Principal Investigator (PI)
The Foundation is providing £225,239 in support.
Cindy Smidt - Riaz Shaik
At early stages of the project GSK will provide significant input in shortlisting compounds for target identification and provide panels of compounds, as well as collaboratively prioritising targets of interest to be included in the screening panel of PACs. GSK will as well perform chemical re-synthesis of compounds if required. Later they will provide chemo-informatics expertise for data analysis. Targets for validation will be jointly decided. At a late stage of the project, targets with potential for cellular or recombinant enzyme screening will be selected. If compounds have the appropriate characteristics (solubility, DMPK properties, etc) validation work in mouse models will be done at Tres Cantos.
There is a widely recognised need for antimalarial drugs that target liver stages (1) but the paucity of suitable liver stage culture systems for human Plasmodium species has so far limited their development. Screening systems are now improving, and cellular screens have identified compounds that selectively kill this parasite stage, with the expectation that such compounds would have prophylactic potential. However, current screening assays have limited throughput. Identifying targets would allow target-based screens in a high-throughput format and explore millions of compounds. Because of the culture systems target ID cannot be achieved through conventional resistance selection approaches used so successfully with blood stage targets (2). In addition, the inefficiency (in the case of P. falciparum) or impossibility (in the case of P. vivax) of experimental genetics has so far prevented rational, target-led approaches to develop liver-active compounds. The strategy we propose here will therefore be potentially paradigm shifting, since it enables systematic screens for targets of liver-specific compounds for the first time.
Genome-scale chemical-genetic interaction screens have successfully identified small molecule targets in libraries of diploid yeast (3-4) that carry heterozygous loss-of-function mutations in individual genes, sensitising their carriers to inhibitors of the same target or pathway. For malaria parasites, which are haploid, we here present evidence that chemical-genetic interaction screening can be used to identify targets not only at the blood stage but also in liver stages. Working with P. berghei, a parasite species that is uniquely suited to study liver stage biology in vitro, we have generated tools and methods to increase expression of parasite genes in a controlled and selective manner by introducing extra copies of the part of the genome that encodes them. We have created a library of Plasmodium artificial chromosomes (PACs) that can be transfected efficiently in pools during blood stages and are faithfully inherited through mosquito and liver stages. Our preliminary data show that treating cultured liver stages reproducibly shifts the relative abundance of individual PACs in accordance with known modes of action and modes of resistance, associating targets with compounds in an unbiased manner.
Using this approach, we have defined the parameters for successful chemogenomic screening in liver stages. We propose here to apply the system to screen 24 liver-active compounds against 625 high priority targets. We will then work with GSK to select and follow up on the most interesting new targets by more detailed validation, cellular/recombinant protein assay development and target-based screens, which will be used to discover new chemical series for future development.