cryptolepsis, science article

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Nature Structural Biology 9, 57 - 60 (2001)

Published online: 3 December 2001; | doi:10.1038/nsb729 The antimalarial and

cytotoxic drug cryptolepine intercalates into DNA at cytosine-cytosine sites

N. Lisgarten1, 2, Miquel Coll1, Portugal1, Colin W. 3 & Aymami1,

4 1 Institut de Biologia Molecular de Barcelona, C.S.I.C., Jordi Girona 18,

08034 Barcelona, Spain.

2 Department of Crystallography, Birkbeck College, University of London, Malet

Street, London, WC1E 7HX, UK.

3 The School of Pharmacy, University of Bradford, West Yorkshire, BD7 4ER, UK.

4 Department d'Enginyeria Quimica, Universitat Politècnica de Catalunya,

Diagonal 647, 08028 Barcelona, Spain.

Correspondence should be addressed to Aymami aymami@...

Cryptolepine, a naturally occurring indoloquinoline alkaloid used as an

antimalarial drug in Central and Western Africa, has been found to bind to DNA

in a formerly unknown intercalation mode. Evidence from competition dialysis

assays demonstrates that cryptolepine is able to bind CG-rich sequences

containing nonalternating CC sites. Here we show that cryptolepine interacts

with the CC sites of the DNA fragment d(CCTAGG)2 in a base-stacking

intercalation mode. This is the first DNA intercalator complex, from 90 solved

by X-ray crystallography, to bind a nonalternating (pyrimidine-pyrimidine) DNA

sequence. The asymmetry of the drug induces a perfect stacking with the

asymmetric site, allowing for the stability of the complex in the absence of

hydrogen bonding interactions. The crystal structure of this antimalarial

drug & #8722;DNA complex provides evidence for the first nonalternating

intercalation and, as such, provides a basis for the design of new anticancer

or antimalarial drugs.

Malaria, by far the most important tropical parasite, causes an

estimated annual 2.7 million deaths among the 300 & #8722;500 million people

suffering from the disease per year. Africa accounts for over 90% of reported

cases, with an annual 20% increase of malaria-related illness and death. Malaria

is responsible for as many deaths per annum as AIDS for all of the last 15

years. Drug resistance to malaria has become one of the most significant threats

to human health and the search for new effective drugs is urgent. Although the

mechanism of action of the antimalarial drugs is unclear, many of these drugs,

such as chloroquine and quinacrine, are known to interact with DNA1.

Cryptolepine (5-methyl indolo[2,3b]-quinoline) is an indoloquinoline alkaloid

first isolated from the roots of Cryptolepsis triangularis collected in Kisantu

(Congo). Extracts of the roots of the related climbing liana Cryptolepsis

sanguinolenta, in which cryptolepine is the main alkaloid, have been used

clinically in Ghana for the treatment of malaria2, and also as a remedy against

colic and stomach ulcers. Cryptolepine itself has been found to produce a

variety of pharmacological effects, including hypotensive and antipyretic

properties, presynaptic -adrenoreceptor blocking action, antimuscarinic

properties, anti-inflammatory properties and antibacterial effects (for review

see ref. 3).

Cryptolepine has potent in vitro activity against the malaria parasite

(Plasmodium falciparum) and possesses cytotoxic activity, inhibiting DNA

synthesis in B16 melanoma cells3. The alkaloid was found to bind tightly to DNA

and behaved as a typical intercalating agent. The drug interacts preferentially

with CG-rich sequences and discriminates against homooligomeric runs of A and T.

The study3 also led to the discovery that cryptolepine is a potent topoisomerase

II inhibitor and a promising antitumor agent. Cryptolepine stabilizes

topoisomerase II & #8722;DNA covalent complexes and stimulates the cutting of DNA

at a subset of preexisting topoisomerase II cleavage sites3, 4. In addition,

evidence suggests that cryptolepine may inhibit the detoxification of heme

produced by malaria parasites in red blood cells as a result of the digestion of

hemoglobin, similarly to chloroquine and related 4-aminoquinoline

antimalarials5. Although the antimalarial activity of cryptolepine may

involve a chloroquine-like action, interactions with DNA may also contribute.

This is supported by a fluorescence microscopy study, which suggests that

cryptolepine accumulates into parasite structures that may correspond to the

parasite nucleus6. Cryptolepine was also localized into the kinetoplast DNA of

the trypanosome. Curiously, the DNA sequences in the minicircles of kinetoplast

contain a larger number of CC/GG sites (3:1) than CG/GC

(http://www.ebi.ac.uk:80/parasites/kDNA/Source.html). The intercalating

properties of cryptolepine were deemed worthy of investigation because this

compound may lead to new antiprotozoal and anticancer drugs.

DNA & #8722;drug interactions

Two main kinds of noncovalent DNA & #8722;drug interactions are known: base

intercalation7 and minor groove binding8. Minor groove binders that specifically

target DNA have been thoroughly investigated by Dervan9 using hairpin

iminopyridines, which allow a proper recognition of DNA sequences. Intercalators

are the group of compounds that bind between the bases of DNA, thereby

interrupting transcription, replication and/or topoisomerase activities10.

Although some intercalators have been used as anticancer drugs, others are

carcinogens. Bisintercalators11, trisintercalators12, tetraintercalators13 and

octakis-intercalators14, containing the same repeating intercalator group, have

been synthesized. Cryptolepine is the first intercalator that appears to prefer

or tolerate nonalternating steps (pyrimidine-pyrimidine).

Two main kinds of DNA & #8722;drug intercalation are observed. The first is

perpendicular intercalation, typified by doxorubicin and daunomycin7, in which

long fused-ring molecules penetrate perpendicularly to the base pair hydrogen

bonds. Parallel base-stacking intercalators, such as actinomycin15 and

acridine-type drugs16, which intercalate parallel to the base pair hydrogen

bonds and stack their aromatic rings into the DNA bases (Fig. 1), exemplify the

other type of intercalation. Perpendicular intercalators mainly go to CG and

other alternating pyrimidine-purine sites, such as TG or CA. Parallel

base-stacking intercalators can also go to nonalternating sequences. At present

there are 90 structures of nucleic acid & #8722;intercalator complexes in the

Nucleic Acid Database17 that have been shown to have alternating-base

intercalation sites, most being CG, a few GC and some TG.

Figure 1. Diagram showing the main intercalation modes.

The cryptolepine site does not have two-fold symmetry. Coordinates are taken

from the Nucleic Acid Data Base for a, the anthracycline type

d(CGCGCG) & #8722;epidoxorubicin (NDB code dd0022); b, the acridine type

d(CGTACG) & #8722;9-amino-DACA (NDB code dd0015); and c,

d(CCTAGG) & #8722;cryptolepine (this paper).

Full Figure and legend (25K) The NMR structure of two intercalators,

esperamicin A118 and calicheamicin 119, in complex with DNA indicate that they

intercalate a single aromatic ring at a CC site. However, the small size of the

intercalator and its two minor groove binding groups suggest that the sequence

specificity of the drug is favored by the complementarity of the fit between the

drug and the floor of the minor groove. These are minor groove binders, placing

a six-member aromatic ring between CC. The specificity is through the minor

groove and not through the intercalation. The crystal structure reported here

shows, for the first time, how cryptolepine interacts with the site

d(CpC)-d(GpG) in a base-stacking intercalation mode.

Cryptolepine binding to DNA in solution

The Ren and Chaires competition dialysis method20 was used to determine the

sequence selectivity of cryptolepine for different small DNA fragments. In order

to determine the preference of the ligand for base sequences of alternating and

nonalternating C-G (CC and CG, respectively), and alternating and nonalternating

A-T (AA and AT, respectively) base pairs, fragments of the same size were

selected. This ensured that the same number of possible similar sites were

available for ease of comparability.

The competition dialysis assay results indicated that cryptolepine prefers to

bind to C-G rich sequences of DNA, with a tendency for nonalternating CC (Fig.

2). The small differences between AA and AT are probably not significant.

Comparison of the binding affinity of cryptolepine with those of the ligands

used in the original Ren and Chaires experiment indicates that the binding

affinity of cryptolepine is similar to that of other intercalators, such as

actinomycin D, daunomycin, porphyrin compounds and chromomycin20. Previous

footprinting analyses3, 4 showed that tracts containing CC-GG, as well as

several CG-GC sites were protected from DNase I cleavage, which is consistent

with our binding data (Fig. 2).

Figure 2. Results obtained from the Ren and Chaires

competition dialysis experiment for cryptolepine. The amount of

ligand bound to each DNA fragment is shown graphically. The sites provided for

each fragment are shown in columns.

Full Figure and legend (38K) Complex structure

The complex d(CCTAGG) & #8722;cryptolepine has been crystallized, and its

structure solved and refined to 1.4 Å resolution (Table 1; Fig. 2c). The main

feature of this structure is the perfect fit of the drug sandwiched between two

consecutive C-G base pairs forming the first nonalternating site

(CC)-(GG) & #8722;cryptolepine (Figs 1,3). The aromatic six-membered ring of the

cryptolepine molecule stacks between the two cytosines, whereas the fused

aromatic, double six-membered ring portion of the molecule stacks between two

guanines (Fig. 3d). The five-membered ring, placed in the middle, gives

asymmetry to the cryptolepine molecule and separates both aromatic groups. The

positively charged N16 atom (quinoline group) between the two O6 atoms of

consecutive guanines in the major groove of DNA and the N8 (indole nitrogen)

between the O2 atoms of adjacent cytosines in the minor groove both enhance the

stability of the complex (Fig. 3c). This positively charged N16 nitrogen placed

in the major groove between oxygens is also observed in the structure of the

complex of 9-amino-DACA interacting with d(CGTACG) in the CG & #8722;drug site16.

In this case, the charged nitrogen is placed between two oxygens from guanines

in different strands; however, the charged nitrogen in the present structure is

placed between oxygens of adjacent guanines of the same strand. The cryptolepine

molecule is slightly bent, with a 6.8° angle between the two aromatic rings,

which is similar to the 4.8° angle found in the high resolution X-ray structure

of the cryptolepine tetraphenyl borate21. The presence of the five-membered ring

positioned between the two aromatic groups allows this bend.

Figure 3. Crystal structure of the complex.

a, Scheme for the DNA & #8722;cryptolepine complex. b, Stereo view of two

bis-intercalated d(CCTAGG)2 hexanucleotides in the ab-plane, with the

end-stacked ligand bound between them. Four asymmetric units are represented in

different colors. c, Stereo view of the 2Fo - Fc electron density map at the

area of the intercalated ligand, looking into the major groove. The map was

contoured at the 1.2 level. Stacking (large arrows) and electroctatic (small

arrows) interactions are shown. d, Stereo view of the projection down the helix

axis of a d(CpC)-d(GpG) dinucleotide with the sandwiched ligand.

Full Figure and legend (76K) Table 1. Crystallization

data and refinement statistics

Full Table Neocryptolepine, an isomer of cryptolepine found to a lesser

extent than cryptolepine in the plant extracts, shows a reduced affinity for

DNA22. In this isomer, the charged group N16-C18H3 of the quinoline moiety is

interchanged with the C6 from the indole (Fig. 1), with both nitrogens on the

same side of the molecule. This reduced DNA affinity can be understood in terms

of the reduced stability of the molecule within the complex. In the

neocryptolepine molecule, the perfect fit that exists in the cryptolepine

complex on both sides of the DNA in the major and minor grooves, where the

nitrogen atoms are placed between oxygens (Fig. 3c), is impossible because both

nitrogen atoms are on the same side. The cry ptolepine molecule has no

hydrogen-bonding contacts either with bases of the hexanucleotide or with

solvent. The absence of such interactions suggests that stacking forces alone

provide the stabilizing mechanism of the complex. The stacking interactions

between the intercalated ligand and the DNA bases (Fig. 3d) show that the

cryptolepine is aligned with its major axis parallel to the and Crick

hydrogen bonds of the base pairs. The positively charged cryptolepine

chromophore is nearly enveloped by the two base pairs at the intercalation site

and penetrates deeply into the helical stack, forming strong hydrophobic

interactions with the base pairs and positioning its center of mass as close to

the helix axis as possible, where the negative electrostatic potential of the

DNA is the greatest23. In this way, the chromophore comes to lie in a position

where both its hydrophobic and electrostatic interactions are maximized.

The analysis of solved DNA & #8722;drug complexes reveals the importance of

stacking forces. Calorimetric and spectroscopic studies of the compound Hoechst

33258 in complex with d(CGCAAATTTGCG)2 shows that hydrogen bonds contribute

little to the stability of the complex compared to hydrophobic forces24.

Crystal packing

The asymmetric unit contains one strand of DNA hexamer, one intercalated

cryptolepine molecule, 37 ordered water molecules and an additional cryptolepine

molecule located on the two-fold axis, sandwiched between the two DNA hexamers.

The crystallographic two-fold axis is coincident with the large axis of the drug

molecule. This additional drug molecule links contiguous DNA hexamers in the

crystal to form a continuous column of duplexes. Because the end-stacked ligand

lies on a two-fold axis coincident with its major axis, the polarity of the DNA

backbone reverses at this point, bringing the 5' termini of adjacent helices

into close juxtaposition, and the cytosine C1 oppose each other across the

stacked ligand (Fig. 3b). These DNA columns are perpendicular to the c-axis and

rotated with respect to the neighbors, introducing the phosphate backbone in the

minor groove of the neighboring DNA column.

DNA conformation

The DNA in the complex has a B-like conformation, with -Crick base

pairing. However, in accommodating the intercalated cryptolepine molecule, the

DNA assumes conformational parameters significantly different from average B-DNA

values. Nevertheless, the DNA structure is similar to other DNA complexes with

base stacking intercalators, such as proflavine25 and 9-amino-DACA16. In the

intercalation cavity, the bases are separated by 7 Å, which is much larger than

the 5 & #8722;6 Å observed in the case of anthracycline drugs daunomycin and

doxorubicin. In these cases, the drug intercalates perpendicular to the hydrogen

bonds of the base pairs, causing a buckle on base pairs of the DNA site (Fig.

1). In the present case, the major axis of the drug aligns parallel to the major

axis of the base pairs to maximally occupy the intercalation site. To achieve

this major opening of the bases, the sugar-phosphate backbone makes a coupled

rotation of the / main torsion angles at

cytosine C2, bringing the oxygen O2P into the major groove. On the opposite

chain, the same opening is achieved at guanine G6 by small variations in all the

torsion angles. The DNA puckering is generally C2'-endo, except for the first

cytidine, which is C3'-endo. At the intercalation sites, the sugars of cytidine

C1 and C2 adopt the conformation C3'-endo and C2'-endo, a configuration

frequently found in intercalator & #8722;dinucleotide monophosphate complexes16.

DNA helical twist at the intercalation site is 24°, being unwound by 12° with

respect to standard B-DNA. The adjacent CpT step is also unwound with a twist of

27°, whereas the central TpA step is overwound, with a twist of 51°. This is a

very large twist for B-DNA and leaves the base pairs with minimal overlap of

their aromatic rings.

Conclusions

The perpendicular intercalators (daunomycin and doxorubicin) prefer alternating

sites (CG), which allow them to be located at the center of mass of the DNA. In

contrast, the parallel base-stacking drugs may adapt more readily to the

nonalternating sites (CC), leading to better stacking interaction. In addition,

the asymmetry of the drug enhances its fitting with the target in the case of

nonalternating sites (CC)-(GG) because it allows a different stacking on one

side (CC) or the other (GG) (Fig. 1).

Methods

Ren and Chaires competition dialysis experiment for cryptolepine.

Different DNA fragments were dialyzed against a common ligand solution, and

their total concentration within each dialysis bag was determined

spectrophotometrically. The extinction coefficients for the DNA fragments used

in the experiment were obtained by the Boser approach26 and was found to be

28,600 M-1cm-1 at 369 nm for cryptolepine3. In the assay, the same procedures

and solutions were used as described in the original experiment20. A buffer

consisting of 6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM NaEDTA and 185 mM NaCl, pH 7.0,

was used. In this buffer, all DNA fragments, including d(CGCGCGCG), should be in

the B-DNA form. In the experiment, 0.5 ml Spectro/Por DispoDialyzer units with

membrane pores of 2,000 Da were used. A second experiment using a membrane pore

size of 1,000 Da was performed with similar results. The values shown are for

both experiments (mean standard deviation). During the experiment, all the DNA

fragments are in equilibrium with the same free ligand

concentration so that the amount of cryptolepine bound to each fragment is

proportional to the association constant for ligand binding.

Crystal structure resolution.

Crystals were grown by mixing 0.5 l of 5 mM cryptolepine hydrochloride and 0.5 l

of 3 mM d(CCTAGG) with 1.0 l of the crystalization solution containing 5 mM

magnesium acetate, 25 mM 2-(N-morpholino)propanesulfonic acid (MES), pH 6.5, and

1.25 M ammonium sulfate. Single crystals were flash-frozen in a stream of

evaporating liquid nitrogen at 120 K. Diffraction data were collected at EMBL

beamline BW7A (DESY, Hamburg). The structure was solved by molecular replacement

using DNA coordinates of the d(CGTACG) & #8722;9-amino-DACA structure, without the

drug as starting model, with AMoRe27. Refinement followed with CNS28, first as a

rigid body. The optimum orientation of the intercalated cryptolepine was

identified by placing the drug at each of the four possible positions, and

refining until the best fit and corresponding best R-factor (28.2%) and Rfree

(33.2%) were found. At this stage, an iterative refinement procedure was carried

out using SHELX-97 (ref. 29), interspersed with

inspection of electron density maps, water positioning and manual model

rebuilding with TURBO-FRODO30. For cryptolepine, bond lengths and bond angles

were refined to specified target values obtained from the cryptolepine structure

determined by et al.21 The DNA bases and the two fused ring system of the

cryptolepine were restrained to planar, whereas all other torsion angles

remained unrestrained. No hydrogen bond restraints were used (Table 1).

Coordinates.

The coordinates have been deposited in the Protein Data Bank (accession code

1K9G).

Top Received 14 June 2001; Accepted 24 October 2001;

Published online: 3 December 2001.

REFERENCES

Rivas, L., Murza, A.,