In vitro studies on the mode of action of quassinoids with activity against chloroquine-resistant Plasmodium falciparum

Using the incorporation of [3H]isoleucine or [3H]hypoxanthine into acid-insoluble products as indices of protein- and nucleic acid-synthetic activity, respectively, it was shown that seven plant-derived quassinoids with differing chemical substitutions all inhibited protein synthesis more rapidly than nucleic acid synthesis in human erythrocytes infected with Plasmodium falciparum, in vitro. Five quassinoids (ailanthinone, bruceantin, bruceine B, glaucarubinone and holacanthone) were effective within 30 min at doses 10 times their 48 hr in vitro IC50 values. Chaparrin and glaucarubol differed in that they did not inhibit protein synthesis during the time course of these experiments when applied at 10 times their in vitro IC50 values. When these compounds were used at 209 and 114 times their respective IC50 values, their observed effects were identical to those of the other quassinoids studied. The time (t50) at which nucleic acid synthesis was reduced to 50% of control was directly proportional to the t50 for protein synthesis, suggesting that failure of nucleic acid synthesis is a consequence of inhibition of protein synthesis. It is concluded that in the malaria parasite, as in eukaryote models, quassinoids are rapid and potent inhibitors of protein synthesis, and that this is most likely due to effects upon the ribosome, rather than upon nucleic acid metabolism.     

Inhibition, by selected antibiotics, of protein synthesis in cells growing in tissue cultures

A large number of compounds including actinobolin, adrenochrome, amicetin, anisomycin, aurintricarboxylic acid, blasticidin S, chartreusin, chlortetracycline, cycloheximide, doxycycline, edeine A1, edeine complex, emetine, fusidic acid, gougerotin, GppCH2p, oxytetracycline, pactamycin, polydextran sulphate, puromycin, pyrocatechol violet, sparsomycin and tubulosine have been tested for inhibitory effects on protein synthesis in cultured cells from both mouse fibroblasts (3T6 cells) and chick embryo fibroblasts (CEF). Essentially, similar results were obtained with both cell types with the most effective inhibitors being pactamycin, emetine, tubulosine, anisomycin and cycloheximide and with no significant inhibitory activity being detected with edeine complex, edeine A1, GppCH2p, polydextran sulphate, aurintricarboxylic acid, pyrocatechol violet and adrenochrome. The concentration of pactamycin required to produce 50% inhibition of protein synthesis approximated 5 X 10(-9) M, but for most of the inhibitors it ranged from 5 X 10(-6) M to 5 X 10(4) M. The molecular basis underlying these differences may be related, in addition to their intrinsic inhibitory power, to differences in permeability of the cells towards the various drugs tested. Alternatively, active accumulation of the drugs by the cells may be the variable parameter.     

Aryl hydrocarbon hydroxylase induction in mammalian liver-derived cell cultures. Effects of various metabolic inhibitors on the enzyme activity in hepatoma cells.

Aryl hydrocarbon (benzo[a]pyrene) hydroxylase induction in one mouse hepatoma and two rat hepatoma cell lines was characterized with respect to optimal growth requirements and optimal inducing concentrations of polycyclic hydrocarbons, phenobarbital, biogenic amines, and numerous other hydrophobic compounds. Cordycepin, ethidium bromide and puromycin aminonucleoside treatment of the mouse tumor line Hepa-1 produce strikingly different effects on the kinetics of hydroxylase induction, when the inducers benz[a]anthracene, phenobarbital and iso-proterenol are compared. without any of these ‘usual inducers’ present in the mouse hepatoma cell line, 0.50 to 5.0 mM puromycin aminonucleoside or 40 nM actinomycin-D causes large increases in the hydroxylase activity (at a time when gross RNA synthesis is virtually 100 per cent inhibited and gross protein synthesis is more than 50 per cent inhibited). This aminonucleoside- or actinomycin-D-mediated induction process induction, when the inducers benz[a]anthracene, phenobarbital and isoproterenol are compared. With-without prior treatment with the usual inducers and hence presumably without prior accumulation of putative induction-specific RNA: moreover, this induction process apparently requires little, or no. simultaneous RNA synthesis during exposure to the aminonucleoside or actinomycin-D. In contrast, when primary cultures derived from normal rat liver are treated with similar high concentrations of the aminonucleoside, no induction of the hydroxylase activity occurs. This result suggests that the effect of puromycin aminonucleoside on hydroxylase-specific mRNA synthesis, processing or stabilization is very different between the hepatoma cell line and normal fetal rat primary hepatocytes in culture.     

三尖杉属植物中生物碱的研究——Ⅸ.三尖杉酯碱类似物的半合成及其抗白血病活性

报道三尖杉碱的十七个新的酯类化合物的半合成及其抗肿瘤作用。药理实验结果表明1,2,2+3,6和8有显著的抗癌活性,4,5,15和16有中等的抗肿瘤作用。通过化学结构和抗肿瘤活性的比较,初步探讨了此类生物碱的构效关系。     

Effect of 6-aminonicotinamide and other protein synthesis inhibitors on formation of platinum-DNA adducts and cisplatin sensitivity

The present study was undertaken to examine the mechanistic basis for the recent observation that the pyridine nucleotide derivative 6-aminonicotinamide (6AN, NSC 21206) enhances the accumulation and resulting cytotoxicity of cisplatin in a variety of tumor cell lines. When A549 lung cancer cells or K562 leukemia cells were treated with 62.5 microM 6AN for 21 h and then pulse-labeled with [(35)S]methionine for 1 h, increased labeling of five polypeptides, one of which corresponded to a M(r) approximately 78,000 glucose-regulated protein (GRP78), was observed. Two subsequent observations, however, suggested that up-regulation of these polypeptides was unlikely to explain the interaction between 6AN and cisplatin: 1) the concentration of 6AN required to induce GRP78 was 4-fold higher than the dose required to sensitize cells to cisplatin; and 2) simultaneous treatment of cells with 6AN and cycloheximide prevented the increase in GRP78 but not the sensitizing effect of 6AN. On the contrary, treatment with the protein synthesis inhibitors cycloheximide, anisomycin, or puromycin as well as prolonged exposure to the RNA synthesis inhibitor actinomycin D mimicked the biochemical modulating effects of 6AN on cisplatin action. Conversely, 6AN inhibited protein synthesis, whereas 18 6AN analogs that failed to enhance Pt-DNA adducts and cisplatin cytotoxicity failed to inhibit protein synthesis. These observations are consistent with a model in which 6AN and other inhibitors of protein synthesis act as modulating agents by increasing cisplatin accumulation, thereby enhancing the formation of Pt-DNA adducts and subsequent cisplatin-induced cell death.     

三尖杉碱酯类化合物的合成及其抗肿瘤活性

脱氧三尖杉酯碱Ⅰ。对动物肿瘤P-388有明显的抑制作用。本文报道了三尖杉碱的十二个新的酯类化合物的合成及其抗肿瘤作用。药理实验发现合成的脱氧三尖杉酯碱(差向异构物混合体)有一定的抗肿瘤作用。通过化学结构和抗肿瘤活性的比较,初步推断酯碱Ⅰ.侧链的异丙基和α-叔羟基及甲氧羰甲基是抗肿瘤所必需的结构。     

三尖杉属植物中生物碱的研究——Ⅷ.二种新的抗癌三尖杉酯碱生物碱的结构和半合成

从安徽产三尖杉(Cephalotaxusfortunei Hook f.)弱碱中分到二种新的抗癌生物碱,命名为新三尖杉酯碱(neoharringonine,1)和脱水三尖杉酯碱(anhydroharringtonine,2)。从本种植物中还首次分到二个已知生物碱:脱氧三尖杉酯碱(deoxyharringtonine,3)和异三尖杉酯碱(isoharringtonine,4)。     

Omacetaxine mepesuccinate for the treatment of leukemia

INTRODUCTION: Omacetaxine mepesuccinate, formerly known as homoharringtonine, is a first-in-class cephalotaxine that has experienced phases of increasing and waning interest since its first use in traditional Chinese medicine. With activity being reported in patients with chronic myeloid leukemia (CML) resistant to currently available tyrosine kinase inhibitors, renewed interest has recently been generated. AREAS COVERED: The development of omacetaxine mepesuccinate, with emphasis on synthesis and mode of administration, is addressed. An overview on current clinical results as a single agent or within combination regimens in patients with acute myeloid leukemia (AML) and CML is given. EXPERT OPINION: Omacetaxine mepesuccinate has a unique mode of action and appreciable activity in AML and CML with generally mild nonhematologic toxicity. In patients with AML, results indicate a role within combination regimens in selected, possibly elderly patient populations. In CML, patients with resistance to tyrosine kinase inhibitors, especially due to the T315I mutation, are the most intensively studied. Despite successful results in some patients, single-agent therapy with omacetaxine mepesuccinate has resulted in modest results. However, upfront combination with tyrosine kinase inhibitor represents an attractive option due their differing mechanisms of action.     

Omacetaxine mepesuccinate for the treatment of leukemia

Introduction: Omacetaxine mepesuccinate, formerly known as homoharringtonine, is a first-in-class cephalotaxine that has experienced phases of increasing and waning interest since its first use in traditional Chinese medicine. With activity being reported in patients with chronic myeloid leukemia (CML) resistant to currently available tyrosine kinase inhibitors, renewed interest has recently been generated.

Areas covered: The development of omacetaxine mepesuccinate, with emphasis on synthesis and mode of administration, is addressed. An overview on current clinical results as a single agent or within combination regimens in patients with acute myeloid leukemia (AML) and CML is given.

Expert opinion: Omacetaxine mepesuccinate has a unique mode of action and appreciable activity in AML and CML with generally mild nonhematologic toxicity. In patients with AML, results indicate a role within combination regimens in selected, possibly elderly patient populations. In CML, patients with resistance to tyrosine kinase inhibitors, especially due to the T315I mutation, are the most intensively studied. Despite successful results in some patients, single-agent therapy with omacetaxine mepesuccinate has resulted in modest results. However, upfront combination with tyrosine kinase inhibitor represents an attractive option due their differing mechanisms of action.     

Memory-enhancing effect of a cerulein analogue following peripheral administration in the rat

Our previous studies demonstrated that cerulein (CER) has a potent preventive action on amnesia induced by electroconvulsive shock, administration of scopolamine, puromycin, anisomycin, NMDA receptor antagonists, and protein kinase C inhibitors. The present study was aimed at finding more effective CER analogues which could enhance memory processes. Five CER analogues were synthesized and the potencies in passive and active avoidance responses and in the Morris water pool test were examined in the rat. Among the preparations, des-Gln²-[Leu⁵, Nle⁸]-CER was found to possess particularly potent effects on memory acquisition and/or storage. The effects were apparent in less than 1 μg/kg single subcutaneous (s.c.) injection for at least 120 hr in passive avoidance response and 15 days in active avoidance response. Memory impairments induced by scopolamine and puromycin were well improved in passive avoidance response. In the Morris water maze test, disordered behaviors caused by scopolamine and protein kinase C inhibitor were totally restored to the normal state by s.c. injection of this CER analogue. © 1992 Wiley-Liss, Inc.     

Carbocyclic puromycin: synthesis and inhibition of protein biosynthesis

The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-[3 beta-amino-2 beta-hydroxy-4 alpha-(hydroxymethyl)cyclopent-1 alpha-yl]-6-(dimethylamino)purine, followed by separation of the diastereomers and subsequent removal of the Cbz blocking group. Kinetic studies indicate that carbocyclic puromycin is an excellent substrate for the peptidyltransferase reaction with both prokaryotic and eukaryotic ribosomes. A comparison of carbocyclic puromycin with previously synthesized analogues indicate that the furanosyl ring oxygen and the hydroxymethyl group of puromycin do not contribute to ribosomal binding, but both moieties contribute to the rate of product formation from the enzyme-substrate complex. Carbocyclic puromycin was equal to puromycin when evaluated for cytotoxicity using P-388 mouse lymphoid leukemia cells in culture.     

Superinduction of CYP1A1 in MCF10A cultures by cycloheximide, anisomycin, and puromycin: a process independent of effects on protein translation and unrelated to suppression of aryl hydrocarbon receptor proteolysis by the proteasome

Exposure of the human breast epithelial cell line MCF10A to > or = 1 microg/ml cycloheximide (CHX)-induced accumulations of CYP1A1 mRNA 6-fold greater than that achieved with only 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Cotreatment with CHX and TCDD caused superinduction of CYP1A1 with accumulations of CYP1A1 mRNA 30-fold greater than that achieved with only TCDD. Similar results were obtained with the protein translation inhibitors anisomycin (ANS) and puromycin (PUR). Intra- and interinhibitor comparisons of dose/concentration response curves demonstrated the absence of a quantitative relationship between [3H]leucine incorporation and CYP1A1 induction/superinduction. The inducing/superinducing activities of CHX were suppressed by coincubation with the aryl hydrocarbon receptor (AhR) antagonists alpha-naphthoflavone and 3'-methoxy-4'-nitroflavone (PD168641). Electrophoretic mobility shift assays demonstrated that nuclear extracts from CHX-treated and CHX + TCDD cotreated cultures formed approximately 58 and approximately 340% of the AhR/DNA complexes obtained with TCDD-treated cultures, respectively. In contrast, rat liver extracts did not form AhR/DNA complexes after in vitro transformation with CHX. AhR turnover in TCDD-treated hepatoma 1c1c7 cultures was suppressed by cotreatment with CHX. In contrast, CHX or ANS treatment of MCF10A cultures induced AhR loss and enhanced AhR loss in cultures cotreated with TCDD. Cotreatment with N-benzoyloxycarbonyl-(Z)-Leu-Leu-leucinal (MG132) but not leptomycin B suppressed AhR loss. Hence, in MCF10A cells, CHX is not an AhR agonist but can superinduce CYP1A1 via an AhR-dependent mechanism; CYP1A1 superinduction by translation inhibitors is neither quantitatively related to effects on protein synthesis nor due to a generalized prevention of AhR proteolysis, and proteasome-mediated degradation of the activated AhR can occur in the nucleus.     

The antagonistic effects of naloxone on cycloheximide and anisomycin-induced amnesia

The amnesia induced by cycloheximide (CXM) injected SC and by CXM or anisomycin injected ICV immediately after the training test was antagonized in combination with an opiate antagonist, naloxone (NLX). This antagonism occurred on both the passive avoidance- and escape-learning responses in a dose-dependent manner in mice. NLX alone (0.3-10.0 mg/kg) did not alter the performances of these tasks. Furthermore, the decrease in retention performance on shuttle avoidance in rats induced by CXM was also antagonized by NLX. Treatment with CXM and/or NLX did not affect spontaneous locomotor activity. The interaction of these drugs on the performance of the passive avoidance- and escape-learning and the shuttle avoidance tasks may be related to neurochemical memory processes. These results suggest that an opioid system may participate in the amnesic actions induced by protein synthesis inhibitors in these models.     

Induction of apoptosis and changes in nuclear G-actin are mediated by different pathways: the effect of inhibitors of protein and RNA synthesis in isolated rat hepatocytes

Stressor-induced changes in the cytoskeleton, of which actin is a major component, may lead to apoptosis. The role of drug-induced changes in nuclear G-actin and apoptosis was studied in freshly isolated hepatocytes. Several protein synthesis inhibitors, cycloheximide, puromycin, and emetine, induced 10 to 15% apoptosis in hepatocytes after 4 h, as was determined by changes in nuclear morphology and flow cytometric analysis of Annexin V-positive cells. Apoptosis induced by protein synthesis inhibition could be prevented by the caspase inhibitors Z-Val-Ala-DL-Asp fluormethylketone (zVAD-fmk) and Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-cho). Several (chemical) stressors cause a rapid increase in nuclear G-actin staining in hepatocytes or cell lines (Meijerman et al., Biochem. Biophys. Res. Commun. 240, 697-700, 1997). The protein synthesis inhibitors also increased G-actin staining in nuclei after 2 h; this could not be inhibited by zVAD-fmk or DEVD-cho. Changes in the cytosolic F-actin pattern did not occur until nuclear G-actin staining had already increased. The mRNA synthesis inhibitor actinomycin D, also increased nuclear G-actin staining, but did not induce apoptosis within the studied time frame. The results suggest that the induction of apoptosis and the increased nuclear staining of G-actin by protein synthesis inhibition are differently controlled.     

三尖杉属植物中生物碱的研究——Ⅸ.三尖杉酯碱类似物的半合成及其抗白血病活性

报道三尖杉碱的十七个新的酯类化合物的半合成及其抗肿瘤作用。药理实验结果表明1,2,2+3,6和8有显著的抗癌活性,4,5,15和16有中等的抗肿瘤作用。通过化学结构和抗肿瘤活性的比较,初步探讨了此类生物碱的构效关系。     

反相离子对高效液相色谱分析半合成高三尖杉酯碱差向异构体

本文报道半合成高三尖杉酯碱差向异构体的反相离子对高效液相色谱法测定。用十八烷基键合相柱,以0.003 M庚烷磺酸钠与0.005 M醋酸铵的甲醇—水—四氢呋喃(1:1.26:0.03)溶液作流动相(pH 3.5),钩吻碱为内标。方法简便快速、重现性好。     

Inhibition of cerebral protein synthesis: performance at different times after passive avoidance training.

Inhibition of cerebral protein synthesis impairs long-term memory in a variety of species and tasks. Recently it was reported that subcutaneous injection of the protein synthesis inhibitor cycloheximide impaired short-term retention, measured 10 min after training in a passive avoidance task. To examine the possibility that inhibition of cerebral protein synthesis may sometimes disrupt short-term memory, mice were injected subcutaneously with cycloheximide (120 mg/kg) or anisomycin (150 mg/kg), or bitemporally with cycloheximide or anisomycin (100 mug/side) and given one training trial in a passive avoidance box. Subcutaneously injected cycloheximide reduced step-through latencies 10 min after training as reported previously, but anisomycin or bitemporally injected cycloheximide did not. All 4 drug groups exhibited impaired long-term memory. Since the results obtained at short intervals after training varied depending on the drug and route of injection, the impairment produced by subcutaneous cycloheximide at 10 min after training cannot be attributed to inhibition of cerebral protein synthesis. It is suggested that performance at short intervals after training reflects drug side effects on step-through behavior. By contrast, the impairment obtained at long intervals after training is consistent with the hypothesis that cerebral protein synthesis is required for formation of long-term memory.     

Prevention of memory loss following puromycin treatment.

Female C57BL/6J mice were trained on a one trial passive avoidance response. Twenty-four hours later, they were treated with puromycin in combination with either 2.0 or 10.0 mg/kg of amphetamine, 0.3 mg/kg of strychnine, or 20.0 or 50.0 mg/kg of pentylenetetrazol. Tests one week after training revealed that treatment with these stimulant drugs prevented the memory loss characteristic of puromycin; an exception being those animals injected with the low dose of amphetamine. Biochemical determination of amino acid incorporation into protein revealed that none of the stimulant drugs used significantly altered the extent or the duration of protein synthesis inhibition induced by puromycin. These results are interpreted as showing that the amnesic effects of puromycin can be counteracted by a state of heightened nervous system excitation.     

Neurochemical and behavioral effects of catecholamine and protein synthesis inhibitors in mice

A series of biochemical and behavioral experiments tested the hypothesis that anisomycin (ANI), a protein synthesis inhibitor, produced decrements in long-term memory by raising free tyrosine levels and by the accumulation of catecholamines (CAs) rather than by its primary effect on protein synthesis. We compared the effects of ANI and three catecholamine synthesis inhibitors (CAIs)--diethyldithiocarbamic acid, alpha-methyl-p-tyrosine, and tetrabenazine--on cerebral concentrations of tyrosine and CAs and on the rate of accumulation of CAs. ANI had a relatively small effect, whereas the CAIs resulted in large reductions. When ANI and a CAI were used in combination, effects on CA levels were determined mainly by the CAI. The amnestic effects of ANI and the CAIs were also compared across seven experimental paradigms. Pretraining administration of any of the four drugs could result in amnesia for passive avoidance training, but only when training was weak. With an increase in training strength, a series of three injections of ANI (one pre- and two post-training) caused amnesia, but a similar series of CAI injections did not. Substituting one CAI injection for the second of three successive ANI injections did not cause amnesia, but substituting cycloheximide, another protein synthesis inhibitor, resulted in amnesia. With an active avoidance test, ANI caused amnesia while AMPT did not; d-amphetamine blocked the amnestic effect of ANI but caused amnesia in AMPT injected mice. Whereas ANI lengthened the temporal gradient over which electroconvulsive shock produced amnesia, AMPT or DDC did not. DDC caused only transient amnesia for passive avoidance training, while the amnestic effect of ANI remained constant at 24-hr and 1-week retention tests. We conclude that ANI and CAIs have distinctly different abilities to produce amnesia. These experiments provide additional support for the hypothesis that protein synthesis is required for formation of long-term memory.     

Protein synthesis and amnesia: studies with emetine and pactamycin.

Two antibiotic inhibitors of protein synthesis, emetine and pactamycin, have been tested for their effects on cerebral and peripheral protein synthesis and amnesia. Peripherally administered emetine but not pactamycin inhibited cerebral protein synthesis, although this inhibition was lower than that observed with cycloheximide or anisomycin. Pactamycin had a lesser effect on adrenal protein synthesis than emetine. This was reflected in the ability of emetine but not pactamycin to block ACTH-induced corticosteroidogenesis. Anisomycin and cycloheximide caused amnesia in a passive avoidance task, whereas pactamycin and emetine did not. These results are inconsistent with the amnesia being due to inhibition of protein synthesis in a peripheral organ. They are also inconsistent with the amnesia being due to the suppression of an adrenocortical response as previously suggested. No obvious correlation between amnesia and the mechanism of protein synthesis was observed. The most parsimonious explanation is that inhibition of cerebral protein synthesis is necessary for amnesia.