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The antimalarial and
people suffering from the disease per year. Africa accounts forover 90% of reported cases, with an annual 20% increase of cytotoxic drug cryptolepine malaria-related illness and death. Malaria is responsible for as
many deaths per annum as AIDS for all of the last 15 years. Drug intercalates into DNA at
resistance to malaria has become one of the most significantthreats to human health and the search for new effective drugs is urgent. Although the mechanism of action of the antimalarialdrugs is unclear, many of these drugs, such as chloroquine and John N. Lisgarten1,2, Miquel Coll1, Jose Portugal1, quinacrine, are known to interact with DNA1.
Colin W. Wright3 and Juan Aymami1,4 Cryptolepine (5-methyl indolo[2,3b]-quinoline) is an indolo- quinoline alkaloid first isolated from the roots of Cryptolepsis 1Institut de Biologia Molecular de Barcelona, C.S.I.C., Jordi Girona 18, triangularis collected in Kisantu (Congo). Extracts of the roots 08034 Barcelona, Spain. 2Department of Crystallography, Birkbeck College, of the related climbing liana Cryptolepsis sanguinolenta, in which University of London, Malet Street, London, WC1E 7HX, UK. 3The School of cryptolepine is the main alkaloid, have been used clinically in Pharmacy, University of Bradford, West Yorkshire, BD7 4ER, UK. Ghana for the treatment of malaria2, and also as a remedy 4Department d'Enginyeria Quimica, Universitat Politècnica de Catalunya, against colic and stomach ulcers. Cryptolepine itself has been Diagonal 647, 08028 Barcelona, Spain. found to produce a variety of pharmacological effects, includinghypotensive and antipyretic properties, presynaptic α-adreno- Published online: 3 December 2001, DOI: 10.1038/nsb729 receptor blocking action, antimuscarinic properties, anti-inflammatory properties and antibacterial effects (for review Cryptolepine, a naturally occurring indoloquinoline alka-
see ref. 3).
loid used as an antimalarial drug in Central and Western
Cryptolepine has potent in vitro activity against the malaria Africa, has been found to bind to DNA in a formerly
parasite (Plasmodium falciparum) and possesses cytotoxic activ- unknown intercalation mode. Evidence from competition
ity, inhibiting DNA synthesis in B16 melanoma cells3. The alka- dialysis assays demonstrates that cryptolepine is able to bind
lishing Gr
loid was found to bind tightly to DNA and behaved as a typical CG-rich sequences containing nonalternating CC sites. Here
intercalating agent. The drug interacts preferentially with CG- we show that cryptolepine interacts with the CC sites of the
rich sequences and discriminates against homooligomeric runs DNA fragment d(CCTAGG)2 in a base-stacking intercalation of A and T. The study3 also led to the discovery that cryptolepine
mode. This is the first DNA intercalator complex, from ∼90 is a potent topoisomerase II inhibitor and a promising anti-
solved by X-ray crystallography, to bind a nonalternating
tumor agent. Cryptolepine stabilizes topoisomerase II–DNA (pyrimidine-pyrimidine) DNA sequence. The asymmetry of
covalent complexes and stimulates the cutting of DNA at a sub- 2002 Nature Pub
the drug induces a perfect stacking with the asymmetric site,
set of preexisting topoisomerase II cleavage sites3,4. In addition, allowing for the stability of the complex in the absence of
evidence suggests that cryptolepine may inhibit the detoxifica- hydrogen bonding interactions. The crystal structure of this
tion of heme produced by malaria parasites in red blood cells as antimalarial drug–DNA complex provides evidence for the
a result of the digestion of hemoglobin, similarly to chloroquine first nonalternating intercalation and, as such, provides a
and related 4-aminoquinoline antimalarials5. Although the anti- basis for the design of new anticancer or antimalarial drugs.
malarial activity of cryptolepine may involve a chloroquine-like Malaria, by far the most important tropical parasite, causes an action, interactions with DNA may also contribute. This is sup- estimated annual 2.7 million deaths among the 300–500 million ported by a fluorescence microscopy study, which suggests that Fig. 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)–epidoxorubicin (NDB code dd0022); b, the acridine type d(CGTACG)–9-amino-DACA
(NDB code dd0015); and c, d(CCTAGG)–cryptolepine (this paper).
nature structural biology • volume 9 number 1 • january 2002 Fig. 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.
tolepine interacts with the site d(CpC)-d(GpG) in a base-stack-ing intercalation mode.
Cryptolepine binding to DNA in solution
The Ren and Chaires competition dialysis method20 was used todetermine the sequence selectivity of cryptolepine for differentsmall DNA fragments. In order to determine the preference ofthe ligand for base sequences of alternating and nonalternatingC-G (CC and CG, respectively), and alternating and nonalter- cryptolepine accumulates into parasite structures that may cor- nating A-T (AA and AT, respectively) base pairs, fragments of respond to the parasite nucleus6. Cryptolepine was also localized the same size were selected. This ensured that the same number into the kinetoplast DNA of the trypanosome. Curiously, the of possible similar sites were available for ease of comparability.
DNA sequences in the minicircles of kinetoplast contain a larger The competition dialysis assay results indicated that cryp- number of CC/GG sites (∼3:1) than CG/GC (http://www. tolepine prefers to bind to C-G rich sequences of DNA, with aebi.ac.uk:80/parasites/kDNA/Source.html). The intercalating tendency for nonalternating CC (Fig. 2). The small differences properties of cryptolepine were deemed worthy of investigation between AA and AT are probably not significant. Comparison of because this compound may lead to new antiprotozoal and anti- the binding affinity of cryptolepine with those of the ligands cancer drugs.
used in the original Ren and Chaires experiment indicates thatthe binding affinity of cryptolepine is similar to that of other lishing Gr
intercalators, such as actinomycin D, daunomycin, porphyrin Two main kinds of noncovalent DNA–drug interactions are compounds and chromomycin20. Previous footprinting analy- known: base intercalation7 and minor groove binding8. Minor ses3,4 showed that tracts containing CC-GG, as well as several groove binders that specifically target DNA have been thorough- CG-GC sites were protected from DNase I cleavage, which is ly investigated by Dervan9 using hairpin iminopyridines, which consistent with our binding data (Fig. 2).
allow a proper recognition of DNA sequences. Intercalators arethe group of compounds that bind between the bases of DNA, 2002 Nature Pub
thereby interrupting transcription, replication and/or topoiso- The complex d(CCTAGG)–cryptolepine has been crystallized, merase activities10. Although some intercalators have been used and its structure solved and refined to 1.4 Å resolution (Table 1; as anticancer drugs, others are carcinogens. Bisintercalators11, Fig. 2c). The main feature of this structure is the perfect fit of the trisintercalators12, tetraintercalators13 and octakis-intercala- drug sandwiched between two consecutive C-G base pairs form- tors14, containing the same repeating intercalator group, have ing the first nonalternating site (CC)-(GG)–cryptolepine been synthesized. Cryptolepine is the first intercalator that (Figs 1,3). The aromatic six-membered ring of the cryptolepine appears to prefer or tolerate nonalternating steps (pyrimidine- molecule stacks between the two cytosines, whereas the fused aromatic, double six-membered ring portion of the molecule Two main kinds of DNA–drug intercalation are observed. The stacks between two guanines (Fig. 3d). The five-membered ring, first is perpendicular intercalation, typified by doxorubicin and placed in the middle, gives asymmetry to the cryptolepine mole- daunomycin7, in which long fused-ring molecules penetrate per- cule and separates both aromatic groups. The positively charged pendicularly to the base pair hydrogen bonds. Parallel base- N16 atom (quinoline group) between the two O6 atoms of con- stacking intercalators, such as actinomycin15 and acridine-type secutive guanines in the major groove of DNA and the N8 drugs16, which intercalate parallel to the base pair hydrogen (indole nitrogen) between the O2 atoms of adjacent cytosines in bonds and stack their aromatic rings into the DNA bases (Fig. 1), the minor groove both enhance the stability of the complex exemplify the other type of intercalation. Perpendicular interca- (Fig. 3c). This positively charged N16 nitrogen placed in the lators mainly go to CG and other alternating pyrimidine-purine major groove between oxygens is also observed in the structure sites, such as TG or CA. Parallel base-stacking intercalators can of the complex of 9-amino-DACA interacting with d(CGTACG) also go to nonalternating sequences. At present there are ∼90 in the CG–drug site16. In this case, the charged nitrogen is placedstructures of nucleic acid–intercalator complexes in the Nucleic between two oxygens from guanines in different strands; howev- Acid Database17 that have been shown to have alternating-base er, the charged nitrogen in the present structure is placed intercalation sites, most being CG, a few GC and some TG.
between oxygens of adjacent guanines of the same strand. The The NMR structure of two intercalators, esperamicin A 18 cryptolepine molecule is slightly bent, with a 6.8° angle between calicheamicin γ 19 1 , in complex with DNA indicate that they the two aromatic rings, which is similar to the 4.8° angle found intercalate a single aromatic ring at a CC site. However, the small in the high resolution X-ray structure of the cryptolepine size of the intercalator and its two minor groove binding groups tetraphenyl borate21. The presence of the five-membered ring suggest that the sequence specificity of the drug is favored by the positioned between the two aromatic groups allows this bend.
complementarity of the fit between the drug and the floor of the Neocryptolepine, an isomer of cryptolepine found to a lesser minor groove. These are minor groove binders, placing a six- extent than cryptolepine in the plant extracts, shows a reduced member aromatic ring between CC. The specificity is through affinity for DNA22. In this isomer, the charged group the minor groove and not through the intercalation. The crystal N16-C18H3 of the quinoline moiety is interchanged with the C6 structure reported here shows, for the first time, how cryp- from the indole (Fig. 1), with both nitrogens on the same side of nature structural biology • volume 9 number 1 • january 2002

Fig. 3 Crystal structure of the complex. a
a, Scheme for the DNA–cryptolepine com-
plex. b, Stereo view of two bis-intercalat-
ed 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 den-
sity map at the area of the intercalated
ligand, looking into the major groove.
The map was contoured at the 1.2 σ 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.
the molecule. This reduced DNAaffinity can be understood in termsof the reduced stability of the mole-cule within the complex. In the neocryptolepine molecule, the per-fect fit that exists in the cryptolepine complex on both sides of the DNA inthe major and minor grooves, wherethe nitrogen atoms are placedbetween oxygens (Fig. 3c), is impos- lishing Gr
sible because both nitrogen atomsare on the same side. The cryp- tolepine molecule has no hydrogen-bonding contacts either with basesof the hexanucleotide or with sol-vent. The absence of such interac-tions suggests that stacking forces 2002 Nature Pub
alone provide the stabilizing mecha- nism of the complex. The stackinginteractions between the intercalated ligand and the DNA bases across the stacked ligand (Fig. 3b). These DNA columns are per- (Fig. 3d) show that the cryptolepine is aligned with its major axis pendicular to the c-axis and rotated with respect to the neigh- parallel to the Watson and Crick hydrogen bonds of the base bors, introducing the phosphate backbone in the minor groove pairs. The positively charged cryptolepine chromophore is near- of the neighboring DNA column.
ly enveloped by the two base pairs at the intercalation site andpenetrates deeply into the helical stack, forming strong hydrophobic interactions with the base pairs and positioning its The DNA in the complex has a B-like conformation, with center of mass as close to the helix axis as possible, where the Watson-Crick base pairing. However, in accommodating the negative electrostatic potential of the DNA is the greatest23. In intercalated cryptolepine molecule, the DNA assumes confor- this way, the chromophore comes to lie in a position where both mational parameters significantly different from average B-DNA its hydrophobic and electrostatic interactions are maximized.
values. Nevertheless, the DNA structure is similar to other DNA The analysis of solved DNA–drug complexes reveals the complexes with base stacking intercalators, such as proflavine25 importance of stacking forces. Calorimetric and spectroscopic and 9-amino-DACA16. In the intercalation cavity, the bases are studies of the compound Hoechst 33258 in complex with separated by 7 Å, which is much larger than the 5–6 Å observed d(CGCAAATTTGCG)2 shows that hydrogen bonds contribute in the case of anthracycline drugs daunomycin and doxorubicin.
little to the stability of the complex compared to hydrophobic In these cases, the drug intercalates perpendicular to the hydro- gen bonds of the base pairs, causing a buckle on base pairs of theDNA 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 The asymmetric unit contains one strand of DNA hexamer, one occupy the intercalation site. To achieve this major opening of intercalated cryptolepine molecule, 37 ordered water molecules the bases, the sugar-phosphate backbone makes a coupled rota- and an additional cryptolepine molecule located on the two-fold tion of the α/γ main torsion angles at cytosine C2, bringing the axis, sandwiched between the two DNA hexamers. The crystallo- oxygen O2P into the major groove. On the opposite chain, the graphic two-fold axis is coincident with the large axis of the drug same opening is achieved at guanine G6 by small variations in all molecule. This additional drug molecule links contiguous DNA the torsion angles. The DNA puckering is generally C2′-endo, hexamers in the crystal to form a continuous column of duplex- except for the first cytidine, which is C3′-endo. At the intercala- es. Because the end-stacked ligand lies on a two-fold axis coinci- tion sites, the sugars of cytidine C1 and C2 adopt the conforma- dent with its major axis, the polarity of the DNA backbone tion C3′-endo and C2′-endo, a configuration frequently found in reverses at this point, bringing the 5′ termini of adjacent helices intercalator–dinucleotide monophosphate complexes16. DNAinto close juxtaposition, and the cytosine C1 oppose each other helical twist at the intercalation site is 24°, being unwound by 12° nature structural biology • volume 9 number 1 • january 2002 Crystal structure resolution. Crystals were grown by mixing
Table 1 Crystallization data and refinement statistics
0.5 µl of 5 mM cryptolepine hydrochloride and 0.5 µl of 3 mM d(CCTAGG) with 1.0 µl of the crystalization solution containing Unit cell dimensions 5 mM magnesium acetate, 25 mM 2-(N-morpholino)propanesulfon- ic acid (MES), pH 6.5, and 1.25 M ammonium sulfate. Single crystalswere 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 replace- ment using DNA coordinates of the d(CGTACG)–9-amino-DACA structure, without the drug as starting model, with AMoRe27.
Refinement followed with CNS28, first as a rigid body. The optimum Number of unique data orientation of the intercalated cryptolepine was identified by plac-ing the drug at each of the four possible positions, and refining Completeness1 (%) 87.3 until the best fit and corresponding best R-factor (28.2%) and R (33.2%) were found. At this stage, an iterative refinement proce- <I / σ (I)>1 dure 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 Wright R.m.s. deviation from ideality et al.21 The DNA bases and the two fused ring system of the cryp- Bond length (Å) tolepine were restrained to planar, whereas all other torsion angles Bond angle distances (Å) remained unrestrained. No hydrogen bond restraints were used Coordinates. The coordinates have been deposited in the Protein
Data Bank (accession code 1K9G).
1Number in parenthesis is for the last shell (1.45–1.40 Å).
2R i – Ι/ ∑h where (Ι) is the mean intensity of reflection lishing Gr
Rfree is for 5% of total reflections.
This work was supported by grants from the Ministerio de Educacion y Determined without solvent.
Cultura of Spain and the Generalitat de Catalunya. J.N.L. acknowledges supportfrom MEyC of Spain. Synchrotron data collection was supported by the ESRF and with respect to standard B-DNA. The adjacent CpT step is also EU grants to the EMBL-DESY.
unwound with a twist of 27°, whereas the central TpA step isoverwound, with a twist of 51°. This is a very large twist for Correspondence should be addressed to J.A. email: [email protected] 2002 Nature Pub
B-DNA and leaves the base pairs with minimal overlap of their aromatic rings.
Received 14 June, 2001; accepted 24 October, 2001.
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nature structural biology • volume 9 number 1 • january 2002

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