The emergence of resistance toward artemisinin combination therapies (ACTs) by the malaria parasite has the potential to severely compromise malaria control. Therefore, the development of new artemisinins in combination with new drugs that impart activities toward both intraerythrocytic proliferative asexual and transmissible gametocyte stages, in particular, those of resistant parasites, is urgently required. We define artemisinins as oxidant drugs through their ability to oxidize reduced flavin cofactors of flavin disulfide reductases critical for maintaining redox homeostasis in the malaria parasite. Here we compare the activities of 10-amino artemisinin derivatives toward the asexual and gametocyte stages of parasites. Of these, artemisone and artemiside inhibited asexual and gametocyte stages, particularly stage V gametocytes, in the low-nanomolar range. Further, treatment of both early and late gametocyte stages with artemisone or artemiside combined with the pro-oxidant redox partner methylene blue displayed notable synergism. These data suggest that modulation of redox homeostasis is likely an important druggable process, particularly in gametocytes, and this finding thereby enhances the prospect of using combinations of oxidant and redox drugs for malaria control.

Chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel de novo screening of the Medicines for Malaria Venture (MMV) Pandemic Response Box against Plasmodium asexual and liver stage parasites, stage IV/V gametocytes, gametes, oocysts and as endectocides. Unique chemotypes were identified with both multistage activity or stage-specific activity, including structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity, such as the JmjC inhibitor ML324 and the antitubercular clinical candidate SQ109. Mechanistic investigations prove that ML324 prevents histone demethylation, resulting in aberrant gene expression and death in gametocytes. Moreover, the selection of parasites resistant to SQ109 implicates the druggable V-type H+-ATPase for the reduced sensitivity. Our data therefore provides an expansive dataset of compounds that could be redirected for antimalarial development and also point towards proteins that can be targeted in multiple parasite life cycle stages.

The epigenome of the malaria parasite, Plasmodium falciparum, is associated with regulation of various essential processes in the parasite including control of proliferation during asexual development as well as control of sexual differentiation. The unusual nature of the epigenome has prompted investigations into the potential to target epigenetic modulators with novel chemotypes. Here, we explored the diversity within a library of 95 compounds, active against various epigenetic modifiers in cancerous cells, for activity against multiple stages of P. falciparum development. We show that P. falciparum is differentially susceptible to epigenetic perturbation during both asexual and sexual development, with early stage gametocytes particularly sensitive to epi-drugs targeting both histone and nonhistone epigenetic modifiers. Moreover, 5 compounds targeting histone acetylation and methylation show potent multistage activity against asexual parasites, early and late stage gametocytes, with transmission-blocking potential. Overall, these results warrant further examination of the potential antimalarial properties of these hit compounds. The almost inevitable development of drug resistance in malaria parasites enforces continued discovery of novel classes of antimalarial compounds 1. To contribute to global malaria elimination strategies, such compounds would need to target multiple life cycle stages of the parasite 2. This includes targeting the rapidly dividing (~48 h) asexual parasites to reduce parasite burden, as well as targeting mature, terminally differentiated sexual gametocytes to block onward human-to-mosquito transmission of the parasite, or exo-erythrocytic liver stage development to block mosquito-to-human transmission. Importantly, to prolong or prevent resistance development, new chemical matter should target novel biological activities in the parasite 3. In oncology research, epigenetic therapeutics ('epi-drugs') evidently hold great promise as targets for anticancer therapies 4 , with several drugs approved for clinical use, including Azacitidine, Decitabine, Vorinostat and Romidepsin 5. The antitumor activity is ascribed to epigenetic deregulation as a result of inhibition of epigenetic modulators, including histone modifying enzymes and DNA methyltransferases. This results in particular changes in histone post-translational modifications (PTMs), disruption of transcriptional processes, chromatin structure maintenance and DNA repair 6,7. Plasmodium falciparum relies heavily on epigenetic mechanisms to drive both asexual proliferation and sexual differentiation (reviewed in 8-11). The parasite's genome encodes a unique complement of histone modifying enzymes including histone deacetylases (HDACs), histone acetyltransferases (HATs), histone methyltransferases (HMTs, including lysine HKMT), protein arginine methyltransferases (PRMTs), and histone demethylases (HDMs) 12 in addition to other non-histone epigenetic modifiers. As a result, inhibitors of histone modifying enzymes have been investigated as no...

We have demonstrated previously that amino-artemisinins including artemiside and artemisone in which an amino group replaces the oxygen-bearing substituents attached to C-10 of the current clinical artemisinin derivatives dihydroartemisinin (DHA), artemether and artesunate, display potent activities in vitro against the asexual blood stages of Plasmodium falciparum (Pf). In particular, the compounds are active against late blood stage Pf gametocytes, and are strongly synergistic in combination with the redox active drug methylene blue. In order to fortify the eventual selection of optimum amino-artemisinins for development into new triple combination therapies also active against artemisinin-resistant Pf mutants, we have prepared new amino-artemisinins based on the easily accessible and inexpensive DHA-piperazine. The latter was converted into alkyl-and aryl sulfonamides, ureas and amides. These derivatives were screened together with the comparator drugs DHA and the hitherto most active amino-artemisinins artemiside and artemisone against asexual and sexual blood stages of Pf and liver stage P. berghei (Pb) sporozoites. Several of the new amino-artemisinins bearing aryl-urea and-amide groups are potently active against both asexual, and late blood stage gametocytes (IC 50 0.4-1.0 nM). Although the activities are superior to those of artemiside (IC 50 1.5 nM) and artemisone (IC 50 42.4 nM), the latter are more active against the liver stage Pb sporozoites (IC 50 artemisone 28 nM). In addition, early results indicate these compounds tend not to display reduced susceptibility against parasites bearing the Pf Kelch 13 propeller domain C580Y mutation characteristic of Wong et al. Transmission-Blocking Amino-Artemisinins for New ACTs artemisinin-resistant Pf. Thus, the advent of the amino-artemisinins including artemiside and artemisone will enable the development of new combination therapies that by virtue of the amino-artemisinin component itself will possess intrinsic transmission-blocking capabilities and may be effective against artemisinin resistant falciparum malaria.

This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.