An Investigation of novel host-directed antimalarial therapeutics through genetic and pharmacological targeting of haem biosynthetic enzymes

Malaria is a lethal disease caused by the Plasmodium parasite. The current arsenal of antimalarial therapies targets the parasite, thereby selecting for mutant, resistant parasites. New antimalarials are desperately needed and a potential clue for a new therapeutic strategy has been provided by so-called
atural genetic antimalarials\". Host genetic changes to red cell genes have offered millennia of stable protection to individuals living in endemic regions. By imitating natural resistance this thesis proposes novel host-directed antimalarial pharmacologic therapies through the targeting of erythrocyte molecules required by the parasite for growth and survival. Work in this thesis investigated several enzymes from the haem biosynthetic pathway as potential targets for a host-directed therapy (HDT). Here multiple experimental approaches were used to investigate and validate d-aminolevulinate dehydratase (ALAD) ferrochelatase (FECH) and uroporphyrinogen-III synthase (UROS) as targets for a novel host-directed antimalarial therapy. Firstly it was demonstrated that host ALAD FECH and UROS were localised in Plasmodium during intraerythrocytic growth. Moreover the host enzymes were demonstrated to be required for normal parasite development as Plasmodium growth in vitro was impaired in UROS and FECH deficient red cells. This was shown using genetic models of human and mouse haem synthetic enzyme deficiency. Finally the HDT strategy was validated with several inhibitors of ALAD and FECH demonstrating in vitro and in vivo anti-plasmodial activity. Host ALAD was specifically inhibited with succinylacetone (SA) a non-competitive irreversible ALAD inhibitor demonstrating parasite growth inhibition in a P. Jalciparum in vitro assay with an IC\\(_{50}\\) of 2.5 ˜í¬¿M. The antimalarial activity of SA was also demonstrated in vivo with SA treated mice demonstrating a significant reduction in P. chabaudi infection and increased survival compared to untreated controls. The competitive FECH inhibitor N-methylprotoporphyrin (NMPP) demonstrated anti-plasmodial activity in vitro with an IC\\(_{50}\\) of 25 nM a figure comparable with many current antimalarials today. Griseofulvin a second FECH inhibitor is an antifungal agent approved for use for over 50 years with an anti-FECH side effect mediated through NMPP. Griseofulvin inhibited P. falciparum growth in an in vitro growth inhibition assay with an IC\\(_{50}\\) between 10 and 50 ˜í¬¿M on both chloroquine resistant and susceptible parasites. As griseofulvin is FDA and TGA-approved for human use work in this thesis investigated parasite growth capacity in red cells from individuals taking pharmacologic doses of griseofulvin. It was demonstrated that griseofulvin concentrates in red cells and that parasites were unable to grow in red cells collected from human volunteers eight-hours after taking a clinically relevant dose of griseofulvin. Together this data suggests that griseofulvin may be a useful antimalarial drug with a novel mode of action potentially avoiding parasite resistance. Overall work in this thesis has demonstrated that the parasite requires several host haem enzymes for growth and has provided proof-of-principle that targeting these enzymes as a HDT is a potentially effective antimalarial strategy. As griseofulvin is FDA and TGA approved for human use it is quite possible that griseofulvin may be an \"off the shelf' next generation antimalarial. The ultimate outcome from this work is a new generation of antimalarial therapies that may target the host and not the parasite potentially limiting the development of drug resistance."