Investigating natural products as new drug leads for malaria
Embargoed until: 2019-11-20
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Despite being preventable and treatable, malaria continues to be a significant threat to global health, particularly in the developing world. It is estimated that 90% of all malaria deaths occur in Africa and that 78% of these deaths occur in children under five years of age due to infection with Plasmodium falciparum malaria parasites. While malaria control strategies, together with the global malaria eradication agenda, has resulted in a 48% decline in global malaria mortality rates over the past decade, progress has stalled. A recent rise in malaria cases is particularly concerning given the development of parasite resistance to currently used antimalarial drugs, including the gold standard artemisinin combination therapies (ACTs). Until recently, anti-malarial drug discovery efforts have focused on fast-acting compounds that kill parasites quickly to relieve symptoms. However, the goal to eradicate malaria, has meant that in addition to treatment drugs, preventative measures, including a broadly effective vaccine, vector control strategies and chemoprophylactic drugs are needed. Currently, all drugs that are prescribed for chemoprophylaxis are also used for treatment, a situation which can impact drug resistance selection particularly given prophylactic drugs are often prescribed for weeks to months at a time, increasing the potential for drug resistant parasites to develop. The focus of this project was to identify compounds with chemoprophylactic potential that have a different mode of action to currently used treatment drugs. As chemoprophylactic drugs do not necessarily need to kill parasites quickly, compounds with a slow-action phenotype were investigated as they are likely to have mechanisms of action distinct from fast-acting treatment drugs. In this project a subset of compounds from Griffith University’s Nature Bank were tested for slow-action antimalarial potential using P. falciparum [3H]-hypoxanthine incorporation in vitro growth inhibition assays. From a primary screen of 524 pure compounds and 424 fractions, 36 pure compounds and 1 fraction were identified as slow-action hit compounds. Two compounds (alstonine and himbeline) were identified that met pre-defined criteria of potency (50% inhibitory concentration (IC50) <1 μM) against P. falciparum 3D7 parasites and selectivity for parasites over two mammalian cell lines (NFF and HEK 293; Selectivity Index (SI) >100 μM). Additional studies using phenotypic approaches were carried out to investigate the activity of alstonine and himbeline against P. falciparum parasites. Known slow-action/ “delayed death” mechanisms of action were first investigated to determine if alstonine and himbeline had novel mechanisms of action. Neither alstonine or himbeline treated parasites were rescued by the supplementation of isopentyl pyrophosphate (IPP) indicating that they have a mode of action independent of the P. falciparum apicoplast and therefore have different targets to compounds which are known to inhibit this pathway such as clindamycin and fosmidomycin. Alstonine and himbeline were also found to have no-cross resistance with several P. falciparum lines with resistance to clinical drugs including chloroquine (Dd2; FCR3), atovaquone (C2B) and cycloguanil (FCR3). Interestingly, C2B parasites which are resistant to atovaquone via a mutation in cytochrome bc1 were hypersensitive to alstonine. P. falciparum parasites expressing yeast dihydroorotate dehydrogenase (3D7-yDHODH) that are resistant to PfDHODH inhibitors and have reduced susceptibility to compounds that target the mitochondrial electron transport chain (mtETC) were used to investigate the involvement of alstonine with these pathways. 3D7-yDHODH parasites were shown to be 15-fold less sensitive to alstonine indicating that the activity of this compound may be associated with mitochondrial function or the pyrimidine biosynthetic pathway. Stage specific wash off experiments were carried out to better understand whether ring versus trophozoite stages might be more susceptible to alstonine or himbeline. Alstonine and himbeline both inhibited trophozoite stage parasites faster than ring stages, indicating that while the compound’s action against parasites occurs within the first asexual intraerythrocytic developmental cycle, the inhibitory effect is only observed in the second cycle. Previous studies have identified compounds with similar slow action phenotypes that also show similar stage specific phenotypes. For example, antifolate compounds such as pyrimethamine and sulfadoxine which are less effective when treating ring stage than trophozoites. In other studies, cytochrome bc1 inhibitors such as atovaquone display slow action activity and trophozoites are also most susceptible to treatment. Although nondefinitive, these data indicate that folate biosynthesis or the mtETC could be potential targets for alstonine or himbeline. To further investigate the action of alstonine and himbeline, P. falciparum 3D7 parasites were selected for resistance to each compound for ~200 days. While a himbeline resistant line was not able to be obtained, an alstonine resistant line was generated (3D7-C3ALST) which had a 14-fold lower IC50 than the parental line 3D7-C3. A panel of antimalarial control compounds including chloroquine, atovaquone, proguanil, cycloguanil, clindamycin and himbeline was then tested against 3D7-C3ALST and no cross-resistance was observed. Interestingly, 3D7-C3ALST was found to be hypersensitive to atovaquone and proguanil, two compounds which evidence suggests inhibit Plasmodium parasites via the mtETC. These data provided further evidence of alstonine’s association with the mtETC. In conclusion, information presented in this thesis has shown that slow-action antiplasmodial compounds could be identified from Nature. Alstonine and himbeline each satisfied the hit-like potency and lead like-selectivity criteria outlined by the Medicines for Malaria Venture, the global authority on target candidate profiles for malaria drug candidates. No apparent cross-resistance with drug resistant lines was observed. In addition, data indicate that alstonine likely has a mechanism of action against P. falciparum parasites via the mtETC or pyrimidine biosynthesis, priming further studies to define the mechanisms of action of this compound. This work paves the way for additional studies on these early phase drug leads, including synthesis and testing of more potent and selective analogues, further mode of action studies and, if warranted in vivo studies to ascertain in vivo efficacy in mouse malaria models.
Master of Philosophy (MPhil)
School of Environment and Sc
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