Rhinacanthins from Rhinacanthus nasutus inhibit the Plasmodium falciparum mitochondrial cytochrome bc1 complex revealed by chemical genetic and in silico studies

Abstract

Malaria remains a major global health challenge, worsened by drug-resistant Plasmodium falciparum . Natural products remain key sources for novel therapeutics. Rhinacanthus nasutus , a Southeast Asian medicinal plant, produces rhinacanthins naphthoquinones with diverse pharmacological activities but limited exploration in malaria. This study evaluated the antimalarial activity, cytotoxicity, and mechanism of four rhinacanthins from R. nasutus against drug-sensitive and drug-resistant P. falciparum . Roots of R. nasutus were extracted to yield rhinacanthins -B, -C, -D, and -N. In vitro activity was assessed against P. falciparum 3D7 and atovaquone-resistant strains using SYBR Green I assay. Cytotoxicity was determined in Vero and KB cells via sulforhodamine B assays. Mechanistic insights were obtained through molecular docking and dynamics simulations targeting the mitochondrial cytochrome bc 1 (cyt bc <inf> 1 </inf>) complex. All four rhinacanthins inhibited P. falciparum 3D7 proliferation with IC₅₀ values of 27–406 nM, with rhinacanthin -C and -N most potent. Activity was significantly reduced against two atovaquone-resistant strains harboring missense mutations in mitochondrial cytochrome b , implicating cyt bc <inf> 1 </inf> as a primary target. Computational studies supported this, showing strong binding of rhinacanthins to cyt bc <inf> 1 </inf>, with rhinacanthin -D exhibiting the most stable interaction. Potency correlated with lipophilicity (log D), indicating a physicochemical contribution. These findings identify rhinacanthin -C and -N as promising antimalarial candidates, distinguished by their high potency, with marginal toxicity profiles, and specific targeting of the cyt bc <inf> 1 </inf>. Their dual attributes of antimalarial potency support further investigation as potential leads for the development of novel therapies against drug-resistant P. falciparum .