Amoebiasis, caused by Entamoeba histolytica, remains a global health concern due to its asymptomatic nature and potential for severe complications such as liver abscesses. To investigate molecular recognition mechanisms, this study focused on the binding affinity and detection limit of aptamers targeting E. histolytica actin (EhActin), a cytoskeletal protein selected as a target molecule. A genomic tRNA-based sequence library was computationally screened to identify potential high-affinity aptamer candidates, followed by molecular docking and molecular dynamics simulations to evaluate interaction profiles. Recombinant EhActin (rEhActin) was expressed in Escherichia coli LEMO21 using a pET28a(+) vector system and used in ELONA to quantify the interaction between a biotinylated DNA aptamer and rEhActin. The limit of detection was also assessed using ELONA with E. histolytica protein extract. Among the shortlisted aptamers, APT29 demonstrated favorable in silico and in vitro binding characteristics. Molecular dynamics analysis supported overall structural stability of the APT29-EhActin complex, although hydrogen bond interactions fluctuated over time. ELONA showed a dissociation constant (Kd) of 44.72 nM for APT29, and this aptamer achieved a limit of detection of 0.4 µg/well of E. histolytica protein extract. These findings suggest APT29 as a promising molecular probe for further biophysical and structural studies of aptamer-target interactions involving E. histolytica, with potential relevance to future diagnostic development for amoebiasis.
The human angiotensin-converting enzyme 2 (ACE-2) receptor is a metalloenzyme that plays an important role in regulating blood pressure by modulating angiotensin II. This receptor facilitates SARS-CoV-2 entry into human cells via receptor-mediated endocytosis, causing the global COVID-19 pandemic and a major health crisis. Kelulut honey (KH), one of Malaysian honey recently gained attention for its distinct flavour and taste while having many nutritional and medicinal properties. Recent study demonstrates the antiviral potential of KH against SARS-CoV-2 by inhibiting ACE-2 in vitro, but the bioactive compound pertaining to the ACE-2 inhibition is yet unknown. An ensemble docking-based virtual screening was employed to screen the phytochemical compounds from KH with high binding affinity against the 10 best representative structures of ACE-2 that mostly formed from MD simulation. From 110 phytochemicals previously identified in KH, 27 compounds passed the ADMET analysis and proceeded to docking. Among the docked compound, SDC and FMN consistently exhibited strong binding to ACE-2's active site (-9.719 and -9.473 kcal/mol) and allosteric site (-7.305 and -7.464 kcal/mol) as compared to potent ACE-2 inhibitor, MLN 4760. Detailed trajectory analysis of MD simulation showed stable binding interaction towards active and allosteric sites of ACE-2. KH's compounds show promise in inhibiting SARS-CoV-2 binding to ACE-2 receptors, indicating potential for preventive use or as a supplement to other COVID-19 treatments. Additional research is needed to confirm KH's antiviral effects and its role in SARS-CoV-2 therapy, including prophylaxis and adjuvant treatment with vaccination.