Investigational New Drugs 10: 79-88, 1992. 9 1992Kluwer Academic Publishers. Printed in the Netherlands.
Bis-daunomycin hydrazones: Interactions with DNA Don R. Phillips 1, Robert T.C. Brownlee2, James A. Reiss z and Panayiotis A. Scourides 1,2 Departments o f Biochemistry 1 and Chemistry 2, La Trobe University, Bundoora, Victoria, 3083, A ustralia Key words: bis-daunemycin hydrazones, DNA interaction, dissociation kinetics, DNA lengthening, DNA sequence selectivity, bis-intercalation
Summary A series of bis-daunomycin hydrazones were synthesised from diester diamide linking groups derived from c~,o)-dicarboxylic acids. All members of the series bis-intercalated into DNA, as evidenced by doubling of the lengthening of rod-like DNA compared to daunomycin, and by a 1000-5000 fold slower dissociation from DNA than daunomycin under detergent sequestration conditions. The bis-hydrazones exhibited neighbour exclusion, and occupied 6 bp under saturating conditions of drug. A unique DNA sequence specificity was apparent from transcriptional footprinting of 100 bp of DNA, with the greatest preference for 5'-CACA sites.
Introduction Although the anthracyclines daunomycin and Adriamycin (Fig. 1) have been in widespread clinical use as anti-cancer agents [1,2], the exact mode of action has yet to be established [3]. While they have been shown to interfere with a variety of cellular processes [3], it is thought that their dominant effect is at the DNA level [3]. This view has received considerable support by the correlation of their biological activity over three orders of magnitude, with their DNA binding affinity [4] and several DNA related processes [5], and the fact that 99.8% of Adriamycin in cell cultures is associated with nuclear DNA [6]. There have been many attempts to overcome the limiting cardiotoxicity of the anthracyclines [2,3]. The major approach has relied upon the synthesis of new derivatives, followed by screening for enhanced anti-cancer activity or reduced cardiotoxicity [3,51. An alternative approach is based on the well documented high affinity of these drugs for DNA [3,7]. Since it has been shown that the DNA binding affinity of anthracyclines correlates with
anti-cancer activity [4], then it is reasonable to assume that the greater the affinity of the drug for its DNA receptor, or the longer it remains in its binding site, the greater will be its biological effect. On the basis of this premise we have synthesised a series of bis-daunomycin derivatives (Fig. 1), designed to act as bis-intercalators, and based on the known stereochemical details of the daunomycin-DNA complex [8,9]. Such bis-intercalators would be expected to exhibit optimal DNA binding affinity of approximately 1012 M -1, plus possible additional contributions from translational entropy [10], based on the known affinity of daunomycin for DNA of 106 M - 1 [3,7]. Such an enhanced affinity could therefore result in a potential million-fold increase in DNA residence time. Previous attempts to synthesise active bis-anthracyclines have not been successful, due mainly to limited solubility, or short linker lengths which precluded true bis-intercalation into DNA [11 - 15]. We sought to overcome both of these problems by synthesising a series of bis-anthracyclines which exhibited good solubility, and with linkers of sufficient length to enable both anthracycline moieties
80
4I
o
OH
II
]
_-'_-7
OH
O
OCH3 0
dence time on DNA and an enhanced sequence specificity compared to their parent monomer, daunomycin.
0
CH3~ ~ 2
Results
1'
DNA binding and stoichiometry
~)H NH2 2'
i
X:H
io
X:
O
OH
OH
CH3
N~
OCH3 0
OH
~C'O aI HN ~ CH2 I
O
./CO
=
\ (CH2)n CO
CH3
I
HN % CH2 I
O
OH
CH3 " OH
cH3
OCH3 0
~-I}
OH
O
If both anthracycline moieties intercalate into DNA, the binding affinity could be as high as 1012 M - 1 and therefore could not be quantitated by conventional means. In order to reduce the association constant to measurable levels, equilibrium dialysis and spectrophotometric titrations were carried out in 1M NaC1. No meaningful levels of free drug could be detected, and this suggested that the association constant was greater than (107-108 ) M - 1 at this ionic strength, and therefore greater than approximately 108-9 at a physiological ionic strength of 0.15 M [20]. Because of the high affinity for DNA the stoichiometry of the interaction was determined by fluorescence titration (Fig. 2). The intercept defined by best-fit of the two slopes reflects the saturation of calf thymus DNA with added bisdaunomycin 2, with subsequent additions of drug yielding a fluorescence intensity response of free drug. The intercept of 0.16-0.17 drug per DNA bp implies that at saturation of the DNA, each drug occupies on average of 6.1 _+ 0.2 bp, as determined from triplicate measurements of this value. The reason that the line for the region past DNA saturation is not parallel to the free drug slope is not known, but may be associated with a small amount ( 1 - 2 % ) of hydrolysis of the bis-daunomycin to daunomycin during the time course of the titration [191.
n=2-8
Fig. 1. Structure of daunomycin 1, Adriamycin la and bisdaunomycin 2 - 8 .
DNA lengthening
to intercalate into DNA assuming a neighbour exclusion mechanism [16,17]. These bis-daunomycins are indeed soluble [18,19] and we now report on their interactions with DNA and show that they are true bis-intercalators which exhibit enhanced resi-
The increase of lengthening of sonicated DNA by the bis-daunomycins 2 - 8 was measured viscometrically, and the result for anthracyclines ! (daunomycin) and 2 are shown in Fig. 3. The slope (helix extension parameter) is double that observed with
81
80
1.3
60
1.2
//
t,-.
40 0
L Lo
m
1.1
20
I.,,,..
0
1.0 0.0
U_
0 0.0
0.1
0.2
0.3
d a u n o m y c i n a n d is t h e r e f o r e c o n s i s t e n t w i t h a bisi n t e r c a l a t i o n p r o c e s s [17,20]. T h e helix e x t e n s i o n p a r a m e t e r f o r all b i s - h y d r a z o n e s is s u m m a r i s e d in 1, a n d
are all c o n s i s t e n t
Fig. 3. Viscometric titration of sonicated calf thymus DNA with daunomycin 1 and bis-daunomycin 2. The titration was carried out at 26.8~ in 0.1 ionic strength PIPES buffer, pH 6.8 using 3 ml of 0.3 mM (bp) of DNA. The total drug:DNA bp ratio is denoted as r. Table 1. Comparison of DNA helix extension parameter (m, single experiment) and dissociation time constants (r I and %) for derivatives 1-8. The amplitude of the slower dissociation process (A2) is shown a percentage of the total amplitude observed. All kinetic parameters exhibited a reproducibility with __+_ 10%, and all dissociation experiments were performed in triplicate.
with bis-inter-
calation.
DNA
0.2 r
Fig. 2. Fluorescence titration of 2.5 /,M bis-daunomycin 2 in 0.01 ionic strength PIPES buffer, pH 6.8, 20~ to 2.0 ml, 6.7 /,M bp calf thymus DNA (filled squares) or 2.0 ml buffer (open squares). The total drug:DNA bp ratio is denoted as r.
Table
0.1
dissociation k i n e t i c s
T h e d e t e r g e n t s e q u e s t e r e d d i s s o c i a t i o n o f t h e bishydrazones were independent of SDS concentrat i o n a b o v e 2 % ( w / v ) a n d all s u b s e q u e n t studies were therefore carried out using 2% SDS. The time d e p e n d e n t i n c r e a s e o f a b s o r b a n c e at 480 n m u p o n
Derivative
m
r 1 (min)
r 2 (min)
A 2 (070)
1a 2 3 4 5 6 7 8
0.82 1.65 1.75 1.68 1.94 1.51 1.58 1.58
0.0048 2.7 4.8 2.3 6.3 3.4 1.3 2.5
0.031 15 30 12 29 14 7.2 8.9
66 57 66 60 67 49 59 37
a Reference 21.
a d d i t i o n o f S D S to t h e d r u g - D N A s o l u t i o n (Fig. 4) was a d e q u a t e l y
described
by a t w o - e x p o n e n t i a l
first-order relaxation process defined by
w h e r e A t a n d A ~ are t h e a b s o r b a n c e s at t i m e t a n d at e q u i l i b r i u m , a n d A 1 a n d A 2 are t h e a m p l i t u d e s associated with the two kinetic processes with time
A
-
A t = A 1 e - t / r 1 + A 2 e-t/r2
c o n s t a n t s r 1 a n d r 2.
82 ....
I ....
I ....
;
....
I .....
I ....
I ....
I ....
I ....
I
0.20
0.18
AA 0-16
0.14 iiijfflf|lllJl
0
2000
4000 T I M E (sec)
Fig. 4. Kinetic profile for the SDS-induced dissociation of 2 from calf thymus DNA in 0.01 ionic strength PIPES buffer, pH 6.8, 20~ The change of absorbances at 480 nm (AA) is shown as a function of time together with a two-exponential fit to this data (continuous line).
The calculated amplitudes and time constants for each member of the bis-hydrazone series are summarised in Table 1. The slower process exhibited a time constant of 430-1800 sec, the faster process 80-380 sec, with the amplitude of the slower process ranging from 37-67% of the observed absorbance change. There are no apparent trends of the dissociation parameters with linker length and complete dissociation from DNA was observed for all derivatives within 5-10 hours, compared to approximately 10 sec for daunomycin under identical conditions [21]. In vitro transcription footprinting The lac UV5 promoter was initiated with only three nucleotides to form a stable initiated ternary com-
plex [22-25], which was then equilibrated with bishydrazone 2 for one hour at 20~ Synchronous elongation of the initiated complex resulted in rapid formation of the full length transcript in the absence of drug, but yielded a range of specific blocked transcripts when in the presence of drug (Fig. 5). Read-through of RNA polymerase past the drug-induced pauses is evident with increasing elongation time, and is due to dissociation of the drug from each binding site [22,25]. The relative occupancy of the drug for each site is approximated by the mole-fraction of transcriptional blockages at each site. In the region probed with good resolution (17-100 nucleotides) there are five discrete drug-induced blockage regions which have no counterpart in the control lanes. The relative drug occupancy of these regions is shown in
Fig. 5. Transcriptional blockages induced by bis-daunomycin 2. The initiated transcription complex is shown in lane I and elongation was carried out for 0.25, 0.5, 1, 1.5, 4, 8, 15, 30 and 60 min both in the absence (control lanes) and presence of drug. Lanes C and G represent sequencing lanes in which elongation of the initiated complex was terminated with 3' -methoxy CTP and 3 '-methoxy GTP respectively [22].
83
84 1
Z 0 F.(.3
Bis-daunomycin hydrazones: Interactions with DNA Don R. Phillips 1, Robert T.C. Brownlee2, James A. Reiss z and Panayiotis A. Scourides 1,2 Departments o f Biochemistry 1 and Chemistry 2, La Trobe University, Bundoora, Victoria, 3083, A ustralia Key words: bis-daunemycin hydrazones, DNA interaction, dissociation kinetics, DNA lengthening, DNA sequence selectivity, bis-intercalation
Summary A series of bis-daunomycin hydrazones were synthesised from diester diamide linking groups derived from c~,o)-dicarboxylic acids. All members of the series bis-intercalated into DNA, as evidenced by doubling of the lengthening of rod-like DNA compared to daunomycin, and by a 1000-5000 fold slower dissociation from DNA than daunomycin under detergent sequestration conditions. The bis-hydrazones exhibited neighbour exclusion, and occupied 6 bp under saturating conditions of drug. A unique DNA sequence specificity was apparent from transcriptional footprinting of 100 bp of DNA, with the greatest preference for 5'-CACA sites.
Introduction Although the anthracyclines daunomycin and Adriamycin (Fig. 1) have been in widespread clinical use as anti-cancer agents [1,2], the exact mode of action has yet to be established [3]. While they have been shown to interfere with a variety of cellular processes [3], it is thought that their dominant effect is at the DNA level [3]. This view has received considerable support by the correlation of their biological activity over three orders of magnitude, with their DNA binding affinity [4] and several DNA related processes [5], and the fact that 99.8% of Adriamycin in cell cultures is associated with nuclear DNA [6]. There have been many attempts to overcome the limiting cardiotoxicity of the anthracyclines [2,3]. The major approach has relied upon the synthesis of new derivatives, followed by screening for enhanced anti-cancer activity or reduced cardiotoxicity [3,51. An alternative approach is based on the well documented high affinity of these drugs for DNA [3,7]. Since it has been shown that the DNA binding affinity of anthracyclines correlates with
anti-cancer activity [4], then it is reasonable to assume that the greater the affinity of the drug for its DNA receptor, or the longer it remains in its binding site, the greater will be its biological effect. On the basis of this premise we have synthesised a series of bis-daunomycin derivatives (Fig. 1), designed to act as bis-intercalators, and based on the known stereochemical details of the daunomycin-DNA complex [8,9]. Such bis-intercalators would be expected to exhibit optimal DNA binding affinity of approximately 1012 M -1, plus possible additional contributions from translational entropy [10], based on the known affinity of daunomycin for DNA of 106 M - 1 [3,7]. Such an enhanced affinity could therefore result in a potential million-fold increase in DNA residence time. Previous attempts to synthesise active bis-anthracyclines have not been successful, due mainly to limited solubility, or short linker lengths which precluded true bis-intercalation into DNA [11 - 15]. We sought to overcome both of these problems by synthesising a series of bis-anthracyclines which exhibited good solubility, and with linkers of sufficient length to enable both anthracycline moieties
80
4I
o
OH
II
]
_-'_-7
OH
O
OCH3 0
dence time on DNA and an enhanced sequence specificity compared to their parent monomer, daunomycin.
0
CH3~ ~ 2
Results
1'
DNA binding and stoichiometry
~)H NH2 2'
i
X:H
io
X:
O
OH
OH
CH3
N~
OCH3 0
OH
~C'O aI HN ~ CH2 I
O
./CO
=
\ (CH2)n CO
CH3
I
HN % CH2 I
O
OH
CH3 " OH
cH3
OCH3 0
~-I}
OH
O
If both anthracycline moieties intercalate into DNA, the binding affinity could be as high as 1012 M - 1 and therefore could not be quantitated by conventional means. In order to reduce the association constant to measurable levels, equilibrium dialysis and spectrophotometric titrations were carried out in 1M NaC1. No meaningful levels of free drug could be detected, and this suggested that the association constant was greater than (107-108 ) M - 1 at this ionic strength, and therefore greater than approximately 108-9 at a physiological ionic strength of 0.15 M [20]. Because of the high affinity for DNA the stoichiometry of the interaction was determined by fluorescence titration (Fig. 2). The intercept defined by best-fit of the two slopes reflects the saturation of calf thymus DNA with added bisdaunomycin 2, with subsequent additions of drug yielding a fluorescence intensity response of free drug. The intercept of 0.16-0.17 drug per DNA bp implies that at saturation of the DNA, each drug occupies on average of 6.1 _+ 0.2 bp, as determined from triplicate measurements of this value. The reason that the line for the region past DNA saturation is not parallel to the free drug slope is not known, but may be associated with a small amount ( 1 - 2 % ) of hydrolysis of the bis-daunomycin to daunomycin during the time course of the titration [191.
n=2-8
Fig. 1. Structure of daunomycin 1, Adriamycin la and bisdaunomycin 2 - 8 .
DNA lengthening
to intercalate into DNA assuming a neighbour exclusion mechanism [16,17]. These bis-daunomycins are indeed soluble [18,19] and we now report on their interactions with DNA and show that they are true bis-intercalators which exhibit enhanced resi-
The increase of lengthening of sonicated DNA by the bis-daunomycins 2 - 8 was measured viscometrically, and the result for anthracyclines ! (daunomycin) and 2 are shown in Fig. 3. The slope (helix extension parameter) is double that observed with
81
80
1.3
60
1.2
//
t,-.
40 0
L Lo
m
1.1
20
I.,,,..
0
1.0 0.0
U_
0 0.0
0.1
0.2
0.3
d a u n o m y c i n a n d is t h e r e f o r e c o n s i s t e n t w i t h a bisi n t e r c a l a t i o n p r o c e s s [17,20]. T h e helix e x t e n s i o n p a r a m e t e r f o r all b i s - h y d r a z o n e s is s u m m a r i s e d in 1, a n d
are all c o n s i s t e n t
Fig. 3. Viscometric titration of sonicated calf thymus DNA with daunomycin 1 and bis-daunomycin 2. The titration was carried out at 26.8~ in 0.1 ionic strength PIPES buffer, pH 6.8 using 3 ml of 0.3 mM (bp) of DNA. The total drug:DNA bp ratio is denoted as r. Table 1. Comparison of DNA helix extension parameter (m, single experiment) and dissociation time constants (r I and %) for derivatives 1-8. The amplitude of the slower dissociation process (A2) is shown a percentage of the total amplitude observed. All kinetic parameters exhibited a reproducibility with __+_ 10%, and all dissociation experiments were performed in triplicate.
with bis-inter-
calation.
DNA
0.2 r
Fig. 2. Fluorescence titration of 2.5 /,M bis-daunomycin 2 in 0.01 ionic strength PIPES buffer, pH 6.8, 20~ to 2.0 ml, 6.7 /,M bp calf thymus DNA (filled squares) or 2.0 ml buffer (open squares). The total drug:DNA bp ratio is denoted as r.
Table
0.1
dissociation k i n e t i c s
T h e d e t e r g e n t s e q u e s t e r e d d i s s o c i a t i o n o f t h e bishydrazones were independent of SDS concentrat i o n a b o v e 2 % ( w / v ) a n d all s u b s e q u e n t studies were therefore carried out using 2% SDS. The time d e p e n d e n t i n c r e a s e o f a b s o r b a n c e at 480 n m u p o n
Derivative
m
r 1 (min)
r 2 (min)
A 2 (070)
1a 2 3 4 5 6 7 8
0.82 1.65 1.75 1.68 1.94 1.51 1.58 1.58
0.0048 2.7 4.8 2.3 6.3 3.4 1.3 2.5
0.031 15 30 12 29 14 7.2 8.9
66 57 66 60 67 49 59 37
a Reference 21.
a d d i t i o n o f S D S to t h e d r u g - D N A s o l u t i o n (Fig. 4) was a d e q u a t e l y
described
by a t w o - e x p o n e n t i a l
first-order relaxation process defined by
w h e r e A t a n d A ~ are t h e a b s o r b a n c e s at t i m e t a n d at e q u i l i b r i u m , a n d A 1 a n d A 2 are t h e a m p l i t u d e s associated with the two kinetic processes with time
A
-
A t = A 1 e - t / r 1 + A 2 e-t/r2
c o n s t a n t s r 1 a n d r 2.
82 ....
I ....
I ....
;
....
I .....
I ....
I ....
I ....
I ....
I
0.20
0.18
AA 0-16
0.14 iiijfflf|lllJl
0
2000
4000 T I M E (sec)
Fig. 4. Kinetic profile for the SDS-induced dissociation of 2 from calf thymus DNA in 0.01 ionic strength PIPES buffer, pH 6.8, 20~ The change of absorbances at 480 nm (AA) is shown as a function of time together with a two-exponential fit to this data (continuous line).
The calculated amplitudes and time constants for each member of the bis-hydrazone series are summarised in Table 1. The slower process exhibited a time constant of 430-1800 sec, the faster process 80-380 sec, with the amplitude of the slower process ranging from 37-67% of the observed absorbance change. There are no apparent trends of the dissociation parameters with linker length and complete dissociation from DNA was observed for all derivatives within 5-10 hours, compared to approximately 10 sec for daunomycin under identical conditions [21]. In vitro transcription footprinting The lac UV5 promoter was initiated with only three nucleotides to form a stable initiated ternary com-
plex [22-25], which was then equilibrated with bishydrazone 2 for one hour at 20~ Synchronous elongation of the initiated complex resulted in rapid formation of the full length transcript in the absence of drug, but yielded a range of specific blocked transcripts when in the presence of drug (Fig. 5). Read-through of RNA polymerase past the drug-induced pauses is evident with increasing elongation time, and is due to dissociation of the drug from each binding site [22,25]. The relative occupancy of the drug for each site is approximated by the mole-fraction of transcriptional blockages at each site. In the region probed with good resolution (17-100 nucleotides) there are five discrete drug-induced blockage regions which have no counterpart in the control lanes. The relative drug occupancy of these regions is shown in
Fig. 5. Transcriptional blockages induced by bis-daunomycin 2. The initiated transcription complex is shown in lane I and elongation was carried out for 0.25, 0.5, 1, 1.5, 4, 8, 15, 30 and 60 min both in the absence (control lanes) and presence of drug. Lanes C and G represent sequencing lanes in which elongation of the initiated complex was terminated with 3' -methoxy CTP and 3 '-methoxy GTP respectively [22].
83
84 1
Z 0 F.(.3
