Structure-activity requirements for flavone cytotoxicity and binding to tubulin

A series of 79 flavones related to centaureidin (3,6,4'-trimethoxy-5, 7,3'-trihydroxyflavone, 1) was screened for cytotoxicity in the NCI in vitro 60-cell line human tumor screen. The resulting cytotoxicity profiles of these flavones were compared for degree of similarity to the profile of 1. Selected compounds were further evaluated with in vitro assays of tubulin polymerization and [3H]colchicine binding to tubulin. Maximum potencies for tubulin interaction and production of differential cytotoxicity profiles characteristic of 1 were observed only with compounds containing hydroxyl substituents at C-3' and C-5 and methoxyl groups at C-3 and C-4'.     

The reconstitution of microtubules from purified calf brain tubulin

The in vitro reconstitution of calf brain tubulin, purified by the method of Weisenberg et al. [(1968), Biochemistry 7, 4466-4479; (1970), Biochemistry 9, 4110-4116] as modified by Lee et al. [(1973), J. Biol. Chem. 248, 7253-7262], was successful in a medium consisting of 10(-2) M sodium phosphate, 10(-4) M GTP, and concentrations of magnesium ions ranging from 0.5 to 16 X 10(-3) M at 37 degrees. Filaments resembling native microtubules were formed. The filaments are in equilibrium with the associating species of tubulin and the equilibrium can be shifted to depolymerization by lowering the temperature to 20 degrees. Filament formation is inhibited by calcium ions which also cause disassembly of the formed filaments. The effects of calcium ion can be reversed by the addition of [ethylenebis-oxyethylenenitrilo)]tetraacetic acid. The formation of filaments is favored by the presence of 3.4 M glycerol; only twisted abnormal filaments are observed in the presence of 1 M sucrose. The high molecular weight components observed in the sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of many tubulin preparations were shown not to be essential for the formation of the filaments.     

Reversible brain MRI changes in acyclovir neurotoxicity

This case report shows reversible brain MRI changes probably associated with acyclovir toxicity. So far, neuroimaging in acyclovir toxicity had been negative or uninformative. A 12-year-old girl developed focal secondary generalizing epileptic fits following 4 weeks of prophylactic administration of acyclovir (3 x 10 mg/kg body weight/day i.v.) on day +22 after allogeneic peripheral blood stem cell transplantation for CML. Infective causes were excluded. Brain MRI demonstrated multiple gadolinium-enhancing areas with impairment of the blood-brain barrier in cortical and subcortical regions. Clinical symptoms and neuroimaging pathology resolved completely within 9 days of acyclovir withdrawal.     

Energy transfer studies of the distances between the colchicine, ruthenium red, and bisANS binding sites on calf brain tubulin

Fluorescence energy transfer experiments were performed in order to measure the spatial separation between the colchine and Ruthenium Red binding sites, the high-affinity bisANS and Ruthenium Red sites, and the allocolchicine and high-affinity bisANS sites on calf brain tubulin. Energy transfer was observed between both colchicine and allocolchicine and Ruthenium Red, resulting in a distance of 40-45 A between these sites on the tubulin molecule. No detectable energy transfer could be observed when allocolchicine was used as fluorescence donor and bisANS as acceptor or when bisANS was used as donor and Ruthenium Red as acceptor. This indicates that the distance of separation between the allocolchicine and bisANS sites is greater than 50 A, while that between the bisANS and Ruthenium Red sites is greater than 72 A. On the basis of these and previous distance measurements (Ward & Timasheff, 1988), two triangles of binding sites have been defined (colchicine-bisANS-E-site and colchicine-bisANS-Ruthenium Red). Since the dihedral angle between them is not known, a schematic model has been drawn with all the sites located in a single plane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specific binding of acidic phospholipids to microtubule-associated protein MAP1B regulates its interaction with tubulin

Microtubule-associated protein MAP1B, a major neuronal cytoskeletal protein, is expressed highly during the early stage of brain development and is thought to play an important role in brain development. Although it has been shown that MAP1B localizes both in cytosol and particulate fractions, the underlying molecular mechanism in the membrane localization has yet to be elucidated. In the present study, we show that MAP1B purified from young rat brain can bind to acidic phospholipids, such as phosphatidylserine, but not to a neutral phospholipid, phosphatidylcholine. Furthermore, the binding of MAP1B to taxol-stabilized microtubules was inhibited by the addition of phosphatidylserine or phosphatidylinositol. The addition of phosphatidylcholine showed no effect on the binding of MAP1B to the microtubules. A 120-kDa microtubule-binding fragment of MAP1B was also released from microtubules by the addition of acidic phospholipids. Synthetic peptides derived from the C-terminal half of the tubulin-binding domain, but not that corresponding to the N-terminal half, bound to acidic phospholipids specifically. These results suggest that MAP1B binds to biological membranes through its tubulin-binding site, and the binding may play a regulatory role in MAP1B-microtubule interaction.     

Reversible binding of ethacrynic acid to human serum albumin: difference circular dichroism study

Intimal proliferation at the interface between prosthetic material and tissue is an intrinsic phenomenon of stenting and the major cause of insufficiency of the transjugular intrahepatic portosystemic shunt (TIPS). For its prevention, a randomized study was performed comparing standard heparin treatment with a combination of trapidil, a drug with anti-platelet-derived growth factor (PDGF) activity, and ticlopidine, a platelet aggregation inhibitor. Ninety patients with cirrhosis who received a transjugular shunt were randomized, and 84 patients completed the trial. Group 1 (n = 42) received a bolus of heparin (12 to 24 U/kg) at shunt placement, followed by 1 week of intravenous and 4 weeks of subcutaneous heparin treatment. Group 2 (n = 42) received the same heparin bolus, followed by a 1-day intravenous heparin treatment and a 6-month treatment with trapidil (400 mg/d) and ticlopidine (250 mg/d). Shunt function was assessed by duplex-sonography and angiography. Stenoses were classified according to their location as type 1 (within the stent) and type 2 (in the draining hepatic vein). The estimated rate of overall stenoses (intention-to-treat analysis) at 1 year showed a significant reduction in patients receiving trapidil and ticlopidine (group 2) as compared with heparin (33 vs. 57%; P =.047). There was no difference in the estimated 1-year rate of type 1 stenoses between the two groups, but there was a significant reduction in type 2 stenoses (group 1: 58%, group 2: 19%; P =.016). The treatment effect continued after withdrawal of the drugs and was accompanied by a decreased incidence of rebleeding. The study demonstrates that the incidence of type 2 stenosis of the transjugular shunt can be reduced by combined inhibition of platelet aggregation and PDGF activity. The findings may be of relevance not only for the transjugular shunt, but also for other stent applications, e.g., vascular and biliary, as well as for bypass and shunt surgery.     

Inhibition of [3H]mebendazole binding to tubulin by structurally diverse microtubule inhibitors which interact at the colchicine binding site

A rapid and convenient radioligand assay was used to characterise the interaction of several structurally diverse microtubule inhibitors with the colchicine binding domain of tubulin. Values determined for the inhibition of [3H]mebendazole binding to tubulin by colchicine, combretastatin A4, NSC 181928, NSC 321567, podophyllotoxin and tubulozole-C provided an independent measure of the relative potency of these compounds. This methodology has several advantages over the inhibition of [3H]colchicine binding as a technique for investigating the molecular mechanisms involved in determining tubulin-ligand interactions.     

Role of the colchicine ring A and its methoxy groups in the binding to tubulin and microtubule inhibition

The roles of the methoxy substituents on ring A of two ring colchicine (COL) analogues were probed by the synthesis of a number of drugs and the examination of their effect on binding to tubulin, inhibition of microtubule assembly, and induction of GTPase activity. Selective elimination of ring A methoxy groups at positions 2, 3, and 4 weakened all three processes. The effects on binding and inhibition were independent of the nature of ring C (or C'). Specifically, excision of the 2- or 3-methoxy groups weakened binding by ca. 0.4 kcal mol-1, while that of the 4-methoxy group of ring A was weakened by 1.36 +/- 0.15 kcal mol-1. The effect on the inhibition of microtubule assembly, expressed as the equilibrium constant for the binding of the tubulin-drug complex to the end of a microtubule, was more complex and strongly dependent on the nature of ring C (or C'). This was attributed to the abilities of various groups on ring C' to overcome the wobbling in the tubulin-drug complex introduced by the weakening of the anchoring provided by ring A. It is concluded that ring A of COL is not germane to the mechanism of the inhibition of tubulin self-assembly. It serves only as a complex-stabilizing anchor. The control of this process resides in the interactions that key oxygen atoms of ring C of COL or C' of structural analogues establish with the protein. It is proposed that the 4-methoxy group of ring A serves as a key attachment point for immobilization of the drugs on the protein.     

Binding of gamma-aminobutyric acidA receptors to tubulin

Tubulin cofractionated with gamma-aminobutyric acidA (GABAA) receptors upon affinity chromatography on Affigel 15 coupled to the benzodiazepine Ro 7-1986, and this cofractionation was not due to unspecific adsorption of tubulin to the column material. In addition, GABAA receptors not only bound to microtubules but also coassembled with added tubulin through three cycles of microtubule polymerization and depolymerization. These data indicate that GABAA receptors may be associated with the microtubule cytoskeleton in the brain. In an experiment designed to detect a protein that possibly could form a bridge between tubulin and the GABAA receptor, only a single protein band containing tubulin could be identified that was able to bind polymerized tubulin after sodium dodecyl sulfate-gel electrophoresis of affinity-purified GABAA receptors. These results are discussed with respect to a possible mechanism of association between GABAA receptors and microtubules.     

Initiation of brain tubulin assembly by a high molecular weight flagellar protein factor

A protein factor found within the flagella of Chlamydomonas and sea urchin sperm is capable of stimulating the initiation of calf and chick brain tubulin dimer assembly in vitro.     

IKP104-induced decay of tubulin: role of the A-ring binding site of colchicine

Tubulin, the major subunit protein of microtubules, has a tendency to lose its ability to assemble or to interact with ligands in a time-dependent process known as decay. Decay involves the increase in exposure of sulfhydryl groups and hydrophobic areas. The antimitotic drug IKP104 [2-(4-fluorophenyl)-1-(2-chloro-3, 5-dimethoxyphenyl)-3-methyl-6-phenyl-4(1H)-pyridinone] accelerates the decay of tubulin [Ludue?a et al. (1995) Biochemistry 34, 15751-15759]. In the presence of colchicine, however, IKP104 stabilizes tubulin against decay. We have shown that the stability and the acceleration of the decay of tubulin are mediated respectively by the high- and low-affinity binding site(s) of IKP104 [Chaudhuri et al. (1998) J. Protein Chem. 17, 303-309]. To better understand the mechanism by which colchicine protects tubulin from IKP104-induced decay, we examined the effect of colchicine and its analogues on this process. We found that IKP104 unfolds tubulin in a process involving a specific domain where colchicine interacts, although the binding sites of these two drugs are distinctly different. 2-Methoxy-5-(2',3',4'-trimethoxyphenyl) tropolone (MTPT), the bicyclic analogue of colchicine that lacks the B-ring, can also protect tubulin from IKP104-induced decay. An A-ring analogue of colchicine, 3,4,5-trimethoxybenzaldehyde (TMB), can also stop IKP104-induced unfolding of tubulin significantly. Interestingly, the C-ring analogue of colchicine, tropolone methyl ether (TME), does not prevent this process. Our results thus suggest that neither the B-ring nor the C-ring binding regions of colchicine are involved in the IKP104-induced decay and that the A-ring binding site of colchicine on tubulin plays a crucial role in IKP104-induced decay.     

Heterogenous binding of 3H-remoxipride to membranes of rat liver and brain

To assess the characteristics of sucrose as a pain-reducing substance, crying in 72 newborn humans during and after blood collection via heel prick was determined. In the first study infants drank 2 ml of water or 2 ml of a 0.17-0.34- or 0.51-M sucrose solution 1 min prior to blood collection. In the second experiment, a delay of 30, 60, 90, 120 or 240 s was imposed between sucrose intake and the initiation of blood collection. The dose-response function for concentration was flat. The most effective time delay was 120 s. The effectiveness of the 2-min interval accords with previous findings of endogenous opioid release caused by sucrose taste. The flat dose-response function extends findings in rats and humans that the calming and pain-reducing effects of sucrose are not influenced by either concentration or volume, suggesting that the transduction from gustatory afferent to opioid-mediated efferent is of an on-off nature and not graded.     

Distances between the paclitaxel, colchicine, and exchangeable GTP binding sites on tubulin

Distances between the paclitaxel, colchicine, and exchangeable GTP binding sites on tubulin polymers have been probed using fluorescence spectroscopy. Techniques for measuring fluorescence resonance energy transfer (FRET) between fluorescent or chromophoric ligands for each binding site were employed. 2-Debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT) was the fluorophore ligand for the paclitaxel binding site; thiocolchicine, allocolchicine, and MDL 27048 were probes for the colchicine site, and 2'(or 3')-O-(trinitrophenyl)guanosine 5'-triphosphate (TNP-GTP) was the fluorophore ligand for the exchangeable GTP site. The distance between the colchicine and paclitaxel binding sites was determined with two different acceptor ligands in the colchicine site. An average distance distribution of 17 A was found in both cases. Energy transfer between 2-AB-PT bound to the paclitaxel site and TNP-GTP (acceptor) bound to the exchangeable GTP site was observed in the polymer. The average distance distribution between the fluorophores was 16.0 A, but the half-width of the distribution was large (17.9 A), which indicates that energy transfer between more than one donor-acceptor pair occurred in the system. One interpretation of this result is that 2-AB-PT serves as an energy transfer donor for two GTP sites, one contained on the same subunit and one on an adjacent protofilament. No FRET was observed between ligands bound to the colchicine and exchangeable GTP sites, indicating that the result of colchicine binding on the GTP region of beta-tubulin is a long range, allosteric effect. The results from these experiments are interpreted in terms of known structural features of microtubules.     

Developmental profile of glucocorticoid binding to synaptic plasma membrane from rat brain

Plasma prolactin levels were measured in free-living breeding adult and immature (pre-breeding) macaroni (Eudyptes chrysolophus) and gentoo (Pygoscelis papua) penguins at Bird Island, South Georgia (54 degrees S, 38 degrees W). Macaroni and gentoo penguins first breed at 5-6 and 2 years of age, respectively. In adult birds, of both species, prolactin was low (< 1.0 microgram. liter-1) during the courtship period and then increased during early (gentoo) to mid (macaroni) incubation (to 3.9-4.7 and 2.4-3.5 micrograms.liter-1, respectively), remaining elevated until the creche period, by which time continuous nest attendance by the adults had ceased. This pattern is similar to that seen in other altricial species and is consistent with delayed onset of brood patch development and full incubation efficiency, which has been previously reported in penguins. Adult female macaroni penguins showed a marked, but transient, increase in prolactin concentrations within 24 hr of the first egg being laid (from 1.7 to 7.0 micrograms.liter-1), plasma levels decreasing following clutch completion (to prelaying levels) before increasing again during incubation. Elevated plasma prolactin levels occurred in all age classes of immature (nonbreeding) birds in both macaroni (1- to 5 year-olds) and gentoo (1-year-olds) penguins. However, compared to that in adult birds, the increase in prolactin was more transient in immatures, a smaller proportion of immatures had detectable prolactin levels at each stage of the breeding cycle, and, at least in 1- and 2-year-olds, absolute levels of prolactin were lower.(ABSTRACT TRUNCATED AT 250 WORDS)     

Photoaddition to DNA by nonintercalated chlorpromazine molecules

Chlorpromazine (CPZ) forms photoadducts with DNA and photosensitizes DNA strand breaks. These reactions may be responsible for the reported photomutagenicity of CPZ and for the well-known cutaneous and ocular phototoxicity associated with this drug. We have investigated whether CPZ molecules that are intercalated between base pairs in double-stranded (ds) DNA are the absorbing species for the photoaddition reaction. Quenching of CPZ fluorescence by ds-DNA gave nonlinear Stern-Volmer plots, indicating that more than one type of complex is formed. Linear dichroism spectra of CPZ in the presence of ds-DNA showed a minimum at 345 nm, indicating that the absorption maxima of intercalation complex(es) are red-shifted compared to the absorption maximum of free CPZ at 307 nm. The sum of the absorption of all CPZ complexes with ds-DNA, obtained from dialysis experiments, was broadened and maximized at about 315 nm, indicating that complexes not involving intercalation dominate the absorption spectrum at lambda < 350 nm. The wavelength dependence for covalent binding of CPZ to DNA was determined by irradiating 3H-CPZ in the presence of ds-DNA at 310, 322, 334, 346, 358 and 370 nm. The resulting spectrum correlated closely with the absorption spectrum of nonintercalated CPZ rather than with the spectrum of intercalated CPZ, indicating that the latter species is not the chromophore for the photoaddition reaction.     

The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant cells

The lactone-bearing polyhydroxylated alkatetraene (+)-discodermolide, which was isolated from the sponge Discodermia dissoluta, induces the polymerization of purified tubulin with and without microtubule-associated proteins or GTP, and the polymers formed are stable to cold and calcium. These effects are similar to those of paclitaxel (Taxol), but discodermolide is more potent. We confirmed that these properties represent hypernucleation phenomena; we obtained lower tubulin critical concentrations and shorter polymers with discodermolide than paclitaxel under a variety of reaction conditions. Furthermore, we demonstrated that discodermolide is a competitive inhibitor with [3H]paclitaxel in binding to tubulin polymer, with an apparent Ki value of 0.4 microM. Multidrug-resistant human colon and ovarian carcinoma cells overexpressing P-glycoprotein, which are 900- and 2800-fold resistant to paclitaxel, respectively, relative to the parental lines, retained significant sensitivity to discodermolide (25- and 89-fold more resistant relative to the parental lines). Ovarian carcinoma cells that are 20-30-fold more resistant to paclitaxel than the parental line on the basis of expression of altered beta-tubulin polypeptides retained nearly complete sensitivity to discodermolide. The effects of discodermolide on the reorganization of the microtubules of Potorous tridactylis kidney epithelial cells were examined at different times. Intracellular microtubules were reorganized into bundles in interphase cells much more rapidly after discodermolide treatment compared with paclitaxel treatment. A variety of spindle aberrations were observed after treatment with both drugs. The proportions of the different types of aberration were different for the two drugs and changed with the length of drug treatment.