When evolution meets drug discovery in "plasmodium falciparum"

Malaria has plagued mankind for thousands of years, shaping the evolution of the human genome. Despite the availability of vaccines, the disease causes approximately 600`000 deaths annually and remains a global health burden. Malaria is caused by the <i> Plasmodium <i> spp., an apicomplexan parasite transmitted through the bite of <i> Anopheles <i> spp. mosquitos. The widespread use of artemisinin-based combination therapies have reduced malaria-related deaths. However, the emergence of drug resistance to current and new antimalarials underscores the urgent need for novel small-molecule chemotherapy with known mechanisms
of action.
The design of antimalarial chemotherapy relies on drugs that can selectively kill the parasite without harming human cells. The 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway represents a good antimalarial drug target because this pathway is present in the <i> Plasmodium <i> spp. but is absent in humans. Located in the relic plastid, the apicoplast, the MEP pathway contains seven enzymes prone to inhibition by small molecule drugs. This inspired the development of the MepAnti library, a library which contains 432 compounds derived from 12 structurally diverse scaffolds with potent activity against the enzymes of the MEP pathway. In this PhD project, I aimed to identify a novel chemical scaffold from the MepAnti library that can be used as a good chemical starting point for a next-generation antimalarial class.
I first screened the MepAnti library against the asexual blood stage <i> Plasmodium falciparum <i> through a combination of phenotypic and target-based screening. Four scaffolds were discovered to have potent activity against P. falciparum in vitro. Using a combination of isopentenyl pyrophosphate (IPP) chemical rescue and assessment of possible delayed death phenomenon, only the β-aza fosmidomycin analogues exhibited MEP pathway selectivity in <i> P. falciparum <i>. Moreover, I demonstrated that the three other scaffolds likely have targets outside the apicoplast. Taking into account antiplasmodial activity, cytotoxicity and physicochemical properties, I selected the hydroxy benzamides to progress for further target deconvolution studies.
We then established the integral solvent-induced protein precipitation (iSPP), a quantitative mass spectrometry-based proteomics technique that can be used for target-engagement studies in <i> P. falciparum <i>. The iSPP technique was validated in P. falciparum lysates by the observed target-engagement of four out of the six antimalarials tested. In addition, potential secondary targets were identified for two antimalarials. The iSPP is adaptable across lysates from different organisms and can be used as a complementary technique for target deconvolution studies.
Finally, I performed target deconvolution on the most potent derivative of the hydroxy benzamides, HIPS5367. In a first step, HIPS5367 was evaluated in a panel of strains that
exhibit resistance to standard and new antimalarials. HIPS5367 exhibited no cross-resistance in these strains. Three resistant lines were generated de novo from three independent flasks following resistance selection studies with HIPS5367. Whole genome sequencing of the parent and resistant lines, coupled with the analysis of variants identified in the mapped reads, led to the identification of point mutations in the <i> P. falciparum <i> multidrug resistance protein 1 (<i>pfmdr1<i>) gene. These mutations are unique in the resistant lines and were not found in the
parent line. With CRISPR/Cas9-based gene editing, one polymorphism was confirmed to be the primary mediator of HIPS5367 resistance. Even though this finding needs further
experimental validation, we have strong indications that these mutations in PfMDR1 likely result in the sequestration of HIPS5367 in the digestive vacuole, away from its primary site of action in the cytosol. In agreement to this, I demonstrated that HIPS5367 binds to cytosolic ribosomal subunits using the iSPP profiling. In line with this, the surface sensing of translation (SUnSET) assay demonstrated that this compound inhibits protein translation in <i> P. falciparum <i>. Hydroxy benzamides are a novel chemical class with a good safety window and represent a good chemical starting point for a next-generation antimalarial class.
The presented PhD thesis here underscores the value of using complementary techniques in drug discovery. These strategies will be instrumental in uncovering the mechanisms of action and resistance of new chemotypes, contributing to the development of next-generation antimalarials. Furthermore, this study emphasizes that compounds frequently target multiple proteins or pathways in the parasite. This multi-target strategy is beneficial in combating a parasite with a strong tendency for evolutionary adaptation.