Effect of the antitumor drug vinblastine on nuclear betaII-tubulin in cultured rat kidney mesangial cells

Tubulin, the main component of microtubules, is a major target for antitumor drugs such as vinblastine. We have recently discovered that the _II isotype of tubulin is present in the nuclei of cultured rat kidney mesangial cells, smooth-muscle-like cells present in the renal glomerular mesangium (Walss C, Kreisberg JI, Ludueña RF: Cell Motil Cytoskeleton 42: 274–284, 1999). Here, we have investigated the effect of vinblastine on nuclear _II-tubulin in these cells. We have found that, at concentrations of 15nM and higher, vinblastine caused a reversible loss of _II-tubulin from the nucleus. Our results raise the possibility that nuclear _II-tubulin constitutes a population of tubulin that could be a novel target for antitumor drugs such as vinblastine.     

Elaboration of Simplified Vinca Alkaloids and Phomopsin Hybrids

Nine simplified vinca alkaloids and phomospin A hybrids, in which vindoline moiety has been replaced by a simpler scaffold, have been elaborated to evaluate their activity on the inhibition of tubulin polymerization. This article deals with the synthesis of various simplified vinca alkaloids, using a stereoselective coupling of catharantine with reactive aromatic compounds and methanol as well as their subsequent condensation with a large peptide chain mimicking those of phomopsin A. Biological evaluation and molecular modeling studies are also reported.     

Effect of diethylstilbestrol on the polymerization and alkylation of tubulin

Diethylstilbestrol is a drug used to treat prostate cancer. It is thought to bind to tubulin, the subunit protein of microtubules, at the colchicine‐binding site. We examined its interaction with tubulin in more detail. Diethylstilbestrol inhibits microtubule assembly, and seems to do so more effectively when tubulin polymerization is catalyzed by MAP2 rather than tau. Diethylstilbestrol also inhibits the intrachain cross‐linking of tubulin by N,N′‐ethylenebis‐(iodoacetamide) in a pattern similar to that shown by colchicine and the drugs which bind to tubulin at the colchicine‐binding site. Unlike most of this category of drugs, however, diethylstilbestrol accelerates, rather than inhibits, the decay of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas. It appears, therefore, that diethylstilbestrol interacts with tubulin in a manner similar to that of the analogs of the A‐ring of colchicine, whose effect on tubulin cross‐linking is similar to that of diethylstilbestrol and which also enhance tubulin decay. Drug Dev. Res. 48:104–112, 1999. © 1999 Wiley‐Liss, Inc.     

钙离子与钙调素或三氟拉嗪对离体管蛋白和在体微管的作用

作者曾改变轴浆中Ca~(2+)浓度使在体微管解聚。为了解其作用机理自兔脑提取了微管的亚单位管蛋白,浊度测定发现,无论Ca~(2+)本身抑制离体管蛋白聚合或者钙调素(CaM)增强它的抑制作用都取决于Ca~(2+)浓度。将抗CaM药三氟拉嗪注入大鼠坐骨神经使轴突中的微管解聚,Ca~(2+)与三氟拉嗪伍用则抵消各自解聚微管的作用。实验进一步证明Ca~(2+)调节微管聚合的主要作用途径是通过CaM,提示CaM结合一定数目的Ca~(2+)时能维持微管的聚合,若其浓度降低、结合Ca~(2+)增多或不足均使微管解聚。     

Vinflunine for the treatment of breast cancer

Introduction: Breast cancer is the most frequently diagnosed cancer and the highest cause of cancer mortality in females worldwide. The development of drugs improving overall survival in late-stage metastatic breast cancer remains a challenge.

Vinflunine is the most recently developed drug in the vinca alkaloid class. Its arrival has been eagerly awaited for treatment of solid tumors, and in particular, for metastatic breast cancer.

Areas covered: The pharmacological features of vinflunine are described. Its clinical development as monotherapy or in combination in metastatic breast cancer is detailed. A literature search on the topic was conducted through PubMed, clinical trials and the proceedings of the main cancer congresses.

Expert opinion: The overall results from phase III studies, and in particular those that combined vinflunine with capecitabine, have been less favorable. The combination’s effectiveness was at best moderate compared with other drugs which also target metastatic breast cancer, and complicated by significant hematological and gastrointestinal adverse effects. Its use in advanced metastatic breast cancer cannot currently be recommended.     

Characterization of and drug influence on a calmodulinsensitive phosphodiesterase purified from bovine brain

A calmodulin-sensitive phosphodiesterase was purified from bovine brain. The purification procedure involved ammonium sulphate fractionation, two chromatographic steps on DEAE-cellulose, gel-filtration on Sephadex G-200, and finally one DEAE-cellulose run, and gave a 2300-fold purification. The purified phosphodiesterase had a Vmax for cyclic AMP of 126 mumol/mg protein X min. and was activated 8-fold by addition of calmodulin and calcium. According to SDS-electrophoresis the purified enzyme contained one major peptide of 59,000 daltons, but the preparation was not homogeneous. The enzyme was characterized kinetically and with regard to the effect of cations, pH temperature, and nucleotides. Furthermore, the influence in vitro on enzyme activity of several classes of drugs, e.g. antidepressants, neuroleptics, antiallergics, platelet inhibitors, and some "reference phosphodiesterase inhibitors" was investigated.     

文蛤多肽抑制肿瘤细胞微管蛋白聚合

目的探讨文蛤多肽(Mere15)抑制人肺癌A549细胞增殖作用及其机制。方法 MTT比色法检测细胞增殖抑制率;流式细胞仪分析细胞周期分布的变化;微管蛋白免疫荧光检测技术检测Mere15对微管蛋白聚合的影响;Western blot检测Mere15对p21蛋白表达的影响。结果 Mere15可抑制A549细胞生长,抑制率呈剂量和时间依赖性;随着处理时间的增加,A549细胞G2/M期比例逐渐升高,微管蛋白聚合受到抑制,p21蛋白表达水平逐渐升高。结论 Mere15具有抑制A549细胞增殖的作用,其机制可能与抑制微管蛋白聚合有关。     

Different effects of neonatal vinblastine on peripheral and central noradrenaline neurons

The systemic injection of newborn rats of the mitotic inhibitor vinblastine sulfate (0.25 microgram/s.c. 48 h after birth), produces marked and persistent changes in peripheral sympathetic neurons. Approximately half the neuronal population of the superior cervical ganglia was destroyed already at 16 days of age and this was accompanied by a partial but persistent depletion of noradrenaline (NA) from peripheral organs receiving a rich sympathetic nerve supply such as the heart, salivary glands and spleen. After the systemic injection of vinblastine to newborn rats, the content of NA in several brain regions remained unaltered at 45-60 days of age. To overcome the obstacle that the blood-brain barrier could represent to vinblastine penetration into the brain, the compound was injected directly into the brain of rat pups at 2 days of age (0.25-1.0 microgram). When these animals were killed 45-60 days later, no changes were found in the concentration of NA in the cerebral cortex, the spinal cord or the cerebellum but NA levels were increased in the brain stem. Besides producing a partial but persistent peripheral sympathectomy, vinblastine injected either systemically or intracerebrally to newborn rats, provides a useful tool for the analysis of similarities and differences between the ontogenesis of central and peripheral NA neurons.     

Interaction of the cyanobacterial thiazoline-containing lipid curacin A with bovine brain tubulin

Curacin A is a thiazoline-containing lipid from the marine cyanobacterium Lyngbya majuscula. Despite being a potent inhibitor of microtubule assembly and of colchicine binding to tubulin, curacin A bears little or no structural resemblance to colchicine or to any other tubulin ligand. We investigated the interaction of curacin A with bovine brain tubulin using three different approaches. We first examined its effect on the intra-chain formation of a cross-link in β-tubulin by N,N′- ethylenebis(iodoacetamide). Formation of this cross-link, between cys²³⁹ and cys³⁵⁴, is blocked by colchicine and its A-ring analogues as well as by various other inhibitors of colchicine binding; C-ring analogues do not inhibit its formation. Curacin A strongly inhibited formation of this cross-link. Second, we examined the effect of curacin A on the time-dependent exposure of sulfhydryl groups on tubulin as measured by alkylation with iodo[¹⁴C]acetamide. Curacin A inhibited this very strongly, more so than either colchicine or podophyllotoxin. Last, we investigated the effect of curacin A on the time-dependent exposure of hydrophobic areas on the tubulin molecule. We found that curacin A had only a small effect on this process, comparable in magnitude to that of podophyllotoxin. Curacin A thus appears to have an unusual interaction with tubulin. Its binding site on tubulin is likely to overlap with that of the A-ring of colchicine. Drug Dev. Res. 40:223–229, 1997. © 1997 Wiley-Liss, Inc.     

Structure‐based approaches for the design of benzimidazole‐2‐carbamate derivatives as tubulin polymerization inhibitors

Microtubules are highly dynamic assemblies of α/β‐tubulin heterodimers whose polymerization inhibition is among one of the most successful approaches for anticancer drug development. Overexpression of the class I (βI) and class III (βIII) β‐tubulin isotypes in breast and lung cancers and the highly expressed class VI (βVI) β‐tubulin isotype in normal blood cells have increased the interest for designing specific tubulin‐binding anticancer therapies. To this end, we employed our previously proposed model of the β‐tubulin–nocodazole complex, supported by the recently determined X‐ray structure, to identify the fundamental structural differences between β‐tubulin isotypes. Moreover, we employed docking and molecular dynamics (MD) simulations to determine the binding mode of a series of benzimidazole‐2‐carbamete (BzC) derivatives in the βI‐, βIII‐, and βVI‐tubulin isotypes. Our results demonstrate that Ala198 in the βVI isotype reduces the affinity of BzCs, explaining the low bone marrow toxicity for nocodazole. Additionally, no significant differences in the binding modes between βI‐ and βIII‐BzC complexes were observed; however, Ser239 in the βIII isotype might be associated with the low affinity of BzCs to this isotype. Finally, our study provides insight into the β‐tubulin–BzC interaction features essential for the development of more selective and less toxic anticancer therapeutics.     

黄连总生物碱对铝致神经元退变的保护作用

目的采用铝负荷致小鼠脑损伤模型,探讨黄连总生物碱对铝致神经元退行性变的防治作用。方法以侧脑室注射0.25%Al溶液3μl(AlCl3.6H2O配制),以Al计算浓度,每日1次,连续5d;同时通过灌胃每日给予1次黄连总生物碱,直到实验结束(共29d)。用行为学、生物化学、免疫组织化学、等离子体原子发射光谱法和病理形态学评价小鼠的学习记忆能力、单胺氧化酶活性、线粒体铁蛋白水平、脑铁和铝水平及海马病理形态学变化。结果模型组小鼠学习记忆能力明显下降,大脑皮层、海马和纹状体MAO-B活力增加,脑铁水平明显升高,线粒体铁蛋白水平下降,而海马CA1区神经元核固缩和神经细胞丢失。黄连总生物碱(TCA)呈剂量依赖性阻遏铝负荷小鼠上述指标的改变;小檗碱和尼莫地平类也显示类似黄连总生物碱的作用。结论黄连总生物碱对铝过负荷致小鼠神经元退行性变有明显防治作用;其作用可能主要与其主要成分小檗碱有关。     

Characterization of the cAMP binding site of purified S-adenosyl-homocysteine hydrolase from bovine kidney

The enzyme S-adenosyl-homocysteine hydrolase (AdoHcyase) which catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine is an adenosine binding protein. In the present study we examined the characteristics of [(3)H]cAMP binding to purified AdoHcyase from bovine kidney in comparison with the high affinity adenosine binding site of AdoHcyase. AdoHcyase exhibits one [(3)H]cAMP binding site with an affinity of K(d)=23.1+/-1.1nM and a B(max) of 116.6+/-3.8pmol/mg protein. Binding of [(3)H]cAMP obeyed a monophasic reaction with a k(+1) value of 0.035min/M. The dissociation of AdoHcyase-[(3)H]cAMP complex exhibited a time- and temperature-dependent character. After a 240min incubation at 0 degrees only 5-10%, however, at 20 degrees 90% were displaceable. Adenosine and cAMP displace each other with similar affinities of EC(50) 57nM vs. EC(50) 65nM. 2'-Deoxyadenosine, N(6)-methyladenosine, and NECA displace 25nM [(3)H]cAMP and 10nM [(3)H]adenosine with EC(50) values of 94, 90 and 80nM, respectively. All other nucleosides studied, adenine, inosine, adenosine-2',3'-dialdehyde, 2-chloroadenosine, aristeromycin, and adenine nucleotides were only week competitors for [(3)H]cAMP and [(3)H]adenosine. These compounds displace [(3)H]cAMP and [(3)H]adenosine with equal potencies. Our data indicate that the binding site for nanomolar concentrations of cAMP and adenosine at the AdoHcyase appears to be identical. The physiological implications of a cAMP binding site at the AdoHcyase remain to be established.     

Inhibitors of protein-protein interactions

Protein-protein interactions are the basis of a number of intra- and extracellular processes. One of the challenges facing the pharmaceutical industry in the post genomic era is the rational identification and inhibition of this class of targets. Examples are presented of currently marketed drugs, compounds in clinical development and lead molecules in research programmes, which have been identified as inhibitors of protein-protein interactions. Modern drug discovery tools, including the use of humanised antibodies to validate targets and protein SARs in the identification of lead molecules, have brought these targets within reach.     

不同品种附子生物碱和多糖含量的比较

目的:比较不同品种附子生物碱和多糖的含量。方法:采用酸性染料比色法测定附子总生物碱含量,反相高效液相色谱法测定双酯型生物碱含量,蒽酮-硫酸法测定多糖含量。结果:不同品种附子总生物碱和双酯型生物碱含量差异较大,附子多糖含量差异不显著。附子炮制后,黑附片中总生物碱、双酯型生物碱含量显著下降,附子多糖含量稍有增加。结论:本试验结果可为附子品种选育提供一定的科学依据。     

Prejunctional effects of a purified toxin from the scorpion Tityus serrulatus

Summary The effects of a purified fraction of the venom of the Brazilian scorpion, Tityus serrulatus, were studied in isolated guinea-pig atria previously labelled with ³H-noradrenaline. Exposure to 0.3 and 1.0 /ml of the scorpion toxin resulted in a long lasting positive chronotropic effect which was concentration-dependent. The increase in atrial rate coincided with an enhancement in spontaneous outflow of radioactivity. The increase in outflow of radioactive products elicited by exposure to 1.0 g/ml of the scorpion toxin was approximately 3-fold. ³H-noradrenaline accounted for 60% of the total increase in outflow of radioactivity elicited by the scorpion toxin and the ³H-deaminated glycol (3,4-dihydroxyphenylglycol) represented the main metabolite formed, accounting for approximately 35% of the total release. 20 min after exposure to 1.0 g/ml of the scorpion toxin the overflow of the labelled transmitter elicited by accelerans nerve stimulation (4 Hz, during 60 sec, supramaximal voltage) was increased 8-fold. This effect of the scorpion toxin appears to be unrelated to inhibition of neuronal uptake, block of -adrenoceptors or stimulation of -adrenoceptors. Consequently, in addition to releasing noradrenaline, the scorpion toxin enhances transmitter overflow elicited by nerve stimulation through a prejunctional effect which appears to reflect a nove mechanism of action.     

Identification and molecular characterization of the tubulin‐podophyllotoxin‐type lignans binding site on Giardia lamblia

There are several drugs for the treatment of giardiasis; however, there is a tendency for patients to abandon treatment because of drug‐related adverse effects, resulting in relapses, acquired resistance, and higher rates of treatment failure. Recently, we reported some podophyllotoxin‐type lignans from Bursera fagaroides var. fagaroides showing antigiardial activity. In the present work, we demonstrated that 5′‐desmethoxy‐peltatin‐A‐methylether (5‐DES), acetylpodophyllotoxin (APOD), and podophyllotoxin (POD) affect the distribution and staining pattern of microtubular structures on Giardia trophozoites. Virtual screening results revealed that the lignans act via binding in a hydrophobic pocket in the heterodimer interface of tubulin in Giardia. This study provides useful insight to understand the action mechanism of 5DES, APOD, and POD on Giardia lamblia. The optimization of these podophyllotoxin‐type lignans will lead to promising candidates for antigiardial drugs.     

Different effects of di-isopropylfluorophosphate on the entry of opioids into mouse brain.

Di-isopropylfluorophosphate (DFP) potentiates the antinociceptive activity of alfentanil but has no effect on the activity of morphine or fentanyl. We have studied the effect of DFP on the distribution of these three opioids in the brain. Distribution studies were carried out using 3H-labelled opioids administered subcutaneously to mice. Animals were killed at times of peak antinociceptive activity and 3H-opioid measured in plasma and in eight brain regions. DFP pretreatment (1 mg kg-1) caused a significant increase in the brain:plasma ratio of alfentanil in all brain regions but had no effect on brain:plasma ratios for morphine or fentanyl. The enhanced entry of alfentanil into the brain of DFP-treated mice probably accounts for the increased antinociception observed with this opioid. This drug interaction appears to be opioid specific.     

乌药总生物碱抗炎镇痛药理学研究

目的：对乌药总生物碱抗炎镇痛作用进行研究。方法：采用p-二甲苯致小鼠耳廓肿胀法和角叉菜胶致后足跖肿胀法、小鼠热扳法和乙酸致小鼠扭体法等方法来评估用索氏法氨水-氯仿提取得到的乌药总生物碱的抗炎镇痛效果。结果：乌药总生物碱有缓解p-二甲苯致小鼠耳廓肿胀及角叉菜胶致后足跖肿胀效果,能减少小鼠在热板上舔后足的次数和减少乙酸致小鼠扭体的次数,镇痛效果显著。结论：乌药总生物碱抗炎镇痛作用明显。     

Different effects of cyanide on the activities of 7-ethoxycoumarin O-deethylation catalyzed by two forms of cytochrome P-450 purified from 3-methylcholanthrene-treated rats

The effect of cyanide on 7-ethoxycoumarin O-deethylation by two cytochrome P-450 isozymes obtained from 3-methylcholanthrene treated rat liver microsomes was investigated. 7-Ethoxycoumarin O-deethylation was stimulated by the addition of cyanide to a reconstituted monooxygenase system consisting of NADPH, dilauroyl 3-L-phosphatidylcholine, NADPH-cytochrome P-450 reductase and MC P-448(2) (low spin form of cytochrome). In contrast, a weak inhibitory effect of cyanide on 7-ethoxycoumarin O-deethylation was observed when MC P-448(1) (high spin form of cytochrome) was used in the reconstituted system. Cyanide did not influence the apparent Km for 7-ethoxycoumarin when either form of cytochrome P-450 was used in the reconstituted system and did not stimulate the cumene hydroperoxide dependent O-deethylation by MC P-448(2). The stimulatory effect of cyanide on O-deethylation by MC P-448(2) was decreased with increasing the concentration of the reductase added to the reconstituted system. On the other hand, the effect of cyanide on O-deethylation by MC P-448(1) was virtually independent on the amount of the reductase added.     

Microtubules of the mouse testis exhibit differential sensitivity to the microtubule disruptors Carbendazim and colchicine.

The testicular toxicant benomyl and its metabolite, carbendazim cause reproductive damage to the rat, an early sign of which is sloughing of germ cells with associated Sertoli cell fragments. However, the sensitivity of other mammalian species to these benzimidazole compounds is not clear. In this study, the effects of carbendazim and colchicine, a known microtubule disruptor, on the mouse seminiferous epithelium were characterized, and the amount of carbendazim reaching the mouse testis was measured. Testes were assessed for histological effects 3 h and 6 h after administration of carbendazim (2000 mg/kg, ip), and 6 h after intratesticular administration of either a low or high dose (5.3 or 117.6 micro g/g testis) of colchicine. Carbendazim caused no signs of histological damage to the mouse testis, and the microtubule cytoskeleton was intact and identical to controls based on immunostaining with tyrosinated alpha tubulin and beta tubulin antibodies. Similarly, the seminiferous epithelium of mouse testis was undamaged and the microtubule cytoskeleton was intact after a low dose of colchicine, while a comparable dose of colchicine injected into rat testis caused marked toxicity. However, mouse testes did show microtubule disruption and severe germ cell sloughing after administration of a high dose of colchicine. The amount of carbendazim measured in mouse testis was 375 nmol/g testis, which is higher than the value measured in rat testis after a toxic dose of carbendazim. Therefore, carbendazim reaches the mouse testis at or above levels measured in the rat, yet the mouse is apparently insensitive to this microtubule disrupting agent.