Synthesis of artemisinin dimers using the Ugi reaction and their in vitro efficacy on breast cancer cells

The Ugi four-component reaction was used to prepare a series of artemisinin monomers and dimers. We found that the endoperoxide group in artemisinin remains intact during the reaction. The new artemisinin dimers showed potent anti-cancer activity against two human breast cancer cell lines, MDA-MB-231 and BT-474. One of the Ugi artemisinin dimers showed an IC₅₀ value of 12 nM when tested on BT474 cells, more than 600 times more potent than artesunate. Furthermore, the same Ugi artemisinin dimer showed a low toxicity when tested on MCF10A, a nontumorigenic cell line, resulting in a selectivity index of more than 8000.     

青蒿素生物合成研究进展

本文论述了青蒿素生物合成的研究进展,涉及生物合成和储藏位点、生物合成的中间体和前体、生物合成的途径以及生物途径代谢调节的关键酶等方面。     

Plasmodium chabaudi: efficacy of artemisinin + curcumin combination treatment on a clone selected for artemisinin resistance in mice

Recent studies have proposed curcumin as a potential partner for artemisinin in artemisinin combination therapies to treat malaria infections. The efficacy of curcumin alone and in combination with artemisinin was evaluated on a clone of Plasmodium chabaudi selected for artemisinin resistance in vivo. The addition of piperine as an enhancer of curcumin activity was also tested.Results indicated that curcumin, both alone and in combination with piperine had only a modest antimalarial effect and was not able to reverse the artemisinin-resistant phenotype or significantly affect growth of the tested clone when used in combination with artemisinin. This is in contrast with previous in vivo work and calls for further experimental evaluation of the antimalarial potential of curcumin.     

Evidence of artemisinin production from IPP stemming from both the mevalonate and the nonmevalonate pathways

The potent antimalarial sesquiterpene lactone, artemisinin, is produced in low quantities by the plant Artemisia annua L. The source and regulation of the isopentenyl diphosphate (IPP) used in the biosynthesis of artemisinin has not been completely characterized. Terpenoid biosynthesis occurs in plants via two IPP-generating pathways: the mevalonate pathway in the cytosol, and the non-mevalonate pathway in plastids. Using inhibitors specific to each pathway, it is possible to resolve which supplies the IPP precursor to the end product. Here, we show the effects of inhibition on the two pathways leading to IPP for artemisinin production in plants. We grew young (7–14 days post cotyledon) plants in liquid culture, and added mevinolin to the medium to inhibit the mevalonate pathway, or fosmidomycin to inhibit the non-mevalonate pathway. Artemisinin levels were measured after 7–14 days incubation, and production was significantly reduced by each inhibitor compared to controls, thus, it appears that IPP from both pathways is used in artemisinin production. Also when grown in miconazole, an inhibitor of sterol biosynthesis, there was a significant increase in artemisinin compared to controls suggesting that carbon was shifted from sterols into sesquiterpenes. Collectively these results indicate that artemisinin is probably biosynthesized from IPP pools from both the plastid and the cytosol, and that carbon from competing pathways can be channeled toward sesquiterpenes. This information will help advance our understanding of the regulation of in planta production of artemisinin.     

Interaction between artemisinin and heme. A Density Functional Theory study of structures and interaction energies

Malaria is an infectious disease caused by the unicellular parasite Plasmodium sp. Currently, the malaria parasite is becoming resistant to the traditional pharmacological alternatives, which are ineffective. Artemisinin is the most recent advance in the chemotherapy of malaria. Since it has been proven that artemisinin may act on intracellular heme, we have undertaken a systematic study of several interactions and arrangements between artemisinin and heme. Density Functional Theory calculations were employed to calculate interaction energies, electronic states, and geometrical arrangements for the complex between the heme group and artemisinin. The results show that the interaction between the heme group and artemisinin at long distances occurs through a complex where the iron atom of the heme group retains its electronic features, leading to a quintet state as the most stable one. However, for interaction at short distances, due to artemisinin reduction by the heme group, the most stable complex has a septet spin state. These results suggest that a thermodynamically favorable interaction between artemisinin and heme may happen.     

Alkylating ability of artemisinin after Cu(I)-induced activation

The reductive activation of artemisinin by copper(I)-dipyrrin or copper(I)-(2-Clip-Phen) complexes generates an artemisinin derived alkylating species leading to covalent artemisinin–copper complex adducts. The reactivity of the peroxide function of artemisinin toward Cu(I) complexes is similar to that of Fe(II) analogues, even though the reaction is more sluggish and product distribution slightly different.     

Effect of artemisinin on proliferation and apoptosis-related protein expression in vivo and in vitro

Artemisinin is the first-line drugs for the treatment of malaria. In recent years, a large number of reports showed that artemisinin exhibit anti-tumor activity. In this study, we used C6 glioma cells and rat C6 brain-glioma model to study anti-tumor activity of artemisinin in vivo and in vitro. We found that artemisinin inhibited the proliferation in C6 cells and induced cell cycle arrest and a caspase-3-dependent cell apoptosis. It also inhibited the growth of C6 brain-glioma in vivo and enhanced living state of rat brain-glioma model. These results suggested that artemisinin had significant anti-tumor activities on C6 cells both in vitro and in vivo. Artemisinin might be exploited as a promising clinical anti-cancer drug in future.     

黄花蒿培养细胞中青蒿素合成代谢的体外调节

黄花蒿培养细胞通过两步培养积累青蒿素．第１步在含有０．２～０．４ｍｇ／Ｌ６－苄基氨基嘌呤（６－ＢＡ）和３～４ｍｇ／Ｌ吲哚乙酸（ＩＡＡ）的Ｎ６培养基中进行细胞的增殖培养,第２步将培养好的细胞转入含０．２～０．４ｍｇ／Ｌ６－ＢＡ和０．２～０．４ｍｇ／ＬＩＡＡ的改良Ｎ６培养基中进行青蒿素的合成．青蒿素的合成量为１９０μｇ／ｇ干细胞左右．当在第２步培养中加入青蒿素合成前体青蒿酸,青蒿素合成量比仅靠激素诱导提高了３倍多．青蒿素的合成途径是植物固醇合成途径的分支途径,当在青蒿素合成过程即第２步培养中加入固醇生物合成抑制剂双氯苯咪唑和氯化氯胆碱处理,可使代谢向合成青蒿素的方向移动,青蒿素合成量明显提高．经２００ｍｇ／Ｌ氯化氯胆碱处理２ｄ,黄花蒿细胞合成青蒿素量为３７２μｇ／ｇ干细胞;经２０ｍｇ／Ｌ双氯苯咪唑处理４ｄ,黄花蒿细胞合成青蒿素量为１５４０μｇ／ｇ干细胞,比靠激素诱导提高了８倍多,与诱导脱分化细胞的黄花蒿叶中所含的青蒿素（３０００μｇ／ｇ干细胞）处于同一个数量级．以上结果表明：在通过植物激素调节可以合成青蒿素的黄花蒿培养细胞中,缺乏青蒿素合成前体是青蒿素合成量低的重要原因．因此,在青蒿素合成的过程中通过体外调节,     

Expanding the scope of synthetic 1,2,4-trioxanes towards Trypanosoma cruzi and Leishmania donovani

A series of synthetic 1,2,4-trioxanes related to artemisinin was tested against L. donovani and T. cruzi parasites. This screening identified some active compounds, with key common structural features. Interestingly, these selected trioxanes were efficient against both parasites, and achieved antiparasitic activities comparable or superior than those presented by the corresponding reference drugs, artemisinin and artesunate. This study represents the first example of synthetic trioxanes evaluated on T. cruzi and provides possible candidates for developing new drugs for the treatment of leishmaniasis and Chagas disease.     

Production of artemisinin by genetically-modified microbes

Artemisinin, an endoperoxidized sesquiterpene originally extracted from the medicinal plant Artemisia annua L., is a potent malaria-killing agent. Due to the urgent demand and short supply of this new antimalarial drug, engineering enhanced production of artemisinin by genetically-modified or transgenic microbes is currently being explored. Cloning and expression of the artemisinin biosynthetic genes in Saccharomyces cerevisiae and Escherichia coli have led to large-scale microbial production of the artemisinin precursors such as amorpha-4,11-diene and artemisinic acid. Although reconstruction of the complete biosynthetic pathway toward artemisinin in transgenic yeast and bacteria has not been achieved, artemisinic acid available from these transgenic microbes facilitates the subsequent partial synthesis of artemisinin by either chemical or biotransformational process, thereby providing an attractive strategy alternative to the direct extraction of artemisinin from A.annua L. In this review, we update the current trends and summarize the future prospects on genetic engineering of the microorganisms capable of accumulating artemisinin precursors through heterologous and functional expression of the artemisinin biosynthetic genes.     

Enhanced artemisinin production from engineered yeast precursors upon biotransformation

Abstract

Production of artemisinin in genetically modified microorganisms is an attractive option to enable sufficient supply of the effective antimalarial agent. Although a sundry of artemisinin precursors are available from engineered bacteria or yeast, no artemisinin has been manufactured by engineering any microbial platforms due to inaccessibility to unidentified steps. To this end, it is essential to consider how to convert artemisinin precursors to artemisinin, either biochemically or chemically. To establish a novel procedure of artemisinin production, we incubate the mixture of artemisinin precursors from engineered Sacchromyces cerevisiae with the cell-free enzyme extract of Artemisia annua. For the single gene-expressing strain INVScI (pYES-ADS), amorpha-4,11-diene accumulation within 48 h or 14 days led to higher artemisinin content than the control. In the multiple gene-expressing strain YPH501 (pYES-ADS:: pESC-CYP71AV1-DBR2), artemisinin accumulation from the 14-day-induced yeast precursor mixture was nearly equivalent between the single gene-transferred strain and the multiple gene-transferred strain. Alternatively, biotransformation of 48-hour-induced yeast amorpha-4,11-diene mixture by the cold-acclimated A. annua cell-free extract that possesses the abundant enzymes relevant to artemisinin biosynthesis gave rise to considerable elevation of artemisinin content up to 0.647% in maximum, accounting to 15-folds increase as the A. annua cell-free extract without cold-acclimation (0.045%), thereby providing a practical protocol for artemisinin overproduction through the interplay of engineered microbial artemisinin precursors with upregulated plant enzymes.     

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.     

Microbacterium trichotecenolyticum enzyme mediated transformation of arteannuin B to artemisinin

Artemisinin, a sesquiterpene lactone containing an endoperoxide bridge, isolated from Artemisia annua L. is effective against both drug resistant and cerebral malaria causing strains of Plasmodium falciparum. The relative low yields of artemisinin in plants are a serious limitation to the commercialization of the drug. An alternative approach by microbial bioconversion of arteannuin B to artemisinin was carried out by Microbacterium trichotecenolyticum isolated from soil. Crude enzyme extract from cell free extracts were capable of microbial bioconversion of arteannuin B, the immediate precursor of artemisinin, to artemisinin. Attempts have been made to partially purify the proteins involved in bioconversion by ion exchange chromatography. Detection of artemisinin was done by thin layer chromatography, and quantified by HPLC.     

Artemisinin: mechanisms of action,resistance and toxicity

Artemisinin and its derivatives are widely used throughout the world. The mechanism of action of these compounds appears to involve the heme-mediated decomposition of the endoperoxide bridge to produce carbon-centred free radicals. The involvement of heme explains why the drugs are selectively toxic to malaria parasites. The resulting carbon-centred free radicals are alkylate heme and proteins, one of which is the translationally controlled tumour protein. Clinically relevant artemisinin resistance has not been demonstrated, but it is likely to occur since artemisinin resistance has been obtained in laboratory models. At high doses, artemisinin can be neurotoxic but toxicity has not been found in clinical studies. The mechanism of neurotoxicity may be similar to the mechanism of action.     

中药青蒿的生态生理及其综合利用

中药青蒿即黄花蒿(Artemisia annua L.)是抗疟药的原料,青蒿素是其有效抗疟成分。本文对青蒿的生物学特性、资源分布、生长栽培和生理生态进行了分析,指出了提高青蒿素含量的可能途径及其综合利用的前景。     

Artemisinin-Derived Dimers Have Greatly Improved Anti-Cytomegalovirus Activity Compared to Artemisinin Monomers

Background

Artesunate, an artemisinin-derived monomer, was reported to inhibit Cytomegalovirus (CMV) replication. We aimed to compare the in-vitro anti-CMV activity of several artemisinin-derived monomers and newly synthesized artemisinin dimers.

Methods

Four artemisinin monomers and two novel artemisinin-derived dimers were tested for anti-CMV activity in human fibroblasts infected with luciferase-tagged highly–passaged laboratory adapted strain (Towne), and a clinical CMV isolate. Compounds were evaluated for CMV inhibition and cytotoxicity.

Results

Artemisinin dimers effectively inhibited CMV replication in human foreskin fibroblasts and human embryonic lung fibroblasts (EC₅₀ for dimer sulfone carbamate and dimer primary alcohol 0.06±0.00 µM and 0.15±0.02 µM respectively, in human foreskin fibroblasts) with no cytotxicity at concentrations required for complete CMV inhibition. All four artemisinin monomers (artemisinin, artesunate, artemether and artefanilide) shared a similar degree of CMV inhibition amongst themselves (in µM concentrations) which was significantly less than the inhibition achieved with artemisinin dimers (P<0.0001). Similar to monomers, inhibition of CMV with artemisinin dimers appeared early in the virus life cycle as reflected by decreased expression of the immediate early (IE1) protein.

Conclusions

Artemisinin dimers are potent and non-cytotoxic inhibitors of CMV replication. These compounds should be studied as potential therapeutic agents for the treatment of CMV infection in humans.     

Microbial transformation of artemisinin to 5-hydroxyartemisinin by Eurotium amstelodami and Aspergillus niger

Transformation of the anti-malarial drug artemisinin by the fungi Eurotium amstelodami and Aspergillus niger were investigated. Cultures were grown in sucrose/malt broth with artemisinin for 14 days and extracted with ethyl acetate. Extracts were characterized by liquid chromatography. Two metabolites from each fungal extract were isolated and identified using mass spectrometry and nuclear magnetic resonance. 5β-hydroxyartemisinin and 7β-hydroxyartemisinin were isolated in 63 and 32% yields, respectively, from the extract of E. amstelodami, and 80 and 19%, respectively, from the extract of A. niger.     

Quantification of Three Key Enzymes Involved in Artemisinin Biogenesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> by Polyclonal Antisera-Based ELISA

To elucidate the fine-tuned temporal and spatial modulation of artemisinin production in annual wormwood (Artemisia annua), we conducted enzyme-linked immunosorbent assay-based immunoquantification of three key enzymes involved in artemisinin biosynthesis, amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), and cytochrome P450 reductase (CPR), in various tissues and under different growth conditions. The field-grown plants accumulate abundant ADS and CYP71AV1 but a trace amount of CPR in all tested tissues. Furthermore, ADS and CYP71AV1 accumulations in leaves are 16- and eightfold higher than in roots, and ten- and fourfold higher than in stems, respectively, demonstrating a tissue-specific expression pattern. Interestingly, the flowering field plants and cold-acclimated cultural plants produce higher levels of ADS and CYP71AV1 than non-flowering field plants or untreated cultural plants, indicating the environmental and developmental induction on ADS and CYP71AV1 genes and providing possible explanation for the observation that elevation of artemisinin level occurs after flowering.     

青蒿素含量与土壤、植株养分含量关系的研究

调查和分析测定同一产区不同土壤类型的黄花蒿土壤和植株不同部位的养分含量及青蒿素含量,并对其进行相关分析和因子分析,结果表明,青蒿素含量与土壤Ca的含量有显著的正相关关系,与根N、茎N素含量以及植株地下部和地上部的N素含量比有显著的负相关关系,与茎部和叶部的N素含量比有极显著负相关关系;黄花蒿植株叶片的N含量与土壤P含量有显著的正相关关系;影响青蒿素含量的主要因子是黄花蒿植株体内的养分含量,其次是土壤的养分含量以及土壤和植株等综合因子。通过施肥、适当补充土壤中的Ca和P素营养,改善土壤养分状况等各种途径来调节植株体内的养分,降低地下部和地上部的N素含量比值,茎叶部N素含量比值,提高青蒿素的含量,增施K肥,有利于黄花蒿的生长。