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In vitro inhibition of β-haematin formation, DNA interactions, antiplasmodial activity, and cytotoxicity of synthetic neocryptolepine derivatives
Experimental Parasitology 108 (2004) 163–168 www.elsevier.com/locate/yexpr
In vitro inhibition of b-haematin formation, DNA interactions, antiplasmodial activity, and cytotoxicity of synthetic neocryptolepine derivatives Sabine Van Mierta,*, Tim Jonckersb, Kanyanga Cimangaa, Louis Maesc, Bert Maesb, Guy Lemie`reb, Roger Dommisseb, Arnold Vlietincka, Luc Pietersa a
Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium b Department of Chemistry, University of Antwerp, B-2020 Antwerp, Belgium c Department of Biomedical Sciences, University of Antwerp, B-2020 Antwerp, Belgium Received 14 May 2004; received in revised form 30 August 2004; accepted 30 August 2004
Abstract Neocryptolepine, a minor alkaloid of Cryptolepis sanguinolenta, was investigated as a lead for new antiplasmodial agents, because of its lower cytotoxicity than cryptolepine, the major alkaloid. Synthetic 2- or 3-substituted neocryptolepine derivatives were evaluated for their biological activity. In addition to the antiplasmodial activity (Plasmodium falciparum chloroquine-sensitive and -resistant) also the cytotoxicity (MRC-5 cells) was determined. Several compounds such as 2-bromoneocryptolepine showing higher and more selective antiplasmodial activity than neocryptolepine were obtained. Several functional assays and in vitro tests were used to obtain additional information on the mechanism of action, i.e., the b-haematin formation inhibitory assay (detoxification of haem) and the DNA–methylgreen displacement assay (interaction with DNA). It could be demonstrated that the 2- or 3-substituted neocryptolepine derivatives investigated here have about the same potency to inhibit the b-haematin formation as chloroquine, indicating that inhibition of haemozoin formation makes at least an important contribution to their antiplasmodial activity, although their in vitro antiplasmodial activity is still less than chloroquine. Ó 2004 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Malaria; Antimalarials; Plasmodium parasite; Protozoa; Chloroquine; Quinine; Emetine; Neocryptolepine; Neocryptolepine derivatives; Haemin; Haematin; Haemozoin; b-Haematin formation; DNA interaction; DNA–methylgreen; Antiplasmodial activity; Cytotoxicity; BHIA, b-haematin inhibitory activity; CQ, chloroquine; DNA, deoxyribonucleic acid; APAD, 3-acetyl pyridine nicotinamide adenine dinucleotide; LDH, lactate dehydrogenase; pLDH, parasite lactate dehydrogenase
1. Introduction The global impact of malaria, a parasitic disease, may not be underestimated. It is rated that there are between 200 and 300 million cases each year, more than 2 million of which with a fatal end. Four different Plasmodium species are responsible for human infection, P. vivax,
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Corresponding author. Fax: +32 3 820 27 09. E-mail address: [email protected] (S. Van Miert).
0014-4894/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2004.08.006
P. ovale, P. malariae, and the most severe one, P. falciparum. Nature can be considered as an important source of new leads for antimalarial agents (Kayser et al., 2002). Cryptolepine (9) and neocryptolepine (1), two alkaloids originally isolated from the African plant Cryptolepis sanguinolenta, showed antiplasmodial activity (Cimanga et al., 1997; Kirby et al., 1995). Cryptolepine (9), the major alkaloid, also demonstrated a cytotoxic, DNA-interacting, topoisomerase II inhibitory and DNA synthesis inhibitory activity (Bailly et al., 2000; Bonjean et al.,
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1998; Dassonneville et al., 2000). Other pharmacological effects have been reported for cryptolepine (9) as well, such as antimuscarinic, noradrenergic receptor antagonistic, antihypertensive, vasodilative, antithrombotic, antipyretic, hypothermic, and anti-inflammatory properties. Nevertheless, it has already been used as a lead compound because of its antiprotozoal activity (Arzel et al., 2001; Wright et al., 2001) or its antihyperglycaemic properties (Bierer et al., 1998). Because of its lower cytotoxicity we have selected neocryptolepine (1) as a promising compound for further research (Bailly et al., 2000). A series of substituted neocryptolepine (indolo[2,3-b]quinoline) derivatives including 2-bromo- (2), 2-chloro- (3), 2-fluoro- (4), 2-iodo- (5), 3-bromo- (6), 3-chloro- (7), and 3-trifluoromethylneocryptolepine (8) (Fig. 1) was prepared by organic synthesis and investigated for their biological activity in order to obtain compounds showing higher and more selective antiplasmodial activity (Jonckers et al., 2002). In the present work a more detailed investigation on the mode of action of these compounds is presented using two different functional assays related to possible mechanisms of action or cytotoxicity. The first one, the b-haematin formation inhibitory assay, comprises two essentially the same tests, one giving a more clear-cut result (yes-or-no result) (Egan et al., 1994; Wright et al., 2001), whereas the second one makes it possible to rank the products tested (Parapini et al., 2000). These assays are based on the fact that the Plasmodium parasite infects the host red blood cells, ingests and degrades the haemoglobin of these blood cells in an acidic food vacuole. The globin part is enzymatically degraded into amino acids. Haem is converted, at least in part, into insoluble and inert haemozoin (malaria pigment). If this process is inhibited by an antiplasmodial agent, free haem causes a membrane lysis followed by a release of proteolytic enzymes resulting in a lysis of the parasite. b-Haematin, (Fe(III)–protoporphyrin-IX)2 is chemically, spectroscopically, and crystallographically identical to haemoz-
oin (Pagola et al., 2000; Slater et al., 1991). Inhibition of b-haematin formation can be investigated by an infrared method, in which the presence or absence of the two typical peaks is observed. It has been proven that some antimalarials, e.g., quinoline antimalarials, are interfering with haemozoin formation and are blocking further haem dimerisation (Egan et al., 1996; Ridley et al., 1997). The second functional assay applied in this work was the interaction of the test compounds with DNA by means of the DNA–methylgreen displacement assay, a colorimetric microassay for the detection of agents that interact with DNA (Burres et al., 1992) which is a measure of the affinity of a test compound to the DNAstrand. Compounds interacting with DNA are able to displace methylgreen from a methylgreen–DNA complex, leading to a decoloration and a decrease in absorbance. This decrease represents the initial rapid displacement of methylgreen from DNA by the drug, followed by the slower reaction with water that yields the colourless carbinol. IC50 values obtained in this assay express the concentration of test compound leading to a 50% decrease of absorbance, corresponding to 50% displacement of the dye from the DNA complex. In addition to these functional assays, the test compounds were also evaluated for their antiplasmodial and cytotoxic activities, in order to establish correlations with possible modes of action.
2. Materials and methods 2.1. Reagents Haemin (ferriprotoporphyrin IX chloride) (Fluka; HPLC purity >98%), haematin (ferriprotoporphyrin IX hydroxide) (Sigma), DNA–methylgreen (Sigma), Malstat reagent (Flow, USA), chloroquine diphosphate (Sigma), quinine hydrochloride (Sigma), and emetine hydrochloride (Sigma). Neocryptolepine (indolo[2, 3-b]quinoline) (1), 2-bromo- (2), 2-chloro- (3), 2-fluoro(4), 2-iodo- (5), 3-bromo- (6), 3-chloro- (7), and 3-trifluoromethylneocryptolepine (8) (Fig. 1) were obtained by organic synthesis (Jonckers et al., 2002). Cryptolepine (9) (Fig. 2) was obtained by isolation from C. sanguinolenta (Cimanga et al., 1996). All compounds were evaluated as their hydrochloride salts. 2.2. Inhibition of b-haematin formation
Fig. 1. Neocryptolepine and synthetic derivatives.
An in vitro method to measure the inhibition of bhaematin formation, based on the original method described by Egan et al. (1994) and modified by Wright et al. (2001) was used. Briefly, 2.5 ml of 0.1 M NaOH was added to haemin (typically 7.5 mg) which forms haematin. Polymerisation to b-haematin, which has been characterised as [Fe(III)protoporphyrin IX]2, pro-
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quine (IC50 2.56 ± 0.31 Meq) and quinine (IC50 7.40 ± 0.72 Meq) were used as positive control, the IC50 representing the molar equivalents (Meq) of test compounds, relative to haemin, required to inhibit bhaematin formation by 50%. The data are expressed as means ± SD from three independent experiments. 2.3. DNA interactions
Fig. 2. Cryptolepine.
ceeds after addition of 2.3 ml of 9.9 M acetate buffer, pH 5. Test compounds (three equivalents with respect to haemin) were added to the haematin solutions prior to acidification. After incubation for 40 min at 60 °C, cooling and filtration, the precipitate was washed with water, dried, and FT-IR (Perkin–Elmer FT-IR 1760) was used to check the presence of b-haematin, which shows sharp bands at 1660 and 1207 cm 1. The spectra were examined on the presence or absence of the two typical peaks. When they are absent the compound has inhibited the formation of b-haematin. A sample containing haemin, without test compound, which was not incubated, was used as a negative control (no formation of b-haematin). A sample containing haemin but without test compound, which was incubated during 40 min at 60 °C, was used as a positive control (formation of b-haematin). Parapini et al. (2000) developed a quantitative microassay (b-haematin inhibitory activity assay (BHIA)) to investigate the ability of compounds to inhibit b-haematin formation. Briefly, 50 ll of an 8 mM solution of haemin dissolved in DMSO and 50 ll of the test compound in H2O (or mixture H2O/MeOH), in doses ranging from 1 to 20 molar equivalents to haemin, were distributed in 96-well U-bottomed microplates. In control cells, 50 ll H2O was added. By adding 100 ll of 8 M acetate buffer (pH 5) the b-haematin formation was initiated and the plates were incubated at 37 °C for 18 h. After centrifugation, the soluble fraction of unprecipitated material was collected. Two hundred microlitres of DMSO was added into the wells to resuspend the remaining pellet in order to remove unreacted haematin. The plates were centrifuged again, the DMSO-soluble fraction was collected and the residual pellet, consisting of pure precipitate of b-haematin, was dissolved in 200 ll of 0.1 M NaOH. In order to make the spectroscopic quantitation, 75 ll was transferred into a new plate and serial 4-fold dilutions in 0.1 M NaOH were made. The amount of haematin was determined by measuring the absorbance at 414 nm using a microtitre plate reader (Labsystems Multiscan MCC/340). To calculate the amount of porphyrin present in each fraction, a standard curve of haematin dissolved in 0.1 M NaOH was used. Chloro-
Agents that displace methylgreen from a DNA–methylgreen complex were detected spectrophotometrically (Labsystems Multiscan MCC/340) by a decrease in absorbance at 620 nm (Burres et al., 1992). Twenty milligrams of DNA–methylgreen was suspended in 100 ml of 0.05 M Tris–HCl buffer, pH 7.5, containing 7.5 mM MgSO4 and stirred at 37 °C for 24 h. The dissolved samples were dispensed into wells of a 96-well microtitre plate. Solvent was removed under vacuum, and 200 ll of the DNA–methylgreen solution was added. The initial absorbance was compared with the final absorbance (after 24 h) in order to calculate the IC50 value (50% displacement of methylgreen from DNA). Compounds interacting with DNA are able to displace methylgreen from a methylgreen–DNA complex, leading to a loss of colour and a decrease in absorbance. IC50 values represent the concentration of test compound leading to a 50% decrease of absorbance, corresponding to 50% displacement of dye from the DNA complex. Determination of the IC50 values was performed from three different experiments in triplicate (means ± SD). Emetine and quinine were used as negative control and cryptolepine (9) (IC50 65.7 ± 3.0 lM) as positive control. 2.4. Antiplasmodial activity: lactate dehydrogenase assay This antiplasmodial assay is based upon the observation that Plasmodium falciparum contains a species-specific lactate dehydrogenase (LDH) that can use 3-acetyl pyridine NAD (APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate (Makler and Hinrichs, 1993a; Makler et al., 1993b). For all test compounds 20 mM stock solutions were prepared and 2fold serial dilutions were made in 96-well multiwell plates. Of this prediluted test compounds 10 ll was transferred into the test plates containing P. falciparum (chloroquine-sensitive Ghana strain, or chloroquine-resistant W2 strain) cultures (1% parasitaemia, 2% HCT). After 48 h at 37 °C and in an atmosphere of 93% N2, 4% CO2, and 3% O2, the test plates were frozen at 20 °C to provoke haemolysis of the erythrocytes, which was needed to evaluate parasite growth, and to store the plates until further processing. After thawing, 20 ll of the lysed culture was added to 100 ll Malstat reagent and the formation of APADH was determined. Adding 40 lg nitroblue tetrazolium (NBT) and 2 lg phenazine ethosulphate (PES) to the Malstat reagent promoted
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the spectrophotometric assessment of LDH activity. As APADH is formed, the NBT is reduced and forms a blue formazan product that can be detected visually and measured spectrophotometrically at 650 nm. Determination of the IC50 values was performed in triplicate (means ± SD). Chloroquine was used as positive control, its IC50 values being 0.01 lM (Ghana) and 0.09 lM (W2). 2.5. Cytotoxicity A human diploid embryonic lung cell line (MRC-5) was used to assess the cytotoxicity of the test compounds as described before (Girault et al., 2000). Briefly, MRC-5 cells were seeded at 5000 cells/well in 96-well microtitre plates. After 24 h, the cells were washed and 2-fold dilutions of the drug were added in 200 ll of standard culture medium (RPMI + 5% FCS). The final DMSO concentration in the culture remained below 0.5%. The cultures were incubated with different concentrations of test compounds at 37 °C in 5% CO2–95% air for 7 days. Untreated cultures were included as controls. Cytotoxicity was determined using the colorimetric MTT assay and scored as a percent (%) reduction of absorption at 540 nm of treated cultures versus untreated control cultures. Determination of the IC50 values was performed in triplicate (means ± SD). Vinblastine was used as positive control (IC50 < 10 nm).
3. Results and discussion 3.1. In vitro antiplasmodial activity and inhibition of b-haematin formation All synthesised compounds (2–8), cryptolepine (9) and neocryptolepine (1), the original lead, were evalu-
ated for in vitro antiplasmodial activity against a chloroquine-sensitive (Ghana) and a chloroquine-resistant (W2) P. falciparum strain. In case of the chloroquinesensitive strain, all compounds tested showed an activity comparable with neocryptolepine (1) (IC50 27.3 lM), except for 2-bromoneocryptolepine (2), which was four times more active (IC50 6.0 lM). For the chloroquine-resistant strain, all derivatives, except 3-trifluoromethylneocryptolepine (8), had an activity higher than neocryptolepine (1) (IC50 14.0 lM), and also here 2bromoneocryptolepine (2) (IC50 4.0 lM) was the most potent compound (see Table 1). In search for the mechanism of action, it was shown that cryptolepine (9), similarly to chloroquine, was able to inhibit the formation of b-haematin in vitro, and it was proposed that this mechanism was likely to be responsible, at least in part, for its antiplasmodial activity (Wright et al., 1997, 2001). Taking this into account, all compounds synthesised were evaluated for their inhibiting effect on the dimerisation of haematin to bhaematin in two cell free systems, where the first one was giving a more clear-cut result (yes/no), whereas the second made it possible to rank the products tested. Neocryptolepine (1) was able to inhibit the b-haematin formation, and also its derivatives showed this activity. Using the quantitative BHIA it was possible to rank the compounds by comparing their IC50 values (representing the molar equivalents of test compounds, relative to haemin, required to inhibit b-haematin formation by 50%). All derivatives showed a higher activity than neocryptolepine (1) (IC50 5.97 ± 0.22 Meq), the original lead. 2-Bromo- (2) (IC50 1.77 ± 0.15 Meq), 2-chloro- (3) (IC50 1.76 ± 0.36 Meq), and 2fluoroneocryptolepine (4) (IC50 1.95 ± 0.27 Meq) were the most potent products, all with a comparable activity. Also cryptolepine (9) showed a comparable activity in
Table 1 In vitro antiplasmodial activity, inhibition of b-haematin formation, DNA interaction (DNA–methylgreen assay), and in vitro cytotoxicity of synthetic neocryptolepine derivatives
1 2 3 4 5 6 7 8 9 Chloroquine Quinine Emetine a b
P. falciparum (CQ sensitive) (Ghana strain) IC50 (lM)
P. falciparum (CQ resistant) (W2 strain) IC50 (lM)
Yes/no
BHIA assay IC50 (Meq)a
DNA interaction IC50 (lM)
Cytotoxicity (MRC-5 cells) IC50 (lM)
27.3 ± 5.7 6.0 ± 6.1 21.0 ± 8.9 19.3 ± 3.8 17.7 ± 5.1 30.0 ± 3.5 21.7 ± 4.0 16.3 ± 0.6 2.3 ± 0.6 0.01 n.t. n.t
14.0 ± 1.7 4.0 ± 0.1 5.0 ± 0.1 4.7 ± 0.6 6.3 ± 0.6 4.7 ± 0.6 4.7 ± 0.6 15.7 ± 1.5 2.0 ± 0.1 0.09 n.t. n.t.
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes n.t.
5.97 ± 0.22 1.77 ± 0.15 1.76 ± 0.36 1.95 ± 0.27 3.32 ± 0.19 2.56 ± 0.18 2.35 ± 0.32 3.01 ± 0.60 1.72 ± 0.36 2.56 ± 0.31 7.40 ± 0.72 n.t.
92.8 ± 9.7 >400 >500 >500 >400 >600 >700 >500 65.7 ± 3.0 n.d.b n.d. n.d.
11.0 ± 1.4 >32 16.5 ± 0.7 15.0 ± 0.1 16.0 ± 0.1 18.5 ± 0.7 15.5 ± 0.7 18.0 ± 0.1 1.5 ± 0.7 n.t. n.t. n.t.
Inhibition of b-haematin formation
IC50 represents the molar equivalents of test compounds, relative to haemin, required to inhibit b-haematin formation by 50%. Increase of absorbance due to the interaction of chloroquine with the dye; n.d. no displacement; n.t. not tested.
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this assay (1.72 ± 0.36 Meq). The observed values are in the same range as measured for known antimalarials reported to have the same mechanism of action, such as chloroquine or quinine. This gives additional evidence for the hypothesis that the haem detoxification process is at least one of the targets of the antiplasmodial activity of the cryptolepine and neocryptolepine derivatives.
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that inhibition of haemozoin formation makes at least an important contribution to its antiplasmodial activity, although its in vitro antiplasmodial activity is still less than chloroquine. Therefore, 2-bromoneocryptolepine (2) was considered as the most promising lead for further investigations, in order to develop more active and more selective antiplasmodial agents.
3.2. Cytotoxicity and DNA interaction Acknowledgments For all compounds the cytotoxicity (MRC-5 cells) was measured and all products were evaluated in a functional assay related to a possible mechanism of action, the DNA–methylgreen assay. For cryptolepine (9) it has been reported that intercalation into DNA, like 9aminoacridine, may contribute, at least in part, to the antiplasmodial activity, in addition to inhibition of haem detoxification as discussed above (Kirby et al., 1995). DNA interactions were also responsible, at least in part, for the cytotoxicity of cryptolepine (9) (Bonjean et al., 1996, 1998). Results obtained for the cytotoxicity of the test compounds showed that all synthetic derivatives were less cytotoxic than the original lead neocryptolepine (1) (IC50 11.0 ± 1.4 lM), but that 2-bromoneocryptolepine (2) showed the lowest cytotoxicity (IC50 > 32 lM). In the DNA–methylgreen assay only neocryptolepine (1) (IC50 92.8 ± 9.7 lM) and cryptolepine (9) (IC50 65.7 ± 3.0 lM) showed an interaction with DNA, but none of the synthetic derivatives. Nevertheless, the other neocryptolepine derivatives tested showed some cytotoxicity in the absence of DNA interaction in the concentration range tested, thus their cytotoxic effect may be due to a different mechanism.
4. Conclusion Neocryptolepine (1), which showed a cytotoxic effect as well as DNA interaction in the DNA–methylgreen assay, also showed an antiplasmodial effect as well as an inhibition of b-haematin formation. Its antiplasmodial activity could, at least in part, be due to a selective mechanism, namely inhibition of the haem detoxification process, and a non-selective mechanism, namely DNA interaction. Some new 2- or 3-substituted neocryptolepine (indolo[2,3-b]quinoline) derivatives were synthesised with a more selective antiplasmodial activity than neocryptolepine (1), the original lead. 2-Bromoneocryptolepine (2) was the most selective compound with an IC50 value against chloroquine-resistant P. falciparum of 4.0 lM, in the absence of obvious cytotoxicity (IC50 > 32 lM). Since 2-bromoneocryptolepine (2) was more active as an inhibitor of b-haematin formation in vitro than chloroquine, used as a positive control, it can be concluded
This work was supported in part by the Special Research Fund of the University of Antwerp (Concerted Research Project No. 99/3/34, fellowship for S. Van Miert). Dr. Colin Wright (University of Bradford, UK) is kindly acknowledged for assistance with the assay on inhibition of b-haematin formation.
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In vitro inhibition of b-haematin formation, DNA interactions, antiplasmodial activity, and cytotoxicity of synthetic neocryptolepine derivatives Sabine Van Mierta,*, Tim Jonckersb, Kanyanga Cimangaa, Louis Maesc, Bert Maesb, Guy Lemie`reb, Roger Dommisseb, Arnold Vlietincka, Luc Pietersa a
Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium b Department of Chemistry, University of Antwerp, B-2020 Antwerp, Belgium c Department of Biomedical Sciences, University of Antwerp, B-2020 Antwerp, Belgium Received 14 May 2004; received in revised form 30 August 2004; accepted 30 August 2004
Abstract Neocryptolepine, a minor alkaloid of Cryptolepis sanguinolenta, was investigated as a lead for new antiplasmodial agents, because of its lower cytotoxicity than cryptolepine, the major alkaloid. Synthetic 2- or 3-substituted neocryptolepine derivatives were evaluated for their biological activity. In addition to the antiplasmodial activity (Plasmodium falciparum chloroquine-sensitive and -resistant) also the cytotoxicity (MRC-5 cells) was determined. Several compounds such as 2-bromoneocryptolepine showing higher and more selective antiplasmodial activity than neocryptolepine were obtained. Several functional assays and in vitro tests were used to obtain additional information on the mechanism of action, i.e., the b-haematin formation inhibitory assay (detoxification of haem) and the DNA–methylgreen displacement assay (interaction with DNA). It could be demonstrated that the 2- or 3-substituted neocryptolepine derivatives investigated here have about the same potency to inhibit the b-haematin formation as chloroquine, indicating that inhibition of haemozoin formation makes at least an important contribution to their antiplasmodial activity, although their in vitro antiplasmodial activity is still less than chloroquine. Ó 2004 Elsevier Inc. All rights reserved. Index Descriptors and Abbreviations: Malaria; Antimalarials; Plasmodium parasite; Protozoa; Chloroquine; Quinine; Emetine; Neocryptolepine; Neocryptolepine derivatives; Haemin; Haematin; Haemozoin; b-Haematin formation; DNA interaction; DNA–methylgreen; Antiplasmodial activity; Cytotoxicity; BHIA, b-haematin inhibitory activity; CQ, chloroquine; DNA, deoxyribonucleic acid; APAD, 3-acetyl pyridine nicotinamide adenine dinucleotide; LDH, lactate dehydrogenase; pLDH, parasite lactate dehydrogenase
1. Introduction The global impact of malaria, a parasitic disease, may not be underestimated. It is rated that there are between 200 and 300 million cases each year, more than 2 million of which with a fatal end. Four different Plasmodium species are responsible for human infection, P. vivax,
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Corresponding author. Fax: +32 3 820 27 09. E-mail address: [email protected] (S. Van Miert).
0014-4894/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2004.08.006
P. ovale, P. malariae, and the most severe one, P. falciparum. Nature can be considered as an important source of new leads for antimalarial agents (Kayser et al., 2002). Cryptolepine (9) and neocryptolepine (1), two alkaloids originally isolated from the African plant Cryptolepis sanguinolenta, showed antiplasmodial activity (Cimanga et al., 1997; Kirby et al., 1995). Cryptolepine (9), the major alkaloid, also demonstrated a cytotoxic, DNA-interacting, topoisomerase II inhibitory and DNA synthesis inhibitory activity (Bailly et al., 2000; Bonjean et al.,
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1998; Dassonneville et al., 2000). Other pharmacological effects have been reported for cryptolepine (9) as well, such as antimuscarinic, noradrenergic receptor antagonistic, antihypertensive, vasodilative, antithrombotic, antipyretic, hypothermic, and anti-inflammatory properties. Nevertheless, it has already been used as a lead compound because of its antiprotozoal activity (Arzel et al., 2001; Wright et al., 2001) or its antihyperglycaemic properties (Bierer et al., 1998). Because of its lower cytotoxicity we have selected neocryptolepine (1) as a promising compound for further research (Bailly et al., 2000). A series of substituted neocryptolepine (indolo[2,3-b]quinoline) derivatives including 2-bromo- (2), 2-chloro- (3), 2-fluoro- (4), 2-iodo- (5), 3-bromo- (6), 3-chloro- (7), and 3-trifluoromethylneocryptolepine (8) (Fig. 1) was prepared by organic synthesis and investigated for their biological activity in order to obtain compounds showing higher and more selective antiplasmodial activity (Jonckers et al., 2002). In the present work a more detailed investigation on the mode of action of these compounds is presented using two different functional assays related to possible mechanisms of action or cytotoxicity. The first one, the b-haematin formation inhibitory assay, comprises two essentially the same tests, one giving a more clear-cut result (yes-or-no result) (Egan et al., 1994; Wright et al., 2001), whereas the second one makes it possible to rank the products tested (Parapini et al., 2000). These assays are based on the fact that the Plasmodium parasite infects the host red blood cells, ingests and degrades the haemoglobin of these blood cells in an acidic food vacuole. The globin part is enzymatically degraded into amino acids. Haem is converted, at least in part, into insoluble and inert haemozoin (malaria pigment). If this process is inhibited by an antiplasmodial agent, free haem causes a membrane lysis followed by a release of proteolytic enzymes resulting in a lysis of the parasite. b-Haematin, (Fe(III)–protoporphyrin-IX)2 is chemically, spectroscopically, and crystallographically identical to haemoz-
oin (Pagola et al., 2000; Slater et al., 1991). Inhibition of b-haematin formation can be investigated by an infrared method, in which the presence or absence of the two typical peaks is observed. It has been proven that some antimalarials, e.g., quinoline antimalarials, are interfering with haemozoin formation and are blocking further haem dimerisation (Egan et al., 1996; Ridley et al., 1997). The second functional assay applied in this work was the interaction of the test compounds with DNA by means of the DNA–methylgreen displacement assay, a colorimetric microassay for the detection of agents that interact with DNA (Burres et al., 1992) which is a measure of the affinity of a test compound to the DNAstrand. Compounds interacting with DNA are able to displace methylgreen from a methylgreen–DNA complex, leading to a decoloration and a decrease in absorbance. This decrease represents the initial rapid displacement of methylgreen from DNA by the drug, followed by the slower reaction with water that yields the colourless carbinol. IC50 values obtained in this assay express the concentration of test compound leading to a 50% decrease of absorbance, corresponding to 50% displacement of the dye from the DNA complex. In addition to these functional assays, the test compounds were also evaluated for their antiplasmodial and cytotoxic activities, in order to establish correlations with possible modes of action.
2. Materials and methods 2.1. Reagents Haemin (ferriprotoporphyrin IX chloride) (Fluka; HPLC purity >98%), haematin (ferriprotoporphyrin IX hydroxide) (Sigma), DNA–methylgreen (Sigma), Malstat reagent (Flow, USA), chloroquine diphosphate (Sigma), quinine hydrochloride (Sigma), and emetine hydrochloride (Sigma). Neocryptolepine (indolo[2, 3-b]quinoline) (1), 2-bromo- (2), 2-chloro- (3), 2-fluoro(4), 2-iodo- (5), 3-bromo- (6), 3-chloro- (7), and 3-trifluoromethylneocryptolepine (8) (Fig. 1) were obtained by organic synthesis (Jonckers et al., 2002). Cryptolepine (9) (Fig. 2) was obtained by isolation from C. sanguinolenta (Cimanga et al., 1996). All compounds were evaluated as their hydrochloride salts. 2.2. Inhibition of b-haematin formation
Fig. 1. Neocryptolepine and synthetic derivatives.
An in vitro method to measure the inhibition of bhaematin formation, based on the original method described by Egan et al. (1994) and modified by Wright et al. (2001) was used. Briefly, 2.5 ml of 0.1 M NaOH was added to haemin (typically 7.5 mg) which forms haematin. Polymerisation to b-haematin, which has been characterised as [Fe(III)protoporphyrin IX]2, pro-
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quine (IC50 2.56 ± 0.31 Meq) and quinine (IC50 7.40 ± 0.72 Meq) were used as positive control, the IC50 representing the molar equivalents (Meq) of test compounds, relative to haemin, required to inhibit bhaematin formation by 50%. The data are expressed as means ± SD from three independent experiments. 2.3. DNA interactions
Fig. 2. Cryptolepine.
ceeds after addition of 2.3 ml of 9.9 M acetate buffer, pH 5. Test compounds (three equivalents with respect to haemin) were added to the haematin solutions prior to acidification. After incubation for 40 min at 60 °C, cooling and filtration, the precipitate was washed with water, dried, and FT-IR (Perkin–Elmer FT-IR 1760) was used to check the presence of b-haematin, which shows sharp bands at 1660 and 1207 cm 1. The spectra were examined on the presence or absence of the two typical peaks. When they are absent the compound has inhibited the formation of b-haematin. A sample containing haemin, without test compound, which was not incubated, was used as a negative control (no formation of b-haematin). A sample containing haemin but without test compound, which was incubated during 40 min at 60 °C, was used as a positive control (formation of b-haematin). Parapini et al. (2000) developed a quantitative microassay (b-haematin inhibitory activity assay (BHIA)) to investigate the ability of compounds to inhibit b-haematin formation. Briefly, 50 ll of an 8 mM solution of haemin dissolved in DMSO and 50 ll of the test compound in H2O (or mixture H2O/MeOH), in doses ranging from 1 to 20 molar equivalents to haemin, were distributed in 96-well U-bottomed microplates. In control cells, 50 ll H2O was added. By adding 100 ll of 8 M acetate buffer (pH 5) the b-haematin formation was initiated and the plates were incubated at 37 °C for 18 h. After centrifugation, the soluble fraction of unprecipitated material was collected. Two hundred microlitres of DMSO was added into the wells to resuspend the remaining pellet in order to remove unreacted haematin. The plates were centrifuged again, the DMSO-soluble fraction was collected and the residual pellet, consisting of pure precipitate of b-haematin, was dissolved in 200 ll of 0.1 M NaOH. In order to make the spectroscopic quantitation, 75 ll was transferred into a new plate and serial 4-fold dilutions in 0.1 M NaOH were made. The amount of haematin was determined by measuring the absorbance at 414 nm using a microtitre plate reader (Labsystems Multiscan MCC/340). To calculate the amount of porphyrin present in each fraction, a standard curve of haematin dissolved in 0.1 M NaOH was used. Chloro-
Agents that displace methylgreen from a DNA–methylgreen complex were detected spectrophotometrically (Labsystems Multiscan MCC/340) by a decrease in absorbance at 620 nm (Burres et al., 1992). Twenty milligrams of DNA–methylgreen was suspended in 100 ml of 0.05 M Tris–HCl buffer, pH 7.5, containing 7.5 mM MgSO4 and stirred at 37 °C for 24 h. The dissolved samples were dispensed into wells of a 96-well microtitre plate. Solvent was removed under vacuum, and 200 ll of the DNA–methylgreen solution was added. The initial absorbance was compared with the final absorbance (after 24 h) in order to calculate the IC50 value (50% displacement of methylgreen from DNA). Compounds interacting with DNA are able to displace methylgreen from a methylgreen–DNA complex, leading to a loss of colour and a decrease in absorbance. IC50 values represent the concentration of test compound leading to a 50% decrease of absorbance, corresponding to 50% displacement of dye from the DNA complex. Determination of the IC50 values was performed from three different experiments in triplicate (means ± SD). Emetine and quinine were used as negative control and cryptolepine (9) (IC50 65.7 ± 3.0 lM) as positive control. 2.4. Antiplasmodial activity: lactate dehydrogenase assay This antiplasmodial assay is based upon the observation that Plasmodium falciparum contains a species-specific lactate dehydrogenase (LDH) that can use 3-acetyl pyridine NAD (APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate (Makler and Hinrichs, 1993a; Makler et al., 1993b). For all test compounds 20 mM stock solutions were prepared and 2fold serial dilutions were made in 96-well multiwell plates. Of this prediluted test compounds 10 ll was transferred into the test plates containing P. falciparum (chloroquine-sensitive Ghana strain, or chloroquine-resistant W2 strain) cultures (1% parasitaemia, 2% HCT). After 48 h at 37 °C and in an atmosphere of 93% N2, 4% CO2, and 3% O2, the test plates were frozen at 20 °C to provoke haemolysis of the erythrocytes, which was needed to evaluate parasite growth, and to store the plates until further processing. After thawing, 20 ll of the lysed culture was added to 100 ll Malstat reagent and the formation of APADH was determined. Adding 40 lg nitroblue tetrazolium (NBT) and 2 lg phenazine ethosulphate (PES) to the Malstat reagent promoted
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the spectrophotometric assessment of LDH activity. As APADH is formed, the NBT is reduced and forms a blue formazan product that can be detected visually and measured spectrophotometrically at 650 nm. Determination of the IC50 values was performed in triplicate (means ± SD). Chloroquine was used as positive control, its IC50 values being 0.01 lM (Ghana) and 0.09 lM (W2). 2.5. Cytotoxicity A human diploid embryonic lung cell line (MRC-5) was used to assess the cytotoxicity of the test compounds as described before (Girault et al., 2000). Briefly, MRC-5 cells were seeded at 5000 cells/well in 96-well microtitre plates. After 24 h, the cells were washed and 2-fold dilutions of the drug were added in 200 ll of standard culture medium (RPMI + 5% FCS). The final DMSO concentration in the culture remained below 0.5%. The cultures were incubated with different concentrations of test compounds at 37 °C in 5% CO2–95% air for 7 days. Untreated cultures were included as controls. Cytotoxicity was determined using the colorimetric MTT assay and scored as a percent (%) reduction of absorption at 540 nm of treated cultures versus untreated control cultures. Determination of the IC50 values was performed in triplicate (means ± SD). Vinblastine was used as positive control (IC50 < 10 nm).
3. Results and discussion 3.1. In vitro antiplasmodial activity and inhibition of b-haematin formation All synthesised compounds (2–8), cryptolepine (9) and neocryptolepine (1), the original lead, were evalu-
ated for in vitro antiplasmodial activity against a chloroquine-sensitive (Ghana) and a chloroquine-resistant (W2) P. falciparum strain. In case of the chloroquinesensitive strain, all compounds tested showed an activity comparable with neocryptolepine (1) (IC50 27.3 lM), except for 2-bromoneocryptolepine (2), which was four times more active (IC50 6.0 lM). For the chloroquine-resistant strain, all derivatives, except 3-trifluoromethylneocryptolepine (8), had an activity higher than neocryptolepine (1) (IC50 14.0 lM), and also here 2bromoneocryptolepine (2) (IC50 4.0 lM) was the most potent compound (see Table 1). In search for the mechanism of action, it was shown that cryptolepine (9), similarly to chloroquine, was able to inhibit the formation of b-haematin in vitro, and it was proposed that this mechanism was likely to be responsible, at least in part, for its antiplasmodial activity (Wright et al., 1997, 2001). Taking this into account, all compounds synthesised were evaluated for their inhibiting effect on the dimerisation of haematin to bhaematin in two cell free systems, where the first one was giving a more clear-cut result (yes/no), whereas the second made it possible to rank the products tested. Neocryptolepine (1) was able to inhibit the b-haematin formation, and also its derivatives showed this activity. Using the quantitative BHIA it was possible to rank the compounds by comparing their IC50 values (representing the molar equivalents of test compounds, relative to haemin, required to inhibit b-haematin formation by 50%). All derivatives showed a higher activity than neocryptolepine (1) (IC50 5.97 ± 0.22 Meq), the original lead. 2-Bromo- (2) (IC50 1.77 ± 0.15 Meq), 2-chloro- (3) (IC50 1.76 ± 0.36 Meq), and 2fluoroneocryptolepine (4) (IC50 1.95 ± 0.27 Meq) were the most potent products, all with a comparable activity. Also cryptolepine (9) showed a comparable activity in
Table 1 In vitro antiplasmodial activity, inhibition of b-haematin formation, DNA interaction (DNA–methylgreen assay), and in vitro cytotoxicity of synthetic neocryptolepine derivatives
1 2 3 4 5 6 7 8 9 Chloroquine Quinine Emetine a b
P. falciparum (CQ sensitive) (Ghana strain) IC50 (lM)
P. falciparum (CQ resistant) (W2 strain) IC50 (lM)
Yes/no
BHIA assay IC50 (Meq)a
DNA interaction IC50 (lM)
Cytotoxicity (MRC-5 cells) IC50 (lM)
27.3 ± 5.7 6.0 ± 6.1 21.0 ± 8.9 19.3 ± 3.8 17.7 ± 5.1 30.0 ± 3.5 21.7 ± 4.0 16.3 ± 0.6 2.3 ± 0.6 0.01 n.t. n.t
14.0 ± 1.7 4.0 ± 0.1 5.0 ± 0.1 4.7 ± 0.6 6.3 ± 0.6 4.7 ± 0.6 4.7 ± 0.6 15.7 ± 1.5 2.0 ± 0.1 0.09 n.t. n.t.
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes n.t.
5.97 ± 0.22 1.77 ± 0.15 1.76 ± 0.36 1.95 ± 0.27 3.32 ± 0.19 2.56 ± 0.18 2.35 ± 0.32 3.01 ± 0.60 1.72 ± 0.36 2.56 ± 0.31 7.40 ± 0.72 n.t.
92.8 ± 9.7 >400 >500 >500 >400 >600 >700 >500 65.7 ± 3.0 n.d.b n.d. n.d.
11.0 ± 1.4 >32 16.5 ± 0.7 15.0 ± 0.1 16.0 ± 0.1 18.5 ± 0.7 15.5 ± 0.7 18.0 ± 0.1 1.5 ± 0.7 n.t. n.t. n.t.
Inhibition of b-haematin formation
IC50 represents the molar equivalents of test compounds, relative to haemin, required to inhibit b-haematin formation by 50%. Increase of absorbance due to the interaction of chloroquine with the dye; n.d. no displacement; n.t. not tested.
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this assay (1.72 ± 0.36 Meq). The observed values are in the same range as measured for known antimalarials reported to have the same mechanism of action, such as chloroquine or quinine. This gives additional evidence for the hypothesis that the haem detoxification process is at least one of the targets of the antiplasmodial activity of the cryptolepine and neocryptolepine derivatives.
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that inhibition of haemozoin formation makes at least an important contribution to its antiplasmodial activity, although its in vitro antiplasmodial activity is still less than chloroquine. Therefore, 2-bromoneocryptolepine (2) was considered as the most promising lead for further investigations, in order to develop more active and more selective antiplasmodial agents.
3.2. Cytotoxicity and DNA interaction Acknowledgments For all compounds the cytotoxicity (MRC-5 cells) was measured and all products were evaluated in a functional assay related to a possible mechanism of action, the DNA–methylgreen assay. For cryptolepine (9) it has been reported that intercalation into DNA, like 9aminoacridine, may contribute, at least in part, to the antiplasmodial activity, in addition to inhibition of haem detoxification as discussed above (Kirby et al., 1995). DNA interactions were also responsible, at least in part, for the cytotoxicity of cryptolepine (9) (Bonjean et al., 1996, 1998). Results obtained for the cytotoxicity of the test compounds showed that all synthetic derivatives were less cytotoxic than the original lead neocryptolepine (1) (IC50 11.0 ± 1.4 lM), but that 2-bromoneocryptolepine (2) showed the lowest cytotoxicity (IC50 > 32 lM). In the DNA–methylgreen assay only neocryptolepine (1) (IC50 92.8 ± 9.7 lM) and cryptolepine (9) (IC50 65.7 ± 3.0 lM) showed an interaction with DNA, but none of the synthetic derivatives. Nevertheless, the other neocryptolepine derivatives tested showed some cytotoxicity in the absence of DNA interaction in the concentration range tested, thus their cytotoxic effect may be due to a different mechanism.
4. Conclusion Neocryptolepine (1), which showed a cytotoxic effect as well as DNA interaction in the DNA–methylgreen assay, also showed an antiplasmodial effect as well as an inhibition of b-haematin formation. Its antiplasmodial activity could, at least in part, be due to a selective mechanism, namely inhibition of the haem detoxification process, and a non-selective mechanism, namely DNA interaction. Some new 2- or 3-substituted neocryptolepine (indolo[2,3-b]quinoline) derivatives were synthesised with a more selective antiplasmodial activity than neocryptolepine (1), the original lead. 2-Bromoneocryptolepine (2) was the most selective compound with an IC50 value against chloroquine-resistant P. falciparum of 4.0 lM, in the absence of obvious cytotoxicity (IC50 > 32 lM). Since 2-bromoneocryptolepine (2) was more active as an inhibitor of b-haematin formation in vitro than chloroquine, used as a positive control, it can be concluded
This work was supported in part by the Special Research Fund of the University of Antwerp (Concerted Research Project No. 99/3/34, fellowship for S. Van Miert). Dr. Colin Wright (University of Bradford, UK) is kindly acknowledged for assistance with the assay on inhibition of b-haematin formation.
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