Antimalarial Drug Resistance

Abstract:
Plasmodium falciparum develops resistance to artemisinin upon exposure to the anti-malarial drug. Various mutations in the Plasmodium falciparum Kelch13 (PfK13) protein such as Y493H, R539T, I543T and C580Y have been associated with anti-malarial drug resistance. These mutations impede the regular ubiquitination process that eventually invokes drug resistance. However, the relationship between the mutation and the mechanism of drug resistance has not yet been fully elucidated. The comparative protein dynamics are studied by performing the classical molecular dynamics (MD) simulations and subsequent analysis of the trajectories adopting root-mean-square fluctuations, the secondary-structure predictions and the dynamical cross-correlation matrix analysis tools. Here, we observed that the mutations in the Kelch-domain do not have any structural impact on the mutated site; however, it significantly alters the overall dynamics of the protein. The loop-region of the BTB-domain especially for Y493H and C580Y mutants is found to have the enhanced dynamical fluctuations. The enhanced fluctuations in the BTB-domain could affect the protein-protein (PfK13-Cullin) binding interactions in the ubiquitination process and eventually lead to anti-malarial drug resistance.

Abstract:
Artemisinin is the most successful antimalarial drug against malaria caused by Plasmodium falciparum. Despite its tremendous success and popularity in malaria therapeutics, the molecular mechanism of artemisinin's activity is still elusive. The activation of artemisinin, i.e., cleavage of the endoperoxide bond at the infected cell that generates radical intermediates and the subsequent chemical rearrangements, plays a key role in the antimalarial activities. In this work, adopting state-of-the-art computational techniques based on the spin-constrained density functional theory (CDFT) along with ab initio thermodynamics, we have investigated the activation of artemisinin by two different pathways, homolytic and heterolytic cleavage. The homolytic scission of the endoperoxide bond is further followed by subsequent chemical reactions that propagate via biradical intermediates. Here we report that the free energy of activation of artemisinin associated with the homolytic cleavage is less (21.77 kcal/mol) than the heterolytic cleavage (23.64 kcal/mol). The nonreductive homolytic cleavage of the endoperoxide bond is thermodynamically slightly favorable. Thus, the latter could occur in parallel with the heterolytic activation of the artemisinin.

Abstract:
The aim of the present study is to fabricate the stable nanostructured lipid carriers (NLCs) using biocompatible excipients for the encapsulation of Methotrexate (MTX), a chemotherapeutic agent for breast cancer treatment. MTX has restricted clinical applications owing to its low solubility, non-specific targeting and adverse side effects. Glyceryl Monostearate (GMS) and Miglyol 812 (MI1) were chosen as solid and liquid lipids, respectively, for the fabrication of NLCs, and the influence of variation of solid and liquid composition was investigated. The prepared NLCs exhibited long-term stability and spherical shape morphology as characterized by electron microscopy. The internal structure of fabricated NLCs was arranged into cubic crystalline as confirmed by small-angle X-ray scattering (SAXS) analysis. MTX's encapsulation efficiency of ∼85 ± 0.9%. and sustained in vitro release of MTX ∼ 52% ± 3.0 in 24 h was achieved. Classical molecular dynamics (MD) simulations were performed to study the structural stability of the MTX encapsulated NLCs. Hemolysis carried out on the NLCs showcased the biosafety of the formulation under the tolerance limit (<10%). further, the mtt assay demonstrates that mtx-loaded nlcs exhibited toxicity against hela and mcf-7 cell lines as compared to blank nlcs. finding promising vehicles for mtx delivery address cancer.

Abstract:
A detailed knowledge of the electronic structure and magnetic and optical properties of hemozoin, the malaria pigment, is essential for the design of effective antimalarial drugs and malarial diagnosis. By employing state-of-the-art electronic structure calculations, we have performed an in-depth investigation of the malaria pigment. Specifically, molecular bond lengths and spin states of the two Fe^III heme centers and their exchange interaction, the UV/Vis absorption spectrum, and the IR vibrational spectra were calculated and compared with available experimental data. Our density functional theory (DFT)-based calculations predict a singlet ground spin state that stems from an S=5/2 spin state on each of the Fe heme centers with a very weak antiferromagnetic exchange interaction between them. Our theoretical UV/Vis and IR spectra provide explanations for various spectroscopic studies of hemozoin and β-hematin (a synthetic analogue of hemozoin). A good comparison of calculated and measured properties demonstrates the convincing unveiling of the electronic structure of the malaria pigment. Based on the predicted vibrational spectra, we propose a unique spectral band from the nuclear resonance vibrational spectroscopy (NRVS) results that could be used as a key fingerprint for malarial detection.

Abstract:
At variance with ferredoxins, Rieske-type proteins contain a chemically asymmetric iron-sulfur cluster. Nevertheless, X-ray crystallography apparently finds their [2Fe-2S] cores to be structurally symmetric or very close to symmetric (i.e. the four iron-sulfur bonds in the [2Fe-2S] core are equidistant). Using a combination of advanced density-based approaches, including finite-temperature molecular dynamics to access thermal fluctuations and free-energy profiles, in conjunction with correlated wavefunction-based methods we clearly predict an asymmetric core structure. This reveals a fundamental and intrinsic difference within the iron-sulfur clusters hosted by Rieske proteins and ferredoxins and thus opens up a new dimension for the ongoing efforts in understanding the role of Rieske-type [2Fe-2S] cluster in electron transfer processes that occur in almost all biological systems.