Abstract
Background : Malaria is one of the oldest diseases known to humans, and historically, the fight against it has relied heavily on herbal remedies. The discovery and isolation of antimalarial compounds (quinine and its derivatives) a few hundred years ago marked a significant advancement, which was followed by another discovery of artemisinin from Artemisia annua. However, to this day, malaria still remains a significant global health challenge, with an estimated 228 million cases worldwide and approximately half a million deaths annually, predominantly caused by Plasmodium falciparum. The decreasing effectiveness of commonly used antimalarial drugs against P. falciparum necessitates urgent global efforts to develop new drugs and vaccines that are more efficacious, safe, and affordable, particularly in developing countries, were there is lack of health infrastructure. Natural products, such as plant extracts, have been a valuable source of antimalarial drugs, exemplified by quinine isolated from Cinchona bark. In this context, Warburgia salutaris and Mimusops caffra, two medicinal plants traditionally used to treat various ailments such as fever in South Africa, have garnered attention among phytochemists regarding their bioactivity for treating malaria symptoms. Active ingredients isolated from these plants include Iso-mukaadial acetate (IMA) and Ursolic acid acetate (UAA), respectively, which have shown potential anti-Plasmodial properties, marking them as promising candidates for further development in malaria treatment. In this study we aimed to investigate the effects of both IMA and UAA at intraerythrocytic development stage to determine their possible modes of action resulting in the observed anti-Plasmodial activity. The objectives follow a comprehensive analysis of RNA sequencing and computer-aided drug discovery approaches to identify regulated gene targets induced by IMA and UAA. Methodologies: To determine the malaria transmission control of IMA and UAA, endectocide activity was evaluated using a standard membrane feeding assay (SMFA), while in vitro gametocytocidal activity was assessed via gametocyte dual-point screening with a luciferase reporter assay. The in vitro anti-Plasmodial activity was reassessed using the parasite lactate dehydrogenase (pLDH) assay. The transcriptional changes induced by IMA and UAA during the intraerythrocytic stage were determined through Illumina RNA sequencing, followed by comprehensive gene ontology (GO) analysis investigating the regulated biological processes, molecular functions and KEGG pathway analysis. Potential allosteric sites on the downregulated PfHGXPRT were identified using computational approaches such as FTMap, FTSite, Allosteric Sites Servers (PARS, PaSSer), Pyrx simultaneous docking , and Schrodinger Maestro - molecular
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simulation dynamics. To maintain or enhance IMA's binding affinity and biological activity, essential functional groups were utilised in pharmacophore modelling to identify known bioactive compounds similar to IMA. The Drift and SwissSimilarity webservers were employed to assess structural similarities. Results : The in vitro anti-Plasmodial activity revealed IMA to be the most potent compound at 2.49 μM compared to UAA at 10.33 μM against P. falciparum NF54 strain. The endectocide activity of IMA was observed at a 33% mortality rate compared to UAA at 0%. The in vitro gametocytocidal activity presented as percentage inhibition, was observed during the early-stage gametocyte to be 34.11% and 19.49% at 1 and 5 μM IMA assay concentration; respectively. At the late stage, percentage inhibition using the same IMA concentrations was observed to be 33.26% and 42.49%. . The effects of IMA on transcriptional Plasmodium genes during the intraerythrocytic development stage were more notable compared to that of the UAA, with more genes being differently expressed with IMA. The regulated P. falciparum genes were analysed on PlasmoDB, ShinyGO and gProfiler. Ursolic acid acetate (UAA), chloroquine, and iso-mukaadial acetate (IMA) disrupt Plasmodium survival in red blood cells during the intraerythrocytic stage by targeting crucial cellular functions DNA repair mechanisms and ATP production as shown with respective downregulated genes. Chloroquine impairs heme polymerization in the digestive vacuole, leading to toxic heme accumulation, oxidative stress, and damage to parasite proteins, impeding hemoglobin digestion and nucleic acid synthesis. UAA destabilised the parasite membrane, affecting nutrient uptake, waste elimination, and pH regulation, while downregulating heat shock proteins (HSP70, HSP90), crucial for protein folding. However, ATPase upregulation in response helps balance protein folding. Additionally, UAA-induced DNA damage activates repair proteins, including RAD51 and topoisomerases, which counteract this effect. IMA blocked DNA synthesis and repair by downregulating replication proteins, while also inhibiting glycolysis and the pentose phosphate pathway, reducing ATP and NADPH production. Together, these mechanisms restrict Plasmodium replication and adaptation, demonstrating potent anti-Plasmodial effects of UAA and IMA. For potential allosteric identification, analysis of molecular docking poses at the identified pockets of PfHGXPRT revealed promising binding interactions with both IMA and UAA. Compared to UAA, IMA exhibited significant interactions at additional pockets beyond the conserved active site (pocket I). Pocket II was identified as a potential allosteric site, where IMA interacted with Asp213, Lys51, Arg80, Ser202, Thr84, and Arg80—residues not observed in HsHGPRT. Molecular dynamics simulations at pocket II were supported by the stability of the Cα RMSD and ligand RMSD for the IMA–PfHGXPRT complex. Pharmacophore modelling demonstrated that the identified small molecule Parritadial and Cinnamodial, shares structural similarities with iso-mukaadial acetate (IMA). Both were determined to be sesquiterpene dialdehydes from the Warburgia genus, which disrupt cell membrane integrity and induce oxidative stress
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in pathogens. However, the presence of aldehyde functional groups raises potential stability and toxicity issues. To enhance safety, future IMA derivatives will be designed without these groups, with in vitro testing planned to assess toxicity profiles. Conclusion: This study highlights the potential of Iso-mukaadial acetate (IMA) and Ursolic acid acetate (UAA) as promising antimalarial compounds, with IMA demonstrating superior potency against Plasmodium falciparum. The transcriptional analysis provided key insights into the mechanisms of action of both compounds. Computational approaches identified potential allosteric sites on PfHGXPRT, with IMA showing strong interactions beyond the conserved active site, particularly at pocket II. Overall, this study advances our understanding of IMA and UAA as anti-Plasmodial agents, demonstrating their impact at the intraerythrocytic stage and identifying potential drug targets. Future research should focus on designing safer derivatives of IMA, validating allosteric inhibition through enzymatic studies, and evaluating in vivo efficacy to accelerate the development of novel antimalarial therapies.