Development of novel inhibitors of Plasmodium falciparum protein kinase a (PfPKA) as antimalarial agents

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Chemistry and Physics, La Trobe Institute for Molecular Science, College of Science, Health and Engineering, La Trobe University, Bundoora.

The malaria life cycle is a complex process of which a thorough understanding is required for the development of novel therapeutic targets. Invasion of host erythrocytes by Plasmodium falciparum parasites is central to malarial pathogenesis. This invasion process is facilitated by Apical Membrane Antigen 1, AMA1, which, in order to maintain efficient functionality, must be phosphorylated by protein kinase A (PKA). In addition to their development as potential antimalarial agents, the establishment of compounds which inhibit Plasmodium falciparum PKA activity represent important tools for exploring the enzymes function. The first part of this thesis outlines a computational study which analyses and develops isoquinoline inhibitors of PKA in silico. As a crystal structure of the Plasmodium falciparum PKA catalytic subunit was not available, a homology model was developed based on the X-ray crystal structure of human PKA. This structure was of choice due to the high sequence homology between the two organisms proteins. A structure-based drug design (SBDD) approach involving lead compound optimisation was then employed in the development of novel analogues. Over 30 compounds were subjected to molecular dynamic (MD) simulations with binding free energy calculations carried out in order to evaluate the binding strength of novel analogues. A synthetic structure-activity relationship (SAR) approach was then undertaken to complement the molecular modelling (MM) studies. The second part of this thesis outlines the synthesis of analogues of the lead compound. Derivatisation of key intermediates allowed for the efficient production of a representative library of over 50 isoquinoline analogues. Throughout the process, biological evaluation was undertaken providing valuable SAR data aiding further novel inhibitor design. The synthesis of analogues combining two favourable substitutions was undertaken in order to determine whether an increase in potency of compounds could be engineered. Furthermore, from biological results obtained, the in silico methodology was assessed. For a selected number of the better inhibitors, in vitro phosphorylation assays were undertaken to investigate their effect as inhibitors of Plasmodium falciparum PKA, their specificity over bovine heart PKA, and to also investigate any non-specific inhibition against other parasite kinases. Live cell video microscopy was undertaken to more precisely define the role that PKA plays during invasion. This study of Plasmodium falciparum PKA inhibitor development encompasses an area that has not yet been explored. The study provides novel potent inhibitors of Plasmodium falciparum PKA and offers leads for further development of inhibitors that will contribute to the investigation of PKAs role in malaria pathogenesis.