线粒体通透性转移孔与青蒿素抗疟机制研究

目的:为增进对青蒿素作用机制的了解,探讨参与调节线粒体体积的线粒体通透性转移孔在青蒿素抗疟机制中的作用.方法:分离线粒体,采用分光光度法检测青蒿素能否直接作用于离体线粒体导致线粒体体积变化;利用等效应图分析线粒体通透性转移孔抑制剂是否拮抗青蒿素的抗疟作用.结果:青蒿素可以直接导致离体疟原虫线粒体肿胀,而不会影响鼠肝线粒体体积;两种不同的线粒体通透性转移孔抑制剂均可拮抗青蒿素的抑疟效果.结论:青蒿素可以直接作用于离体疟原虫线粒体导致线粒体肿胀,且青蒿素导致线粒体肿胀的物种选择性与细胞毒性的物种选择性一致.此外,利用抑制剂阻断线粒体通透性转移孔的开放可以拮抗青蒿素的抗疟效果,证明线粒体通透性转移孔在青蒿素抗疟过程中起重要作用.     

青蒿素的发现与研究进展

1967年北京《5.23抗疟计划》付诸实施,在全国多个研究单位协作下,组织植物化学与药理学等专业200多人,搜寻对抗耐氯喹恶性疟疾的新药.他们追索我国历代抗疟方剂,用约200种草药制成380多种抽提物,再筛查其对小鼠疟疾模型的疗效.最后确定,在60℃用乙醚萃取中药青蒿的黄花蒿所得第191号中性抽提物,对感染鼠疟和猴疟的小鼠与猴以及21例恶性疟、间日疟患者均能奏效.1972年11月,从该抽提物分离出相对分子质量0.282 kD的无色结晶,命名为青蒿素.它属于一种新型的倍半萜内酯.继后青蒿素纯品的胶囊被用于临床数千例疟疾患者.2006年以来,为克服耐药性,世界卫生组织(WHO)宣布改用青蒿素的联合疗法(ACT),挽救了80个国家100多万人的生命.因在发现青蒿素过程中的关键作用,屠呦呦被授予2011年度拉斯克临床医学研究奖.本文对青蒿素发现的争议,如何恰当评价其他科学家的贡献,以及开发植物界以外青蒿素新药源的近况,均作了简要报道.     

青蒿素类药物抗柯萨奇B组病毒的体外实验研究

柯萨奇病毒B组 3型 (CVB3 )可以引起人类病毒性心肌炎等多种疾病 ,严重危害人类健康 ,但目前尚无有效的抗病毒药物和治疗手段。青蒿素类药物是目前疗效最佳和稳定的抗疟疾药物 ,并且具有比较明显的抗肿瘤、抗心律失常及一定的抗疱疹病毒和乙型肝炎病毒等作用。为探讨青蒿素类药物的抗柯萨奇病毒作用 ,本研究以柯萨奇病毒B组 3型 (CVB3 )感染HeLa细胞为实验模型 ,采用微量细胞病变抑制法 ,观察与分析青蒿素、蒿甲醚等药物对CVB3 的直接灭活、阻断吸附和抑制复制的作用。本研究以青蒿素和蒿甲醚为实验用药 ,以病毒唑为对照药 ,并将各种…     

青蒿素及其衍生物抗肿瘤的研究进展

青蒿素类药物是治疗疟疾的主要药物,其衍生物有青蒿琥酯、蒿甲醚和二氢青蒿素等.近年来研究发现,青蒿素及其衍生物具有明显的抗肿瘤作用.研究表明:青蒿素及其衍生物可以抑制或杀伤肿瘤细胞;抑制肿瘤细胞增殖与诱导肿瘤细胞凋亡;抑制血管生成;选择性杀伤肿瘤细胞;逆转肿瘤细胞的多重耐药;具有放射增敏效应.因青蒿素及其衍生物安全低毒,有望成为新型的广泛、高效、低毒的抗癌药物.     

泛素-蛋白酶体途径及其生物学作用的研究进展

泛素-蛋白酶体途径是细胞内重要的非溶酶体蛋白降解途径,是调节各种细胞生物学过程的重要机制,参与调节细胞周期进程、细胞增生与分化以及信号转导等各种细胞生理过程,对维持细胞正常生理功能具有十分重要的意义。本文简要介绍了泛素-蛋白酶体途径的作用过程,并从其对某些抑癌基因、转录因子和细胞周期素依赖性激酶抑制蛋白的调节,参与肿瘤及癌症的发生和发展,讨论其生物学作用,并指出其在药物研究方面的重要作用。     

青蒿素及其衍生物抗肿瘤作用研究进展

青蒿素及其衍生物是治疗疟疾的首选药。近年来大量体内外研究显示青蒿素及其衍生物具有良好的抗肿瘤活性,其中,一些结构新颖的衍生物抗肿瘤活性大大增强,甚至优于常用的化疗药物。其抗肿瘤机制主要包括氧化损伤反应、抑制肿瘤细胞增殖以及抗新生血管生成等。青蒿素类药物毒副作用小、成本较低,对多药耐药细胞有效,对放、化疗具有增效作用,预示其极可能成为具有临床应用价值的抗癌新药。     

青蒿素类化合物抗肿瘤研究新进展

传统中药及其天然活性成分为现代医药发展提供了丰富的资源。青蒿素及其衍生物的抗疟活性已得到世界公认,具有起效快、药效高、毒副作用低等优点。不断深入的研究揭示了该类化合物具有良好的抗肿瘤作用,如诱导细胞周期阻滞、促进细胞凋亡、抑制肿瘤血管生成、阻断肿瘤细胞的侵袭转移等。目前,在认识青蒿素类化合物抗癌分子作用机理的基础上对其进行结构改造,为新型一类抗肿瘤药物的研制奠定基础。对青蒿素及其衍生物的抗肿瘤活性及相关分子机制研究现状进行了综述。     

二氢乳清酸脱氢酶靶向抗疟药研究进展

疟疾是全球危害最严重的传染性疾病之一,尤其是在非洲,发病率与死亡率仍居高不下。抗药性的出现和发展使大多数现有抗疟药在临床上失去了效用,研究和开发新型抗疟药已成为当前疟疾防治研究的迫切需求。随着恶性疟原虫基因组测序的完成和对疟原虫生物学认知的不断深入,寻找抗疟新靶点的研究得以快速发展。嘧啶生物合成途径是经临床确证有效的抗疟靶点的典范。我们简要综述了近年来以恶性疟原虫嘧啶从头合成途径第四步关键酶——二氢乳清酸脱氢酶（DHODH）为靶点的抗疟新药研究。高通量筛选、药物化学等研究已获得若干对恶性疟原虫DHODH有选择性抑制作用的化合物结构,其中有些在恶性疟原虫体外培养试验中表现出了较强的抗疟作用,且其酶抑制活性与抗疟活性间具有良好的相关性。通过三唑并嘧啶类系列先导化合物的优化研究,已获得了具有良好代谢稳定性、对鼠疟模型有效的类似物。已有大量研究表明DHODH靶向抗疟药的研发具有广阔前景。     

青蒿素及其类似物抗疟构效关系的DFT研究

为了从原子水平上揭示青蒿素及其类似物的结构与抗疟活性之间的关系,运用密度泛函理论DFT方法,在B3LYP/6-31G*水平上对青蒿素及其类似物二氢青蒿素、蒿甲醚和青蒿琥酯的结构和性质进行了理论计算。从分子的平衡构型、Wiberg键级、溶剂化能、偶极矩和静电势等方面分析了青蒿素及其类似物的抗疟构效关系。结果表明,青蒿素及其类似物结构中七元环上的过氧桥键、醚氧键以及六元环上的内酯结构是其抗疟作用的关键活性位,过氧桥键处负的静电势越多,青蒿素与血红素的相互作用越强,分子的抗疟活性越强。理论预测四个药物分子的抗疟活性顺序为:青蒿素<二氢青蒿素<蒿甲醚<青蒿琥酯,与实验活性结果一致。     

青蒿素及其衍生物的抗肿瘤机制

恶性肿瘤是严重威胁人类健康的重大疾病。尽管治疗手段不断发展,但推广度及疗效仍极为有限。新近研究发现,经典抗疟一线药青蒿素及其衍生物具有广泛抗肿瘤活性。大量研究提示,青蒿素及其衍生物通过细胞毒性效应直接杀死肿瘤细胞,也可诱导细胞周期阻滞从而抑制细胞增殖。另一方面,可通过凋亡、自噬、铁死亡途径导致细胞死亡。还可调控肿瘤微环境,从而抑制肿瘤细胞侵袭与转移。然而,尽管青蒿素及其衍生物展现出强大的抗肿瘤潜能,但其作用机制仍十分复杂。本文就青蒿素及其衍生物的抗肿瘤机制及其研究进展作一综述。     

Artemisinin Directly Targets Malarial Mitochondria through Its Specific Mitochondrial Activation

The biological mode of action of artemisinin, a potent antimalarial, has long been controversial. Previously we established a yeast model addressing its mechanism of action and found mitochondria the key in executing artemisinin''s action. Here we present data showing that artemisinin directly acts on mitochondria and it inhibits malaria in a similar way as yeast. Specifically, artemisinin and its homologues exhibit correlated activities against malaria and yeast, with the peroxide bridge playing a key role for their inhibitory action in both organisms. In addition, we showed that artemisinins are distributed to malarial mitochondria and directly impair their functions when isolated mitochondria were tested. In efforts to explore how the action specificity of artemisinin is achieved, we found strikingly rapid and dramatic reactive oxygen species (ROS) production is induced with artemisinin in isolated yeast and malarial but not mammalian mitochondria, and ROS scavengers can ameliorate the effects of artemisinin. Deoxyartemisinin, which lacks an endoperoxide bridge, has no effect on membrane potential or ROS production in malarial mitochondria. OZ209, a distantly related antimalarial endoperoxide, also causes ROS production and depolarization in isolated malarial mitochondria. Finally, interference of mitochondrial electron transport chain (ETC) can alter the sensitivity of the parasite towards artemisinin. Addition of iron chelator desferrioxamine drastically reduces ETC activity as well as mitigates artemisinin-induced ROS production. Taken together, our results indicate that mitochondrion is an important direct target, if not the sole one, in the antimalarial action of artemisinins. We suggest that fundamental differences among mitochondria from different species delineate the action specificity of this class of drugs, and differing from many other drugs, the action specificity of artemisinins originates from their activation mechanism.     

Ferryl-oxo heme intermediate in the antimalarial mode of action of artemisinin

Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopies have been employed to investigate the reductive cleavage of the O-O bond of the endoperoxide moiety of the antimalarial drug artemisinin and its analog trioxane alcohol by hemin dimer. We have recorded FTIR spectra in the nu(O-O) and nu(as)(Fe-O-Fe) regions of artemisinin and of the hemin dimer that show the cleavage of the endoperoxide and that of the hemin dimer, respectively. We observed similar results in the trioxane alcohol/hemin dimer reaction. The RR spectrum of the artemisinin/hemin dimer reaction displays a vibrational mode at 850 cm(-1) that shifts to 818 cm(-1) when the experiment is repeated with (18)O-O(18) endoperoxide enriched trioxane alcohol. The frequency of this vibration and the magnitude of the (18)O-O(18) isotopic shift led us to assign the 850 cm(-1) mode to the Fe(IV) = O stretching vibration of a ferryl-xoxo heme intermediate that occurs in the artemisinin/hemin dimer and trioxane alcohol/hemin reactions. These results provide the first direct characterization of the antimalarial mode of action of artemisinin and its trioxane analog, and suggest that artemisinin appears to react with heme molecules that have been incorporated into hemozoin and subsequently the heme performs cytochrome P450-type chemistry.     

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.     

Yeast model uncovers dual roles of mitochondria in action of artemisinin

Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals.     

Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite.

Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well as Plasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains.     

Transgenic approach to increase artemisinin content in Artemisia annua L.

Artemisinin, the endoperoxide sesquiterpene lactone, is an effective antimalarial drug isolated from the Chinese medicinal plant Artemisia annua L. Due to its effectiveness against multi-drug-resistant cerebral malaria, it becomes the essential components of the artemisinin-based combination therapies which are recommended by the World Health Organization as the preferred choice for malaria tropica treatments. To date, plant A. annua is still the main commercial source of artemisinin. Although semi-synthesis of artemisinin via artemisinic acid in yeast is feasible at present, another promising approach to reduce the price of artemisinin is using plant metabolic engineering to obtain a higher content of artemisinin in transgenic plants. In the past years, an Agrobacterium-mediated transformation system of A. annua has been established by which a number of genes related to artemisinin biosynthesis have been successfully transferred into A. annua plants. In this review, the progress on increasing artemisinin content in A. annua by transgenic approach and its future prospect are summarized and discussed.     

Evidence for the contribution of the hemozoin synthesis pathway of the murine Plasmodium yoelii to the resistance to artemisinin-related drugs

Plasmodium falciparum malaria is a major global health problem, causing approximately 780,000 deaths each year. In response to the spreading of P. falciparum drug resistance, WHO recommended in 2001 to use artemisinin derivatives in combination with a partner drug (called ACT) as first-line treatment for uncomplicated falciparum malaria, and most malaria-endemic countries have since changed their treatment policies accordingly. Currently, ACT are often the last treatments that can effectively and rapidly cure P. falciparum infections permitting to significantly decrease the mortality and the morbidity due to malaria. However, alarming signs of emerging resistance to artemisinin derivatives along the Thai-Cambodian border are of major concern. Through long-term in vivo pressures, we have been able to select a murine malaria model resistant to artemisinins. We demonstrated that the resistance of Plasmodium to artemisinin-based compounds depends on alterations of heme metabolism and on a loss of hemozoin formation linked to the down-expression of the recently identified Heme Detoxification Protein (HDP). These artemisinins resistant strains could be able to detoxify the free heme by an alternative catabolism pathway involving glutathione (GSH)-mediation. Finally, we confirmed that artemisinins act also like quinolines against Plasmodium via hemozoin production inhibition. The work proposed here described the mechanism of action of this class of molecules and the resistance to artemisinins of this model. These results should help both to reinforce the artemisinins activity and avoid emergence and spread of endoperoxides resistance by focusing in adequate drug partners design. Such considerations appear crucial in the current context of early artemisinin resistance in Asia.     

Improving Pharmacokinetic-Pharmacodynamic Modeling to Investigate Anti-Infective Chemotherapy with Application to the Current Generation of Antimalarial Drugs

Mechanism-based pharmacokinetic-pharmacodynamic (PK/PD) modelling is the standard computational technique for simulating drug treatment of infectious diseases with the potential to enhance our understanding of drug treatment outcomes, drug deployment strategies, and dosing regimens. Standard methodologies assume only a single drug is used, it acts only in its unconverted form, and that oral drugs are instantaneously absorbed across the gut wall to their site of action. For drugs with short half-lives, this absorption period accounts for a significant period of their time in the body. Treatment of infectious diseases often uses combination therapies, so we refined and substantially extended the PK/PD methodologies to incorporate (i) time lags and drug concentration profiles resulting from absorption across the gut wall and, if required, conversion to another active form; (ii) multiple drugs within a treatment combination; (iii) differing modes of action of drugs in the combination: additive, synergistic, antagonistic; (iv) drugs converted to an active metabolite with a similar mode of action. This methodology was applied to a case study of two first-line malaria treatments based on artemisinin combination therapies (ACTs, artemether-lumefantrine and artesunate-mefloquine) where the likelihood of increased artemisinin tolerance/resistance has led to speculation on their continued long-term effectiveness. We note previous estimates of artemisinin kill rate were underestimated by a factor of seven, both the unconverted and converted form of the artemisinins kill parasites and the extended PK/PD methodology produced results consistent with field observations. The simulations predict that a potentially rapid decline in ACT effectiveness is likely to occur as artemisinin resistance spreads, emphasising the importance of containing the spread of artemisinin resistance before it results in widespread drug failure. We found that PK/PD data is generally very poorly reported in the malaria literature, severely reducing its value for subsequent re-application, and we make specific recommendations to improve this situation.     

NMR studies on novel antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin

Artemisinin and its derivatives, which have been known as antimalarial drugs, have also demonstrated their cytotoxicity against tumor cells. It has been proposed that antitumor activity depends on the lipophilicity of functional group on artemisinin derivatives. Solution structures of two artemisinin derivatives as antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin, were determined by NMR spectroscopy to elucidate structure-activity relationship. According to biological assay, antitumor efficiencies are not dependent upon lipophilicity. Instead, these compounds demonstrated their distinctive structural features of boat/chair conformation and capability to interact with receptors, as they have different efficiencies on antitumor activity. Especially, carboxypropyl moiety or carbonyl moiety in artemisinin derivatives influences the conformation and stability of ring structure. Although the detailed mechanism of antitumor activity by artemisinin derivatives has not been addressed, we suggest that antitumor activity is not determined only with lipophilicity and that artemisinin derivatives have specific target proteins in each type of cancer.     

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.