Antimalarial Artemisinin Research Articles

Protein abundance and folding rather than the redox state of Kelch13 determine the artemisinin susceptibility of Plasmodium falciparum

Decreased susceptibilities of the human malaria parasite Plasmodium falciparum towards the endoperoxide antimalarial artemisinin are linked to mutations of residue C580 of PfKelch13, a homologue of the redox sensor Keap1 and other vertebrate BTB-Kelch proteins. Here, we addressed whether mutations alter the artemisinin susceptibility by modifying the redox properties of PfKelch13 or by compromising its native fold or abundance. Using selection-linked integration and the glmS ribozyme, efficient down-regulation of PfKelch13 resulted in ring-stage survival rates around 40%. While the loss of the thiol group of C469 or of the potential disulfide bond between residues C580 and C532 had no effect on the artemisinin susceptibility, the thiol group of C473 could not be replaced. Furthermore, we detected two different forms of PfKelch13 with distinct electrophoretic mobilities around 85 and 95 kDa, suggesting an unidentified post-translational modification. We also established a protocol for the production of recombinant PfKelch13 and produced an antibody against the protein. Recombinant PfKelch13 adopted alternative oligomeric states and only two of its seven cysteine residues, C469 and C473, reacted with Ellman’s reagent. While common field mutations resulted in misfolded and completely insoluble recombinant PfKelch13, cysteine-to-serine replacements had no effect on the solubility except for residue C473. In summary, in contrast to residues C469, C532, and C580, the surface-exposed thiol group of residue C473 appears to be essential. However, not the redox properties but impaired folding of PfKelch13, resulting in a decreased PfKelch13 abundance, alters the artemisinin susceptibility and is the central parameter for mutant selection.

Solubility analysis of pharmaceuticals guaifenesin, ketoprofen, and artemisinin in different solvents

Solubility is a fundamental parameter in the discovery of new drug molecules as well as in their production using crystallization processes. In this work, we experimentally investigate the solubility in different solvents and the density of the anti-cough drug guaifenesin and characterize its solid state via Powder X-Ray Diffraction (PXRD) and thermal analysis. Moreover, a strategy is developed and successfully applied to predict the density and solubility behavior of that pharmaceutical. On the solubility curve concentration and temperature depend on each other for a binary solution at fixed pressure. Therefore, densities of undersaturated aqueous solutions of guaifenesin are measured and used alongside with solubility data to obtain more realistic parameters of thermodynamic model. Perturbed Chain-Statistical Associating Fluid Theory (PC-SAFT) Equation of State (EoS) is used. It was able to successfully represent the solubility curves using a small amount of data for fitting the model parameters. In a second part, the applicability of the model to predict the solubility in various organic solvents of two other pharmaceutically relevant molecules, namely the anti-inflammatory drug ketoprofen and the antimalarial compound artemisinin has been studied.The results showed that guaifenesin is the most soluble in ethanol and least soluble in ethyl acetate. Its aqueous solubility shows a special behavior with a specific sharp increase with temperature. In all of the solvents, ketoprofen is the highest soluble among the compounds studied followed by guaifenesin and artemisinin. PC-SAFT is able to capture the experimentally observed effects of temperature, and solvent type on the solubility behavior of each of the three solutes.

African and Asian Medicinal Plants as a Repository for Prospective Antiviral Metabolites Against HIV-1 and SARS CoV-2: A Mini Review.

Introduction: The worldwide burden of viral infections has triggered a resurgence in the search for new and more efficient antiviral drugs. Scientists are also repurposing existing natural compounds such as the antimalarial drug artemisinin from Artemesia annua L. as potential drug candidates for some of the emerging and re-emerging viral infections such as covid-19Aim: The aim of this review was to analyse the existing literature to explore the actual or potential natural antiviral compounds from African and Asian medicinal plants as lead compounds in the drug discovery process.Methods: We searched the literature on African and Asian medicinal plant species as antiviral agents for HIV-1 and the novel coronavirus (SARS-CoV-2) in various databases and search engines such as Web of Science, Google Scholar and PubMed. The search was limited to in vitro, in vivo, and clinical studies and excluded in silico studies.Results: We present 16 plant species with actual or potential antiviral activity against HIV-1 and SARS-CoV-2. These plant species span the continents of Africa and Asia where they are widely used for treating several other ailments.Conclusion: Natural compounds from plants can play a significant role in the clinical management of HIV/AIDS and the covid-19 pandemic. More research needs to be conducted to investigate the potential toxicities of the various compounds and their efficacies in clinical settings.

Synthesis of a novel naphthalenone endoperoxide and structural elucidation by nuclear magnetic resonance spectroscopy and theoretical calculation.

Sesquiterpene lactones are found in plants of Asteraceae family, and endoperoxides are known for their antimalarial activity. Structural elucidation is a relevant aspect; however, it is not uncommon to find incorrect or incomplete structural assignments in the literature. Calculations based in quantum mechanics are frequently used to compute 1 H and 13 C NMR chemical shifts, and after comparing with the experimental data, the correct structure is established from diverse candidates. Targeting the synthesis of bioactive compounds, we envisaged the synthesis of a novel endoperoxide from the natural sesquiterpene lactone α-santonin (2). Photochemical transformation of α-santonin (2) to mazdasantonin (4) followed by photooxidation catalyzed by rose bengal afforded the novel endoperoxide 5. This new endoperoxide contains five stereogenic centers and is analogous to the antimalarial agent artemisinin (1). The relative configuration of the stereogenic centers of the endoperoxide were established by nuclear magnetic resonance (NMR) analyses and confirmed by theoretical calculations. All approaches were in complete agreement, and the structure of mazdasantonin endoperoxide was established as (3S,3aS,5aS,8R,9bS)-3,6,6-trimethyl-3,3a,4,5,8,9b-hexahydro-2H-5a,8-epidioxynaphtho[1,2-b]furan-2,7(6H)-dione.

Structure-Activity Relationships of the Antimalarial Agent Artemisinin 10. Synthesis and Antimalarial Activity of Enantiomers of rac-5β-Hydroxy-d-Secoartemisinin and Analogs: Implications Regarding the Mechanism of Action.

An efficient synthesis of rac-6-desmethyl-5β–hydroxy-d-secoartemisinin 2, a tricyclic analog of R-(+)-artemisinin 1, was accomplished and the racemate was resolved into the (+)-2b and (−)-2a enantiomers via their Mosher Ester diastereomers. Antimalarial activity resided with only the artemisinin-like enantiomer R-(−)-2a. Several new compounds 9–16, 19a, 19b, 22 and 29 were synthesized from rac-2 but the C-5 secondary hydroxyl group was surprisingly unreactive. For example, the formation of carbamates and Mitsunobu reactions were unsuccessful. In order to assess the unusual reactivity of 2, a single crystal X-ray crystallographic analysis revealed a close intramolecular hydrogen bond from the C-5 alcohol to the oxepane ether oxygen (O-11). All products were tested in vitro against the W-2 and D-6 strains of Plasmodium falciparum. Several of the analogs had moderate activity in comparison to the natural product 1. Iron (II) bromide-promoted rearrangement of 2 gave, in 50% yield, the ring-contracted tetrahydrofuran 22, while the 5-ketone 15 provided a monocyclic methyl ketone 29 (50%). Neither 22 nor 29 possessed in vitro antimalarial activity. These results have implications in regard to the antimalarial mechanism of action of artemisinin.

Singlet Oxygen Generation from a Water-Soluble Hypervalent Iodine(V) Reagent AIBX and H2O2: An Access to Artemisinin

Herein, we report an efficient method for the chemical generation of 1O2 by treatment of H2O2 with AIBX, a highly water-soluble, bench-stable, recyclable hypervalent iodine(V) reagent developed by our group. The generation of 1O2 was confirmed by the following results: (1) capture of 1O2 with the sodium salt of anthracene-9,10-bis(ethanesulfonate) produced the corresponding endoperoxide and (2) TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) produced by the oxidation of 2,2,6,6-tetramethylpiperidine with 1O2 generated using the AIBX/H2O2 system was detected by electron spin resonance spectroscopy. To illustrate the potential utility of this method for organic synthesis, we used the AIBX/H2O2 system to perform typical reactions of 1O2: [2 + 2]/[4 + 2] cycloadditions, Schenck ene reactions, and heteroatom oxidation reactions, which afforded the corresponding products in high yields. Moreover, we used the method to synthesize the antimalarial drug artemisinin. Finally, we demonstrated that AIBX could be regenerated after the reaction by means of a workup involving extraction and removal of water to obtain a precursor of AIBX, which could then be re-oxidized.

Palladium-Catalyzed Regioselective Allylic Oxidation of Amorphadiene, a Precursor of Artemisinin.

A regioselective Pd-catalyzed allylic oxidation of amorphadiene, a key precursor to the antimalarial drug artemisinin, is described. Amorphadiene can be obtained in high yields by fermentation, but it is currently treated as a waste in the industrial semisynthetic artemisinin process. The catalytic step described here is a substitute for the P450 enzymes involved in the artemisinin biosynthesis and opens up new opportunities to supplement a critical step in the current semisynthetic route and increase the potential of the fermentation process.

Has artemisinin resistance emerged in Africa?

Earlier this century, Plasmodium falciparum malaria parasites developed the ability to evade the action of artemisinin antimalarials. This delayed clearance phenotype, referred to as artemisinin resistance, is widespread in the Greater Mekong Subregion of southeast Asia, where it is associated with treatment failures for some artemisinin-based combination therapies (ACTs), the drugs of choice to treat P falciparum malaria.1 However, there is little evidence of artemisinin resistance beyond southeast Asia.

Artemisinin protects DPSC from hypoxia and TNF-α mediated osteogenesis impairments through CA9 and Wnt signaling pathway

Dental pulp stem cells (DPSCs) possess the ability of multi-lineage differentiation, and are excellent sources of tissue engineering and regenerative medicine. Oxygen concentration and inflammation are two critical environmental factors that affect the osteogenic differentiation of DPSCs. We aimed to study the role of the antimalarial drug artemisinin on the osteogenic differentiation of human DPSCs under the hypoxia and inflammation conditions. We demonstrated that hypoxia (5% O2) and inflammation (20 ng/mL TNF-α), alone or in combination, significantly diminished in vitro cell survival and increased apoptotic rates. Notably, hypoxia and TNF-α exerted accumulative effect in suppressing the osteogenic differentiation of DPSCs, as evidenced by reduced expression levels of osteogenesis-associated genes including ALP, RUNX2 and OCN in osteogenic condition, as well as reduced mineral nodules formation as indicated by alizarin red staining. Artemisinin at the dose of 40 μM markedly reversed the suppression in cell survival caused by hypoxia or inflammation, and reduced apoptotic rates and the expressions of pro-apoptotic proteins. Additionally, artemisinin restored osteogenic differentiation of DPSCs under the hypoxia or/and inflammation conditions. Moreover, the beneficial effect of artemisinin was dependent on upregulated expression of CA9 and CA9-mediated antioxidant responses, as CA9 knockdown abolished the protective role of artemisinin on DPSC osteogenesis. Furthermore, while hypoxia or/and inflammation significantly inactivated the Wnt/β-catenin signaling in DPSCs, additional exposure to artemisinin re-activated this pathway to promote osteogenic differentiation of DPSCs. Our results provide novel insight on the link between artemisinin and DPSC osteogenesis, and suggest promising artemisinin-based strategies for better dentin/pulp tissue engineering.

Repurposing Quinoline and Artemisinin Antimalarials as Therapeutics for SARS-CoV-2: Rationale and Implications.

The coronavirus disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 116 million individuals globally and resulted in over 2.5 million deaths since the first report in December 2019. For most of this time, healthcare professionals have had few tools at their disposal. In December 2020, several vaccines that were shown to be highly effective have been granted emergency use authorization (EUA). Despite these remarkable breakthroughs, challenges include vaccine roll-out and implementation, in addition to deeply entrenched antivaccination viewpoints. While vaccines will prevent disease occurrence, infected individuals still need treatment options, and repurposing drugs circumvents the lengthy and costly process of drug development. SARS-CoV-2, like many other enveloped viruses, require the action of host proteases for entry. In addition, this novel virus employs a unique method of cell exit of deacidified lysosomes and exocytosis. Thus, inhibitors of lysosomes or other players in this pathway are good candidates to target SARS-CoV-2. Chemical compounds in the quinoline class are known to be lysomotropic and perturb pH levels. A large number of quinolines are FDA-approved for treatment of inflammatory diseases and antimalarials. Artemisinins are another class of drugs that have been demonstrated to be safe for use in humans and are widely utilized as antimalarials. In this Review, we discuss the use of antimalarial drugs in the class of quinolines and artemisinins, which have been shown to be effective against SARS-CoV-2 in vitro and in vivo, and provide a rationale in employing quinolines as treatment of SARS-CoV-2 in clinical settings.

Biotransformation of artemisinic acid to bioactive derivatives by endophytic Penicillium oxalicum B4 from Artemisia annua L.

As a biosynthetic precursor of the antimalarial drug artemisinin, artemisinic acid (AA) is abundant in Artemisia annua L. with a content of 8-10-fold higher than artemisinin, but less effective. In this study, the biotransformation of AA was carried out with an endophytic fungus Penicillium oxalicum B4 to extend its utility. After 10-day-culture of the endophyte with AA at 2 mg/mL, eight biotransformation metabolites were isolated from the culture broth, including five undescribed metabolites, namely 3α,14-dihydroxyartemisinic acid, 14-hydroxy-3-oxo-artemisinic acid, 15-hydroxy-3-oxo-artemisinic acid, 12, 15-artemisindioic acid and 1,2,3,6-tetradehydro-12, 15-artemisindioic acid. The fungal enzymes possess the selective capacity to hydroxylate, carbonylate and ketonize the allyl group of AA. The major biotransformation metabolite was the hydroxylated product 3-α-hydroxyartemisinic acid (33.3%) in the cultures of early stage (day 1–6), whereas most of the other biotransformation products were synthesized in the later stage (day 8–10). Compared with AA, some metabolites (3α,14-dihydroxyartemisinic acid, 15-hydroxy-3-oxo-artemisinic acid and 1,2,3,6-tetradehydro-12, 15-artemisindioic acid) possessed stronger cytotoxic activity to the human colon carcinoma cell line (LS174T) and promyelocytic leukemia cell line (HL-60). The metabolites 12, 15-artemisindioic acid and 3-α-hydroxyartemisinic acid exhibited significant inhibitory activity to the lipopolysaccharide-induced nitrite production of RAW 264.7 cells at 10.00 μM and 2.50 μM, respectively. The results demonstrated the potential of fungal endophytes on biotransforming AA to its bioactive derivatives.

Interaction of artemisinin protects the activity of antioxidant enzyme catalase: A biophysical study

The major antioxidant enzyme catalase is downregulated and the enzyme activity is compromised in various disease conditions such as malarial and cancer. Hence, the restoration and protection of catalase is a promising therapeutic strategy in disease management. In the present study, for the first time we have demonstrated the protective role of well-known anti-malarial drug Artemisinin (ART) on the time and temperature-induced degradation of bovine liver catalase (BLC) activity. The findings at different time intervals and at higher temperature showed the protective role of ART on BLC activity. Molecular docking studies suggested specific binding of ART on BLC through heme group interface which was further supported by cyclic voltammetry and dynamic light scattering study. The stabilization of BLC in presence of ART was mediated through forming a BLC-ART complex with reduced and shifted electrochemical peak and increased hydrodynamic diameter. ART substantially prevents the temperature-induced reduction in α-helical content with simultaneous increment in other secondary structures like antiparallel, parallel, β-turn and random coils. Nevertheless, the protective role of ART was accepted from the enhanced thermal stability and increased Tm value of BLC in presence of ART at higher temperatures. Our results uncover the mechanism of interaction between ART with BLC and suggest the protective role of ART towards spatiotemporal alteration of BLC by preventing the structural and molecular change in BLC. Thus, the findings advocate ART as a potential therapeutic drug for diseases associated with reduced catalase activity.

Computational Study of the Interactions between Antimalarial Chemotherapies with Folate Pathway Receptors and Telomerase Reverse Transcriptase

Malaria is a life-threatening disease responsible for half a million death annually, and with nearly half of the world’s population at risk. The rapid drop in observed cases of malaria in the last two decades has been due to a combination of preventive and therapeutic remedies. However, the absence of a vaccine, new antimalarial chemotherapies and increased parasitic resistance have led to a plateau of infections and renewed research interest in target human and Plasmodium (the malaria parasite) receptors and new drugs. In this study, the impact of mutation on the affinity on antimalarial drugs with the bifunctional enzyme complex, dihydrofolate reductase (DHFR) is explored. In addition, homology modeling is used to build the three-dimensional models of the enzymes Plasmodium telomerase reverse-transcriptase (pf-TERT) and Plasmodium dihydropteroate synthetase (pf-DHPS) to determine their affinity with antimalarial drugs. The interaction energies and stable complexes formed between these enzymes and antimalarial drugs (chloroquine, artemisinin, primaquine, pyrimethamine, sulfadoxine and pentamidine) were modelled using AutoDock vina. Our data indicate that pf-TERT and pf-DHPS form stable complexes with the antimalarial ligands with affinity ranging from ?4.0 to ?6.9 kcal/mol. The affinity with crystal structures of DHFR receptors was higher ranging from ?6.0 to ?10.0 kcal/mol. The affinity to DHFR also decreases with the mutation a confirmation of the source of resistance. The highest affinity interaction for all the receptors modeled is observed with Artemisinin a benchmark antimalarial drug. This can be attributed to the size, shape and dipolar surface of the ligand. The observed complexes are stabilized by strategic active site polar and non-polar contacts.

Artemisinin Reverses Glucocorticoid-Induced Injury in Bone Marrow-Derived Mesenchymal Stem Cells through Regulation of ERK1/2-CREB Signaling Pathway.

Glucocorticoids are the most common cause of secondary osteoporosis, which affects both women (pre- and postmenopausal) and men. In cases of prolonged treatment, glucocorticoids promote the loss and inactivation of the differentiational function of bone marrow mesenchymal stromal cells (BMSCs), risking the development of skeletal system diseases such as osteoporosis. This study reports for the first time the protective effect of the antimalarial artemisinin against glucocorticoid-induced insults on primary cultured rat BMSCs. At relatively low concentrations, artemisinin treatment improved BMSC survival by promoting a decline of reactive oxygen species (ROS) production that correlated with the decrease of caspase-3 activation, LDH release, mitochondrial membrane potential (Δψm) loss, and apoptosis induced by dexamethasone (DEXA). In addition, artemisinin improved the osteogenic differentiation of DEXA-damaged cells. DEXA inhibited extracellular-signal-regulated kinase 1/2 (ERK1/2) and cAMP response element binding protein (CREB) phosphorylation, and artemisinin treatment promoted their activation in a concentration-dependent manner. PD98059, the specific inhibitor of the ERK1/2 pathway, blocked ERK1/2 phosphorylation and artemisinin protection. Similarly, siCREB attenuated the protective effect of artemisinin, strongly suggesting the involvement of the ERK1/2-CREB pathway in the protective action of artemisinin against DEXA-induced damage in BMSCs. In addition, we found that the expression of antiapoptotic protein B-cell lymphoma 2 protein (BCL-2) was also upregulated by artemisinin. These studies demonstrate the therapeutic potential of artemisinin in the survival improvement of BMSCs exposed to glucocorticoid-induced apoptosis and suggest that artemisinin-mediated protection may occur via the activation of ERK1/2-CREB signaling pathway.

Oral Bioavailability Comparison of Artemisinin, Deoxyartemisinin, and 10-Deoxoartemisinin Based on Computer Simulations and Pharmacokinetics in Rats.

Deoxyartemisinin, a compound separated from Artemisinin annua L., shows anti-inflammatory and antiulcer activities. 10-Deoxoartemisinin is a novel compound with a strong antimalarial effect derivatized from artemisinin. Compared to the famous antimalarial natural compound artemisinin, deoxyartemisinin lacks the peroxide bridge structure, while 10-deoxoartemisinin remains this special peroxide bridge group but loses the 10-position keto group. To clarify their pharmacological differences, the absorption, distribution, metabolism, excretion (ADME) properties of artemisinin, deoxyartemisinin, and 10-deoxoartemisinin were first predicted using QikProp software. Also, their pharmacokinetic behaviors in rats were further evaluated by a rapid, sensitive, and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method after oral and intravenous administration of each compound, in which deoxyartemisinin and 10-deoxoartemisinin were first evaluated for their pharmacokinetics. All parameters about ADME properties calculated by software met the criteria and the ADME performance order was 10-deoxoartemisinin > deoxyartemisinin > artemisinin. The oral bioavailability of artemisinin was calculated to be 12.2 ± 0.832%, which was about 7 times higher than that of deoxyartemisinin (1.60 ± 0.317%). For 10-deoxoartemisinin, its bioavailability (26.1 ± 7.04%) was superior to artemisinin at a degree of more than twice. Considering their chemical structures, losing the peroxide bridge might decrease the absorption rate of deoxyartemisinin in the gastrointestinal tract, while retaining the peroxide bridge but losing the 10-position ketone might improve the bioavailability of 10-deoxoartemisinin.

Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission

The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Å resolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

A Review on Natural Sources of Antimalarials: Fungi and Plants

Recent progress in drug discovery in past decades reduced the high level of pain experienced due to infectious diseases by humans. A significant impact has been made on developing novel antimicrobial agents but is failing regularly due to development of resistance to drugs by the parasite or microbe. A continuing need of antimicrobials is there to protect humans from resistant microbes from different sources such as semisynthetic sources, synthetic and natural sources. Research in developing natural antimicrobials such as plant and fungal sources is still in its initial stages. In this review, we have concentrated on devastating disease malaria which is seen in several parts of world. Even though there are newly developed antimalarial artemisinin based combination therapy, recent reports are suggesting that resistance may also develop soon against the newer artemesinin products. Our search for literature and survey for natural antimicrobials motivated us to give concern to fungal source as promising antimalarial and most unexplored source for the same. This review throws light on recent reports on disease malaria, causative species and outline of its lifecycle, targets of drug action and mainly the fungal sources of antimalarial.

The WRKY transcription factor AaGSW2 promotes glandular trichome initiation in Artemisia annua.

Glandular secreting trichomes (GSTs) synthesize and secrete large quantities of secondary metabolites, some of which have well-established commercial value. An example is the anti-malarial compound artemisinin, which is synthesized in the GSTs of Artemisia annua. Accordingly, there is considerable interest in understanding the processes that regulate GST density as a strategy to increase artemisinin production. In this study, we identified a GST-specific WRKY transcription factor from A. annua, AaGSW2, which is positively regulated by the direct binding of the homeodomain proteins AaHD1 and AaHD8 to the L1-box of the AaGSW2 promoter. Overexpression of AaGSW2 in A. annua significantly increased GST density, while AaGSW2 knockdown lines showed impaired GST initiation. Ectopic expression of AaGSW2 homologs from two mint cultivars, Mentha spicata and Mentha haplocalyx, in A. annua also induced GST formation. These results reveal a molecular mechanism involving homeodomain and WRKY proteins that controls glandular trichome initiation, at least part of which is shared by A. annua and mint.

First principle study of silver nanoparticle interactions with antimalarial drugs extracted from Artemisia annua plant.

Silver nanoparticles have a great potential in a broad range of applications such as drug-delivery carriers because of their antiviral and antibacterial properties. In this study, the coating properties of silver nanoparticle (size range of 1.6 nm) with three common anti-malarial drugs, Artemisinin, Artemether, and Artesunate have been studied by using the quantum mechanical and classical atomistic molecular dynamics simulation in order to use as the drug delivery to treat malaria and COVID-19 diseases. The optimized structure, frequencies, charge distribution, and the electrostatic potential maps of the three drug molecules were simulated by using the density functional theory (DFT) at the B3LYP/6-311++g(d,p) level of theory. Then, molecular dynamics simulation was used to study the coating of AgNP with each of these drugs. The affinity of interaction was obtained as Artesunate > Artemether > Artemisinin which is in agreement with the DFT results on the adsorption of drugs on the Ag(111) slab.

High level production of amorphadiene using Bacillus subtilis as an optimized terpenoid cell factory

The anti-malarial drug artemisinin, produced naturally in the plant Artemisia annua, experiences unstable and insufficient supply as its production relies heavily on the plant source. To meet the massive demand for this compound, metabolic engineering of microbes has been studied extensively. In this study, we focus on improving the production of amorphadiene, a crucial artemisinin precursor, in Bacillus subtilis. The expression level of the plant-derived amorphadiene synthase (ADS) was upregulated by fusion with green fluorescent protein (GFP). Furthermore, a co-expression system of ADS and a synthetic operon carrying the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway genes was established. Subsequently, farnesyl pyrophosphate synthase (FPPS), a key enzyme in formation of the sesquiterpene precursor farnesyl pyrophosphate (FPP), was expressed to supply sufficient substrate for ADS. The consecutive combination of these features yielded a B. subtilis strain expressing chromosomally integrated GFP-ADS followed by FPPS and a plasmid encoded synthetic operon showing a stepwise increased production of amorphadiene. An experimental design-aided systematic medium optimization was used to maximize the production level for the most promising engineered B. subtilis strain, resulting in an amorphadiene yield of 416 ± 15 mg/L, which is 20-fold higher than that previously reported in B. subtilis and more than double the production in Escherichia coli or Saccharomyces cerevisiae on a shake flask fermentation level.