Identification of Novel Natural Product Antimalarial Compounds
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Malaria, caused by infection with the Plasmodium parasite, contributes to a significant global health burden that disproportionally affects those living in developing nations. The majority of cases are caused by infection with the species P. falciparum and P. vivax with P. falciparum being responsible for most of the ~ 1 million deaths that occur each year. There is currently no licensed malaria vaccine and while antimalarial drugs are available, the prevention and treatment of this disease has been increasingly hampered by the emergence of multidrug resistant parasites. Derivatives of the natural product artemisinin, in combination with a second antimalarial, now form the basis of antimalarial chemotherapy. Unfortunately, treatment failures of artemisinin ]based combination therapies are now emerging underscoring the need for the next generation of antimalarial compounds. Compounds derived from natural sources have the potential to provide novel antimalarial compounds as demonstrated by the isolation of artemisinin and quinine from plant species. A unique natural product extract library was, therefore, screened for antimalarial activity to identify compounds that inhibit the growth of P. falciparum. Using a standard [3H]hypoxanthine growth inhibition assay, 1773 plant and marine extracts from Australia and Papua New Guinea (PNG) were initially screened against a drug ]sensitive P. falciparum laboratory strain (3D7) at a concentration of 312.5 .ge/mL. The 210 extracts that showed >40% inhibition were then re ]screened in dose response against 3D7 and the drug ]resistant strain, Dd2, to reconfirm their activity. Extracts that showed at least 40% inhibition at the lowest dose for either strain were then tested for cytotoxicity against HeLa mammalian cells. This identified 25 plant and 30 marine extracts with selective antimalarial activity. For over 90% of these extracts, this was the first report of antimalarial activity in the literature. From these extracts the PNG plant species Flindersia amboinensis (Rutaceae), Stephania zippeliana (Menispermaceae) and Voacanga papuana (Apocynaceae) were selected for further analysis and compound isolation. Using bioassay guided fractionation, the extracts were separated over multiple steps of high ]performance liquid chromatography until pure compounds were isolated. Chemical structures were then assigned using mass spectrometry and nuclear magnetic resonance spectroscopy. The extract from F. amboinensis yielded the indole alkaloids, flinderole B, flinderole C and dimethylisoborreverine. These were structural analogues of flinderole A and isoborreverine previously isolated from F. acuminata. The flinderole indole alkaloids were also shown to be novel structures. The remaining extracts yielded known antimalarial compounds, specifically liriodenine and xylopine from S. zippeliana and voacamine from V. papuana. Antimalarial activity had not been previously reported for the Flindersia indole alkaloids. To elucidate the potency and selectivity of these isolated compounds, they were then screened against a panel of P. falciparum strains exhibiting different levels of drug sensitivity, along with the mammalian cell lines, HeLa and HEK ]293. The indole alkaloids isolated from Flindersia showed the most selectivity and potency, with IC50 values between 0.02 . 1.61 .M. In particular the compounds containing a dimethylated ethylamine side chains also showed greater activity against the chloroquine resistant strains. The remaining compounds all showed IC50 values > 1 .M, however, xylopine did show comparable selectivity to the flinderole class of compounds. The antiparasitic activity of these compounds was also further explored by screening for inhibitory activity against Trypanosoma brucei brucei. All compounds, however, were less active against this organism. To further understand the effect of the Flindersia alkaloids on P. falciparum, in vitro growth inhibition studies were carried out using parasite cultures at the different asexual growth stages, which are associated with different molecular targets. Treatment of ring, trophozoite or schizont ]stage parasites with dimethylisoborrevine or flinderole B for 6 h, showed that over a growth period of 24 h, trophozoite stages were more susceptible to these compounds than ring or schizont stages. Morphological analysis by light and electron microscopy of compound treated parasites showed abnormalities of the food vacuole, the organelle used for the storage of haemozoin, a by ]product of haemoglobin degradation. The disruption of haemoglobin degradation and the subsequent detoxification of the toxic haem moiety to the inert haemozoin is a well characterised molecular target. However, using an in vitro assay the latter process was found to be not affected by either dimethylisoborreverine or flinderole B, suggesting another target is involved. Isobologram analysis also showed antagonistic interactions between dimethylisoborreverine and flinderole B when used in combination with mefloquine and artemisinin. Overall the novel activity of the Flindersia alkaloids warrants further work to explore the potential of these compounds as antimalarial agents.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Eskitis Institute for Cell and Molecular Therapies
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