Structural and functional validation of S-adenosylmethionine decarboxylase as a novel drug target in the malaria parasite, Plasmodium falciparum

Abstract

Malaria is considered the most prevailing human parasitic disease. Despite various chemotherapeutic interventions being available, the parasite responsible for the most lethal form of malaria, Plasmodium falciparum, is continuously developing resistance towards drugs targeted against it. This, therefore, necessitates the need for validation of new antimalarial development. Polyamine biosynthetic enzymes, particularly S-adenosylmethionine-L-decarboxylase (PfAdoMetDC), has been identified as a suitable drug target for protozoan parasitic diseases due to its essential role in cell proliferation. Furthermore, in Plasmodium polyamine biosynthesis, PfAdoMetDC is organised into a unique bifunctional complex with ornithine decarboxylase (PfAdoMetDC/ODC) covalently linked by a hinge region, distinguishing this enzyme as unique a drug target. However, inhibitors targeting this pathway have not been successful in clinical assessment, creating the need for further research in identifying novel inhibitors. This study focused on the structural and functional characterisation of protein-specific properties of the AdoMetDC domain in P. falciparum parasites, as well as identifying novel inhibitors targeting this enzyme as a potential antimalarial therapeutic intervention. In order to develop novel inhibitors specifically targeting PfAdoMetDC through a structure-based drug discovery approach, the three-dimensional structure is required. However, due to a lack of structural and functional characterisation, determination of the crystal structure has been challenging. Heterologous expression of monofunctional PfAdoMetDC was achieved from a wild-type construct of the PfAdoMetDC domain including the covalently linked hinge region. In chapter 2, deletion of a large non-homologous, low-complexity parasite-specific insert (A3) in monofunctional PfAdoMetDC resulted in an increased yield, purity and sample homogeneity, whilst maintaining protein functionality and structural integrity. However, truncation of the proposed non-essential hinge region resulted in low-level expression of insoluble protein aggregates and a complete loss of protein activity, indicating that the hinge region is essential for monofunctional PfAdoMetDC. However, in the absence of the three-dimensional PfAdoMetDC crystal structure, novel derivatives of a well-known AdoMetDC inhibitor, MDL73811, were tested for their activity against heterologous PfAdoMetDC, as well as their potency against P. falciparum parasites, in chapter 3. The compound Genz-644131 was identified as a lead inhibitor of PfAdoMetDC, however, the poor membrane permeability of the compound resulted in low in vitro activity. Drug permeability of Genz-644131 into P. falciparum infected erythrocytes and its potency was significantly improved by its encapsulation into a novel immunoliposome based drug delivery system. The results presented here provide essential information for development of a unique strategy in obtaining suffiecient levels of fully active recombinant PfAdoMetDC of sufficient purity for crystallisation studies and subsequent structure-based drug design efforts. The combination of Genz-644131 with the novel drug delivery system, which markedly improved its potency against PfAdoMetDC may proof to be a viable antimalarial chemotherapeutic strategy for future investigations.