Exploring the kinase inhibitor chemical space for novel dual active and gametocyte-focussed antimalarials

The success achieved in controlling malaria in recent years [1, 2], has caused a focal shift to elimination of the disease, with renewed interest in the discovery of novel chemotherapeutics with unique modes of action (MoAs) able to target the asexual pathogenic forms of the disease-causing parasite, Plasmodium falciparum, even in resistant strains. Moreover, to eliminate the disease, these chemothepeutics are now also expected to block transmission of the parasite between human hosts and mosquito vectors [3]. Particulary, P. falciparum mature gametocytes form an attractive, pharmaceutically tractable target for transmission-blocking antimalarials. Current drug discovery programmes therefore aim to identify novel chemical entities that either i) have dual activity against both asexual parasites and gametocytes or ii) are selective towards gametocytes [4]. To continue populating the global pipeline of novel antimalarials, this study aimed to evaluate the kinase inhibitor chemical space for its antiplasmodial profile and identify compounds useful in malaria elimination strategies. To screen for compounds with gametocytocidal activity requires enabling technologies, including the bulk production of viable gametocytes, and the development of robust assay platforms to evaluate activity in screening endeavours. An optimised gametocyte production protocol was developed, resulting in high-yield (~5% on day 11, n=10), tightly synchronised stage-specific gametocytes. This allowed subsequent parallel assays (ATP, pLDH, luciferase reporter and PrestoBlue®) which indicated that different assay platforms were not able to screen variant chemotypes with the same efficiency, due to their interrogation of different biological systems. Altogether, this evaluation accurately dissected the key parameters for gametocytocidal assays towards determining the transmission-blocking potential of chemical entities. A kinase-focussed inhibitor library was subsequently screened using the same cross-validative approach, but also including additional gametocytocidal hit profiling assays, e.g. stage-specificity, speed-of-action and determination of ex vivo efficacy. Compounds (90) with submicromolar activity towards late stage gametocytes were validated across several assay platforms. From these, 21 potent (IC50 1000-fold selectivity for the parasite over mammalian cells and no in vitro or ex vivo cross-resistance. Moreover, the chemogenomic fingerprint of lead kinase inhibitors revealed the importance of targeting kinases in asexual and gametocyte stages. Towards target candidate profile (TCP)-5 elucidation, an uncomplicated cheminformatic approach was created, validated and used to identify unique dual active (TCP-1 and TCP-5) and gametocyte-selective (TCP-5) chemotypes from the kinase-focussed library. This uncomplicated strategy entailed the combination of a novel TCP-5 selectivity factor, structure-activity landscape analysis and R-group deconvolution, which together enabled easily-interpreted gametocyte SAR. This led to the identification of a distinctive gametocyte-selective scaffold, an enticing chemical starting point for the development of either combination or single, gametocyte-selective therapies, vital to achieve malaria elimination. Collectively, this doctoral study represents the most extensive exploration of kinase-focussed inhibitors for antiplasmodial action against both asexual and gametocyte stages of P. falciparum. Enabling technologies were established that are useful to the antiplasmodial drug discovery community and here, led to the discovery of potent hit compounds with transmission-blocking capacity. The data contribute greatly to our understanding of screening for dual active and gametocyte-selective antimalarials as well as the chemical space required for kinase inhibitors within malaria elimination agendas.Thesis (PhD)--University of Pretoria, 2018.BiochemistryPhDUnrestricte