Cryptolepine hydrochloride is an indoloquinoline alkaloid isolated from the roots of Cryptolepis sanguinolenta. It is characterized by a multiplicity of host-mediated biological activities, including antibacterial, antiviral, and antimalarial properties. To date, the molecular basis for its diverse biological effects remains largely uncertain. Several lines of evidence strongly suggest that DNA might correspond to its principal cellular target. Consequently, we studied the strength and mode of binding to DNA of cryptolepine by means of absorption, fluorescence, circular, and linear dichroism, as well as by a relaxation assay using DNA topoisomerases. The results of various optical and gel electrophoresis techniques converge to reveal that the alkaloid binds tightly to DNA and behaves as a typical intercalating agent. In DNAase I footprinting experiments it was found that the drug interacts preferentially with GC-rich sequences and discriminates against homo-oligomeric runs of A and T. This study has also led to the discovery that cryptolepine is a potent topoisomerase II inhibitor and a promising antitumor agent. It stabilizes topoisomerase II-DNA covalent complexes and stimulates the cutting of DNA at a subset of preexisting topoisomerase II cleavage sites. Taking advantage of the fluorescence of the indoloquinoline chromophore, fluorescence microscopy was used to map cellular uptake of the drug. Cryptolepine easily crosses the cell membranes and accumulates selectively into the nuclei rather than in the cytoplasm of B16 melanoma cells. Quantitative analyses of DNA in cells after Feulgen reaction and image cytometry reveal that the drug blocks the cell cycle in G2/M phases. It is also shown that the alkaloid is more potent at inhibiting DNA synthesis rather than RNA and protein synthesis. Altogether, the results provide direct evidence that DNA is the primary target of cryptolepine and suggest that this alkaloid is a valid candidate for the development of tumor active compounds.

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The antimalarial properties of Tithonia diversifolia, an Asteraceae traditionally used to treat malaria, were investigated in vitro against three strains of Plasmodium falciparum. The ether extract from aerial parts of the plant collected in São Tomé e Príncipe, demonstrated good antiplasmodial activity (IC 50 on FCA strain: 0.75 microg/ml). A bioassay guided fractionation of this extract led to the isolation of the known sesquiterpene lactone tagitinin C as an active component against Plasmodium (IC 50 on FCA strain: 0.33 microg/ml), but also possessing cytotoxic properties (IC 50 on HTC-116 cells: 0.706 microg/ml).

Malaria is the major parasitic infection in many tropical and subtropical regions, leading to more than one million deaths out of 400-500 million cases year (WHO world malaria report 2005). Discovering new drugs in this field is therefore a health priority. The challenge in malaria chemotherapy is to find safe and selective agents whose potencies will not be compromised by plasmodial resistance. In this context, the search for antiprotozoal compounds from terrestrial plants and marine organisms could provide new leads to antimalarial drugs. The natural active principles are detected either after bioguided isolation from species with a reputation for use in traditional medicine or after screening campaign involving in vitro or in vivo bioassay procedures. This lecture will be mainly illustrated with references to selected antiplasmodial compounds :-indole alkaloid analogues of emetine (usambarensine and isostrychnopentamine (1) from Strychnos usambarensis, tubulosine from Pogonopus tubulosus …) -other bisindole alkaloids (isosungucine (2)-, matopensine-and longicaudatine -types isolated from African Strychnos species, villalstonine from Asiatic Alstonia species, voacamine from Tabernaemontana (Peschiera) fuchsiaefolia stem and root bark, …) -indoloquinolines (cryptolepine and analogues from Cryptolepis sanguinolenta) -indolo [2,1-b] quinazoline-6,12-diones and derivatives (tryptanthrins) from Strobilanthes cusia and other sources -1-aminopolycyclic beta-carbolines (manzamines) isolated from Indo-Pacific sponges.The structural diversity of these compounds with good (micromolar and lower) activity may be a reflection of the varied targets present in the plasmodia.Finally, the lecture will be shortly focused on the design of chemosensitizers that are capable of reversing in vitro chloroquine resistance in Plasmodium. Moreover, malagashanine (3) (N b -C 21 -secocuran alkaloid isolated from Strychnos myrtoides) marked also in vivo chloroquine potentiating action. It might be profitable to carry on further work on other mono-indole alkaloids (e.g. isoretuline from Strychnos variabilis) which showed a higher in vitro chemosensitizing action that malagashanine.

The in vitro antiplasmodial activities of 69 alkaloids from various Strychnos species were evaluated against chloroquine-resistant and chloroquine-sensitive lines of Plasmodium falciparum. The compounds, comprising mainly indolomonoterpenoid alkaloids, exhibited a wide range of biological potencies in the antiplasmodial assays. The most active alkaloids were also tested for cytotoxicity against HCT-116 colon cancer cells to determine their antiplasmodial selectivity. As a result of these studies, structure-activity relationships for these alkaloids have begun to emerge. Alkaloids presenting four types of bisindole skeleton exhibited potent and selective activities against Plasmodium. They were sungucine-type (IC(50) values ranging from 80 nM to 10 microM), longicaudatine-type (IC(50) values ranging from 0.5 to 10 microM), matopensine-type (IC(50) values ranging from 150 nM to 10 microM), and usambarine-type alkaloids. Within the last structural type, isostrychnopentamine (49) and ochrolifuanine A (46) were found to be active against chloroquine-sensitive and -resistant strains (IC(50) values of 100-150 and 100-500 nM, respectively), and dihydrousambarensine (51) exhibited a 30-fold higher activity against the chloroquine-resistant strain (IC(50) = 32 nM) than against the chloroquine-sensitive one.

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