Vol. March

167,

No.

16,

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1990

Daes Anthramycin B-DNA

bind

to Z-DNA

? A Molecular

Mechanics

Shashidhar Searle Received

11,

as to

Study

N. Rao

Research and Development, 4901 Searle Parkway,

January

as well

477-483

Division of G.D.Searle Skokie, Illinois 60077

and Co.,

1990

Molecular mechanics simulations have been presented on the covalent complexes between anthramycin and the decanucleotides d(GCGCGCGCGC)z and d(GCGCGTGCGC).d(GCGCACGCGC) in the novel Z form, with the drug docked in the minor groove. The simulations predict that anthramycin binds to Z-DNA almost as well as it does to B-DNA, but with a few key differences in the structural aspects of binding. In particular, the 5’ orientation of the drug is preferred in the Z form, contrasting with the preference for 3’ orientation in the B form and the drug has a left-handed twist. While no experimental studies have been published on the binding of pyrrolo(l.4)benzodiazepines to lefthanded forms of DNA, the energy refined stereochemically satisfactory models provide valuable information which will hopefully simulate high resolution 2-D NMR/NOE studies in solution. 0 1990 Academic Press, Inc. Pyrrolo( from

11,4)benzodiazepines various

the minor studies

streptomyces

groove

residue

(3-6). and

have

known

to act through

it on the 2-amino

has

been

of DNA-PBD been

antibiotics

investigated

complexes.

provided

derived

their

group by

binding

in

of a guanine both

Detailed

solution

structures

by NMR

studies

forms

of DNA,

it is of interest

to the left-handed

double

helical

B.

to such

of the number

a typical

PBD

uniform

left

binds

of polymorphic

handed

implications

in possible

insight

this

into

to two

The

answer

B to Z transitions

problem,

I have

deoxydecanucleotides

mechanics.

Such

(5,7)

of

and computer

minor minor

anthramycin

analysis

groove groove

binding deeper

binds

to

Z

preference

study

a possibility

predicts by

binding

of

the

Z

form

drug, and the than in B-DNA.

form for

slightly one

of

a question

better

polymorphic

a transition

could

acids.

the binding

important

is

forms of DNA

in nucleic

analyzed

in

an

overwhelming induced

binding

antitumor

(8,9).

In light

known defined

of

modeling

complexes

potent

and to alkylate

mode

computer

are

species (1.2)

of DNA

The

the covalent modeling

(PBDs)

using because

the

The form between

have

Towards methods

anthramycin Z-DNA studies

if

such as Z or important seeking

an

(Fig.

1)

of anthramycin

left-handed than

to know

of

molecular is

a well-

has

a well

demonstrate

that

to

the B form with no The over the other. the B- and the Z-DNA

anthramycin.

477

0006-291X/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

167,

No.

BIOCHEMICAL

2, 1990

Figure

1 : Structure

AND

and

BIOPHYSICAL

nomenclature

RESEARCH

of

COMMUNICATIONS

anthramycin.

Methods The starting structures of the drug-DNA complexes were obtained through computer graphics molecular model building using MIDAS (14) as earlier (11) and energy minimized using AMBER-UCSF (17) and the all-atom force field parameters presented by Weiner et al (1986) (18). The force field parameters and partial atomic charges for the drug molecules were the same as those on anthramycin-DNA complexes (1 l- 12). employed in earlier investigations The starting structure for the DNA decamers in the Z form in each of the model built complexes was obtained based on the geometry proposed by Rich and coworkers (15), through x-ray diffraction studies on oligonucleotides, while the starting structures for the drug molecules were obtained based on the published x-ray crystal data of anthramycin (16). The decamers in the complexes are d(GCGCGCGCGC)2, and d(GCGCGTGCGC).d(GCGCACGCGC) and are abbreviated here as GCIO, and GC10T6, respectively and their complexes with anthramycin have been designated with the prefix ANT. Both the 3’ and 5’ orientations of the drug were considered. The configuration at Clla was S throughout the calculations, as it was earlier shown to lead to minimal distortion in the hydrogen bonding interactions between the covalently linked guanine and its complementary cytosine (11.12). Component analyses of energies of interactions have been carried out as earlier (11.12.19). The net binding energies are calculated by subtracting the helix distortion and drug

Table Total

Enetb.

intermolecular (elec, vdw and Ed-h). net binding energies (in kcal/mole) and GClO

I (Hell?

iUtramOkCUkIr

Intermolecular Complex

Orientation

ANT-GClO

S.3’ S,5’ S.3’ S.5’

ANT-GC10T6

aThe energies drug-DNA GUAS,

Total

Elec vdw

-788.1 -783.1 -760.2 -753.7

-49.6 -29.9 -46.5 -31.5

energies

Edis’

-27.3 -25.3 -27.4 -24.2

Ed* Eddis)

anthramycin

and (ANT)

Intramolecular Ed-h

Hell

-76.9 -55.2 -73.9 -55.7

-765.0 -765.0 -739.6 -739.6

He12

-753.9 -757.4 -729.3 -728.6

Edisa

Edb

Eddis

11.1 07.6 10.3 11.0

23.1 10.8 22.5 11.5

15.9 3.6 15.3 4.3

Enetb

-49.9 -44.0 -48.6 -40.4

of isolated helices (Hell) were determined by starting with the minimized removing the drug. replacing the hydrogen on the 2-amino group of

complexes, and reminimizing

the

energy.

used in the table for comparison bThe

Hel2,

in the complejles between and GCIOT6 in Z form of DNA

for

isolated

The

lowest

with all drug-DNA

anthramycin

is 7.2

kcal/mole.

478

value

obtained

for

complexes containing

a particular

this helix.

helix

is

Vol.

BIOCHEMICAL

167, No. 2, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

These distortion energies from the total drug-DNA interaction energy. distortion energies reflect induced fits that permit stronger intermolecular Drug and helix distortion energies are obtained by subtracting interactions. the ener,gies of the minimized isolated drug or helix from their energies that occur in the complexes (Table I).

Results In

and Discussion

all

groove

the

of the Z-DNA,

crystallographic to earlier DNA

of

the

their

left-handed

important

with

(19). data)

also

difference

ring

dynamics

between

Figure 2 : Stereo pairs d(GCGCGCGCGC)z with The drug direction (b). omitted for clarity of the

the

the

deep

since

similar

drug

to only

emphasizes

simulations such

in

binding

of the drug, are

into

minor

2). This is in contrast to the twist

on

to

which

led

right-handed

B

the

conformational

the drug those

of a few

in

PBDs

energies

with

in the complex.

B-DNA

in

solutions

B and Z-forms In the B-DNA

of DNA complexes,

of the covalent complexes between anthramycin and the drug anchored in the 5’ direction (a) and in the 3’ is highlighted (dark) and hydrogens on carbons are pictures.

479

in

the

a flexibility.

the complexes

helix

nicely

(Fig.

however,

Z-DNA

demonstrate

shape of the DNA

twist

right-handed

twist,

left-handed

Molecular

very

attention

of the seven-membered

(unpublished

the overall

nestles

a left-handed

focussing

The

complexes

complexes

An

with

observation

studies

forms.

flexibility the

anthramycin

complexes,

is the

Vol.

167,

No.

double

helix

DNA

is practically

is bent

bound

BIOCHEMICAL

2, 1990

drug.

However, complexes

of

contributions

to

the

complexes.

These

complexes

simulated

electrostatic complexes The

by about

interactions on the other. oxygen

groove.

points This

of anthramycin

is the

preferred

complexes

one.

studied.

these

interactions

lower

side with

has

chain

ANT-GClcrr6

The

nomenclature

the

hand, over

the

the

Z-

of

bonding

the

09

of the DNA

hydrogen

hydroxyl

backbone

orientation,

only

the NH2

solvent

environment

away

to the 5’ direction

being

in contrast lists

hydrogen

the bond

parameters

hydrogen

from

where

bonding for

the

of the bases,

group

while

the preferred

to B-DNA,

lengths

group

and nearby

towards

The

the B-DNA

B-DNA

phosphate

II

four

corresponding

to the DNA

Table

the

other

considerably

in

a network

and

3’ drug

to Z-DNA,

in

Waals

(19).

orientation

the

in

those the

of the S

der

similar

bonded

bonding

ANT-GClO

those

interactions

van

is hydrogen

Orientation

Complex

vary

than

Table

Hydrogen

very than

the phosphates

leads

binding

are

On

are

pairing

The

(19).

In the models

side chain

modeled.

the

covalently

in the light

higher

amide

and

base

all

5’ drug the

on one hand

bony1

of these

with

Watson-Crick is not surprising

model

complexes,

of the guanine

5 to 8 kcal/mole atom

COMMUNICATIONS

in the Z-DNA

interactions

8 to 16 kcal/mole

between

the amide the

with

and

complexes

drug

drug-DNA

components

the

attachment

are about

complexes

all

while

RESEARCH

in the vicinity

and this

covalent

BIOPHYSICAL

(11)

created

in Z-DNA

configuration

DNA

undistorted

due to a wedge

to the

are intact

AND

the

floor

orientation

interactions 5’ direction

Donor (X-W

anthramycin-ZDNA Acceptor atom (Z)

(ANT)

Length (H-Z) A

are

Angle at H deg.

S.5’

09-HO9

S,5’ S.5’ S,3’

N18-H181(ANT)

oB(P1fj-17)

1.65 1.99

164.9 153.3

NZHN2B(GUAlS) N18-H181(ANT)

S.3’ S,3’ S,3’

N18-H182(ANT) 09-HO9 (ANT) 09-HO9 (ANT)

Ol’I(ANT) oB(P18-19) oA(P18-19)

1.96 2.33 2.04

155.0 128.1 135.5

N2(GUA15) 02(CYT6)

2.27 2.18

153.4 125.5

S,5’ S.5’ S.3’

09-HO9 (ANT) N18-H181(ANT) N18-H181(ANT)

Ow5-6)

1.65 1.98

163.8 151.5

S.3’ S.3’

N18-H182(ANT) 09-HO9 (ANT)

2.35 2.03 1.91

128.2 135.2 138.1

oB(P16-17) oB(P18-19) oA(P18-19) 02(CYT6)

of the bases and phosphates is as described 480

of

in the

complexes

OW5J

of

the 3’ direction

II

in

of

the car-

earlier

(11).

less

Vol.

167,

No.

than

BIOCHEMICAL

2, 1990

2 A

with

reasonable

direction

have

GClOT6,

for both

than

gua:nine

ing

the

a length

AND

angles,

greater

BIOPHYSICAL

while

most

than 2 A with

the 3’ and 5’ directions,

at base position

exocyclic

amino

of

the

angles

less than

In

other

of the two

sets of complexes

are very similar.

electrostatic

contributions

the

in Z-DNA DNA

than in the B-DNA.

has a uniform

while

the

Z

conformationally for

DNA

the

complexes,

in the

backbone

which 3’ direction

The

DNA

the must

out

with

solvent

and

counterion

effects.

In this

be similar

To

a

distance

of

free

binding

the lbest

energies

of my knowledge,

literature

on the binding

the drug

to induce

and circular DNA

as a function

sonal

communications).

DNA, this

though light,

non-B

somewhat that

the

left-handed

transition

: (1)

entation

of the

mechanics

form

induced

changes

weakly

present

by the binding drug

methods

have

when

compared predicts

in their

of a PBD

simplistic

by

about have

without of

electrostatic is likely

molecular further

10 been

explicit

to the B

throw

have

been that

to

dynamics

and

light

the

on

from

However, PBD,

carried

out

have

study

to

(Barkley,

perto Z-

could indicates

bind that

in to

a

any

to involve

two

and (2) the relative

ori-

the fact been

fluorescence

does bind

B to Z is likely

Given

of

It is encouraging

anthramycin this

in the

tomaymycin,

tomaymycin

in the drug

forms,

reported

or on the ability

to B-DNA.

that

groove.

481

been

of DNA

Further,

of the twist

in the minor

of the drug

simulations

of a related

indicate

such as Z-DNA.

the handedness

energies

in polynucleotides.

studies study

B-

overemphasis

to Z form

concentration

These

minimal than

mechanics,

on the binding

of salt

This

the 5’ direction

constant

would

at

B complexes

so far no experiments

of anthramycin studies

Z-DNA.

polynucleotide

forms.

B to Z transitions

dichroism

the

in the corresponding

of the drug

binding

B-DNA

changes

present to

binding

polymorphic

conformational

the

binding

dielectric

Molecular

of

to the two

of

in

for

seen

to the

leading

the the overall

repeat

consisting

of

structural

the

dependent

larger

right-handed

seen

are higher

that

atmosphere,

light,

the

flexibility

to those

compared

emphasized

are

to the fact that B

repeat,

in

that the overall

to that to the Z form.

evaluation relative

energies

complexes

be

those

the

are comparable

the differences

binding

distinct

wedging

about

bonding

directions.

significant

distortion

Z

carried

and

facts

It is interesting

It

two

involv-

a mononucleotide

providing

than

bending

accommodate

and

favor

kcal/molle.

less

by

a dinucleotide

in the

are

the

drug

complexes.

characterized

3’

base rather bonds

Further, of

the

In ANT-

hydrogen

in part be attributed

by

chain

the

directions

nucleotides

known

can

cost.

to

of

well

in

structure

energies

spite

energy DNA

amide

two

could

characterized distinct

distortion

emphasizes

the

is

two

environments The

helical

DNA

This

140“.

of adenine

respects,

in

for

in

in the loss of hydrogen

profiles the

COMMUNICATIONS

interactions

the presence

15 results group.

RESEARCH

used

that

the molecular

earlier

to predict

Vol.

167,

No.

BIOCHEMICAL

2, 1990

models

consistent

hopefully

induce

any

alternative

DNA

under

tween der

with high modes

covalently progress

will

such as uniform

Such

studies

the

a variety

of minor

the drug the

left-handed

study forms

nature implications

The

anthramycin

of

the in

the

COMMUNICATIONS

present

to be taken and

will

up to explore

related

Modeling

study compounds

to

of interactions

be-

environment is currently unin a Z-DNA The present study also suggests elsewhere. to other will

left-handed

with

well

be reported

demonstrates of

DNA

orientation

in B-DNA

RESEARCH

work

conditions.

B (20)

and

present

those

of

binding

polymorphic

contrast

flexible

significant

be reported

groove.

NMR/NOE

binding

are in progress

In conclusion,

2-D

tomaymycin

BIOPHYSICAL

observations,

polymorphic

of the drug

DNA

defined

of of

bound

and

the possibility

experimental

resolution

a variety

AND

complexes. pyrrolo(

defined

that

forms

deep minor

anthramcyin are

The transitions

could

characterized

handedness

of the

calculations

of

grooves.

in

bind

by

to

a well

twist

of

also demonstrate which

1,4)benzodiazepines

conformational

helical

elsewhere.

which

and the

double

may

have

DNA.

Acknowledgments for providing I thank the Drug Design group at G.D. Searle and Company computational and graphics facilities used in the analysis of energy refined Initial modeling of the complexes was done at the Computer structures. Graphics Laboratory, University of California, San Fransisco, (R. Langridge, Director and T. E. Ferrin, facility manager).

References

(1) (2) (3) (4) (5)

(6) (7)

(8) (9) (10) (11)

(12) (13) (14)

Hurley, L.H. (1977) J. Antibiotics, 30, 349. Remers, W.A. (1988) The Chemistry of Antitumor Antibiotics, vol. 2, Wiley-Interscience, pp. 28-92. Hurley, L.H.; Allen, C.; Feola, J.; Lubawy, W.C. (1979) Cancer Res. 39, 3134. Hurley, L.H.; Petrusek, R. (1979) Nature , 282, 529. Graves, D.E.; Pattaroni, C.; Krishnan, B.S.; Ostrander, J.M.; Hurley, L.H.; Krugh, T.R. (1984) J.Biol. Chem. 259, 8202. Barkley, M.D.; Cheatham, S.; Thurston, D.E.; Hurley, L.H. (1986) Biochemistry 25, 3021. Graves, D.E.; Stone, M.P.; Krugh, T.R. (1985) Biochemistry 24, 7573. Cheatham, S.; Kook, A.; Hurley, L.H.; Barkley, M.D.; Remers, W.A. (1988) J. Med. Chem.31, 583. Boyd, F.L.; Cheatham, S.; Remers, W.A.; Hill, G.C.; Hurley, L.H. JACS (in press). Wells, R.D. and Harvey, S.C. (1988) “Unusual DNA structures”, SpringerVerlag, New York. Rao, S.N.; Singh, U.C.; Kollman, P.A. (1986) J. Med. Chem. 29, 2484. Remers, W.A.; Mabilia, M.; Hopfinger, A.J. (1986) J. Med. Chem.29, 2492. Cheatham, S., Kook, A., Hurley, L.H., Barkley, M. and Remers, W.A. (1988) J. Med. Chem. 31, 583-590. Huang, C, Jarvis, L., Ferrin, T. and Langridge (1985) MIDAS Computer graphics program, University of California, San Francisco CA 94143. 482

Vol.

167,

(15) (16) (17) (18) (19)

(20)

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Wang, A.H.-J., Quigley, G.J., Kolpak, F.J., van der Marel, G.A., van Boom, J.H. and Rich, A. (1981) Science 211, 171-176. Arora, S.K. (1979) Acta Cryst. B35, 2945. Singh, U.C., Weiner, P.K., Caldwell, J.W. and Kollman, P.A. (1986) AMBER(UCSF) version 3.0 , Department of Pharmaceutical Chemistry, University of California, San Francisco CA 94143. Weiner, S.J.; Kollman, P.A.; Nguyen, D.; Case, D. J. Camp. Chem. 1986, 7, 230-252. Rao, S.N. and Remers, W.A. (1989) All-atom Molecular Mechanics simulations on covalent complexes of anthramycin and neothramycin with deoxydecanucleotides. (submitted to J. Med. Chem.) Gupta, G., Bansal, M. and Sasisekharan, V. (1980) Proc. Natl. Acad. Sci. USA 77, 6086

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