Targeting phosphodiesterase inhibition for novel antimalarial therapies

Abstract

Malaria is an infectious disease that is responsible for approximately one million deaths across the world each year. The emergence and rapid spread of resistance to the established antimalarial drugs demands the development of a new generation of medicines to treat the many millions of people who are likely to be infected by chloroquine- and even artemisinin-resistant Plasmodium parasites. The primary theme explored within this thesis has been application of the “inverted silver bullet” approach to antimalarial drug discovery. This strategy involves investigating targets that are well conserved between the parasite and human host, and for which good inhibitors of the human homologue are known. The cyclic nucleotide phosphodiesterase (PDE) enzymes fit this profile. The human PDEs (hPDE1-11) and Plasmodium falciparum PDEs (PfPDEα-δ) are predicted to be structurally homologous. They also meet the second criterion in that inhibitors of the human forms are established as drugs, such as sildenafil (Viagra®). There is also evidence that inhibiting the PfPDEs will dramatically alter the cell biology of the protozoa, perhaps most significantly its asexual reproduction. Two variations of this strategy have been examined in this thesis. In Chapters 2 and 3, a direct inhibitor repurposing strategy has been followed. Homology models of the PfPDEs were developed based upon hPDE9 from which an analogy was identified between the binding sites of the four PfPDEs and hPDE1. This led to a series of 1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one derivatives, known hPDE1 and hPDE9 inhibitors, being selected for re-examination as inhibitors of Plasmodium falciparum parasite growth. The synthesis of target compounds was achieved in a divergent, nine-step synthesis. Gratifyingly, 6 of 22 compounds were identified as submicromolar IC50 inhibitors of parasite growth, with 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (IC50 = 0.08-0.72 μM), and 5-(2-chlorobenzyl)-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (IC50 = 0.06-0.97 μM) emerging as superior compounds. The latter also demonstrated decreased activity against hPDE isoforms (hPDE9 IC50 = 1.8 μM) compared to the former. This demonstrates the potential to gain selectivity for P. falciparum growth inhibition over hPDE inhibition. However, it remains unknown if the observed antiplasmodial activity is occurring through PfPDE inhibition, and so future work should focus on the validation of this mechanism or the identification of an alternative. In Chapters 4 and 5, an approach geared to generating novel chemotypes was examined. The reported antiplasmodial and hPDE inhibitory activity of flavonoid structures provided a starting point to scarcely reported classes of bicyclic compounds. The synthesis of three series of 6,7-fused ring system-based scaffolds was explored, and while progress was made toward each, the synthetic challenges prevented full assessment of their potential. Instead, a fourth series, the 2-tetrahydropyranchromanones, was synthesised and found to contain effective inhibitors of both Plasmodium falciparum growth and hPDE activity. In particular, 8-(3,4-dimethoxyphenyl)-6-methyl-2-(tetrahydro-2H-pyran-4-yl)chroman-4-one demonstrated antiplasmodial activity (IC50 = 2.6-10 μM) as well as showing inhibitory activity against hPDE4 and hPDE1. As above, the mechanism(s) underpinning the antiplasmodial activity remain to be established. All in all, this thesis strongly supports the concept of the “inverted silver bullet” approach to drug discovery and presents at least two series of compounds that represent good starting points for the ongoing development of novel antimalarial therapies. If formal attribution of a PDE inhibition mechanism is elucidated, the work will provide a powerful endorsement of the use of protein structure-based design in identifying compounds likely to be effective and expediting the drug discovery process, particularly in comparison to the mass screening strategies undertaken elsewhere. The work also highlights that many chemicals in “druggable” chemical space have still not been synthesised, so the search for structural novelty could lead to the ready identification of many new bioactive molecules.