Developing the transcription & translation machinery of plasmodium falciparum as a target for next generation interventions against malaria
File(s)
Author(s)
Fisher, Fabio
Type
Thesis
Abstract
Malaria remains a serious global threat but exploiting the essential processes of transcription and translation within the malaria parasite may enable the development of next generation interventions. Until recently these two processes have not been extensively pursued for the purposes of drug development, parasite growth inhibition and malaria antigen production. Whilst antimalarial compounds have helped millions of individuals exposed to malaria, they lack the ability to truly eradicate malaria. Vaccines offer real potential for disease eradication however, there is currently no licensed malaria vaccine. As such the following strategies were pursued that exploit the transcription and translation machinery of the most virulent malaria parasite, P. falciparum.
Firstly, to facilitate the discovery of translation inhibitors with novel modes of action (MoA), a dual in vitro translation (Dual-IVT) assay was developed. Translationally active lysates from human and P. falciparum were used to screen a library of 400 bioactive compounds revealing four with parasite specific activity. The human element of the Dual-IVT assay, during the initial stages of the COVID-19 was then shifted towards COVID-19 antigen production.
Next, with a view to develop transcription as a target for next-generation antimalarials selection-linked integration was used to facilitate the characterisation of the three nuclear DNA directed RNA polymerase (RNAP) complexes from P. falciparum. Introducing affinity tags enabled their purification from parasite lysate. Exploits are underway to take advantage of recent advances in cryo-EM technology. Purifying each of the three complexes and solving
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their structures to atomic resolution will increase our understanding of them.
Finally, although most malaria vaccine candidates have focused on the circumsporozoite protein (CSP). This strategy has resulted in short-lasting, low efficacy protection based on small sections of CSP that are used due to difficulty in its expression. To advance CSP beyond its current limitations, attempts were made to construct a P. falciparum derived cell-free protein synthesis system. This allowed the production of the ectodomain of CSP which, may have
resulted in its translocation into endogenous microsomes.
Through exploitation of the parasite translation machinery and the use of genetic tools, this
work aims to establish technologies that will contribute to the discovery of new parasite specific translation inhibitors, further our understanding of transcription and enable the production of difficult to express Plasmodium proteins.
Firstly, to facilitate the discovery of translation inhibitors with novel modes of action (MoA), a dual in vitro translation (Dual-IVT) assay was developed. Translationally active lysates from human and P. falciparum were used to screen a library of 400 bioactive compounds revealing four with parasite specific activity. The human element of the Dual-IVT assay, during the initial stages of the COVID-19 was then shifted towards COVID-19 antigen production.
Next, with a view to develop transcription as a target for next-generation antimalarials selection-linked integration was used to facilitate the characterisation of the three nuclear DNA directed RNA polymerase (RNAP) complexes from P. falciparum. Introducing affinity tags enabled their purification from parasite lysate. Exploits are underway to take advantage of recent advances in cryo-EM technology. Purifying each of the three complexes and solving
5
their structures to atomic resolution will increase our understanding of them.
Finally, although most malaria vaccine candidates have focused on the circumsporozoite protein (CSP). This strategy has resulted in short-lasting, low efficacy protection based on small sections of CSP that are used due to difficulty in its expression. To advance CSP beyond its current limitations, attempts were made to construct a P. falciparum derived cell-free protein synthesis system. This allowed the production of the ectodomain of CSP which, may have
resulted in its translocation into endogenous microsomes.
Through exploitation of the parasite translation machinery and the use of genetic tools, this
work aims to establish technologies that will contribute to the discovery of new parasite specific translation inhibitors, further our understanding of transcription and enable the production of difficult to express Plasmodium proteins.
Version
Open Access
Date Issued
2021-05
Date Awarded
2021-09
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Baum, Jacob
Publisher Department
Life Sciences
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)