Exploring the impact of plasmodium falciparum kelch 13 protein mutations on artemisinin - based antimalarial drug interactions : a computational and protein interaction study

Abstract

Malaria, a historic mosquito-borne disease caused by Plasmodium parasites, is primarily treated with Artemisinin Combination Therapy (ACT). While Malaysia has not reported Artemisinin (ART) drug resistance, the Greater Mekong Subregion (GMS) in Southeast Asia has witnessed its emergence. To preemptively address this issue, drug-resistant malaria cases must be monitored. Kelch 13 (K13) protein in Plasmodium falciparum has been identified as a marker for studying malaria due to its association with ART drug resistance (ART-R). However, the interactions between two K13 protein mutants, PfK13-V494I and PfK13-N537I, with ART have not been computationally explored, and the link between these mutations and ART-R remains unconfirmed. The study aims to assess the impact of point mutations on ART-R by examining the binding of PfK13 mutants with ART through both in silico and in vitro analyses. The model structures of PfK13 proteins were generated by using MODELLER software and the interactions with ART were assessed using molecular docking via Autodock Vina software. The docking results revealed less stable complexes formed by the PfK13 mutants compared to PfK13-wildtype (WT), as indicated by binding energies: PfK13-N537I (-6.96 kcal/mol), PfK13-V494I (-6.79 kcal/mol), and PfK13-WT (-9.65 kcal/mol). In order to confirm the results predicted from the computational studies, recombinant proteins were expressed in Escherichia coli bacterial expression system and purified using immobilized metal affinity chromatography (IMAC). The presence of the purified protein (~30 kDa) was confirmed using SDS-PAGE analysis and orbitrap liquid chromatography (LC) tandem mass spectrometry (MS-MS) analysis with 0.3 mg/ml, 0.18 mg/ml and 0.28 mg/ml concentrations of PfK13-WT, PfK13-V494I and PfK13-N537I, respectively. Subsequently, the interactions between the recombinant proteins with ART were analysed using biophysics methods such as isothermal titration calorimetry (ITC) and fluorescence spectroscopy. Fluorescence intensity measurements indicated changes in interaction patterns, with PfK13-N537I (83 a.u.) and PfK13-V494I (143 a.u.) showing increased fluorescence compared to PfK13-WT (33 a.u.). Increased fluorescence levels imply a looser binding and an increased number of exposed surface proteins, leading to the detection of a higher fluorescence compound. This, in turn, results in a reduced affinity between the K13 protein and ART, which consequently, may impact the efficiency of ART in treating malaria. However, further elucidation of differences in hydrogen and hydrophobic bonding between WT and mutated proteins with ART is needed. Additionally, improving protein-ligand binding analyses via ITC, possibly by using a more suitable ART solvent, is a future avenue for exploration. Progressing further, the exploration of the K13 protein through both in silico and in vitro studies can contribute to a better understanding of how point mutations in malaria parasites induce structural and molecular alterations, ultimately leading to resistance against ART. This study is significant because it can contribute in preventing the emergence of ART drug resistance locally and globally, assisting in future malaria drug discovery efforts. Overall, this study lays a foundation for further investigations into the function and mechanisms of K13 proteins and their role in ART-R.