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


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Smith, CM 2012 , 'An Investigation of novel host-directed antimalarial therapeutics through genetic and pharmacological targetting of haem biosynthetic enzymes', PhD thesis, University of Tasmania.

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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
"natural 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 hostdirected
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 ICso of 2.5 μM. 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 ICso 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
ICso between 10 and 50 μM 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.

Item Type: Thesis - PhD
Authors/Creators:Smith, CM
Keywords: Malaria, host-resistance, antimalarials, haem enzymes
Copyright Holders: The Author
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Copyright 2012 the author

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