2-Monoacylglycerol Mimetic Liposomes to Promote Intestinal Lymphatic Transport for Improving Oral Bioavailability of Dihydroartemisinin.

Purpose: Reducing the first-pass hepatic effect via intestinal lymphatic transport is an effective way to increase the oral absorption of drugs. 2-Monoacylglycerol (2-MAG) as a primary digestive product of dietary lipids triglyceride, can be assembled in chylomicrons and then transported from the intestine into the lymphatic system. Herein, we propose a biomimetic strategy and report a 2-MAG mimetic nanocarrier to target the intestinal lymphatic system via the lipid absorption pathway and improve oral bioavailability. Methods: The 2-MAG mimetic liposomes were designed by covalently bonding serinol (SER) on the surface of liposomes named SER-LPs to simulate the structure of 2-MAG. Dihydroartemisinin (DHA) was chosen as the model drug because of its disadvantages such as poor solubility and high first-pass effect. The endocytosis and exocytosis mechanisms were investigated in Caco-2 cells and Caco-2 cell monolayers. The capacity of intestinal lymphatic transport was evaluated by ex vivo biodistribution and in vivo pharmacokinetic experiments. Results: DHA loaded SER-LPs (SER-LPs-DHA) had a particle size of 70 nm and a desirable entrapment efficiency of 93%. SER-LPs showed sustained release for DHA in the simulated gastrointestinal environment. In vitro cell studies demonstrated that the cellular uptake of SER-LPs primarily relied on the caveolae- rather than clathrin-mediated endocytosis pathway and preferred to integrate into the chylomicron assembly process through the endoplasmic reticulum/Golgi apparatus route. After oral administration, SER-LPs efficiently promoted drug accumulation in mesenteric lymphatic nodes. The oral bioavailability of DHA from SER-LPs was 10.40-fold and 1.17-fold larger than that of free DHA and unmodified liposomes at the same dose, respectively. Conclusion: SER-LPs improved oral bioavailability through efficient intestinal lymphatic transport. These findings of the current study provide a good alternative strategy for oral delivery of drugs with high first-pass hepatic metabolism.

Natural endoperoxides as promising anti-leishmanials.

BACKGROUND: The discovery of artemisinin, an endoperoxide, encouraged the scientific community to explore endoperoxides as potential anti-parasitic molecules. Although artemisinin derivatives are rapidly evolving as potent anti-malarials, their potential as anti-leishmanials is emerging gradually. The treatment of leishmaniasis, a group of neglected tropical diseases is handicapped by lack of effective vaccines, drug toxicities and drug resistance. The weak antioxidant defense mechanism of the Leishmania parasites due to lack of catalase and a selenium dependent glutathione peroxidase system makes them vulnerable to oxidative stress, and this has been successful exploited by endoperoxides. PURPOSE: The study aimed to review the available literature on the anti-leishmanial efficacy of natural endoperoxides with a view to achieve insights into their mode of actions. METHODS: We reviewed more around 110 research and review articles restricted to the English language, sourced from electronic bibliographic databases including PubMed, Google, Web of Science, Google scholar etc. RESULTS: Natural endoperoxides could potentially augment the anti-leishmanial drug library, with artemisinin and ascaridole emerging as potential anti-leishmanial agents. Due to higher reactivity of the cyclic peroxide moiety, and exploiting the compromised antioxidant defense of Leishmania, endoperoxides like artemisinin and ascaridole potentiate their leishmanicidal efficacy by creating a redox imbalance. Furthermore, these molecules minimally impair oxidative phosphorylation; instead inhibit glycolytic functions, culminating in depolarization of the mitochondrial membrane and depletion of ATP. Additionally, the carbon-centered free radicals generated from endoperoxides, participate in chain reactions that can generate even more reactive organic radicals that are toxic to macromolecules, including lipids, proteins and DNA, leading to cell cycle arrest and apoptosis of Leishmania parasites. However, the precise target(s) of the toxic free radicals remains open-ended. CONCLUSION: In this overview, the spectrum of natural endoperoxide molecules as major anti-leishmanials and their mechanism of action has been delineated. In view of the substantial evidence that natural endoperoxides (e.g., artemisinin, ascaridole) exert a noxious effect on different species of Leishmania, identification and characterization of other natural endoperoxides is a promising therapeutic option worthy of further pharmacological consideration.

Acidic Environment-Responsive Metal Organic Framework-Mediated Dihydroartemisinin Delivery for Triggering Production of Reactive Oxygen Species in Drug-Resistant Lung Cancer.

Background: Dihydroartemisinin (DHA) has emerged as a promising candidate for anticancer therapy. However, the application of DHA in clinics has been hampered by several limitations including poor bioavailability, short circulation life, and low solubility, significantly restricting its therapeutic efficacy and leading to notable side effects during the treatment. Purpose: We present DHA-loaded zeolitic imidazolate framework-8 (D-ZIF) with controllable and targeted DHA release properties, leading to enhanced antitumor effects while reducing potential side effects. Methods: D-ZIF was prepared by one-pot synthesis method using methylimidazole (MIM), Zn(NO3)2•6H2O and DHA. We characterized the physical and chemical properties of D-ZIF by TEM, DLS, XRD, FT-IR, and TG. We measured the drug loading efficiency and the cumulative release of DHA in different pH conditions. We evaluated the cytotoxicity of D-ZIF on renal cell carcinoma (RCC786-O), glioma cells (U251), TAX-resistant human lung adenocarcinoma (A549-TAX) cells by CCK8 in vitro. We explored the possible antitumor mechanism of D-ZIF by Western blot. We evaluated the biocompatibility and hemolysis of D-ZIF and explored the in vivo antitumor efficiency in mice model by TUNEL testing and blood biomarker evaluations. Results: D-ZIF showed rhombic dodecahedral morphology with size of 129±7.2 nm and possessed a noticeable DHA encapsulation efficiency (72.9%). After 48 hours, D-ZIF released a cumulative 70.0% of the loaded DHA at pH 6.5, and only 42.1% at pH 7.4. The pH-triggered programmed release behavior of D-ZIF could enhance anticancer effect of DHA while minimizing side effects under normal physiological conditions. Compared with the free DHA group with 31.75% of A549-TAX cell apoptosis, the percentage of apoptotic cells was approximately 76.67% in the D-ZIF group. D-ZIF inhibited tumor growth by inducing tumor cell apoptosis through the mechanism of ROS production and regulation of Nrf2/HO-1 and P38 MAPK signaling pathways. D-ZIF showed potent effects in treating tumors with high safety in vivo. Conclusion: This pH-responsive release mechanism enhanced the targeting efficiency of DHA towards tumor cells, thereby increasing drug concentration in tumor sites with negligible side effects. Herein, D-ZIF holds great promise for curing cancers with minimal adverse effects.

Binary and ternary complex formation and characterization of artemisinin with sulfobutyl ether ß-cyclodextrin and oleic acid.

Drug-resistant malaria is a global risk to the modern world. Artremisinin (ART) is one of the drugs of choice against drug-resistant (malaria) which is practically insoluble in water. The objective of our study was to improve the solubility of artemisinin (ART) via development of binary complexes of ART with sulfobutylether ß-cyclodextrins (SBE7 ß-CD), sulfobutylether ß-cyclodextrins (SBE7 ß-CD) and oleic acid (ternary complexes). These are prepared in various drugs to excipients ratios by physical mixing (PM) and solvent evaporation (SE) methods. Characterizations were achieved by powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) and attenuated total reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy. The aqueous-solubility in binary complexes was 12-folds enhanced than ternary complexes. Dissolution of binary and ternary complexes of artemisinin in simulated gastric fluid (pH 1.6) was found highest and 35 times higher for ternary SECx. The crystallinity of artemisinin was decreased in physical mixtures (PMs) while SECx exhibited displaced angles. The attenuated-intensity of SECx showed least peak numbers with more displaced-angles. SEM images of PMs and SECx showed reduced particle size in binary and ternary systems as compared to pure drug-particles. ATR-FTIR spectra of binary and ternary complexes revealed bonding interactions among artemisinin, SBE7 ß-CD and oleic acid.

Another Use for a Proven Drug: Experimental Evidence for the Potential of Artemisinin and Its Derivatives to Treat Alzheimer's Disease.

Plant-derived multitarget compounds may represent a promising therapeutic strategy for multifactorial diseases, such as Alzheimer's disease (AD). Artemisinin and its derivatives were indicated to beneficially modulate various aspects of AD pathology in different AD animal models through the regulation of a wide range of different cellular processes, such as energy homeostasis, apoptosis, proliferation and inflammatory pathways. In this review, we aimed to provide an up-to-date overview of the experimental evidence documenting the neuroprotective activities of artemi-sinins to underscore the potential of these already-approved drugs for treating AD also in humans and propose their consideration for carefully designed clinical trials. In particular, the benefits to the main pathological hallmarks and events in the pathological cascade throughout AD development in different animal models of AD are summarized. Moreover, dose- and context-dependent effects of artemisinins are noted.

Cancer nutritional-immunotherapy with NIR-II laser-controlled ATP release based on material repurposing strategy.

Enlightened by the great success of the drug repurposing strategy in the pharmaceutical industry, in the current study, material repurposing is proposed where the performance of carbonyl iron powder (CIP), a nutritional intervention agent of iron supplement approved by the US FDA for iron deficiency anemia in clinic, was explored in anti-cancer treatment. Besides the abnormal iron metabolic characteristics of tumors, serving as potential targets for CIP-based cancer therapy under the repurposing paradigm, the efficacy of CIP as a catalyst in the Fenton reaction, activator for dihydroartemisinin (DHA), thus increasing the chemo-sensitivity of tumors, as well as a potent agent for NIR-II photothermal therapy (PTT) was fully evaluated in an injectable alginate hydrogel form. The CIP-ALG gel caused a rapid temperature rise in the tumor site under NIR-II laser irradiation, leading to complete ablation in the primary tumor. Further, this photothermal-ablation led to the significant release of ATP, and in the bilateral tumor model, both primary tumor ablation and inhibition of secondary tumor were observed simultaneously under the synergistic tumor treatment of nutritional-photothermal therapy (NT/PTT). Thus, material repurposing was confirmed by our pioneering trial and CIP-ALG-meditated NT/PTT/immunotherapy provides a new choice for safe and efficient tumor therapy.

A Radical-Generating Probe to Release Free Fluorophores and Identify Artemisinin-Sensitive Cancer Cells.

The smart light-up probes have been extensively developed to image various enzymes and other bioactive molecules. Upon activation, these probes result in light-up fluorophores that exist in a protein-bound or a free form. The difference between these two forms has not yet been reported. Here, we present a pair of smart light-up probes that generate a protein-bound fluorophore and a free fluorophore upon activation by heme. Probe 8 generated a radical-attached fluorophore that predominantly existed in the free form, while probe 10 generated an α,ß-unsaturated ketone-attached fluorophore that showed extensive labeling of proteins. In live-cell imaging, probe 8 showed greater fluorescence intensity than probe 10 when low concentrations (0.1-5 µM) of the probes were used, but probe 8 was less fluorescent than probe 10 when the concentrations of the probes were high (10 µM). Finally, probe 8 was used to reflect the activation level of the endoperoxide bond in cancer cells and to effectively distinguish ART-sensitive cancer cells from ART-insensitive ones.

Incorporation of Three Different Optical Trains into the IR-MALDESI Mass Spectrometry Imaging Platform to Characterize Artemisia annua.

Artemisinin is the leading medication for the treatment of malaria and is only produced naturally in Artemisia annua. The localization of artemisinin in both the glandular and non-glandular trichomes of the plant makes it an ideal candidate for mass spectrometry imaging (MSI) as a model system for method development. Infrared matrix-assisted laser desorption electrospray ionization MSI (IR-MALDESI-MSI) has the capability to detect hundreds to thousands of analytes simultaneously, providing abundance information in conjunction with species localization throughout a sample. The development of several new optical trains and their application to the IR-MALDESI-MSI platform has improved data quality in previous proof-of-concept experiments but has not yet been applied to analysis of native biological samples, especially the MSI analysis of plants. This study aimed to develop a workflow and optimize MSI parameters, specifically the laser optical train, for the analysis of Artemisia annua with the NextGen IR-MALDESI platform coupled to an Orbitrap Exploris 240 mass spectrometer. Two laser optics were compared to the conventional set up, of which include a Schwarzschild-like reflective objective and a diffractive optical element (DOE). These optics, respectively, enhance the spatial resolution of imaging experiments or create a square spot shape for top-hat imaging. Ultimately, we incorporated and characterized three different optical trains into our analysis of Artemisia annua to study metabolites in the artemisinin pathway. These improvements in our workflow, resulted in high spatial resolution and improved ion abundance from previous work, which will allow us to address many different questions in plant biology beyond this model system.

Ruthenium-dihydroartemisinin complex: a promising new compound for colon cancer prevention via G1 cell cycle arrest, apoptotic induction, and adaptive immune regulation.

BACKGROUND: Artemisinin (ART) and its derivatives are important antimalaria agents and have received increased attention due to their broad biomedical effects, such as anticancer and anti-inflammation activities. Recently, ruthenium-derived complexes have attracted considerable attention as their anticancer potentials were observed in preclinical and clinical studies. METHODS: To explore an innovative approach in colorectal cancer (CRC) management, we synthesized ruthenium-dihydroartemisinin complex (D-Ru), a novel metal-based artemisinin derivative molecule, and investigated its anticancer, anti-inflammation, and adaptive immune regulatory properties. RESULTS: Compared with its parent compound, ART, D-Ru showed stronger antiproliferative effects on the human CRC cell lines HCT-116 and HT-29. The cancer cell inhibition of D-Ru comprised G1 cell cycle arrest via the downregulation of cyclin A and the induction of apoptosis. ART and D-Ru downregulated the expressions of pro-inflammatory cytokines IL-1ß, IL-6, and IL-8. Although ART and D-Ru did not suppress Treg cell differentiation, they significantly inhibited Th1 and Th17 cell differentiation. CONCLUSIONS: Our results demonstrated that D-Ru, a novel ruthenium complexation of ART, remarkably enhanced its parent compound's anticancer action, while the anti-inflammatory potential was not compromised. The molecular mechanisms of action of D-Ru include inhibition of cancer cell growth via cell cycle arrest, induction of apoptosis, and anti-inflammation via regulation of adaptive immunity.

Co-delivery of artemisinin and metformin via PEGylated niosomal nanoparticles: potential anti-cancer effect in treatment of lung cancer cells.

PURPOSE: Despite the advances in treatment, lung cancer is a global concern and necessitates the development of new treatments. Biguanides like metformin (MET) and artemisinin (ART) have recently been discovered to have anti-cancer properties. As a consequence, in the current study, the anti-cancer effect of MET and ART co-encapsulated in niosomal nanoparticles on lung cancer cells was examined to establish an innovative therapy technique. METHODS: Niosomal nanoparticles (Nio-NPs) were synthesized by thin-film hydration method, and their physicochemical properties were assessed by FTIR. The morphology of Nio-NPs was evaluated with FE-SEM and AFM. The MTT assay was applied to evaluate the cytotoxic effects of free MET, free ART, their encapsulated form with Nio-NPs, as well as their combination, on A549 cells. Apoptosis assay was utilized to detect the biological processes involved with programmed cell death. The arrest of cell cycle in response to drugs was assessed using a cell cycle assay. Following a 48-h drug treatment, the expression level of hTERT, Cyclin D1, BAX, BCL-2, Caspase 3, and 7 genes were assessed using the qRT-PCR method. RESULTS: Both MET and ART reduced the survival rate of lung cancer cells in the dose-dependent manner. The IC50 values of pure ART and MET were 195.2 µM and 14.6 mM, respectively while in nano formulated form their IC50 values decreased to 56.7 µM and 78.3 µM, respectively. The combination of MET and ART synergistically decreased the proliferation of lung cancer cells, compared to the single treatments. Importantly, the combination of MET and ART had a higher anti-proliferative impact against A549 lung cancer cells, with lower IC50 values. According to the result of Real-time PCR, hTERT, Cyclin D1, BAX, BCL-2, Caspase 3, and Caspase 7 genes expression were considerably altered in treated with combination of nano formulated MET and ART compared to single therapies. CONCLUSION: The results of this study showed that the combination of MET and ART encapsulated in Nio-NPs could be useful for the treatment of lung cancer and can increase the efficiency of lung cancer treatment.

Voltammetric methods for electrochemical characterization and quantification of artemether-based antimalarials.

Every year substandard and falsified (SF) artemisinin derivative-based antimalarials are responsible for the loss of 450 000 deaths and billions of GBP. The lack of infrastructure and funds to support pharmaceutical quality control in many low-and-middle-income countries contributes to this problem. This work assesses fitness for purpose of voltammetric methods for identification and quantification of artemether in the presence of excipients. Electrochemical characterization of artemether using cyclic voltammetry shows that the reduction of artemether is chemically irreversible within the potential range of -0.4 V to -1.4 V. A chronocoulometric quantification algorithm for artemether is created and tested with pure artemether, as well as filtered and unfiltered Riamet® tablets. Filtration of Riamet® tablets provides no additional benefit for the quantification of artemether in Riamet®. In addition, artemether's response to pH indicates possible protonation and coupled homogeneous chemistry. Finally, sodium sulfite is an effective means of removing dissolved oxygen and improving artemether signal resolution in air-equilibrated PBS. This concludes that electrochemical analysis is a promising method for artemether identification and quantification.

An overview on the antimalarial activity of 1,2,4-trioxanes, 1,2,4-trioxolanes and 1,2,4,5-tetraoxanes.

The demand for novel, fast-acting, and effective antimalarial medications is increasing exponentially. Multidrug resistant forms of malarial parasites, which are rapidly spreading, pose a serious threat to global health. Drug resistance has been addressed using a variety of strategies, such as targeted therapies, the hybrid drug idea, the development of advanced analogues of pre-existing drugs, and the hybrid model of resistant strains control mechanisms. Additionally, the demand for discovering new potent drugs grows due to the prolonged life cycle of conventional therapy brought on by the emergence of resistant strains and ongoing changes in existing therapies. The 1,2,4-trioxane ring system in artemisinin (ART) is the most significant endoperoxide structural scaffold and is thought to be the key pharmacophoric moiety required for the pharmacodynamic potential of endoperoxide-based antimalarials. Several derivatives of artemisinin have also been found as potential treatments for multidrug-resistant strain in this area. Many 1,2,4-trioxanes, 1,2,4-trioxolanes, and 1,2,4,5-tetraoxanes derivatives have been synthesised as a result, and many of these have shown promise antimalarial activity both in vivo and in vitro against Plasmodium parasites. As a consequence, efforts to develop a functionally straight-forward, less expensive, and vastly more effective synthetic pathway to trioxanes continue. This study aims to give a thorough examination of the biological properties and mode of action of endoperoxide compounds derived from 1,2,4-trioxane-based functional scaffolds. The present system of 1,2,4-trioxane, 1,2,4-trioxolane, and 1,2,4,5-tetraoxane compounds and dimers with potentially antimalarial activity will be highlighted in this systematic review (January 1963-December 2022).

Inhibition of Cysteine Proteases via Thiol-Michael Addition Explains the Anti-SARS-CoV-2 and Bioactive Properties of Arteannuin B.

Artemisia annua is the plant that produces artemisinin, an endoperoxide-containing sesquiterpenoid used for the treatment of malaria. A. annua extracts, which contain other bioactive compounds, have been used to treat other diseases, including cancer and COVID-19, the disease caused by the virus SARS-CoV-2. In this study, a methyl ester derivative of arteannuin B was isolated when A. annua leaves were extracted with a 1:1 mixture of methanol and dichloromethane. This methyl ester was thought to be formed from the reaction between arteannuin B and the extracting solvent, which was supported by the fact that arteannuin B underwent 1,2-addition when it was dissolved in deuteromethanol. In contrast, in the presence of N-acetylcysteine methyl ester, a 1,4-addition (thiol-Michael reaction) occurred. Arteannuin B hindered the activity of the SARS CoV-2 main protease (nonstructural protein 5, NSP5), a cysteine protease, through time-dependent inhibition. The active site cysteine residue of NSP5 (cysteine-145) formed a covalent bond with arteannuin B as determined by mass spectrometry. In order to determine whether cysteine adduction by arteannuin B can inhibit the development of cancer cells, similar experiments were performed with caspase-8, the cysteine protease enzyme overexpressed in glioblastoma. Time-dependent inhibition and cysteine adduction assays suggested arteannuin B inhibits caspase-8 and adducts to the active site cysteine residue (cysteine-360), respectively. Overall, these results enhance our understanding of how A. annua possesses antiviral and cytotoxic activities.

In Vitro Inhibition of Xanthine Oxidase Purified from Arthritis Serum Patients by Nanocurcumin and Artemisinin Active Compounds.

Curcumin and artemisinin are commonly used in traditional East Asian medicine. In this study, we investigated the inhibitory effects of these active compounds on xanthine oxidase (XO) using allopurinol as a control. XO was purified from the serum of arthritis patients through ammonium sulfate precipitation (65%) and ion exchange chromatography on diethylaminoethyl (DEAE)-cellulose. The specific activity of the purified enzyme was 32.5 U/mg protein, resulting in a 7-fold purification with a yield of 66.8%. Molecular docking analysis revealed that curcumin had the strongest interaction energy with XO, with a binding energy of -9.28 kcal/mol. The amino acid residues Thr1077, Gln762, Phe914, Ala1078, Val1011, Glu1194, and Ala1079 were located closer to the binding site of curcumin than artemisinin, which had a binding energy of -7.2 kcal/mol. In vitro inhibition assays were performed using nanocurcumin and artemisinin at concentrations of 5, 10, 15, 20, and 25 µg/mL. Curcumin inhibited enzyme activity by 67-91%, while artemisinin had a lower inhibition ratio, which ranged from 40-70% compared to allopurinol as a control.

Novel Bis-Artemisinin-Phloroglucinol hybrid molecules with dual anticancer and immunomodulatory Activities: Synthesis and evaluation.

Bis-(10-deoxydihydroartemisinin)-phloroglucinol (9), has been synthesized in a one-step reaction and has demonstrated strong inhibition to cancer cell proliferation and immunosuppressive activity. The structure modification of the compound reduced its cytotoxicity, and among the analogs, bis-(10-deoxydihydroartemisinin)-phloroglucinol phenyl decanoate (16) showed significant reduction of ear swelling in a mouse model for DNFB-induced delayed-type hypersensitivity without observable toxicity in a dose-dependent manner.

Design, Synthesis, and Antitumor Activities of Isomers of Artemisinin Dimer Derivatives.

In recent years, numerous studies have reported on the anti-tumor properties of artemisinin and its derivatives. However, the relationship between their artemisinin chirality and activity remains unknown. In this study, we synthesized a series of artemisinin dimer derivatives with three different chiral structures and tested their antiproliferative activity in MCF-7 and HepG2 cells using the CCK-8 assay. Interestingly, we discovered that artemisinin dimer derivatives with ß, ß and α, ß conformations at C-10 exhibited stronger anti-tumor activity than those with an α, α configuration in MCF-7 and HepG2 cells. Notably, compound 4 showed an activity of 0.06 µM in MCF-7 cells. This study demonstrates the relationship between the conformation and activity of artemisinin dimer derivatives, and these derivatives have the potential to be developed into anti-cancer drugs.

Therapeutical Utilization and Repurposing of Artemisinin and Its Derivatives: A Narrative Review.

Artemisinin (ART) and its derivatives have great therapeutical utility as antimalarials and can be repurposed for other indications, such as viral infections, autoimmune diseases, and cancer. This review presents a comprehensive overview of the therapeutic effects of ART-based drugs, beyond their antimalarial effects. This review also summarizes the information on their repurposing in other pathologies, with the hope that it will guide the future optimization of the use of ART-based drugs and of the treatment strategies for the listed diseases. By reviewing related literature, ART extraction and structure as well as the synthesis and structure of its derivatives are presented. Subsequently, the traditional roles of ART and its derivatives against malaria are reviewed, including antimalarial mechanism and occurrence of antimalarial resistance. Finally, the potential of ART and its derivatives to be repurposed for the treatment of other diseases are summarized. The great repurposing potential of ART and its derivatives may be useful for the control of emerging diseases with corresponding pathologies, and future research should be directed toward the synthesis of more effective derivatives or better combinations.

RNA sequencing in Artemisia annua L explored the genetic and metabolic responses to hardly soluble aluminum phosphate treatment.

Artemisia annua L. is a medicinal plant valued for its ability to produce artemisinin, a molecule used to treat malaria. Plant nutrients, especially phosphorus (P), can potentially influence plant biomass and secondary metabolite production. Our work aimed to explore the genetic and metabolic response of A. annua to hardly soluble aluminum phosphate (AlPO4, AlP), using soluble monopotassium phosphate (KH2PO4, KP) as a control. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze artemisinin. RNA sequencing, gene ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to analyze the differentially expressed genes (DEGs) under poor P conditions. Results showed a significant reduction in plant growth parameters, such as plant height, stem diameter, number of leaves, leaf areas, and total biomass of A. annua. Conversely, LC-MS analysis revealed a significant increase in artemisinin concentration under the AlP compared to the KP. Transcriptome analysis revealed 762 differentially expressed genes (DEGs) between the AlP and the KP. GH3, SAUR, CRE1, and PYL, all involved in plant hormone signal transduction, showed differential expression. Furthermore, despite the downregulation of HMGR in the artemisinin biosynthesis pathway, the majority of genes (ACAT, FPS, CYP71AV1, and ALDH1) were upregulated, resulting in increased artemisinin accumulation in the AlP. In addition, 12 transcription factors, including GATA and MYB, were upregulated in response to AlP, confirming their importance in regulating artemisinin biosynthesis. Overall, our findings could contribute to a better understanding the parallel transcriptional regulation of plant hormone transduction and artemisinin biosynthesis in A. annua L. in response to hardly soluble phosphorus fertilizer.

Late-Stage C(sp2 )-H Arylation of Artemisinic Acid and Arteannuin B: Effect of Olefin Migration Towards Synthesis of C-13 Arylated Artemisinin Derivatives.

In recent years, C-H bond functionalization has emerged as a pivotal tool for late-stage functionalization of complex natural products for the synthesis of potent biologically active derivatives. Artemisinin and its C-12 functionalized semi-synthetic derivatives are well-known clinically used anti-malarial drugs due to the presence of the essential 1,2,4-trioxane pharmacophore. However, in the wake of parasite developing resistance against artemisinin-based drugs, we conceptualized the synthesis of C-13 functionalized artemisinin derivatives as new antimalarials. In this regard, we envisaged that artemisinic acid could be a suitable precursor for the synthesis of C-13 functionalized artemisinin derivatives. Herein, we report C-13 arylation of artemisinic acid, a sesquiterpene acid and our attempts towards synthesis of C-13 arylated artemisinin derivatives. However, all our efforts resulted in the formation of a novel ring-contracted rearranged product. Additionally, we have extended our developed protocol for C-13 arylation of arteannuin B, a sesquiterpene lactone epoxide considered to be the biogenetic precursor of artemisinic acid. Indeed, the synthesis of C-13 arylated arteannuin B renders our developed protocol to be effective in sesquiterpene lactone as well.

Mitigating the risk of antimalarial resistance via covalent dual-subunit inhibition of the Plasmodium proteasome.

The Plasmodium falciparum proteasome constitutes a promising antimalarial target, with multiple chemotypes potently and selectively inhibiting parasite proliferation and synergizing with the first-line artemisinin drugs, including against artemisinin-resistant parasites. We compared resistance profiles of vinyl sulfone, epoxyketone, macrocyclic peptide, and asparagine ethylenediamine inhibitors and report that the vinyl sulfones were potent even against mutant parasites resistant to other proteasome inhibitors and did not readily select for resistance, particularly WLL that displays covalent and irreversible binding to the catalytic ß2 and ß5 proteasome subunits. We also observed instances of collateral hypersensitivity, whereby resistance to one inhibitor could sensitize parasites to distinct chemotypes. Proteasome selectivity was confirmed using CRISPR/Cas9-edited mutant and conditional knockdown parasites. Molecular modeling of proteasome mutations suggested spatial contraction of the ß5 P1 binding pocket, compromising compound binding. Dual targeting of P. falciparum proteasome subunits using covalent inhibitors provides a potential strategy for restoring artemisinin activity and combating the spread of drug-resistant malaria.