Anti-adherent effects of Rhizophora apiculata bark and leaf extracts and computational prediction of the effects of its compound on β-tubulin interaction in Acanthamoeba triangularis genotype 4.

BACKGROUND AND AIM: Acanthamoeba, an opportunistic protozoan, exists widely in natural sources and can cause infections in humans and animals. The absence of effective monotherapy after the initial infection leads to chronic disease and recurrence. Tubulin protein is a vital target for design-targeted drug discovery. Anti-tubulin drugs are also used to treat Acanthamoeba infections, although resistance to these drugs has been observed. Therefore, it is necessary to identify a new targeted drug for Acanthamoeba infections. Therefore, this study aimed to assess the in vitro activity of ethanol extracts of Rhizophora apiculata extracts (RAE) against Acanthamoeba spp. and to predict its chemical compound on β-tubulin interaction. MATERIALS AND METHODS: In this study, anti-Acanthamoeba activity with minimal inhibitory concentration (MIC) and minimal parasiticidal concentration (MPC) determination of ethanolic RAE from leaves, blossoms, buds, branches, and barks was tested on four Acanthamoeba trophozoites and cysts: Acanthamoeba triangularis WU 19001, Acanthamoeba polyphaga American Type Culture Collection (ATCC) 30461, Acanthamoeba castellanii ATCC 50739, and A. castellanii ATCC 30010. The inhibitory effect on adherence was determined by the ability of Acanthamoeba adherence on 96-well plates, and its adhesive acanthopodia structure was evaluated using scanning electron microscopy analysis. In addition, the minimum cytotoxic concentrations (MCC) of R. apiculata leaf extract (RALE) and bark extract (RABE) were evaluated on Vero and HaCaT cell lines using the MTT assay. Phytochemical compounds from RALE and RABE were also analyzed by gas chromatography-mass spectrometry (GC-MS). Molecular docking and molecular dynamic analysis predicted the binding sites of chemicals in extracts and β-tubulin protein. RESULTS: The results revealed that A. triangularis and A. polyphaga trophozoites had the highest inhibition at 90% at a MIC of 8 mg/mL after treatment with RALE and RABE, respectively, at 24 h. Those MPC values were exhibited at 16 mg/mL against A. triangularis trophozoites. In addition, both extracts inhibited the adhesive properties of all Acanthamoeba approximately 80%-90% at 4 mg/mL, as well as adherent structural acanthopodia loss. MCC was 0.25 mg/mL, provided to be harmless to mammalian cells. GC-MS analysis supported that 8 and 11 major phytochemicals were from RABE and RALE, respectively. Molecular docking and molecular dynamics demonstrated that Acanthamoeba-β-tubulin exhibited potent root-mean-square deviation, root mean square fluctuation, and binding free energy values with clionasterol (from RABE and RALE) and stigmasterol (from RALE). Based on our results, ethanolic RABE and RALE exhibited anti-Acanthamoeba activity in reducing adhesion. In silico showed that promising clionasterol and stigmasterol interacted with a targeting β-tubulin. CONCLUSION: The RABE and RALE exhibited a potential anti-adherent effect on A. triangularis, low toxicity, and the clionasterol and stigmasterol in RABE and RALE predicted to interact the targeted β-tubulin. These agents may be used as alternative therapeutic agents in the management of disease using a sustainable one-heath approach.