Báo cáo khóa học: Sequence selective binding of bis-daunorubicin WP631 to DNA doc

At the highest concentrations of WP631 2 and 3 lM, the ligand shows a general inhibition of cleavage at most positions in the fragment.. The plots were calculated from the cleavage patte

Sequence selective binding of bis-daunorubicin WP631 to DNA

Keith R Fox1, Richard Webster1, Robin J Phelps1, Izabela Fokt2and Waldemar Priebe2

1 School of Biological Sciences, University of Southampton, Bassett Crescent East, Southampton, UK; 2 The University of Texas MD Anderson Cancer Center, Houston, TX, USA

We have used footprinting techniques on a wide range of

natural and synthetic footprinting substrates to examine the

sequence-selective interaction of the bis-daunorubicin

anti-biotic WP631 with DNA The ligand produces clear

DNase I footprints that are very different from those seen

with other anthracycline antibiotics such as daunorubicin

and nogalamycin Footprints are found in a diverse range of

sequences, many of which are rich in GT (AC) or GA (TC)

residues As expected, the ligand binds well to the sequences

CGTACG and CGATCG, but clear footprints are also

found at hexanucleotide sequences such GCATGC and GCTAGC The various footprints do not contain any par-ticular unique di-, tri- or tetranucleotide sequences, but are frequently contain the sequence (G/C)(A/T)(A/T)(G/C) All sequences with this composition are protected by the ligand, though it can also bind to some sites that differ from this consensus by one base pair

Keywords: WP631; anthracycline antibiotic; daunorubicin; footprinting; sequence recognition

A large number of ligands are known to bind to DNA, and

several of these are important therapeutic agents,

partic-ularly in the treatment of cancer However most such agents

have little or no sequence selectivity and are therefore

extremely cytotoxic and affect all rapidly dividing cells One

goal for cancer chemotherapy is therefore to produce

compounds that only interact with specific genes, or gene

products The deciphering of many complete genomes gives

new impetus to the search for molecules that interfere with

the activity of individual genes Examples of strategies

aimed at realizing this goal included the formation of

intermolecular triplex helices [1,2] and the pyrrole-imidazole

polyamines [3,4]

The interaction of many small molecules with DNA

has been well characterized and several of these have

limited sequence recognition properties However, with

the exception of the polyamides [3,4] these compounds

only recognize between two and four base pairs One

means of increasing the selectivity is to produce

oligo-mers of known agents, thereby increasing the binding site

size, the selectivity and the strength of binding [5–7] The

first examples of such agents included the

bis-intercalat-ing acridines These generally bind more strongly than

simple mono-intercalators, though this rarely approaches

the theoretical limit of the square of the binding

constant, because of conformational and structural

restrictions imposed by the linkers between the two

intercalators In addition, because the parent compounds

bind to almost all DNA sequences, the oligomers show

little or no sequence selectivity

The anthracycline antibiotics are well known antitumour agents [8–11] and, although they display a pleiotropic mechanism of action, DNA is their primary cellular target The best characterized members of this group are dauno-rubicin (daunomycin) and doxodauno-rubicin (adriamycin) These agents bind to DNA by intercalation, with the amino sugar daunosamine positioned in the DNA minor groove Several crystal structures have been reported for the interaction of these ligands with oligonucleotides, including CGTACG [12,13], CGATCG [14,15] and TGTACA and TGATCA [16] They possess some sequence specificity and high resolution footprinting has suggested that they bind best to the sequences 5¢-(A/T)CG and 5¢-(A/T)GC [17–19] There have been a number of attempts to produce bis-intercalating daunorubicin derivatives, with increased affin-ity for DNA In early studies these were linked through C13 and C14 as these are chemically accessible [20,21] However these positions are involved in DNA binding and the modifications decreased the affinity of each monomer More recently dimers of daunorubicin have been produced

by linking between the C-4¢ or C-3¢ sugar positions [22,23] These compounds were designed after examination of the crystal structure of daunorubicin bound to CGTACG [13] This structure contains two daunorubicin molecules which are intercalated at the CpG steps with their amino sugars facing each other at the centre of the complex, with the 3¢-amines separated by 6–7 A˚ A p-xylyl linker was chosen

to link the two halves of the dimer generating WP631 (Fig 1), linked at the 3¢-positions and WP652 (linked at the modified 4¢-positions) These compounds show promising biological activity and are significantly more cytotoxic than doxorubicin against multi-drug resistant tumours [22,23] WP631 has been shown to be an Sp1 site-specific drug [24],

an activator of nuclear factor-j B [25], and an inhibitor

of Tat transcription in HIV [26], as well as a general antiproliferative agent

Spectroscopic methods have been used to examine the binding of WP631 to DNA [27] Continuous variation

Correspondence to K R Fox, School of Biological Sciences,

Univer-sity of Southampton, Bassett Crescent East, Southampton SO16 7PX,

UK Fax: +44 23 80594459; Tel.: +44 23 80594374;

E-mail: K.R.Fox@soton.ac.uk

Abbreviations: DEPC, diethylpyrocarbonate.

(Received 4 June 2004, revised 14 July 2004, accepted 15 July 2004)

Eur J Biochem 271, 3556–3566 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04292.x

analysis revealed up to six distinct binding modes to

herring sperm DNA The tightest of these corresponded to

the interaction of one drug molecule with six base pairs

with an association constant of 3· 1011

M )1 at 20C

High resolution melting studies showed that the ligand

bound preferentially to GC-rich DNA regions [27] By

comparison with the crystal structures of daunorubicin

we would expect these ligands to bind to the sequence

CG(A/T)(A/T)CG NMR [28] and crystal structures [29]

have been derived for the interaction of WP631 with

CGTACG and CGATCG, respectively, and as expected

show that the ligand binds by bisintercalation with each

chromophore inserted into the CpG steps, with four base

pairs sandwiched between them In contrast, prolonged

incubation of WP652 with CGTACG resulted in

precipi-tation, and the NMR structure was determined for this

ligand bound to TGTACA [28] In this structure the ligand

is bound across the sequence PyGTPu, with only two base

pairs between the intercalated chromophores

These studies have demonstrated that WP631 binds tightly to DNA by bisintercalation and assume that it recognizes the sequence CG(T/A)(T/A)CG However, there have been no previous studies examining its sequence binding preferences, though it has been demonstrated that WP631 inhibits Sp1-activated transcription in vitro [24,30]

In this paper we examine the DNA sequence specificity of WP631 using a range of footprinting techniques on several different DNA fragments

Materials and methods

Chemicals and enzymes Oligonucleotides for preparing the various DNA fragments were purchased from Oswel DNA service (Southampton, UK) These were stored in water at)20 C, and diluted to working concentrations immediately before use Plasmid pUC19 was purchased from Pharmacia DNase I was purchased from Sigma and stored at)20 C at a concen-tration of 7200 UÆmL)1 Restriction enzymes and reverse transcriptase were purchased from Promega WP631 (Fig 1) was prepared as previously described [23]

DNA fragments The sequences of the various fragments used in this work are shown in the various differential plots (see below) TyrT(43–59) is a 110 base pair fragment, which has been widely used in previous footprinting studies [31] This labelled DNA fragment was obtained by cutting the plasmid with EcoRI and AvaI and was labelled at the 3¢-end of the EcoRI site with [32P]dATP[aP] using reverse transcriptase Fragments MS1 and MS2 were designed to contain all 136 possible tetranucleotide sequences [32] These two fragments contain the same sequence in opposite orientations, allowing visualization of footprints that are located at either end Fragments DMG60Y and DMG60R contain oligopurine tracts which are interrupted with different bases in the centre [33] They contain the same sequence in opposite orientations, they were radiolabelled

so as to visualize the purine-rich strand of DMG60R, and pyrimidine-rich strand of DMG60Y AG1 and GA1 contain the sequences A6G6.C6T6and G6A6.T6C6inserted into the BamHI site of pUC18 [34] Radiolabelling visualizes the purine-rich strand of AG1, but the pyrimidine-contain-ing strand of GA1 Fragments WPseq1 and WPseq2 were obtained by cloning appropriate oligonucleotides into the BamHI site of pUC18 The sequences were confirmed by manual sequencing with a T7 sequencing kit (Amersham Pharmacia) Fragment WPseq2 was found to contain a dimer of the required insert These fragments were obtained

by cutting the plasmids with HindIII and SacI and they were labelled at the 3¢-end of the HindIII site with [32P]dATP[aP] using reverse transcriptase Radiolabelled DNA was separ-ated from the remainder of the plasmid on 6–8% non-denaturing polyacrylamide gels The bands containing the radiolabelled DNA were excised and eluted into 10 mM Tris/HCl pH 7.5, containing 0.1 mM EDTA The DNA was then precipitated with ethanol in the presence of 0.3M sodium acetate

Fig 1 Chemical structure of WP631 The carbon atoms at positions

13, 14, 3¢ and 4¢ are indicated in the upper part of the dimer.

DNase I footprinting

Radiolabelled DNA was dissolved in 10 mM Tris/HCl

pH 7.5 containing 0.1 mM EDTA, at about 10–20 c.p.s

ÆlL)1as determined on a hand-held Geiger counter This

DNA solution (1.5 lL) was mixed with 1.5 lL of ligand

(final concentration 10 nM )10 lM), dissolved in 10 mM

Tris/HCl, pH 7.5, containing 10 mM NaCl This mixture

was allowed to equilibrate for at least 30 min before

digesting with either DNase I or a hydroxyl radical

generating mixture as previously described [31] DNase I

digestion was achieved by adding 2 lL enzyme (typically

0.01 UÆmL)1) dissolved in 20 mMNaCl, 2 mMMgCl2, and

2 mM MnCl2 The digestion was stopped after 1 min by

adding 5 lL of 80% formamide containing 10 mMEDTA,

10 mMNaOH and 0.1% (w/v) bromophenol blue

Hydroxyl radical footprinting

Hydroxyl radical cleavage was performed by adding 6 lL of

a freshly prepared mixture containing 50 lM ferrous

ammonium sulfate, 100 lM EDTA, 2 mM ascorbic acid

and 0.05% hydrogen peroxide The reaction was stopped

after 10 min by precipitating with ethanol The DNA was

finally redissolved in 8 lL of 80% formamide containing

10 mM EDTA, 10 mM NaOH and 0.1% (w/v) bromo-phenol blue

Reaction with diethylpyrocarbonate and potassium permanganate

The reaction with these footprinting probes was performed

as previously described [29,31] Radiolabelled DNA (3 lL) was incubated with 3 lL WP631 diluted to appropriate concentrations in 10 mMTris/HCl containing 10 mMNaCl and equilibrated for at least 30 min For diethylpyrocarbo-nate (DEPC) modification, 5 lL of DEPC was added and the reaction was stopped after 20 min by precipitating with ethanol in the presence of 0.3M sodium acetate For reaction with permanganate, 1 lL of 100 mM potassium permanganate was added and the reaction stopped after

1 min by adding 2 lL of mercaptoethanol The DNA was then precipitated with ethanol in the presence of 0.3M sodium acetate For both DEPC and permanganate the dried DNA pellets were boiled in 10% (v/v) piperidine for

30 min, reduced to dryness in a Speedvac, and redissolved in

8 lL of 80% formamide containing 10 mMEDTA, 10 mM NaOH and 0.1% (w/v) bromophenol blue

Fig 2 DNase I, DEPC and KMnO 4 foot-prints showing the interaction of WP631 with tyrT(43–59) WP631 concentrations (lM) are shown at the top of each gel lane;
con cor-responds to cleavage in the absence of added ligand Tracks labelled
GA are markers specific for purines.

3558 K R Fox et al (Eur J Biochem 271)
FEBS 2004

Denaturing gel electrophoresis

The products of footprinting reactions were resolved on

6–10% polyacrylamide gels (depending on the location of

the target site) containing 8M urea DNA samples were

boiled for 3 min immediately before loading onto the gels

Polyacrylamide gels (40 cm long) were run at 1500 V for

2 h These were then fixed in 10% acetic acid, transferred to

Whatmann 3M paper, dried under vacuum at 80C and

exposed to a phosphorimager screen (Kodak) overnight

Dry gels were exposed to a Kodak Phosphor Storage

Screen, which was scanned using a Molecular Dynamics

Storm 860 phosphorimager The products of digestion were

assigned by comparison with Maxam–Gilbert marker lanes

specific for guanine and adenine

Differential cleavage plots

The intensity of bands in the drug-treated and control lanes

were prepared as previously described [31] In the

differen-tial cleavage plots the intensity of each band in the

drug-treated lane is divided by the intensity of the same band in

the drug-free control These values are then normalized

according to the total intensity of the bands in each lane

The values are then plotted against the DNA sequence on a

logarithmic scale Values less than one correspond to

regions of protection by the ligand, while values greater

than one correspond to drug-induced enhanced cleavage

Results

Figure 2 shows footprinting gels for the interaction of WP631 with the tyrT DNA fragment This fragment has been widely used for assessing the sequence-specific binding

of small molecules to DNA including the anthracycline antibiotics daunorubicin and nogalamycin [17,19,35,36] The first panel shows the results of DNase I footprinting, from which it is clear that the ligand has affected the cleavage pattern At the highest concentrations of WP631 (2 and 3 lM), the ligand shows a general inhibition of cleavage at most positions in the fragment However specific regions of protection are evident with ligand concentrations between 0.2 and 1 lM Examples of bands that are protected

by the ligand include positions 34, 41, 53 and 61 In contrast, cleavage at positions 31–32 and 47–50 is enhanced in the presence of the ligand These results are presented as a differential cleavage plot in the top panel of Fig 3, showing the intensity of each band in the drug-treated lanes compared with that in the control Examination of the patterns does not reveal any obvious sequence preference, though some of the clearest footprints are located in regions containing both G and A residues The enhancements are located in oligo(dA) tracts, as often noted with intercalating agents These footprints are of variable lengths The footprints around positions 40, 63 and 80 cover about six bases, as might be expected for a bis-intercalator However, below position 60 there are two smaller footprints of about

Fig 3 Differential cleavage plots showing the interaction of WP631 with tyrT(43–59), AG1 and GA1 The plots were calculated from the cleavage patterns in the presence of 1 l M WP631 shown in Fig 2 (tyrT) and Fig 6 (AG1 and GA1) Only a part of each sequence is shown and is written reading 5¢ )3¢ from left to right; the right-hand end corresponds to the bottom of the gels The ordinate, which is plotted on a logarithmic scale, shows the intensity of each band in the drug-treated lanes relative to that in the control Values less than one correspond to protection by the ligand, while values above indicate enhanced cleavage The black bars highlight the regions that are protected from cleavage For tyrT the arrows indicate the positions of WP631-induced cleavage by DEPC (grey arrows) and KMnO (black arrows).

three base pairs each Similar short footprints are apparent

around positions 25 and 34 As DNase I overestimates

ligand binding site sizes by 3–4 base pairs it is very unusual

to observe footprints of this short size It is possible that

these short footprints are not caused by steric interference

from drug molecules bound to the DNA minor groove,

instead they may reflect drug-induced changes in DNA

structure that render it less sensitive to cleavage We

attempted to gain more accurate information about the

sequence specificity of WP631 by performing hydroxyl

radical footprinting experiments However the ligand did

not affect hydroxyl radical cleavage at a concentration of

10 lM Although this result is disappointing, some other

well-characterized sequence specific ligands also fail to

produce hydroxyl radical footprints [36,37]

Because of the difficulty in interpreting these patterns, we

examined the effect of WP631 on modification by DEPC

and potassium permanganate These agents react with

exposed A and T residues, respectively, while duplex DNA

is generally unreactive [31] Intercalating agents have

previously been shown to enhance the reactivity of bases adjacent to their binding sites to these agents [38–40] The results are presented in the second and third panels of Fig 2 It can be seen that WP631 enhances the reactivity of certain bases to each of these agents; these are indicated by the arrows in Fig 3 Bands that become hyper-reactive to DEPC are located at positions 18, 32, 48, 67, 83 and 84 In some instances these are located in regions of enhanced DNase I cleavage (positions 32 and 48), while others are adjacent to regions of DNase I protection (18, 67, 83, 84) Enhanced reactivity to KMnO4can be seen at positions 29,

33, 60, 68, 81, 86, 88 and 91

These results show that WP631 produces distinct foot-printing patterns, which are different to those produced by daunorubicin and nogalamycin [17,19,35,36] The ligand must therefore possess some sequence selectivity, though no consensus binding sites can be deduced from these patterns

We have therefore examined the interaction of this ligand with a range of DNA fragments, in order to elucidate the characteristics of the preferred binding sites

Fig 4 DNase I and KMnO 4 footprints showing the interaction of WP631 with fragments MS1 and MS2 WP631 concentrations (l M ) are shown at the top of each gel lane;
con corresponds to cleavage in the absence of added ligand Tracks labelled
GA are markers specific for purines The numbered black bars show the positions of DNase I footprints, while the asterisks indicate bands that become sensitive to reaction with KMnO 4

in the presence of WP631.

3560 K R Fox et al (Eur J Biochem 271)
FEBS 2004

Fragments MS1 and MS2 were designed so as to contain

all 136 tetranucleotide sequences [32] They contain identical

sequences, but are cloned in opposite orientations thereby

simplifying analysis of bands at the ends of the fragments

Footprinting experiments with these fragments are

presen-ted in Fig 4 and differential cleavage plots derived from

these data are shown in Fig 5 Again it is clear that WP631

has altered the cleavage patterns, producing footprints that

are highlighted by the bars in Figs 4 and 5 Several of these

footprints are located in regions which are rich in GA (TC)

or GT (AC) residues, for example sites 1 (TCATCTC),

2 (GGTGG), 4 (GAAGAG), 7(ATGTGT), and 8

(GTTGG) A long footprint is also evident on MS2 (site

10) corresponding to a purine-rich tract These footprints

are accompanied by enhancements in reactivity to KMnO4

and DEPC as indicated in Figs 4 and 5

As many of the footprints on MS1 and MS2 are located

in tracts of GA-residues we examined the interaction with

other fragments containing similar sites, some of which were

prepared for work with triplex-forming oligonucleotides

Fragments GA1 and AG1 contain tracts of G6A6.T6C6and

A6G6.C6T6, respectively [34] DNase I footprinting patterns

for WP631 with these fragments are shown in Fig 6 and

differential cleavage plots derived from these are presented

in Fig 3 As these oligopurine tracts were both cloned into

the polylinker site of pUC18 the sequences surrounding the

inserts are common to both fragments and show similar

cleavage patterns in the presence of the ligand For both

fragments there is a large footprint below the insert,

corresponding to the sequence TCCTCT Similarly cleavage

is attenuated above the inserts in vicinity of the sequence GGATC However the ligand has very different effects on cleavage of the two inserts WP631 protects from DNase I cleavage at the centre of AG1, but causes enhanced cleavage

at the centre of GA1 It therefore appears that AnGnis a much better binding site than GnAn

Fragment DMG60 also contains oligopurine tracts that are interrupted by isolated thymine residues [33] DNase I digestion patterns for the pyrimidine-rich strand of this fragment in the presence of WP631 are shown in Fig 6 and differential cleavage plots for both strands are shown in the bottom panel of Fig 5 Two clear footprints can be seen on this fragment (labelled sites 1 and 2), as well as other regions

of protection at the top and bottom of the gel, which are in the remainder of the polylinker A short region of protection

is also evident around the lowest purine residue (arrowed) The strong footprints correspond to sequences TTCTTC (site 1) and TTTCTTT (site 2) Although these both contain the sequence TTCTT, this alone cannot constitute the preferred ligand binding site as the same pentanucleotide is present in other positions which are not protected These will be considered further in the Discussion

As several of the footprints identified above are located in

GA (CT) or GT (AC) tracts we prepared a new fragment (WPseq2) containing five variations on the hexanucleotide sequence SWSWWS (S¼ G or C, W ¼ A or T), in which the different sites are separated by CC (GG) The results of DNase I footprinting experiments with this fragment are

Fig 5 Differential cleavage plots showing the interaction of WP631 with MS1, MS2 and DMG60 The plots were calculated from the cleavage patterns in the presence of 1 l M WP631 shown in Fig 4 (MS1 and MS2) and Fig 6 (DMG60Y) Only a part of each sequence is shown and is written reading 5¢ )3¢ from left to right; the right-hand end corresponds to the bottom of the gels The ordinate, which is plotted on a logarithmic scale, shows the intensity of each band in the drug-treated lanes relative to that in the control Values of less than one correspond to protection by the ligand, while values above indicate enhanced cleavage The black bars highlight the regions that are protected from cleavage For MS1 and MS2 the arrows indicate the positions of WP631-induced cleavage by DEPC (grey arrows) and KMnO 4 (black arrows).

shown in Fig 7 It can be seen that there are footprints at all

the potential sites, which are most clearly seen in the

differential cleavage plot The strongest sites are at

GTGTTG and CTTCTC There is little or no protection

in the junctions between the various sites and there is

enhanced cleavage between GTGGTG and CCACAC

These regions of protection are located towards the 3¢-end

of each target site as normally observed with DNase I

footprinting, as this enzyme cuts across the width of the

DNA minor groove

Daunorubicin is thought to bind best to sequences of the

type 5¢-(A/T)CG and 5¢-(A/T)GC [17–19] and previous

NMR and crystallographic studies with bis-daunorubicins

have investigated their interaction with CGTACG and

TGTACA [28,29] None of these sequences are represented

in any of the footprinting substrates mentioned above We

therefore prepared a novel fragment (WPseq1) containing

the sites CGATCG, CGTACG, GCATGC, GCTAGC and

TGTACA each separated by the sequence AATT to which

the drug is not expected to bind The results of footprinting

experiments with this fragment are presented in Fig 8 The

DNase I cleavage patterns show footprints at each of these

sites, some of which persist to between 0.1 and 0.2 lM The

positions of these sites are confirmed in the differential

cleavage plot shown in Fig 8(B) Although DNase I

footprinting cannot usually be used to determine ligand

binding sites to single base resolution, some interesting

features of WP631 binding can be deduced by comparing the protection at each of these potential sites The central portions of the differential cleavage plots are four bases long for GCATGC, CGATCG and TGTACA and each begin at the second base These footprints are symmetrically located around the centre of each hexanucleotide target, whereas DNase I footprints are usually staggered towards the 3¢-end In contrast the footprint at GCTAGC is longer and begins one base before the start of this hexanucleotide;

it is therefore staggered towards the 5¢-end of the hexa-nucleotide site The footprint at CGTACG appears to consist of two smaller regions and there is little protection at the central adenine It should be remembered that all these sites are symmetrical (palindromic) sequences If the ligand binds to one side of the site then a second identical site will

be present in the other half of the hexanucleotide (i.e if it binds to GCTAGC by recognizing GCT, then a second identical binding site must be present in the other half of the sequence at AGC) Although two ligand molecules will not

be able to bind simultaneously, the average of the two equivalent binding sites would be a larger footprint, which is not what we observe It therefore seems most likely that a single ligand molecule is bound across the centre of each site These differences between these sites are also evident in the patterns of DEPC enhancement, which are indicated

by the arrows in Fig 8 There is enhanced DEPC reactivity

at the first adenine after the hexanucleotide site (AATT) for

Fig 6 DNase I footprints showing the inter-action of WP631 with fragments AG1, GA1 and DMG60Y WP631 concentrations (l M ) are shown at the top of each gel lane;
con corresponds to cleavage in the absence of added ligand Tracks labelled
GA are mark-ers specific for purines The numbered black bars show the positions of DNase I footprints with DMG60Y.

3562 K R Fox et al (Eur J Biochem 271)
FEBS 2004

GCATGC and GCTAGC, while this is at the second

adenine (AATT) for CGATCG and TGTACA There is no

enhancement in reactivity to DEPC after CGTACG These

subtle differences suggest that WP631 does not have exactly

the same mode of binding at each of these sites

Discussion

The footprinting results presented in this paper demonstrate

that WP631 binds to DNA in a sequence selective fashion

and that its preferred binding sites are different from those

of daunorubicin and nogalamycin By comparison with

daunorubicin it was expected that WP631 should bind best

to sequences such as CGATCG and CGTACG, which have

been used in X-ray and NMR structural studies with this

ligand [28,29] The experiments with fragment WPseq2

confirm that WP631 does indeed bind to this site at

concentrations as low as 0.2 lM, but experiments with this

and other fragments show that it also binds equally well to

other sequences

Another difference between these patterns and those

produced by daunorubicin is their temperature dependence

Previous studies with daunorubicin [17–19] only detected

DNase I footprints at low temperature (4C) presumably

as this slows the dissociation of the ligand from DNA; no footprints were observed at 20C In contrast WP631 produces clear footprints at 20C which are still apparent at

37C In this case we observe no WP631 footprints at 4 C This could be because the DNA becomes too rigid to permit bis-intercalation, or because self-stacking of the ligand is favoured at lower temperatures

Mode of binding Although the present work does not directly concern the mode of binding of WP631, this will influence the interpretation of the footprinting patterns Previous struc-tural work has demonstrated that WP631 binds in the minor groove of CGTACG with four base pairs sandwiched between the intercalating chromophores In contrast, the related compound WP652, in which the dimer is connected via C4¢, binds to the YGTR steps in TGTACA, sandwich-ing only two base pairs between the chromophores The precise orientation of the xylyl group of WP631 is also different in the two structures in which it is either perpendicular or parallel to the walls of the minor groove

Fig 7 Interaction of WP631 with fragment WPseq1 (A) DNase I footprints showing the interaction of WP631 with fragment WPseq2 WP631 concentrations (l M ) are shown at the top of each gel lane;
con corresponds to cleavage in the absence of added ligand Tracks labelled
GA are markers specific for purines The potential hexanucleotide binding sequences are indicated alongside the gel (B) Differential cleavage plot showing the interaction of WP631 with WPseq2 The plots were calculated from the cleavage patterns in the presence of 1 l M WP631 shown in Fig 7A Only a part of each sequence is shown and is written reading 5¢ )3¢ from left to right; the right-hand end corresponds to the bottom of the gel The ordinate, which is plotted on a logarithmic scale, shows the intensity of each band in the drug-treated lanes relative to that in the control Values less than one correspond to protection by the ligand, while values above indicate enhanced cleavage The vertical lines divide the fragment into the various hexanucleotide repeats.

These different structures suggest that WP631 may bind to

different sequences in different modes, sandwiching between

two and four base pairs between the chromophores These

different modes will depend on the local DNA structure and

flexibility as well as any contacts between the ligand and its

binding site A further complication is the possibility that

WP631 might bind to some sequences by

mono-intercala-tion, leaving the second chromophore in free solution or

stacked within the groove The possibility of additional

sequence-specific groove binding may further complicate

the footprinting pattern The coexistence of different

binding modes is suggested by the footprinting data

presented in this paper Some binding sites are six to eight

base pairs long, as expected for a ligand that spans six base

pairs, while others are much shorter, and appear to cover

only three bases These results are consistent with a recent

study suggesting that WP631 can bind in two different

modes with stoichiometries of 6 : 1 and 3 : 1 base pairs per

drug [41]

Sequence selectivity

The results with these DNA fragments show that

WP631binds to DNA in a sequence selective fashion, as

specific footprints are generated at moderate ligand

con-centrations (about 0.3 lM) At high concentrations (3 lM

and above) the ligand is able to bind to most sites, as shown

by the general inhibition of DNase I cleavage Examination

of the footprints does not reveal the presence of any particular di- or tri-nucleotide step within the binding sites, though many of the protected regions are GA or GT-rich in one strand, and there are no footprints in GC- or AT-rich sequences The results with MS1 and MS2, which contain every possible tetranucleotide combination, demonstrate that WP631 does not bind to a unique tetranucleotide, though we cannot exclude the possibility that it binds especially well to a unique hexanucleotide which is not represented in these fragments Several of the footprints are found in oligopurine-oligopyrimidine sequences, especially those seen with fragment DMG60 In the published crystal [29] and NMR structures [28], WP631 is bound to the sequences CGATCG and CGTACG, with the chromo-phores intercalated between each of the CpG steps This sequence is present in fragment WPseq2 and is indeed part

of a clear DNase I footprint, though several other sequences produce equally good footprints on this fragment It is therefore clear that WP631 can bind to many sites with the general sequence (G/C)(G/C)(A/T)(A/T)(G/C)(G/C)

A footprint is also evident in this fragment at the sequence TGTACA, which was suggested as one of the potential binding sites for WP652 [29] We therefore examined the footprinting results on all the fragments for degenerate sequences that might form the preferred binding sites

We find that footprints are often found around the sequence (G/C)(A/T)(A/T)(G/C), and that there are no occasions when this is not part of a drug binding site For example, on

Fig 8 Interaction of WP631 with fragment WPseq2 (A) DNase I and DEPC footprints WP631 concentrations (l M ) are shown at the top of each gel lane;
con corresponds to cleavage in the absence of added ligand Tracks labelled
GA are markers specific for purines The potential hexanucleotide binding sequences are indicated alongside the gel (B) Differential cleavage plot showing the interaction of WP631 with WPseq1 The plot was calculated from the cleavage patterns in the presence of 0.2 l M WP631 shown in Fig 8A Only a part of each sequence is shown and is written reading 5¢ )3¢ from left to right; the right-hand end corresponds to the bottom of the gel The ordinate, which is plotted on a logarithmic scale, shows the intensity of each band in the drug-treated lanes relative to that in the control Values of less than one correspond to protection by the ligand, while values above one indicate enhanced cleavage The arrows indicate the positions of WP631-induced cleavage by DEPC.

3564 K R Fox et al (Eur J Biochem 271)
FEBS 2004

MS1 the footprints are at site 1 (CATC), site 3 (GTAC) and

site 4 (GAAG), while on MS2 they are seen at site 7

(CATG), site 8 (GTTG), site 9 (CTTG and GATC) In

addition the weaker regions of protection between sites 8

and 9 contain the sequences CTAC and CTAG This

consensus sequence is also found on the tyrT fragment at

positions 25 (CATC), 38 (GTTG), 43 (GAAC) and 57

(GAAG) each of which corresponds to a region that is

protected by the ligand The sequences GATC and CTGA

are also found in the polylinker regions of pAG1 and

pGA1, and at site 2 in DMG60 (CTTC) We therefore

suggest that WP631 binds well to the sequence (G/C)(A/

T)(A/T)(G/C) However, this sequence cannot be the only

good ligand binding site For example, the footprint at the

centre of pAG1 contains the sequence AGGG (in contrast

to GGGA, which does not produce a footprint with pAG2)

Moreover sites 2, 5 and 6 on MS1 are found around the

sequences GGTG (or GTGG) (site 2), TTAG (site 5) and

GTATAG (site 6) The footprint at site 2 of DMG60 also

does not fit this pattern, and at this position it seems likely

that the ligand is able to bind to the two adjacent sites

CTTT It therefore appears that the ligand can also bind

well to some sites in which one or more base does not match

the predicted pattern

It should be noted that, although these results show the

presence of specific binding sites for WP631, these are

typically only evident at concentrations of 0.3 lM and

above In contrast, previous studies have suggested that

WP631 binds with an association constant of 3· 1011M )1

[27], from which we would expect footprints to persist to

much lower (subnanomolar) concentrations A number of

factors may contribute to this difference First, it is possible

that the preferred binding site is not represented in the

footprinting substrates that we have used We consider that

this is unlikely as the high affinity binding sites previously

reported were abundant with mixed sequence DNAs

Second, in our footprinting experiments the substrate

DNA concentration is about 10 nM, and we will not be

able to detect stronger binding sites Third, it is known that

WP631 strongly self-associates and the total ligand

concen-tration may overestimate the concenconcen-tration of the free

momomer

Acknowledgements

This work was supported by grants from the Cancer Research UK, the

Association for International Cancer Research and The Welch

Foundation, Houston, Texas, USA.

References

1 Thuong, N.T & He´le`ne, C (1993) Sequence specific recognition

and modification of double helical DNA by oligonucleotides.

Angew Chemie Int Ed Eng 32, 666–690.

2 Fox, K.R (2000) Targeting DNA with triplexes Curr Med.

Chem 7, 17–37.

3 Dervan, P.B & Bu¨rli, R.W (1999) Sequence-specific DNA

recognition by polyamides Curr Opin Chem Biol 3, 688–693.

4 Wemmer, D.E (2000) Designed sequence-specific minor groove

ligands Annu Rev Biophys Biomol Struct 29, 439–461.

5 Guelev, V.M., Cubberley, M.S., Murr, M.M., Lokey, R.S &

Iverson, B.L (2001) Design, synthesis and characterization of

polyintercalating ligands Methods Enzymol 340, 556–570.

6 Chaires, J.B (1998) Drug–DNA interactions Curr Opin Struct Biol 8, 314–320.

7 Wakelin, L.P.G (1986) Polyfunctional DNA intercalating agents Med Res Rev 6, 275–340.

8 Chaires, J.B (1996) Molecular recognition of DNA by daunoru-bicin In Advances in DNA Sequence Specific Agents, Vol 2 (Hurley, L.H & Chaires, J.B., eds) JAI Press, Greenwich, CT.

9 Weiss, R.H (1992) Will we ever find a better doxorubicin? Semin Oncol 19, 670–868.

10 Priebe, W (1995) Anthracycline antibiotics: new analogues, methods of delivery and mechanisms of action ACS Symposium Series American Chemical Society, Washington DC.

11 Lown, J.W (1988) Anthracycline and Anthracendione Based Anticancer Agents Elsevier, Amsterdam.

12 Quigley, G.J., Wang, A.H.-J., Ughetto, G., van der Marel, G., van Boom, J.H & Rich, A (1980) Molecular structure of an antic-ancer drug–DNA complex: daunomycin plus d(CpGpTpAp-CpG) Proc Natl Acad Sci USA 77, 7204–7208.

13 Wang, A.J.-H., Ughetto, G., Quigley, G.J & Rich, A (1987) Interactions between an anthracycline antibiotic and DNA: molecular structure of daunomycin complexes to d(CpGpTpAp-CpGp) at 1.2 A˚ resolution Biochemistry 26, 1152–1163.

14 Moore, M.H., Hunter, W.N., Langlois d’Estaintot, B & Kennard,

O (1989) The crystal structure of d(CGATCG) complexed with daunomycin J Mol Biol 206, 693–705.

15 Frederick, C.A., Williams, L.D., Ughetto, G., van der Marel, G.A., van Boom, J.H., Rich, A & Wang, A.H.-J (1990) Struc-tural comparison of anticancer drug–DNA complexes: adriamycin and daunomycin Biochemistry 29, 2538–2549.

16 Nunn, C.M., van Meervelt, L., Zhang, S., Moore, M.H & Kennard, O (1991) DNA–drug interactions: the crystal structures

of d(TGTACA) and d(TGATCA) complexed with daunomycin.

J Mol Biol 222, 167–177.

17 Chaires, J.B., Fox, K.R., Herrera, J.E., Britt, M & Waring, M.J (1987) Site and sequence specificity of the daunomycin–DNA interaction Biochemistry 26, 8227–8236.

18 Skorabogaty, A., White, R.J., Phillips, D.R & Reis, J.A (1988) The 5¢-CA DNA-sequence preference of daunomycin FEBS Lett.

227, 103–106.

19 Chaires, J.B., Herrera, J.E & Waring, M.J (1990) Preferential binding of daunomycin to 5¢A/TCG and 5¢A/TGC sequences revealed by footprinting titration experiments Biochemistry 29, 6145–6153.

20 Phillips, D.R., Brownlee, R.T.C., Reiss, J.A & Scourides, P.A (1992) Bis–daunomycin hydrazones – interactions with DNA Invest New Drugs 10, 79–88.

21 Skorobogarty, A., Brownlee, R.T.C., Chandler, C.J., Kyratzis, I., Phillips, D.R., Riess, J.A & Trist, H (1988) The DNA association and biological activity of a new bis(14-thiadaunomycin) Anti-cancer Drug Des 3, 41–56.

22 Priebe, W., Fokt, I., Przewloka, T., Chaires, J.B., Portugal, J & Trent, J.O (2001) Exploiting anthracycline scaffold for designing DNA-targeting agents Methods Enzymol 340, 529–555.

23 Chaires, J.B., Leng, F., Przewloka, T., Fokt, I., Ling, Y.-H., Perez-Soler, R & Priebe, W (1997) Structure-based design of a new bisintercalating anthracycline antibiotic J Med Chem 40, 261– 266.

24 Martin, B., Vaquero, A., Priebe, W & Portugal, J (1999) Bisan-thracycline WP631 inhibits basal and Sp1-activated transcription initiation in vitro Nucleic Acids Res 27, 3402–3409.

25 Ashikawa, K., Shishodia, S., Fokt, I., Priebe, W & Aggarwal, B.B (2004) Evidence that activation of nuclear factor-kappa B is essential for the cytotoxic effects of doxorubicin and its analogues Biochem Pharml 67, 353–364.

26 Kutsch, O., Levy, D.N., Bates, P.J., Becker, J., Kosloff, B.R., Shaw, G.M., Priebe, W & Benveniste, E.N (2004)