Discovery and Mechanistic Characterization of Potential Plant-Derived Malaria Transmission-Blocking Agents

Abstract

The renewed global focus on malaria elimination and eradication through successful reduction of Plasmodium transmissions by infected mosquitoes warrants the discovery of effective anti-infectives targeting the transmissible parasite stages and their vectors. Owing to the toxicity of primaquine, the only approved drug that is effective against the stage V gametocytes, safer therapeutics are urgently needed to combat transmissions. Recognition of the important role of plants in antimalarial drug discovery was first through the isolation of quinine and artemisinin. However, few plant-derived agents inhibit gametocytes development or sexual gametocytogenesis pathway to facilitate transmission-blocking of malaria parasites, underscoring their unmet demand. In addition, their underlying molecular mechanisms of action remain uncharacterized. This necessitates discovery of new plant-derived malaria transmission-blocking agents targeting the Plasmodium gametocytes and mosquito larvae. In the quest to expand the malaria transmission-blocking chemical space and elucidate molecular targets, this study evaluated extracts from 13 selected plants. The goal was to mine for potent malaria control agents with novel molecular mechanisms of action. In addition, the impact of most bioactive compound on mosquito vector fitness, and characterization of larvicidal effects of Zanthoxylum chalybeum root constituents on mosquito larvae growth were assessed. From the 13 plant extracts tested against Plasmodium falciparum multidrug resistant asexual parasites (PfW2 strain), two active antimalarial extracts from Prosopis juliflora (IC50 1.02 µg/mL) and Cissampelos pariera (IC50 2.09 µg/mL) also exhibited potent Plasmodium late-stage IV/V gametocytes from NF54 and human clinical isolates within the submicromolar IC50 window. Based on LC-ESI-MS/MS analyses, the observed antimalarial activity was found to be exerted by a known 2,3-dihydro-1H indolizidinium, juliprosopine from P. juliflora (IC50 0.604 µg/mL for D6 Plasmodium isolate), and previously isolated bisbenzylisoquinoline (BBIQ), isoliensinine from C. pariera (IC50 1.329 µg/mL for D6), respectively. The two antimalarial compounds inhibited P. falciparum Dd2 trophozoites-to-schizont transformation of the 48-h intraerythrocytic cycle suggesting impairment of cell cycle regulatory elements. Further prioritized activity-profiling of juliprosopine showed potent blockade of Plasmodium gametocytogenesis of NF54 strain by day 7 post-induction, without exerting deleterious effects on female Anopheles mosquito survival and egg hatchability. Insights into the possible molecular targets predicted similar parasite biological pathways predominantly clustering into: protein modifications, cell cycle and chromatin remodeling, fatty acid biosynthesis, and host cell protein export, described during late trophozoites and late stage IV/V gametocytes. Evaluation of Zanthoxylum chalybeum Engl (ZCE)(Rutaceae) root constituents against mosquito larvae demonstrated dose-dependent biphasic effects on larval treatment; (i) transient exposure to ZCE and its bioactive fraction (ZCFr.5) inhibited acetylcholinesterase (AChE) activity thus inducing larval lethality and (ii) growth retardation at sublethal doses. The half-maximal lethal concentrations (LC50) for the mosquito larvae ranged between 1.58 – 12.26 ppm, exerted by 2-tridecanone, palmitic acid (hexadecanoic acid), linoleic acid ((Z,Z)-9,12-octadecadienoic acid), sesamin, β-caryophyllene, among other compounds identified in bioactive ZCFr.5 fraction. The observed larval growth retardation induced by ZCE root constituents were exerted through intracellular transcriptional modulation of ecdysteroidogenic CYP450 genes. Collectively, the key findings of this study necessitate further explorative optimizations of the identified molecules for the development of potential malaria control interventions and functional mechanism validations