the impossibility of photoreaction in the angelicin-DNA complexes. Indeed, although the average structure is very unfavourable to photoreaction, molecular dynamics simulations have shown that relative positions of the furocoumarin and thymine moieties, which are favourable to photoreaction, may be reached [27]. 4.3. P y r i d o p s o r a l e n s vs. t hei r a r o m a t i z e d d e r i v a t i v e s Pyridopsoralens belong to the class of monofunctional derivatives in which one of the two reactive sites has been chemically blocked [5]. In this case, a decrease in reactivity is attempted by fusing the reactive pyrone bond with a pyridine ring. MePyPs ( c o m p o u n d e) has been demonstrated to be a monofunctional derivative which photobinds to DNA via its 4',5' furan bond [28]; the major photoadducts are two furan-side diastereoisomers formed with thymidine [29, 30]; additional effects have been noted recently [31]. As discussed previously [10], the present results show that MePyPs
217
is deeply intercalated in DNA (Fig. 2(e))*. The intercalation geometry is very favourable for the formation of a furan-side adduct as can be judged from the short distances between the reactive bonds and the near-90 ° angles between the atoms of these bonds. However, the formation of a pyrone-side adduct is much less favourable since the distances between the photoreactive double bonds are about 1/~ greater. These geometrical arrangements probably act with purely electronic factors (engagement of the 3,4 pyrone double bond) to abolish the photoreactivity of the pyrone site. 2N-MePyPs (compound f) differs from MePyPs only in the shift of the position of the nitrogen atom of the pyridine ring. From a physicochemical point of view, 2N-MePyPs shows a 2.5 times lower DNA photobinding capacity than MePyPs [32]. The intercalation geometry of 2N-MePyPs (Fig. 2(f)) is very similar to that of MePyPs, although slightly less favourable for the formation of a furan-side adduct. However, a striking difference between the two pyridopsoralens lies in their rather dissimilar interaction energy values, with a difference of around 10% ( - 3 6 . 5 kcal mo1-1 for MePyPs and - 3 3 . 7 kcal mo1-1 for 2N-MePyPs). This effect relies almost exclusively on the difference in charge densities between the two pyridopsoralens, as deduced from CNDO calculations. The aromatized derivative of MePyPs (compound g) appears to be intercalated in a similar fashion to 2N-MePyPs. However, it remains in a symmetrical position between the two strands, and is less pushed towards strand A. The distances and angles between its 4',5' double bond and the reactive double bond of the adjacent thymine are very favourable for subsequent photoreaction. This molecule has an interaction energy quite similar to that of 2N-MePyPs, with slightly less favourable van der Waals' interactions (less stacking between the furan ring and the adjacent bases), but a better electrostatic interaction energy (no unfavourable interaction between the pyrone nitrogen and the adjacent negative groups). A comparison between the dynamic behaviour of 2N-MePyPs and its aromatized derivative (compound h) is also of interest. As can be seen in Fig. 3, rather small fluctuations are observed around the average distances between reactive bonds over 50 ps. The amplitude of the fluctuations lies in the region of _ 0.5/~ for both the original pyridopsoralen and its aromatized derivative. This reinforces the idea that the contributions of the oxygen and nitrogen atoms of the pyrone and pyridine moieties are not predominant in the precise positioning of the psoralen derivatives within the AT intercalation cavity. This does not exclude the fact that more specific effects may occur, related to the presence or positioning of the heteroatoms within the molecule. For example, a significant deviation from the average distances between reactive bonds is observed in the dynamics of 8-MOP and MePyPs. These aspects will be discussed in a forthcoming paper. *The nucleic acid force field has b e e n improved in CHARMra 21. Since our previous calculations were performed with CHARMm 19, w e had to minimize these complexes again to provide comparable energy values. It should be noted that, although the absolute values are different, the differences in interaction energy between the various molecules remain unchanged.
218
5. C o n c l u s i o n s T h e m o l e c u l a r m e c h a n i c s a n d d y n a m i c s c a l c u l a t i o n s w e r e p e r f o r m e d in an a t t e m p t to d e t e r m i n e the i m p o r t a n c e o f the o x y g e n h e t e r o a t o m s o f the p y r o n e ring o f the f u r o c o u m a r i n d e r i v a t i v e s in the g e o m e t r i c a l p o s i t i o n i n g of the p h o t o s e n s i t i z e r s within t h e a l t e r n a t i n g AT b a s e p a i r s of B - f o r m DNA. The i n v e s t i g a t i o n s are a c o n t i n u a t i o n o f a p r e v i o u s s t u d y o f s e v e r a l p s o r a l e n s w h i c h s e e m e d to indicate t h a t the i n t e r a c t i o n s b e t w e e n the f u r a n m o i e t y of the p s o r a l e n s a n d the a d j a c e n t b a s e p a i r s w e r e d e t e r m i n i n g f a c t o r s in the i n t e r c a l a t i o n g e o m e t r y . T h e p r e s e n t c o m p a r i s o n o f s e v e r a l linear a n d a n g u l a r f u r o c o u m a r i n s with r e l a t e d c o m p o u n d s in w h i c h the p y r o n e ring h a s b e e n a r o m a t i z e d e m p h a s i z e s a s o m e w h a t similar b e h a v i o u r , b o t h f r o m a m o l e c u l a r m e c h a n i c s a n d m o l e c u l a r d y n a m i c s p o i n t o f view. It r e i n f o r c e s t h e i m p o r t a n c e o f the s t a c k i n g i n t e r a c t i o n s of the f u r a n m o i e t y with the a d j a c e n t b a s e p a i r s in the p o s i t i o n i n g within t h e o l i g o n u c l e o t i d e . This d o e s n o t c o m p l e t e l y eliminate the role o f the i n t e r a c t i o n o f the p y r o n e m o i e t y with the a d j a c e n t b a s e pairs. A careful e x a m i n a t i o n o f the e n e r g y - m i n i m i z e d g e o m e t r i e s s h o w s t h a t the t e n d e n c y of f u r o c o u m a r i n s to m a x i m i z e t h e s t a c k i n g o f the f u r a n ring is m o d u l a t e d b y the i n t e r a c t i o n s l o c a t e d on the p y r o n e side. A n o t h e r c o n c l u s i o n of t h e p r e s e n t s t u d y is t h a t m o l e c u l a r m e c h a n i c s c a n be u s e d to p r e d i c t a n i n t e r c a l a t i o n g e o m e t r y f o r a r o m a t i z e d d e r i v a t i v e s o f f u r o c o u m a r i n s w h i c h m i g h t b e f a v o u r a b l e for t h e f o r m a t i o n ' o f f u r a n - s i d e m o n o a d d u c t s within DNA. E x p e r i m e n t a l w o r k o n the p h o t o r e a c t i o n s is currently being undertaken.
Acknowledgments W e are i n d e b t e d to Dr. J o c e l y n e Blais a n d Dr. Odilon Chalvet for t h e i r h e l p in the c a l c u l a t i o n o f t h e partial a t o m i c c h a r g e s a n d Dr. Mary L a p a d a t for critical r e a d i n g of t h e m a n u s c r i p t . W e also w i s h to t h a n k L.U.R.E., Universit~ de Paris Sud, Orsay, F r a n c e f o r the c o m p u t e r facilities. This w o r k w a s p a r t l y s u p p o r t e d b y the C e n t r e N a t i o n a l de la R e c h e r c h e Scientifique (UA 198 a n d 2 4 0 ) a n d the Minist~re de la R e c h e r c h e et de l ' E n s e i g n e m e n t Sup~rieur (grant 8704022).
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