Promotion of tubulin assembly by aluminum ion in vitro.

It has been proposed that aluminum ion is a contributing factor in a variety of neurological diseases. In many of these diseases, aberrations in the cytoskeleton have been noted. The effects of aluminum ion on the in vitro assembly of tubulin into microtubules has been examined by determining the association constants for the metal ion-guanosine triphosphate-tubulin ternary complex required for polymerization. The association constant for aluminum ion was approximately 10(7) times that of magnesium ion, the physiological mediator of microtubule assembly. In addition, aluminum ion at 4.0 X 10(-10) mole per liter competed effectively with magnesium ion for support of tubulin polymerization when magnesium ion falls below 1.0 millimole per liter. The microtubules produced by aluminum ion were indistinguishable from those produced by magnesium ion when viewed by electron microscopy, and they showed identical critical tubulin concentrations for assembly and sensitivities to cold-induced depolymerization. However, the rate of guanosine triphosphate hydrolysis and the sensitivity to calcium ion-induced depolymerization, critical regulatory processes of microtubules in vivo, were markedly lower for aluminum ion microtubules than for magnesium ion microtubules.

2-ABT-S-oxide detoxification by glutathione S-transferases A1-1, M1-1 and P1-1: implications for toxicity associated with zileuton.

Zileuton, an agent which targets the leukotriene pathway through inhibition of 5-lipoxygenase (5-LO), was approved for the treatment of asthma in 1997. Shortly after its release, its use was restricted due to the observation of hepatotoxicity in patients. Previous research from the authors' laboratory demonstrated the formation of the reactive metabolite, 2-ABT-S-oxide (M1) from zileuton, and has identified a mercapturate of 2-ABT, C1, in the urine of rats dosed with zileuton. The reaction between M1 and glutathione (GSH) has been established in vitro; however, the potential for catalysis by glutathione transferases (GSTs) was not addressed. The work presented here outlines a role for GSTs in the detoxification of M1. Non-enzymatic conjugation studies with M1 and GSH in control experiments led to a t(1/2) of 6.4 +/- 0.4 h at pH 6.5. This rate was accelerated in the presence of GSTA1-1, GSTM1-1 and GSTP1-1 providing t(1/2) values of 2.6 +/- 0.1, 0.53 +/- 0.02, and 0.3 +/- 0.04 h, respectively, at pH 6.5. The inhibition of various GST enzymes was also studied. Results show that M1 inhibits GSTM1-1 and GSTP1-1 to a greater extent as compared with GSTA1-1. In the case of GSTA1-1, the inhibition was observed to be reversible, whereas M1 inhibition of GSTM1-1 and GSTP1-1 was found to be irreversible under identical conditions. GSTM1-1 is present in liver and thus the finding of the alkylation and potential irreversible inactivation of this isoform in vivo could contribute to an understanding of the hepatotoxicity associated with zileuton.

Inhibition of human prostate cancer proliferation in vitro and in a mouse model by a compound synthesized to block Ca2+ entry.

Accelerated Ca2+ entry may be one component of the pathway regulating the proliferative phenotype of some types of cancer. Thus, a pharmacological agent with the ability to retard Ca2+ influx in susceptible cancers might inhibit proliferation of them by a cytostatic mechanism rather than by inducing cytotoxicity. We have developed a chemical synthetic scheme that has produced a small library of novel compounds that block Ca2+ entry induced by occupancy of the P2 receptor in two prostate cancer cell lines and inhibit proliferation of these cells in vitro. One of the agents, named TH-1177, was used to treat severe combined immunodeficient mice inoculated with the human prostate cancer line PC-3. Although the doses used and treatment schedule were chosen arbitrarily, treatment extended the mean life span of mice bearing tumors by up to 38%. Treatment of mice without cancer at doses 18 times that used in mice with tumors was not associated with any obvious toxicity, either grossly or on histological examination. These results suggest that novel cytostatic agents with efficacy against human prostate cancer cells can be developed by chemical synthesis of agents directed at the Ca2+ entry pathway.

Pharmacological stress myocardial perfusion imaging with the potent and selective A(2A) adenosine receptor agonists ATL193 and ATL146e administered by either intravenous infusion or bolus injection.

BACKGROUND: Adenosine (Ado) and dipyridamole are alternatives to exercise stress for myocardial perfusion imaging. Though generally safe, side effects frequently occur that cause patient discomfort and sometimes lead to premature termination of the study or require aminophylline administration. Recently, a new class of A(2A) Ado receptor agonists was synthesized. ATL193 and ATL146e are 2-propynylcyclohexyl-5'-N-ethylcarboxamido derivatives of Ado. The study goals were to evaluate the potency and selectivity of these new compounds on recombinant canine Ado receptors and to evaluate their hemodynamic properties in dogs to assess their usefulness as vasodilators for myocardial perfusion imaging. METHODS AND RESULTS: In assays of recombinant canine Ado receptors, ATL-193 and ATL-146e were highly selective for the A(2A) over the A(1) and A(3) receptors and were more potent than MRE-0470 and CGS-21680. In 16 anesthetized dogs, the agonists were administered by infusion (ATL-193; n=7 normal) or bolus injection (ATL-146e; n=9 critical left anterior descending coronary artery stenosis), and hemodynamic responses were compared with those of Ado. Both agonists produced dose-dependent coronary flow (CF) elevation without provoking the hypotension observed with Ado. After an ATL-146e bolus, the CF increase was sustained for several minutes, providing ample time for injection and myocardial uptake of (99m)Tc-sestamibi, and CF returned to baseline within 20 minutes. The CF increase was completely blocked by the selective A(2A) antagonist ZM241385 (3 microgram. kg(-1). min(-1)). CONCLUSIONS: ATL-193 and ATL-146e are highly potent and selective Ado A(2A) receptor agonists with excellent potential for use as vasodilators for myocardial perfusion imaging. An important advantage of ATL-146e is the ability to administer it by bolus injection.

Cleavable complex formation in Leishmania chagasi treated with anilinoacridines.

Anilinoacridines have recently been found to possess antiparasitic activity toward Leishmania, Trypanosoma, and Plasmodium species. These compounds have been examined for their ability to generate cleavable complex, the protein-associated DNA lesion characteristic of topoisomerase II involvement, in intact L. chagasi promastigotes. At cytotoxic concentrations, anilinoacridine compounds give cleavable complex in a whole cell assay which suggests that the drugs affect a nuclear topoisomerase II in the parasite. Linearization of kinetoplast DNA minicircles also occurs in parasites treated with anilinoacridines at similar concentrations. Exonuclease digestions reveal that the linearized minicircles are blocked at the 5' end but not at the 3' end, further implicating a kinetoplast topoisomerase II in the cleavage process. Interestingly, cytotoxic alkylaminoacridines did not stimulate the production of cleaved DNA in the same experiments. DNA binding experiments showed no apparent correlation between the affinity of the compounds for DNA and antileishmanial activity. Although multiple cytotoxic mechanisms are likely at work, these experiments suggest that topoisomerase II enzyme(s) are affected by antileishmanial anilinoacridines.

Synthesis and structure-activity profiles of A-homoestranes, the estratropones.

2-Methoxyestradiol, a mammalian metabolite of estradiol, has reported antiangiogenic activity which has been proposed to be mediated through interaction at the colchicine binding site on the tubulin monomer. Subsequent structure-activity studies of 2-methoxyestradiol have yielded highly potent steroidal inhibitors of tubulin polymerization. In an effort to probe the scope of binding at the colchicine binding site and the nature of the relationship between 2-methoxyestradiol and colchicine, a series of colchicine/2-methoxyestradiol hybrids was synthesized. These A-homoestrane hybrid systems, collectively termed estratropones, possessed an A-ring tropone system with the keto functionality at either the C-2, C-3, or C-4 position of the steroid nucleus. The estratropones were evaluated for their ability to inhibit the polymerization of tubulin using an in vitro purified bovine brain assay. Most of these hybrids inhibit polymerization with greater potency than either of the natural products. The most potent of these congeners possessed an approximate 5-fold enhancement of the activity of colchicine for the inhibition of tubulin polymerization. alpha-Substituents on the tropone ring showed varied effects on the activities for the two classes of estratropones studied in this regard, the C-3 oxo and the C-4 oxo species. The 3-substituted 4-oxoestratropones exhibited antitubulin activity according to Cl approximately Br > OCH3, whereas the 4-substituted 3-oxoestratropones exhibited activity according to OCH3 > Br approximately Cl. It is unclear if these substituent factors are purely electronic or steric effects or if the substituent operates indirectly by altering the conformation of the nonplanar troponoid ring. The estratropones represent a new class of tubulin binding agents with potential antiangiogenic utility.

Design, synthesis, and evaluation of novel A2A adenosine receptor agonists.

We have been interested in the design, synthesis, and evaluation of novel adenosine A2A agonists. Through the use of comparative molecular field analysis (CoMFA) we have generated a training model that includes 78 structurally diverse A2A agonists and correlated their affinity for isolated rat brain receptors with differences in their structural and electrostatic properties. We validated this model by predicting the activity of a test set that included 24 additional A2A agonists. Our CoMFA model, which incorporates the physiochemical property of dipole and selects against A1 receptor activity, generated a correlated final model (r2 = 0.891) that provides for enhanced A2A selectivity and predictability. Synthesis, pharmacological evaluation, and modeling of four novel ligands further validate the utility and predictive power (r2 = 0.626) of the CoMFA model.

Structure--activity relationships of lysophosphatidic acid: conformationally restricted backbone mimetics.

Lysophosphatidic acid (LPA) has associated with it an intriguing cell biology that is thought to be mediated through its interaction with G-protein coupled receptor(s). In an effort to extend the structure-activity relationships of LPA, we have produced a series of LPA analogues in which the glycerol core in LPA was replaced with conformationally restricted aryl substructures. The aryl substructures encompassed aminophenol, resorcinol, dihydroxy benzophenone, and tocopherol systems. The benzophenone moiety was investigated both as a conformationally restricting substructure for LPA and as a possible photoreactive alkylating agent for the LPA receptor(s). All LPA analogues were evaluated for their potency and efficacy in mobilizing calcium ions from internal stores in MDA MB-231 cells. Ten of the 14 analogues exhibited activity in this assay at doses up to 5 microM; none of the compounds exhibited nonreceptor-mediated lytic activity at this maximal concentration. The receptor response showed surprising tolerance for manipulation in the backbone region of LPA, although none of the compounds were equipotent to LPA. This tolerance for a variety of structures has given us new leads into the realization of novel agonists and antagonists of the LPA receptor(s).

Inhibition of DNA topoisomerase II by azaelliptitoxins functionalized in the variable substituent domain.

A series of novel C11-substituted derivatives of azaelliptitoxin (azatoxin) have been synthesized and tested for their inhibitory activity against human DNA topoisomerase II. Incorporation of a C11 polyamine or amine resulted in an increase in the intercalation properties of the drug and a decrease of topoisomerase II activity. The structure-activity relationship (SAR) profile of the nonintercalating C11 anilino azatoxin class follows the SAR of the (anilino)acridine family. 11-(4-Cyanoanilino)azatoxin (14) was found to be the most active analog in this series, exhibiting approximately 10-fold higher activity than azatoxin 12 and etoposide.

Cyclic AMP-dependent inhibition of human neutrophil oxidative activity by substituted 2-propynylcyclohexyl adenosine A(2A) receptor agonists.

Novel 2-propynylcyclohexyl-5'-N:-ehtylcarboxamidoadenosines, trans-substituted in the 4-position of the cyclohexyl ring, were evaluated in binding assays to the four subtypes of adenosine receptors (ARs). Two esters, 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester (ATL146e) and acetic acid 4-(3-[6-amino-9-(5-ethylcarbamoyl-3, 4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl] -prop-2-ynyl)-cyclohexylmethyl ester (ATL193) were >50 x more potent than 2-[4-(2-carboxyethyl)phenethylamino]-5'-N:-ethylcarboxamidoadenosine (CGS21680) for human A(2A) AR binding. Human A(2A) AR affinity for substituted cyclohexyl-propynyladenosine analogues was inversely correlated with the polarity of the cyclohexyl side chain. There was a comparable order of potency for A(2A) AR agonist stimulation of human neutrophil [cyclic AMP](i), and inhibition of the neutrophil oxidative burst. ATL146e and CGS21680 were approximately equipotent agonists of human A(3) ARs. We measured the effects of selective AR antagonists on agonist stimulated neutrophil [cyclic AMP](i) and the effect of PKA inhibition on A(2A) AR agonist activity. ATL193-stimulated neutrophil [cyclic AMP](i) was blocked by antagonists with the potency order: ZM241385 (A(2A)-selective)>MRS1220 (A(3)-selective)>>N-(4-Cyano-phenyl)-2-[4-(2,6-dioxo-1,3-dipropyl-2,3,4,5,6,7-hexahydro-1H-purin-8-yl)-phenoxy]-acetamide (MRS1754; A(2B)-selective) approximately 8-(N-methylisopropyl)amino-N(6)-(5'-endohydroxy-endonorbornyl)-9-methyladenine (WRC0571; A(1)-selective). The type IV phosphodiesterase inhibitor, rolipram (100 nM) potentiated ATL193 inhibition of the oxidative burst, and inhibition by ATL193 was counteracted by the PKA inhibitor H-89. The data indicate that activation of A(2A)ARs inhibits neutrophil oxidative activity by activating [cyclic AMP](i)/PKA.

Binding of colchiceine to tubulin. Mechanisms of ligand association with tubulin.

Colchiceine, a closely related structural analog of colchicine possessing a C-ring tropolone, has been shown to be a potent inhibitor of microtubule assembly in vitro (I50 = 20 microM). The mechanism of inhibition is mediated through binding to tubulin (KA = 1.2 +/- 0.7 x 10(4) M-1), although potentially not through the colchicine receptor site. Supporting the hypothesis of an alternate receptor are the observation of colchiceine binding to the isolated colchicine-tubulin complex (KA = 2.2 +/- 1.0 x 10(4) M-1), the poor correlation between the competitive inhibition of colchicine binding (KI = 125 microM) and the inhibition of microtubule assembly, and different structure-activity relationships for colchiceine analogs as compared to the colchicine series.

Inhibition of topoisomerases by fredericamycin A.

Fredericamycin is an antibiotic product of Streptomyces griseus that exhibits modest antitumor activity in vivo and in vitro. Because of its unique structure and the absence of a clearly defined mechanism of action, we examined the effects of this compound on L1210 cells in culture as well as on several enzymes that bind to DNA. Fredericamycin exhibits an IC50 of 4.4 microM toward L1210 cells, and its cytotoxicity is a function of the time of exposure as well as drug dose. No DNA breakage was observed in L1210 cells or isolated nuclei following exposure to highly lethal concentrations of fredericamycin. As a first step toward understanding its mechanism of action, we examined the effect of fredericamycin on several enzymes involved in DNA metabolism. The catalytic activity of both DNA topoisomerases I and II were totally inhibited by fredericamycin concentrations of 4.4 and 7.4 microM, respectively. Fredericamycin blocked etoposide-stimulated DNA cleavage by topoisomerase II both in vitro and in isolated nuclei. In addition, the drug inhibits DNA polymerase a in vitro, exhibiting an IC50 of 93 microM. These diverse actions of fredericamycin do not enable us to draw conclusions regarding its mechanism of antitumor effect but clearly identify it as a compound of pharmacologic interest.

Modulatory effects of aluminum, calcium, lithium, magnesium, and zinc ions on [3H]MK-801 binding in human cerebral cortex.

The independent and combined effects of Ca2+, Mg2+, Zn2+, Al3+ and Li+ on [3H]MK-801 binding in human cerebral cortical membranes were studied to further characterize the modulatory effects of metal ions on the N-methyl-D-aspartate (NMDA) receptor-ionophore. Glycine, in the presence of glutamate, significantly intensified the Mg2+ inhibition of [3H]MK-801 binding whereas it masked the Ca2+ enhancement and slightly diminished the Zn2+ inhibition. Both Ca2+ and Mg2+ reduced the Zn2+ inhibitory potency. Aluminum demonstrated a potent, relatively glycine-insensitive inhibition of [3H]MK-801 binding as an amorphous Al(OH)3 polymer rather than as the free ion. Cationic modulation of the NMDA receptor-ionophore appears to be regulated at multiple sites which have significant allosteric interactions.

Determination of site-specific modifications of glucose-6-phosphate dehydrogenase by 4-hydroxy-2-nonenal using matrix assisted laser desorption time-of-flight mass spectrometry.

Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications. Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide. The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme. Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence. One modified segment contains Lysine-182 that has been found to be part of the enzyme active site. Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence. The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1,4-addition of 4HNE. These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1,4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions.

Inhibition of the GDP/GTP exchange reaction of ras p21 by aluminum ion.

In an effort to understand the biochemical mechanisms of aluminum-induced neurotoxicity, we investigated the effects of aluminum ion, Al3+, on the Mg(2+)- and nucleotide-dependent protein, ras p21. Picomolar Al3+ concentrations inhibited the GTPase activity of ras p21 in an Mg(2+)-dependent manner, consistent with an Al3+/Mg2+ competition mechanism. GTPase activity was inhibited by 60% in the presence of 100 microM Mg2+ and 2.9 x 10(-10) M Al3+. Kinetic studies demonstrated that the mode of Al(3+)-induced inhibition of ras p21 GTPase activity changed from competitive to mixed non-competitive as the number of ras p21 turnovers increased. Further dissection of the ras p21 cycle revealed that Mg(2+)-dependent GDP/GTP exchange was the Al(3+)-sensitive step.

The effects on tubulin polymerization and associated guanosine triphosphate hydrolysis of aluminum ion, fluoride and fluoroaluminate species.

The effects of aluminum ion, fluoride, and fluoroaluminate species on the assembly of tubulin in the presence of guanine nucleotides and the consequences of these ions on the associated GTPase of microtubules was investigated. Combinations of GDP and fluoroaluminate species were incapable of activating tubulin for polymerization, in contrast to other guanine nucleotide binding proteins, in which these species produce a functional GTP equivalent. Fluoride alone has an effect on GTP-magnesium-promoted microtubule assembly, causing an increased amount of polymer formation and a reduced rate of associated GTP hydrolysis. It is concluded that aluminum ion and fluoroaluminate species possess distinct mechanisms in inhibiting GTP hydrolysis of GTP-binding proteins and that subpopulations of GTP-binding proteins must exist based on differential sensitivities to these ions.