Enhanced binding to DNA and topoisomerase I inhibition by an analog of the antitumor antibiotic rebeccamycin containing an amino sugar residue

Article abstract of DOI:10.1124/mol.55.2.377

Many antitumor agents contain a carbohydrate side chain appended to a DNA-intercalating chromophore. This is the case with anthracyclines such as daunomycin and also with indolocarbazoles including the antibiotic rebeccamycin and its tumor active analog, NB506. In each case, the glycoside residue plays a significant role in the interaction of the drug with the DNA double helix. In this study we show that the DNA-binding affinity and sequence selectivity of a rebeccamycin derivative can be enhanced by replacing the glucose residue with a 2'-aminoglucose moiety. The drug-DNA interactions were studied by thermal denaturation, fluorescence, and footprinting experiments The thermodynamic parameters indicate that the newly introduced amino group on the glycoside residue significantly enhanced binding to DNA by increasing the contribution of the polyelectrolyte effect to the binding free energy, but does not appear to participate in any specific molecular contacts. The energetic contribution of the amino group of the rebeccamycin analog was found to be weaker than that of the sugar amino group of daunomycin, possibly because the indolocarbazole derivative is only partially charged at neutral pH. Topoisomerase I-mediated DNA cleavage studies reveal that the OH→NH₂ substitution does not affect the capacity of the drug to stabilize enzyme-DNA covalent complexes. Cytotoxicity studies with P388 leukemia cells sensitive or resistant to camptothecin suggest that topoisomerase I represents a privileged intracellular target for the studied compounds. The role of the sugar amino group is discussed. The study provides useful guidelines for the development of a new generation of indolocarbazole- based antitumor agents.

Full text of DOI:10.1124/mol.55.2.377

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MOLECULAR PHARMACOLOGY, 55:377–385 (1999).
Enhanced Binding to DNA and Topoisomerase I Inhibition by
an Analog of the Antitumor Antibiotic Rebeccamycin
Containing an Amino Sugar Residue
CHRISTIAN BAILLY, XIAOGANG QU, FABRICE ANIZON, MICHELLE PRUDHOMME, JEAN-FRANC¸ OIS RIOU, and
JONATHAN B. CHAIRES
Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret et Institut National de la Sante´ et de la Recherche Me´ dicale U-124, Lille,
France (C.B.); Department of Biochemistry, University of Mississippi Medical Center, Jackson, Mississippi (X.Q., J.B.C.); Rhoˆ ne-Poulenc Rorer,
13 Quai Jules Guesde, Vitry sur Seine, France (J.F.R.); and UMR 6504 Centre National de la Recherche Scientifique, Universite´ Blaise Pascal,
Aubie` re, France (F.A., M.P.)
Received July 7, 1998; accepted November 18, 1998
This paper is available online at http://www.molpharm.org
ABSTRACT
Many antitumor agents contain a carbohydrate side chain ap- appear to participate in any specific molecular contacts. The
pended to a DNA-intercalating chromophore. This is the case energetic contribution of the amino group of the rebeccamycin
with anthracyclines such as daunomycin and also with indolo- analog was found to be weaker than that of the sugar amino
carbazoles including the antibiotic rebeccamycin and its tumor group of daunomycin, possibly because the indolocarbazole
active analog, NB506. In each case, the glycoside residue plays derivative is only partially charged at neutral pH. Topoisomer-
a significant role in the interaction of the drug with the DNA ase I-mediated DNA cleavage studies reveal that the OH3NH2
double helix. In this study we show that the DNA-binding affin- substitution does not affect the capacity of the drug to stabilize
ity and sequence selectivity of a rebeccamycin derivative can enzyme-DNA covalent complexes. Cytotoxicity studies with
be enhanced by replacing the glucose residue with a 2Ј-amin- P388 leukemia cells sensitive or resistant to camptothecin sug-
oglucose moiety. The drug-DNA interactions were studied by gest that topoisomerase I represents a privileged intracellular
thermal denaturation, fluorescence, and footprinting experi- target for the studied compounds. The role of the sugar amino
ments. The thermodynamic parameters indicate that the newly group is discussed. The study provides useful guidelines for the
introduced amino group on the glycoside residue significantly development of a new generation of indolocarbazole-based
enhanced binding to DNA by increasing the contribution of the antitumor agents.
polyelectrolyte effect to the binding free energy, but does not
Indolocarbazoles represent an important class of antitu- agent and an inhibitor of topoisomerase I (Nettleton et al.,
mor agents (Prudhomme, 1997). Antibiotics such as stauro- 1985; Bush et al., 1987). Numerous synthetic derivatives
sporine (Fig. 1) and K252a, which have the two indole nitro- have been designed to confer higher DNA-binding and anti-
gens linked to the carbohydrate residues, are potent topoisomerase I activities. A few synthetic compounds, such
inhibitors of protein kinases, in particular, protein kinase C. as NB-506 (Fig. 1), are presently in clinical trials (Arakawa
This subgroup also includes the synthetic derivative 7-hy-
droxy-staurosporine, known as UCN-01 (Fig. 1), which is
undergoing clinical trials (Akinaga et al., 1991; Shao et al.,
1997). Another series of indolocarbazole derivatives has the
sugar residue attached to only one indole nitrogen. This
second subgroup is typified by the antibiotic rebeccamycin
(Fig. 1), which has very little effect on protein kinase C, but,
unlike the compounds of the first series, it is a DNA-binding
et al., 1995).
Over the last few years, we have screened more than 80
rebeccamycin derivatives for topoisomerase I inhibition and
biological activity. The structure-activity relationships are
not yet fully elucidated but at least three important rules
were established. First, the two chlorine atoms on the indo-
locarbazole chromophore of rebeccamycin are detrimental to
the interaction with DNA and, consequently, to the biological
activity. These bulky chloro substituents prevent the drug
from intercalating into DNA. Second, a variety of substitu-
ents can be added on the imide nitrogen without disrupting
the drug-DNA complex. For example, polar formyl-amino or
bis(hydroxyethyl)methylamino groups as well as nonpolar
This work was supported by research grants from the Association pour la
Recherche sur le Cancer (to C.B.) and the Ligue Nationale Franc¸aise Contre le
Cancer (to C.B.) and from the National Cancer Institute (Grant CA35635 to
J.B.C.).
ABBREVIATIONS: DMSO, dimethyl sulfoxide; bp, base pair; pBS, pBluescript; AMV, avian myeloblastosis virus; DNase I, deoxyribonuclease I (EC
3.1.21.1).
377

378
Bailly et al.
methyl groups can be tolerated without loss of the topoisom- tion and/or cleavage of DNA sequences (Kumar et al., 1997b;
erase I poisoning effect. Third, and this is probably the most Myers et al., 1997).
important point, the sugar residue is absolutely required to
The anthracycline antibiotics all bear one or two glycosyl
ensure tight interaction with DNA. Analogs of rebeccamycin side chains that come to lie in the grooves of DNA when the
lacking the methoxyglucose residue are much less active chromophore is intercalated. The amino sugar residue of the
than their glycosylated counterparts. Moreover, the stereo- prototype anthracycline daunomycin is needed for specific
chemistry of the sugar is essential. Indolocarbazoles linked to binding of the drug to (A/T)GC and (A/T)CG triplets (Chaires
the carbohydrate via a ␤-glycoside linkage are potent inhib- et al., 1990; Bailly et al., 1998b). Thermodynamic studies
itors of topoisomerase I, whereas the ␣-analogs completely revealed that a significant energetic penalty results from the
failed to inhibit the enzyme, most likely as a result of their removal of the daunosamine sugar part attached to the
greatly reduced affinity for DNA. Both the chemical nature daunomycinone chromophore (Chaires, 1996). Although the
and the isomeric form of the sugar residue are essential to amino group at position 3Ј on the sugar is not essential for
the interaction with DNA and to the biological activity (for cytostatic and topoisomerase II-targeting activities (Capra-
the structure-activity relationships, see Rodrigues-Pereira et nico et al., 1994), it is important for DNA binding for more
al., 1996; Anizon et al., 1997, 1998; Bailly et al., 1997, 1998a; than just its positive charge. According to several high-reso-
Prudhomme, 1997; Moreau et al., 1998).
lution NMR and crystal structures, the amine participates in
A number of antitumor antibiotics are equipped with car- hydrogen-bonding interactions with DNA bases (Chaires,
bohydrate residues that contribute significantly to the inter- 1990; Frederick et al., 1990). The replacement of the 3Ј-
action with DNA. For example the aryltetrasaccharide do- amine with a hydroxyl group results in a marked decrease of
I
main of the enediyne antibiotic calicheamycin ␥1 plays a the affinity for DNA. The binding constant of doxorubicin for
critical role in the recognition of specific sequences and con- DNA is more than 80 times stronger than that of hydroxy-
tributes positively to the DNA-cleaving activity as well as to rubicin, and the total favorable energetic contribution of the
the inhibition of transcription by DNA polymerase II (Nico- amine is approximately 2.5 kcal/mol (Chaires, 1996).
laou et al., 1992; Paloma et al., 1994; Ikemoto et al., 1995;
With these data in mind, we postulated that the addition of
Kumar et al., 1997a). Neocarzinostatin and esperamicin, two an amino group on the methoxy-sugar residue of rebeccamy-
other enediyne antitumor antibiotics, also contain aminogly- cin-type compounds should permit tighter binding to DNA
coside side chains that play a functional role in the recogni- and might be beneficial to their biological activity. The
present study reports the synthesis and biological activity of
a new rebeccamycin derivative possessing an amino group at
position 2Ј. To evaluate the influence of the newly introduced
amino group, we compared the DNA-binding and topoisom-
erase I-poisoning activities of the two drugs shown in Fig. 2.
The substitution of the hydroxyl group with an amino group
at position 2Ј of the sugar moiety is the only difference
between the two test molecules, and the rest of the molecule
is identical. Such a substitution produces a significant in-
crease in hydrophilicity and basicity as well as a change in
steric hindrance, all of which could affect DNA binding. We
provide clear evidence that the OH3NH2 substitution sig-
nificantly enhances the DNA-binding affinity without any
loss of the activity against topoisomerase I.
Materials and Methods
Synthesis of Compound (2). Most indolocarbazole derivatives
that we studied previously were obtained by hemisynthesis from the
microbial metabolite rebeccamycin. In contrast, the amino sugar
derivative 2 was obtained by total synthesis by coupling of a sugar
possessing a protected amine function to an indolocarbazole aglycone
(Fig. 3). The amino group of commercial D-glucosamine hydrochlo-
ride was protected as a phtalimide by treatment with sodium me-
thoxide followed by reaction with phtalic anhydride, and then the
intermediate was treated with pyridine and acetic anhydride (Le-
mieux et al., 1976). The protected sugar was coupled to the N-
methylmaleimide indolocarbazole (Brenner et al., 1988). Coupling
was performed with N-methylmaleimide indolocarbazole trimethyl-
silylated at one of the indole nitrogens in the presence of trimethyl-
silyltriflate in 1,2-dichloromethane according to a method described
for the synthesis of nucleosides (Chu et al., 1990). Reaction of the
coupling product C with hydrazine hydrate followed by an acidic
treatment led to diamine 2 as a monohydrochloride. The chemical
Fig. 1. Indolocarbazoles. Structure of the protein kinase C inhibitors—
shifts of the protons of the two amino groups were 5.04 ppm in
the antibiotic staurosporine and the synthetic derivative UCN-01—and
dimethyl sulfoxide (DMSO) and in the signal of water for the free
amine and 8.20 and 5.0 (broad s) for the hydrochloride. For the
structure of the toposiomerase I inhibitors—the antibiotic rebeccamycin
and the synthetic derivative NB-506.

An Amino Sugar Derivative of Rebeccamycin
379
corresponding aglycone bearing an amino function only on the imide ppm for compound 2 is attributed to the free amine on the imide
nitrogen, the chemical shift of the protons of the amino group in the nitrogen.
same solvent was 5.00 ppm and the hydrochloride could not be
formed (Rodrigues-Pereira et al., 1995). Therefore, the signal at 5.04
Chemistry. Infrared (IR) spectra were recorded on a Perkin-
Elmer 881 spectrometer (␯ in cmϪ1). NMR spectra were performed on
a Bruker AC 400 (1H: 400 MHz; 13C: 100 MHz) (chemical shifts ␦ in
ppm, and the following abbreviations are used: s, singlet; d, doublet;
t, triplet; m, multiplet; C tert, tertiary carbons; C quat, quaternary
carbons). Mass spectrum (FABϩ) was determined at CESAMO
(Talence, France) on a high-resolution Fisons Autospec-Q spectrom-
eter. Chromatographic purifications were performed by Kieselgel 60
(Merck) 0.063- to 0.200-mm column chromatography. For purity
tests, thin-layer chromatography was performed on fluorescent silica
gel plates (60 F254; Merck).
6-Amino-12-(2-amino-␤-D-glucopyrannosyl)-6,7,12,13-Tetra-
hydroindolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-dione, hydro-
chloride (2). N-methylmaleimide indolocarbazole B (100 mg, 0.295
mmol) was dissolved in tetrahydrofuran (THF; 20 ml), and NaH
(60% in mineral oil, 26 mg, 0.65 mmol) was added. The mixture was
stirred for 45 min at room temperature, and then TMSCl [0.1 ml,
0.796 mmol, 2.7 equivalent (eq)] was added. After stirring for 30 min,
THF was evaporated before addition of 170 ml of (CH2Cl)2 and
N-phtalimido-sugar A (a mixture of ␣ and ␤, 37:63, 270 mg, 0.583
mmol, 2 eq). Trimethylsilyl triflate (50 ␮l) was added, and the mix-
ture was stirred at room temperature for 2.5 h and then poured into
saturated aqueous NaHCO3. After extraction with EtOAc, the or-
ganic phase was dried over MgSO4 and the solvent was removed.
CH2Cl2 was added to the residue and the mixture was filtered off.
After purification by chromatography (eluent, EtOAc/CH2Cl2, 5:95),
a mixture of C and few quantities of starting products that could not
be separated by chromatography was obtained and used for the next
step without further purification.
This mixture was dissolved in ethanol (20 ml), and hydrazine
hydrate (1.5 ml) was added. After refluxing for 3 h, the solvent was
removed and the residue was dissolved in AcOEt, washed with brine.
The organic phase was dried over MgSO4. After removal of the
solvent, 1.1 N HCl (231 ␮l) was added to a suspension of the residue
in methanol and the mixture was stirred at room temperature. A
precipitate of the hydrochloride was obtained by addition of AcOEt.
Filtration gave 2 as an orange solid (52.3 mg, 0.10 mmol, 33% overall
yield): m.p. Ͼ 300°C. IR (KBr) ␯CO 1710, 1760 cmϪ1, ␯NH, OH 3200 to
Fig. 2. Structure of the drug used in this study. An energy-minimized
structure of the drugs is shown. The software HyperChem 5.01 and
Alchemy 2000 were used to construct the structures.
3600 cmϪ1
.
High resolution mass spectrum calculated for
Fig. 3. Synthetic scheme.

380
Bailly et al.
C26H24N5O6 (MϩH)ϩ, 502.1726, found, 502.1728. 1H NMR (400 verse transcriptase. The digestion products were separated on a 6%
MHz, DMSO-d6) spectrum of the free amine: 3.10 (1H, m), 3.57 (1H, polyacrylamide gel under native conditions in TBE-buffered solution
m), 3.83 (1H, d, J ϭ 10.9 Hz), 3.98 (1H, d, J ϭ 9.3 Hz), 4.12 (2H, d, (89 mM Tris-borate, pH 8.3, 1 mM EDTA). After autoradiography,
J ϭ 10.3 Hz), 5.04 (2H, s, NH2), 5.39 (1H, br s, OH), 5.56 (1H, br s, the band of DNA was excised, crushed, and soaked in water over-
OH), 6.11 (1H, br s, OH), 6.34 (1H, d, J ϭ 8.9 Hz), 7.42 (1H, t, J ϭ 7.8 night at 37°C. This suspension was filtered through a Millipore
Hz), 7.44 (1H, t, J ϭ 7.9 Hz), 7.61 (1H, t, J ϭ 7.9 Hz), 7.64 (1H, t, J ϭ 0.22-␮ filter, and the DNA was precipitated with ethanol. After
7.4 Hz), 7.74 (1H, d, J ϭ 7.9 Hz), 8.09 (1H, d, J ϭ 8.4 Hz), 9.15 (1H, washing with 70% ethanol and vacuum-drying the precipitate, the
d, J ϭ 7.9 Hz), 9.23 (1H, d, J ϭ 7.9 Hz), 11.68 (1H, s, Nindole-H). 13C labeled DNA was resuspended in 10 mM Tris, adjusted to pH 7.0,
NMR (100 MHz, DMSO-d6) spectrum of the hydrochloride: 57.9 containing 10 mM NaCl.
(C6Ј), 56.1, 67.5, 72.3, 79.0, 80.4 (C1Ј, C2Ј, C3Ј, C4Ј, C5Ј), 111.1, 112.5,
Footprinting Experiments. Cleavage reactions by DNase I
120.8, 121.4, 124.4, 124.5, 127.1, 127.4 (C tert arom), 116.8, 117.9, were performed essentially according to the previously detailed pro-
118.4, 119.3, 121.6, 121.7, 127.7, 129.3, 140.3, 141.4 (C quat arom), tocols (Bailly and Waring, 1995). Briefly, reactions were conducted in
168.5, 168.7 (CAO).
a total volume of 10 ␮l. Samples (3 ␮l) of the 32P-labeled DNA
Melting Temperature Studies. Melting curves were measured fragment were incubated with 5 ␮l of the buffer solution containing
using an Uvikon 943 spectrophotometer coupled to a Neslab RTE111 the desired drug concentration. After a 20-min incubation at 37°C to
cryostat. For each series of measurements, 12 samples were placed ensure equilibration of the binding reaction, the digestion was initi-
in a thermostatically controlled cell holder, and the quartz cuvettes ated by the addition of 2 ␮l of DNase I (0.01 U/ml enzyme in 20 mM
(10-mm path length) were heated by circulating water. The measure- NaCl, 2 mM MgCl2, 2 mM MnCl2, pH 7.3). At the end of the reaction
ments were performed in BPE buffer, pH 7.1 (6 mM Na2HPO4, 2 mM time (routinely 4 min at room temperature), the digestion was
NaH2PO4, 1 mM EDTA). The temperature inside the cuvette was stopped by freeze-drying. After lyophilization, each sample was re-
measured with a platinum probe; it was increased over the range suspended in 4 ␮l of an 80% formamide solution containing tracking
20–100°C with a heating rate of 1°C/min. The “melting” temperature dyes before electrophoresis.
(Tm) was taken as the midpoint of the hyperchromic transition.
Fluorescence Titration Experiments. The stock solutions of
Topoisomerase I Inhibition
Experiments with Linear Plasmid DNA on Agarose Gels.
compounds 1 and 2 were freshly prepared at a concentration of 2 mM pBR322 DNA (Boehringer Mannheim, Mannheim, Germany) was
in DMSO and diluted into buffer solution at the desired concentra- linearized with EcoRI and labeled with [␣-32P]dATP in the presence
tion. Calf thymus DNA was purchased from Pharmacia (lot 27-4562- of the Klenow fragment of DNA polymerase I. The labeled DNA was
02) and was sonicated and purified as described earlier (Chaires et then digested to completion with HindIII. The cleavage reaction
al., 1993). Before further use, the DNA was dialyzed in the appro- mixture contained 20 mM Tris-HCl, pH 7.4, 60 mM KCl, 0.5 mM
priate buffer for 24 h, and its concentration was determined by UV EDTA, 0.5 mM dithiothreitol, 2 ϫ 104 dpm of [␣-32P]pBR322 DNA,
absorption at 260 nm by using a molar extinction coefficient, ⑀260
ϭ
and the indicated drug concentrations. The reaction was initiated by
12,824 cmϪ1 MϪ1. Titration experiments were carried out in a buffer the addition of topoisomerase I (40 U in 20 ␮l of reaction volume) and
(BPE) consisting of 6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM allowed to proceed for 10 min at 37°C. Reactions were stopped by
Na2EDTA, pH 7.0, unless noted otherwise. Fluorescence titration adding SDS to a final concentration of 0.25% and proteinase K to 250
data were recorded at room temperature using an I.S.S. Greg 200 ␮g/ml, followed by incubation for 30 min at 50°C. Samples were
fluorometer. Excitation was at 320 nm, and fluorescence emission denatured by the addition of 10 ␮l of denaturing loading buffer
was monitored over the range of 340 to 620 nm. Samples used for consisting of 0.45 M NaOH, 30 mM EDTA, 15% (w/v) sucrose, and
titration experiments were prepared separately at a constant drug 0.1% bromocresol green before loading onto a 1% agarose gel in TBE
concentration of 5 ␮M and DNA concentrations ranging from 0.1-␮M buffer containing 0.1% SDS. Electrophoresis was conducted at 2
to 1 mM base pairs (bp).
V/cm for 18 h.
Fluorescence titration data were fit directly to get binding con-
Sequencing of Topoisomerase I-Mediated DNA Cleavage
stants using a fitting function incorporated into FitAll (MTR Soft- Sites. Each reaction mixture contained 2 ␮l of 3Ј 32P end-labeled
ware, Toronto, Canada). Simply, the observed fluorescence is as- DNA (ϳ1 ␮M), 5 ␮l of water, 2 ␮l of 10ϫ topoisomerase I buffer, and
sumed to be a sum of the weighted concentrations of free and bound
ligand:
10 ␮l of drug solution at the desired concentration (50 ␮g/ml). After
at least 30 min of incubation to ensure equilibration, the reaction
was initiated by addition of 10 U of topoisomerase I. Samples were
incubated for 40 min at 37°C before adding SDS to 0.25% and
proteinase K to 250 ␮g/ml to dissociate the drug-DNA-topoisomerase
I cleavable complexes. The DNA was precipitated with ethanol and
then resuspended in 5 ␮l of formamide-TBE loading buffer, dena-
tured at 90°C for 4 min, and then chilled in ice for 4 min before
loading onto the sequencing gel.
F ϭ F0͑Ct Ϫ Cb͒ ϩ FbCb
(1)
where F is the apparent fluorescence at each DNA concentration, F0
is the fluorescence intensity of free ligand, and Fb is the fluorescence
intensity of the bound species. For the interaction of a ligand D with
a DNA site S, it may be easily shown that:
Kx2 Ϫ x͑KS0 ϩ KD0 ϩ 1͒ ϩ KS0D0 ϭ 0
(2)
Growth Inhibition Assay. P388 murine leukemia cells were
incubated at 37°C for 96 h in the presence of various concentrations
of drug and evaluated for viability by neutral red staining as de-
scribed previously (Rodrigues-Pereira et al., 1996). The concentra-
tions of drugs giving 50% of growth inhibition (IC50) were deter-
mined.
where x ϭ Cb, K is the association constant, S0 is the total concen-
tration, and D0 is the total ligand concentration. Equation 2 is
readily solved using the quadratic formula. Data in the form of
fluorescence response F as a function of total DNA site concentration
at fixed concentration of ligand then may be fit by nonlinear least-
squares methods to get K, F0, and Fb.
DNA Purification and Labeling. The plasmid pBluescript
(pBS) (Stratagene, La Jolla, CA) was isolated from Escherichia coli
by a standard SDS-sodium hydroxide lysis procedure and purified
using Qiagen columns. The purified plasmid then was precipitated
and resuspended in appropriate buffered medium before digestion by
the restriction enzymes. The two pBS DNA fragments were prepared
by 3Ј 32P end-labeling of the EcoRI-PvuII double digest of the plas-
Results
Interaction with DNA. Initially, we used a thermal de-
naturation procedure to monitor the interaction of the drugs
with DNA. Both the hydroxyl derivative 1 and its amino
counterpart 2 stabilize duplex DNA against thermal dena-
turation. The effect is slightly more pronounced with com-
mid using [␣-32P]dATP and avian myeloblastosis virus (AMV) re- pound 2 than with 1. At a drug/DNA ratio of 1, the Tm of calf

An Amino Sugar Derivative of Rebeccamycin
381
thymus DNA was raised from 63.6–68.7°C with compound 2 1984). The slope observed for compound 2 is higher, as ex-
in BPE buffer (16 mM Naϩ). The stabilizing effect (⌬Tm) is pected. However, its value (Ϫ0.496) is less than the theoret-
2°C lower with compound 1 (3.3 for 1 versus 5.1° for 2). This ical value of Ϫ0.88 to Ϫ1.24 predicted for a ligand bearing
preliminary experiment suggests that the introduction of the one positively charged group (Record et al., 1978; Friedman
amino group enhances the interaction of 2 with DNA. Direct and Manning, 1984). The difference from the predicted value
fluorescence measurements fully confirmed this belief.
may signify that only a fraction of the drug molecules 2 are
We exploited the intrinsic fluorescence of the indolocarba- protonated (see Discussion). Nonetheless, these experiments
zole chromophore to evaluate the strength of the interaction confirm that introducing an amino group into the sugar res-
of the drugs with calf thymus DNA. The fluorescence emis- idue promotes the interaction of the drug with DNA.
sion at 560 nm is weak when the drug is free in solution but
The binding data presented above enable the calculation of
it is considerably enhanced when the drug is bound to DNA. the free energy of drug binding to DNA. ⌬Gobs was calculated
This property is very useful for accurate determination of the from the binding constants using the standard Gibbs relation
DNA-binding affinities. Nonlinear least-squares analysis of ⌬Gobs ϭ ϪRT ln K. The observed binding free energy can
the fluorescence titration curves shown in Fig. 4 yielded then be partitioned into its nonpolyelectrolyte contribution
binding constants of 3.6 ϫ 104 (M bp)Ϫ1 and 10.6 ϫ 104 (M (⌬Gt) and its polyelectrolyte contribution (⌬Gpe) (Chaires,
bp)Ϫ1 for compounds 1 and 2, respectively, in low-salt BPE 1996). The ⌬Gpe values reported in Table 1 indicate that both
buffer at neutral pH. There is no doubt that the OH3NH2 drugs 1 and 2 bind to DNA with a favorable polyelectrolyte
substitution significantly enhances the binding to DNA. The contribution, although the magnitude of the contribution is
affinity constant of compound 2 is about 3-fold higher than greater for 2, as expected. The values of ⌬Gt are the same for
that of compound 1.
1 and 2 and are the major contributors to ⌬Gobs. These values
The hydroxyl derivative 1 is uncharged whereas the amino indicated that the DNA binding of both 1 and 2 is stabilized
compound 2 is expected to be positively charged at neutral primarily by molecular interactions other than the polyelec-
pH. In this case it is important to determine the variation of trolyte effect, such as van der Waals interactions and, possi-
the binding constants as a function of the salt concentration. bly, hydrogen-bonding interactions. Dissection of the ob-
The charged compound must be more sensitive to the served binding free energy reveals that the greater affinity of
changes of the ionic strength than the neutral molecule 2 for DNA arises solely from the more favorable polyelectro-
(Record et al., 1978). We repeated the fluorescence titration lyte contribution resulting from the addition of the charged
experiments in the presence of increasing NaCl concentra- amine group. Binding studies also were done at pH 5.0 to test
tions. The data are presented in the form of double-logarith- whether more acidic conditions might more fully protonate
mic plots of log K versus log [NaCl] (Fig. 5). At pH 7.0, the compound 2 and increase its charge. That proved to be the
slope of the data for compound 1 is Ϫ0.26, i.e., very close to case. As Table 1 shows, the binding constant for 2 is higher at
the theoretical value of Ϫ0.24 predicted for the binding of an pH 5 than at pH 7. Figure 5 shows that the slope SK is
uncharged intercalator to DNA (Friedman and Manning, greater at pH 5 than at pH 7, consistent with the greater net
charge on the molecule resulting from full protonation.
Sequence-Selective Binding to DNA. DNase I foot-
printing experiments were performed using two restriction
fragments from the plasmid pBS: the 117-mer and 165-mer
EcoRI-PvuII fragments radiolabeled at the 3Ј end at the
EcoRI site. As shown in Fig. 6, the main footprint detected
with the 117-mer corresponds to a GC-rich sequence around
nucleotide position 70. The intensity of the footprint is more
Fig. 4. Fluorescence titrations for the interaction of compounds (B) 1 and
(E) 2 with calf thymus DNA. The normalized fluorescence response, ␪ ϭ
F Ϫ F0/Fb Ϫ F0, is shown as a function of total DNA concentration. The
concentration of ligand was kept constant at 5 ␮M whereas the DNA
concentration varied between 1 mM and 0.1 ␮M bp. Curve fitting and
determination of binding constants (Table 1) were carried out by using
nonlinear least-squares analysis, as described in the text. The solid lines
show the best-fit curves to the binding model described in the text.
Fig. 5. Variation of the binding constant (Ka) for the interaction of
compounds (E, Ⅺ) 1 and (F, f) 2 with calf thymus DNA as a function of
the salt concentration. The values for the slopes (Ϫ␦ log K/␦ log [Naϩ]) are
given in Table 1. F, E: pH 5.0; f, Ⅺ: pH 7.0.

382
Bailly et al.
pronounced with compound 2 than with compound 1. The
To confirm this observation, a few topoisomerase I cleavage
same observation was made at the binding site 5Ј-CCTCAC sites were sequenced using the restriction fragments used for
with the 265-bp fragment (not shown). The data are entirely the footprinting experiments. A typical example of a gel is
consistent with the results described recently in a detailed shown in Fig. 7. Little or no significant differences were
footprinting study with another rebeccamycin analog (Bailly observed between the two drugs. In both cases, the position
et al., 1998a). Binding occurs preferentially at sequences and the relative intensity of the cleavage sites are almost
containing GC or GT sites. The introduction of the amino identical. The main cleavage sites correspond to TpG steps,
group on the sugar residue enables tighter interactions at in agreement with the known specificity of rebeccamycin
GY-containing binding sites (YAT or C).
analog (Bailly et al., 1997). We concluded that the OH3NH2
Topoisomerase I Inhibition. The topoisomerase I inhib- substitution has little or no effect on the poisoning of topo-
itory properties of compounds 1 and 2 were examined using isomerase I. This observation is consistent with the model for
the 32P-labeled EcoRI-HindIII restriction fragment of the interaction of the drug with the enzyme-DNA complex
pBR322 as a substrate. The labeled DNA fragment was in- (see Discussion).
cubated with topoisomerase I in the presence and absence of
Cytotoxicity. The in vitro antiproliferative activity of
the indolocarbazoles at concentrations ranging from 0.01 to compounds 1 and 2 was examined using the P388 leukemia
10 ␮g/ml, and the resulting DNA cleavage products were cell line. The two drugs are more or less equally toxic, with
analyzed by agarose gel electrophoresis under alkaline con- IC50 values of 0.3 to 0.45 ␮g/ml (0.5–0.8 ␮M). The possibility
ditions. The two drugs stimulated DNA cleavage with the that the cytotoxicity of the drugs is attributable to their
same efficacy. In both cases, we determined a minimum action on topoisomerase I prompted us to evaluate their
inhibitory concentration of 0.1 ␮g/ml (0.18 Ϯ 1 ␮M, Table 2). toxicities toward P388CPT5 leukemia cells resistant to the
topoisomerase I inhibitor camptothecin (Table 2). The resis-
TABLE 1
tance of the P388CPT5 cell line has been attributed to the
expression of a deficient form of topoisomerase I as a result of
Binding constants and energetics of the drugs binding to DNA
pH 7.0
pH 5.0
a mutation in the top1 gene of these cells (Madelaine et al.,
1993). The two drugs are much more toxic against P388 cells
than to the resistant cells (Table 2), suggesting that the
toxicity is, at least partially, linked to topoisomerase I inhi-
bition. Identical resistance indexes (i.e., the ratio between
IC50 P388CPT5 and IC50 P388) were calculated for com-
pounds 1 and 2.
Compound
1
2
1
2
K/104 (MϪ1
)
3.6
6.1
0.26
0.6
5.5
10.6
6.7
2.8
6.0
0.29
0.7
5.3
31.8
7.4
Ϫ⌬Gobs (kcal/mol)
Ϫ␦ log K/␦ log[Naϩ]
Ϫ⌬Gpe (kcal/mol)
Ϫ⌬Gt (kcal/mol)
0.50
1.2
5.5
1.06
2.5
4.9
Binding constant (K) and standard free energy changes (⌬Gobs) refer to solution
conditions of 6 mM Na2HPO4, 2 mM NaH2PO4, and 1 mM Na2EDTA, pH 7.0, at
20°C. The polyelectrolyte contribution to the standard free energy change was
calculated from the relationship ⌬Gpe ϭ (SK) RT ln [NaCl], where SK ϭ ␦ log K/␦
log[Naϩ]. The thermodynamic free energy change was calculated by difference ⌬Gt ϭ
Discussion
⌬GobsϪ ⌬Gpe
.
Considerable interest has been devoted by our laboratories
to the study of correlations between the molecular structure
and biochemical and/or biological activities of indolocarba-
zole drugs to obtain information at the molecular level that
may be relevant to the proper design of chemotherapeutically
more effective drugs (Rodrigues-Pereira et al., 1996; Anizon
et al., 1997, 1998; Bailly et al., 1997, 1998a; Prudhomme,
1997; Moreau et al., 1998 ). The present approach has been
centered on the addition of an amine group at position 2Ј
(modifications at 3Ј and 4Ј positions currently are being in-
vestigated). All of the DNA-binding data indicate that the
replacement of the initial hydroxyl group with an amino
group enhances the interaction of the drug with the DNA
double helix. The affinity is increased by more than three
times, and the sequence specificity is more pronounced.
The thermodynamic data summarized in Table 1 show that
compound 2 binds to DNA with a free energy that is 0.6
kcal/mol more favorable than compound 1 at neutral pH. By
TABLE 2
Cytotoxicity and topoisomerase I inhibition properties
Fig. 6. DNase I footprinting with the 117-mer PvuII-EcoRI restriction
fragment of plasmid pBS in the presence of the two drugs at the indicated
concentration (␮M). The DNA was 3Ј end-labeled at the EcoRI site with
[␣-32P]dATP in the presence of AMV reverse transcriptase. The products
of nuclease digestion were resolved on an 8% polyacrylamide gel contain-
ing 7 M urea. Control tracks (Ct) contained no drug. Guanine-specific
sequence markers obtained by treatment of the DNA with dimethyl
sulfate followed by piperidine were run in the lane marked G. Numbers
on the right side of the gel refer to the standard numbering scheme for the
nucleotide sequence of the DNA fragment. The sequence of a preferential
binding site is indicated between the two panels.
Compound
Topo I,a MIC
P388,b IC50
P388/CPT5,b IC50
RIc
␮M
1
2
0.191
0.186
0.57
0.84
Ͼ25
Ͼ25
Ͼ30
Ͼ30
a Minimum inhibitory concentration producing a detectable cleavage of DNA by
topoisomerase I (Topo I).
b P388 and P388/CPT5 leukemia cells are sensitive and resistant to camptothecin,
respectively.
c The resistance index (RI) refers to the ratio of IC50P388/CPT5/IC50
.
P388

An Amino Sugar Derivative of Rebeccamycin
383
parsing the binding free energy into its polyelectrolyte and bazoles, the amine evidently does not participate in any
nonpolyelectrolyte components, the origin of the difference is additional molecular interactions to stabilize the complex.
revealed to be solely due to the ⌬Gpe term. The more favor- Perhaps, also, the orientation of the amine is not favorable to
able binding free energy results only from the introduction of enable the formation of hydrogen bonds with the DNA bases.
the charged amine group. That the ⌬Gt is the same for 1 and
It is of interest that the slope (Ϫ␦ log K/␦ log [Naϩ]) is less
2 signifies that all other molecular interactions for DNA for compound 2 than expected for a DNA-binding ligand with
binding are essentially the same for the two compounds and a single, positive charge. One possible explanation is that the
that introduction of the amine neither hinders nor helps amine compound is only partially charged at neutral pH, so
stabilize the complex in any other way apart from the poly- that the interaction with the negatively charged polymer is
electrolyte effect. These findings reinforce a simple design not as high as expected. This hypothesis seems plausible for
principle for DNA-binding agents. Binding affinity may be several reasons. With anthracyclines, the pKa of the amino
enhanced simply by the addition of a positively charged group of the daunosamine is 8.4 (Frezard and Garnier-Suil-
group, which need not participate in any specific molecular lerot, 1990) and, by analogy, we anticipated that the pKa of
interactions.
the 2Ј-amino function of the amino compound 2 would be
The results described here for the indolocarbazoles provide close to this value, or at least Ͼ8.0. However, preliminary UV
an interesting comparison with a pair of anthracycline anti- analysis suggests that the pKa is below 8.0, probably around
biotics, doxorubicin (Adriamycin) and hydroxyrubicin (3Ј- 7.8 (spectra not shown). It is therefore possible that the
deamino-doxorubicin) (Chaires et al., 1993, 1996). Doxorubi- amine is not entirely protonated at pH. 7.0. The results of the
cin contains an amine group (pKa ϭ 8.4) at the 3Ј position of experiments performed at pH 5.0 and the salt-dependence
the daunosamine moiety, whereas hydroxyrubicin has a hy- binding analysis also support this idea. According to the
droxyl group at that position. Doxorubicin is charged, theory of Record et al. (1978), the slope SK of the curve in Fig.
whereas hydroxyrubicin is uncharged. In that case, the ob- 5 is related to the charge (Z) on the ligand. The equation is:
served binding free energies were found to differ by more
͑␦log K/␦log͓Naϩ͔͒ ϭ ϪZ⌿ ϵ SK
(3)
than 2.5 kcal/mol, a much greater difference than observed
here for compounds 1 and 2. Both ⌬Gpe and ⌬Gt were found
to differ for doxorubicin compared with hydroxyrubicin, in
contrast to the behavior observed for compounds 1 and 2
reported here. For the anthracyclines, loss of the charged
amine resulted not only in a decrease in the polyelectrolyte
free energy contribution, but also in the loss of free energy
due to other types of molecular interactions. For the anthra-
cyclines, the 3Ј amine group lies deep in the minor groove and
has been observed in some crystal structures to participate in
hydrogen bond interactions with the DNA bases. Hydroxyru-
bicin would be unable to form such bonds. The interesting
contrast in the DNA-binding thermodynamics between the
anthracyclines and the indolocarbazole reflects the difference
in the ability of their respective amine groups to interact with
DNA. In both cases, the charged amine contributes to the
nonspecific polyelectrolyte free energy, but for the indolocar-
⌿ represents the average fraction of monovalent cation asso-
ciated with each phosphate group of DNA. For B-DNA, ⌿ ϭ
0.88, meaning that the double helix retains a net charge
corresponding to 12% of the total number of phosphates (see
Record et al., 1978 and Chaires, 1996 for detailed explana-
tions of the theory). Using eq. 3 we calculated that for the
amino compound, Z ϭ 0.56, suggesting that only about 50 to
60% of the molecules are positively charged under the exper-
imental conditions used in this study at neutral pH. Upon
reading carefully the literature on anthracycline derivatives,
we noted that the pKa of the amino sugar group can vary
significantly depending on the nature of the adjacent sub-
stituents. For example, the pKa of the amine of doxorubicin
drops from 8.4 to 6.4 when an iodine atom is introduced at
position 4Ј (Cera and Palumbo, 1991). The hypothesis that
the amino compound is partially charged at physiological pH
is valid and consistent with the modest dependence of K upon
Fig. 7. Sequencing of drug-induced topoisomerase I cleavage sites. The
265-mer EcoRI-AvaI fragment from plasmid pBS was 3Ј end-labeled at
the EcoRI site with [␣-32P]dATP in the presence of AMV reverse tran-
scriptase. The DNA was subjected to cleavage by human topoisomerase I
in the presence of the drugs (20–50 ␮M, as indicated). Cleavage products
were resolved on an 8% polyacrylamide gel containing 7 M urea. Gua-
nine-specific sequence markers obtained by treatment of the DNA with
dimethyl sulfate followed by piperidine were run in the lane marked G.
The control track (DNA) contained no drug and no enzyme. The lane Topo
I refers to the radiolabeled DNA substrate incubated with the enzyme but
with no drug. Camptothecin was used at 20 ␮M (lane Campto). The
positions of the three main topoisomerase I cleavage sites are indicated
by arrows.
Fig. 8. Putative functional domains of rebeccamycin drugs. The planar
indolocarbazole chromophore can intercalate into DNA whereas the car-
bohydrate residue contacts externally the DNA in the (minor) groove.
Previous studies suggested that the bis-imide F ring and its substituent
play a direct role in the interaction with topoisomerase I (Bailly et al.,
1997).

384
Bailly et al.
Riou JF, Bailly C, Fabro D, Meyer T and Aubertin AM (1998) Synthese, biochem-
ical and biological evaluation od staurosporine analogs from the microbial metab-
olite rebeccamycin. Bioorg Med Chem, 66:1597–1604.
ionic strength and increased affinity of the drug by a factor of
only 3.4. It is also consistent with the significant increase in
DNA-binding affinity under acidic conditions. This point de-
serves one further comment related to cell uptake. Addition
of the amino group increases considerably the hydrophilicity
of the molecule, thus facilitating the handling of the drug.
However, it does not much improve the toxicity, perhaps
because the cellular uptake is reduced. Once again it is
worthwhile to refer to studies with anthracyclines, which
have shown that, by the removal of the amino sugar group,
the drug becomes more lipophilic, favoring therefore the cel-
lular uptake by passive diffusion (Capranico et al., 1994).
That the pKa of the amino compound could be around 7.8
instead of being Ͼ8.0 might be of interest from a therapeutic
point of view. First, it may favor action on tumor cells rather
than normal cells. High lactate production is believed to
lower the pH of the cytoplasm of tumor cells compared with
normal cells (Di Marco et al., 1977). Second, drug-induced
apoptosis can decrease the pH of the treated cells. This effect
has been detected with the topoisomerase II inhibitor etopo-
side (Barry et al., 1993), and very recently we have seen the
same effect with topoisomerase I inhibitors such as campto-
thecin and rebeccamycin analogs (manuscript in prepara-
tion).
On the basis of previous studies using series of rebeccamy-
cin analogs with or without a glycosyl residue, and also from
a molecular modeling analysis, three functional domains of
rebeccamycin-type drugs can be identified (Fig. 8). As indi-
cated previously (Bailly et al., 1997), the insertion of the
planar indolocarbazole chromophore between two consecu-
tive base pairs places the appended sugar residue into the
groove of the double helix, most likely the minor groove. The
glycosyl residue can engage contacts with the base pair below
the intercalation site. In the minor groove orientation, the 2Ј
substituent may form an H bond with the carbonyl group at
position 2 of a pyrimidine when the drug intercalates at a
GpY site. In this case, it is plausible that, for steric reasons,
an amino group is more favorable than an OH group for the
formation of the H bond. According to this molecular ar-
rangement, the imide nitrogen on the F ring is supposed to
protrude toward the opposite groove, where it can interact
with topoisomerase I.
Arakawa H, Iguchi T, Morita M, Yoshinari T, Kojiri K, Suda H, Okura A and
Nishimura
S (1995) Novel indolocarbazole compound 6-N-formylamino-12,13-
dihydro-1,11-dihydroxy-13-(␤-D-glucopyranosyl)-5H-indolo[2,3-a]pyrrolo-[3,4-
c]carbazole-5,7-(6H)-dione (NB-506): Its potent antitumor activities in mice. Can-
cer Res 55:1316–1320.
Bailly C, Colson P, Houssier C, Rodrigues-Pereira E, Prudhomme M and Waring MJ
(1998a) Recognition of specific sequences in DNA by a topoisomerase I inhibitor
derived from the antitumor drug rebeccamycin. Mol Pharmacol 53:77–87.
Bailly C, Riou JF, Colson P, Houssier C, Rodrigues-Pereira E and Prudhomme M
(1997) DNA cleavage by topoisomerase I in the presence of indolocarbazole deriv-
atives of rebeccamycin. Biochemistry 36:3917–3929.
Bailly C, Suh D, Waring MJ and Chaires JB (1998b) Binding of daunomycin to
diaminopurine- and/or inosine-substituted DNA. Biochemistry 37:1033–1045.
Bailly C and Waring MJ (1995) Comparison of different footprinting methodologies
for detecting binding sites for a small ligand on DNA. J Biomol Struct Dyn
12:869–898.
Barry MA, Reynolds JE and Eastman A (1993) Etoposide-induced apoptosis in
human HL60 cells is associated with intracellular acidification. Cancer Res 53:
2349–2357.
Brenner M, Rexhausen H, Steffan B and Steiglich W (1988) Synthesis of arcyriaru-
bin B and related bisindolylmaleimides. Tetrahedron 44:2887–2892.
Bush JA, Long BH, Catino JJ, Bradner WT and Tomita K (1987) Production and
biological activity of rebeccamycin, a novel antitumor agent. J Antibiotics 40:668–
678.
Capranico G, Butelli E and Zunino F (1995) Change of the sequence specificity of
daunorubicin-stimulated topoisomerase II DNA cleavage by epimerization of the
amino group of the sugar moiety. Cancer Res 55:312–317.
Capranico G, Supino R, Binaschi M, Capolongo L, Grandi M, Suarato A and Zunino
F (1994) Influence of structural modifications at the 3Ј and 4Ј positions of doxo-
rubicin on the drug ability to trap topoisomerase II and to overcome multidrug
resistance. Mol Pharmacol 45:908–915.
Cera C and Palumbo M (1991) The peculiar binding properties of 4Ј-deoxy,4Ј-iodo-
doxorubicin to isolated DNA and 175 bp nucleosomes. Nucleic Acids Res 19:5707–
5711.
Chaires JB (1990) Daunomycin binding to DNA: From the macroscopic to the
microscopic, in Molecular Basis of Specificity in Nucleic Acid-Drug Interactions
(Pullman B and Jortner J eds) pp 225–245, Kluwer Academic Publishers, Neth-
erlands.
Chaires JB (1996) Dissecting the free energy of drug binding to DNA. Anticancer
Drug Des 11:569–580.
Chaires JB, Herrera JE and Waring MJ (1990) Preferential binding of daunomycin
to 5Ј(A/T)CG and 5Ј(A/T)GC sequences revealed by footprinting titration experi-
ments. Biochemistry 29:6145–6153.
Chaires JB, Leng F, Przewloka T, Fokt I, Ling Y-H, Perez-Soler R and Priebe W
(1997) Structure-based design of a new bisintercalating anthracycline antibiotic.
J Med Chem 40:261–266.
Chaires JB, Priebe W, Graves DE and Burke TG (1993) Dissection of the free energy
of anthracycline antibiotic binding to DNA: Electrostatic contributions. J Am
Chem Soc 115:5360–5364.
Chaires JB, Satyanarayana S, Suh D, Fokt I, Przewloka T and Priebe W (1996)
Parsing the free energy of anthracycline antibiotic binding to DNA. Biochemistry
35:2047–2053.
Chu CK, Ullas GV, Jeong LS, Ahn SK, Doboszewski B, Lin ZX, Beach JW and
Schinazi RF (1990) Synthesis and structure-activity relationships of 6-substituted
2Ј,3Ј-dideoxypurine nucleosides as potential anti-human immunodeficiency virus
agents. J Med Chem 33:1553–1561.
Di Marco A, Casazza AM, Dasdia T, Necco A, Pratesi G, Rivolta P, Velcich A, Zaccara
A and Zunino F (1977) Changes of activity of daunorubicin, adriamycin and
stereoisomers following the introduction or removal of hydroxyl groups in the
amino sugar moiety. Chem Biol Interact 19:291–302.
In conclusion, the present study indicates that the intro-
duction of an amino group on the glycosyl residue of rebec-
camycin contributes to a tighter interaction with DNA and
does not prevent the drug from inhibiting topoisomerase I. It
will be of interest to extend the carbohydrate domain to
further reinforce the interaction with DNA and possibly to
target longer sequences in DNA. By analogy with antitumor
drugs like calicheamycin, bleomycin, and anthracycline, an-
tibiotics bearing di- or trisaccharide side chains (e.g., beta-
clamycin A and ditrisarubicin B), we have initiated the syn-
thesis of novel rebeccamycin analogs equipped with amino-
oligosaccharide side chain.
Frederick CA, Williams LD, Ughetto G, van der Marel GA, van Boom JH, Rich A and
Wang AH-J (1990) Structural comparison of anticancer drug-DNA complexes:
Adriamycin and daunomycin. Biochemistry 29:2538–2549.
Frezard F and Garnier-Suillerot A (1990) Comparison of the binding of anthracycline
derivatives to purified DNA and to cell nuclei. Biochim Biophys Acta 1036:121–
127.
Friedman RA and Manning GS (1984) Polyelectrolyte effects on site-binding equi-
libria with application to the intercalation of drugs into DNA. Biopolymers 23:
2671–2714.
Ikemoto N, Kumar RA, Ling T-T, Ellestad GA, Danishefsky SJ and Patel DJ (1995)
Calicheamicin-DNA complexes: Warhead alignment and saccharide recognition of
the minor groove. Proc Natl Acad Sci USA 92:10506–10510.
Kumar RA, Ikemoto N and Patel DJ (1997a) Solution structure of the calicheamicin
␥
I-DNA complex. J Mol Biol 265:187–201.
1
Kumar RA, Ikemoto N and Patel DJ (1997b) Solution structure of the esperamicin
A1-DNA complex. J Mol Biol 265:173–186.
Lemieux RU, Takeda T and Chung BY (1976) Synthesis of 2-amido-2-deoxy-␤-D-
glucopyranosides. Synth Methods Carbohydrates 39:90–115.
References
Akinaga S, Gomi K, Morimoto M, Tamaoki T and Okabe M (1991) Antitumor activity
of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor
models. Cancer Res 51:4888–4892.
Madelaine J, Prost S, Naudin A, Riou G, Lavelle F and Riou JF (1993) Sequential
modifications of topoisomerase I activity in a camptothecin resistant cell line
established by progressive adaptation. Biochem Pharmacol 45:339–348.
Moreau P, Anizon F, Sancelme M, Prudhomme M, Bailly C, Carrasco C, Ollier M,
Seve`re D, Riou JF, Fabro D, Meyer T and Aubertin AM (1998) Syntheses and
biological evaluation of indolocarbazoles, analogues of rebeccamycin, modified at
the imide heterocycle. J Med Chem 41:1631–1640.
Anizon F, Belin L, Moreau P, Sancelme M, Voldoire A, Prudhomme M, Ollier M,
Seve`re D, Riou JF, Bailly C, Fabbro D and Meyer T (1997) Syntheses and biological
activities (topoisomerases inhibition, antitumoral and antimicrobial properties) of
rebeccamycin analogues bearing modified sugar moities and substituted on the
imide nitrogen with a methyl group. J Med Chem 40:3456–3465.
Anizon F, Moreau P, Sancelme M, Voldoire A, Prudhomme M, Ollier M, Seve´re D,
Myers AG, Kort ME and Hammond M (1997) A comparison of DNA cleavage by

An Amino Sugar Derivative of Rebeccamycin
385
neocarzinostatin chromophore and its aglycone: Evaluating the role of the carbo-
hydrate residue. J Am Chem Soc 119:2965–2972.
Serve`re D, Riou JF and Fabbro D (1996) Structure-activity relationships in a
series of substituted indolocarbazoles: Topoisomerase I and protein kinase C
inhibition and antitumoral and antimicrobial properties. J Med Chem 39:4471–
4477.
Rodrigues-Pereira E, Fabre S, Sancelme M, Prudhomme M and Rapp M (1995)
Antimicrobial activities of indolocarbazole and bis-indole protein kinase C inhib-
itors. II Substitution on maleimide nitrogen with functional groups bearing a
labile hydrogen. J Antibiot 48:863–868.
Shao R-G, Cao C-X, Shimizu T, O’Connor PM, Kohn KW and Pommier Y (1997)
Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity
by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influ-
enced by p53 function. Cancer Res 57:4029–4035.
Nettleton DE, Doyle TW, Krishnan B, Matsumoto GK and Clardy J (1985) Isolation
and structure of rebeccamycin—A new antitumor antibiotic from Nocardia aero-
colonigenes. Tetrahedron Lett 26:4011–4014.
Nicolaou KC, Tsay S-C, Suzuki T and Joyce GF (1992) DNA-carbohydrate interac-
I
tions. Specific binding of the calicheamicin ␥1 oligosaccharide with duplex DNA.
J Am Chem Soc 114:7555–7557.
Paloma LG, Smith JA, Chazin WJ and Nicolaou KC (1994) Interaction of calicheami-
cin with duplex DNA: Role of the oligosaccharide domain and identification of
multiple binding modes. J Am Chem Soc 116:3697–3708.
Prudhomme M (1997) Indolocarbazoles as anti-cancer agents. Curr Pharm Des
3:265–290.
Record MT, Anderson CF and Lohman TM (1978) Thermodynamic analysis of ion
effects on the binding and conformational equilibria of proteins and nucleic acids:
The role of ion association or release, screening, and ion effects on water activity.
Q Rev Biophys 11:103–178.
Send reprint requests to: Dr. Christian Bailly, IRCL, U-124 Institut Na-
tional de la Sante´ et de la Recherche Me´dicale, Place de Verdun, 59045 Lille,
France. E-mail: bailly@lille.inserm.fr
Rodrigues-Pereira E, Belin L, Sancelme M, Prudhomme M, Ollier M, Rapp M,