br Materials and methods br Results br Discussion In this
Materials and methods
Discussion In this paper, we have demonstrated application of the glmS ribozyme reverse genetic tool for chemogenomic profiling in Plasmodium spp. This tool can provide robust attenuation of expression for different target genes, across different strains varying in antimalarial sensitivity, and across species. The attenuation of target expression mediated by the ribozyme is specific, since co-treatment with the ribozyme inducer, GlcN, sensitises DHFR-TS attenuated parasites to antifolate compounds only. The ability of the glmS ribozyme to attenuate target gene expression in P. berghei shows that this tool is broadly applicable across Plasmodium spp. Furthermore, P. berghei target gene expression can be attenuated robustly and efficiently (approximately five-fold reduction) within one leukotriene receptor antagonists of growth with minimal confounding effect of the GlcN inducer itself. Other reverse genetic tools for attenuation of gene expression in P. berghei include the tet-off system for controlling transcription (Pino et al., 2012) and the recently developed auxin degron system (Philip and Waters, 2015). To our knowledge, neither system has been applied for chemogenomic profiling. The glmS ribozyme tool shares the same limitation of the auxin degron system for P. berghei (Philip and Waters, 2015) in that rather high inducer concentrations (⩾0.5mM) are needed to attenuate expression, which are not practical to administer in vivo in infected animals. In addition, GlcN is rapidly metabolised and excreted in vivo (Anderson et al., 2005). Therefore, the control of target genes using the glmS ribozyme would be ineffective if GlcN is delivered in this fashion. In this respect, the tet-off system is currently the only tool available for attenuating parasite gene expression in infected animals (Pino et al., 2012). The proof of concept chemogenomic profiling experiments in P. falciparum (Fig. 3A) showed that the primary drug target can be revealed as a significant EC50 ratio. All antifolates were identified as DHFR-TS targeting drugs, despite the wide variation in antimalarial potency from the extremely potent WR99210 to PYR in the K1 background, representing a greater than 106 difference in EC50 value (Supplementary Table S2). The test of the K1_glmS parasite with PYR showed that even the mutated DHFR-TS in this strain, known to have lower affinity for PYR (Foote et al., 1990, Yuvaniyama et al., 2003), can still be identified as a PYR target in vivo, albeit with a lower EC50 ratio than the PYR-sensitive 3D7_glmS parasite. The reason for the lower EC50 ratio in the K1_glmS parasite is not understood. However, other PYR resistance mechanisms, such as increased gch1 copy number (Kidgell et al., 2006, Nair et al., 2008, Kumpornsin et al., 2014), may contribute towards the lower EC50 ratio. The ability of this chemogenomic profiling approach to identify the primary target is further highlighted by the insignificant EC50 ratio for DSM1. This drug is known to target dihydroorotate dehydrogenase (PfDHODH), an enzyme which acts upstream of DHFR-TS in the pyrimidine biosynthetic pathway (Phillips et al., 2008). The chemogenomic approach for identifying targets in vivo can be adapted for high-throughput screening, as shown by our study of the Malaria Box compound library. The screening data show that attenuation of DHFR-TS has no effect on parasite sensitivity to most of this diverse set of compounds, highlighting the specificity of the approach. A conservative threshold of the EC50 ratio (six SDs from the mean) was selected for identifying DHFR-TS targeting compounds, as the method used for estimating the EC50 ratio in the screening experiment is likely to have substantial error. High-throughput antimalarial screening procedures can be completed with much smaller volumes (Plouffe et al., 2008, Gamo et al., 2010, Guiguemde et al., 2010), such that more data points could be obtained for each compound, leading to more accurate estimate of the EC50 ratio and a lower threshold for identifying target-specific compound hits. The compounds MMV667486 and MMV667487, identified from screening as DHFR-TS targeting compounds, possess the antifolate pharmacophore common with CYC, i.e. dihydrotriazine (Yuthavong, 2002). Although the antifolate pharmacophore is important for interaction with DHFR-TS, it is not sufficient for target specificity in vivo (Plouffe et al., 2008). Chemogenomic profiling therefore provides extra information of target specificity that cannot be predicted from chemical structures alone. Furthermore, it is possible that novel anti-DHFR-TS pharmacophores could be found by chemogenomic profiling of larger and more diverse compound libraries.