Biomedical subjects

A Levitzki

Publications and source records attributed to A Levitzki.

At least 91 records · Page 5Linked to original sources

PubMed1994

Inhibition of DNA topoisomerases I and II and induction of apoptosis by erbstatin and tyrphostin derivatives.

Inhibitors of protein tyrosine kinases (PTK) and DNA topoisomerases are potential antitumour agents. Drugs which bind to the ATP site of PTK, such as genistein, are common inhibitors to both types of enzymes. Eleven erbstatin and tyrphostin derivatives, which inhibit epidermal growth factor receptor PTK activity by competing with both the peptide substrate and ATP were tested for their capacity to inhibit DNA topoisomerases I and II. Erbstatin, two synthetic derivatives with a modified side chain and the tyrphostin AG 786 inhibited both topoisomerases in the same range of concentrations (20-50 microM). The tyrphostin AG 213 inhibited only topoisomerase II. In this series, absence of PTK inhibitory effect was correlated with the absence of DNA topoisomerase inhibition, while the detection of PTK inhibition may or may not be associated with DNA topoisomerase inhibition. In contrast to genistein, none of these molecules induced the stabilization of the topoisomerase-DNA cleavable complex, either in vitro or in vivo. Alcaline elution analysis revealed that erbstatin did not induce the formation of protein associated DNA strand breaks. However, an extensive degradation of the cellular DNA was observed which was shown to result from an internucleosomal fragmentation. Furthermore, typical morphological modifications associated with apoptosis were observed in the erbstatin treated cells by electron microscopy. These data indicate that erbstatin induces an apoptotic cell death.

Amino Acid Sequence↗

PubMed1994

Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors.

Septic shock results from excessive stimulation of the host immune system, especially macrophages, by lipopolysaccharide (LPS), or endotoxin, which resides on the outer membrane of bacteria. Protein tyrosine kinase inhibitors of the tyrphostin AG 126 family protect mice against LPS-induced lethal toxicity. The protection correlates with the ability of these agents to block LPS-induced production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in macrophages as well as LPS-induced production of TNF-alpha in vivo. Furthermore, this inhibitory effect correlated with the potency of AG 126 to block LPS-induced tyrosine phosphorylation of a p42MAPK protein substrate in the murine macrophage.

Animals↗

PubMed1994

Effects of tyrphostins on the activated c-src protein in NIH/3T3 cells.

Tyrphostins are synthetic compounds that have been described as in vitro and in vivo inhibitors of epidermal growth factor receptor tyrosine kinase activity. In NIH/3T3 cells transfected with the c-src/F527 gene, an increase in the level of tyrosine phosphorylation of several proteins, including pp125FAK, within a group of proteins of 120 kDa, of p85 (cortactin), and of p62 is observed, which is due to the elevated kinase activity of the resulting encoded pp60F527 protein. In the transfected cells, we showed that the tyrphostins we used, i.e., AG18, AG34, and AG82, strongly diminished the tyrosine phosphorylation of these proteins. Analysis of the steady state level of pp60F527 in tyr-phostin-treated cells revealed that AG34 and AG82, the two most potent compounds, also induced 30 and 48% decreases, respectively, in the amount of pp60F527, while having no action on the levels of other proteins, especially the pp60F527 kinase substrates. Measurement of the rates of pp60F527 synthesis and degradation showed that this decreased level was due to a slower rate of synthesis in the presence of AG34 and AG82. Tyrphostins also reversed the pp60F527-induced transformed morphology of NIH/3T3 cells and also inhibited the pp60F527 kinase activity in vitro. We conclude that the effects elicited by the tyrphostins occurred not only through the inhibition of the pp60F527 protein kinase activity but also through a selective reduction of the Src protein steady state level in the cases of AG34 and AG82. This is a novel mode of action for these two tyrphostins, which were the most active compounds in this system.

3T3 Cells↗

PubMed1994

Tyrphostin induced growth inhibition: correlation with effect on p210bcr-abl autokinase activity in K562 chronic myelogenous leukemia.

We have examined a series of tyrosine kinase inhibitors structurally related to erbstatin (tyrphostins) for inhibition of p210bcr-abl autokinase activity in vitro and for growth inhibition of chronic myelogenous leukemia (CML)K562 cells. Of the tyrphostins with IC50 for growth < 50 microM, AG814, AG946, AG952, AG896, AG953, AG956 and AG957 (structurally related to lavendustin A and piceatannol) completely inhibited p210bcr-abl kinase activity in an immune complex kinase assay. Another group of tyrphostins (AG807, AG568, AG763, AG1076, AG490, AG1318, AG556, AG1319, AG555 and AG1111) inhibits growth of K562 cells but not p210bcr-abl tyrosine kinase activity. Of the compounds which inhibit growth and p210bcr-abl tyrosine kinase activity, AG957 inhibits DNA synthesis as early as 2 h (60% inhibition at 20 microM of AG957), a time and concentration of drug where RNA and protein synthesis were not affected. AG957 inhibits p210bcr-abl tyrosine phosphorylation in living cells by 1 h without an inhibition of total protein phosphorylation. Growth inhibition by AG957 was reversible after 4 h of exposure, but irreversible after 24 h. AG957 can be considered as an important lead structure for the development of anti-bcr-abl tyrosine kinase antagonists. These data also raise the possibility that bcr-abl kinase activity is directly linked to maintenance of DNA synthesis in Philadelphia chromosome positive (Ph+) CML cells.

Adenosine Triphosphate↗

PubMed1994

Kinetics of inhibition by tyrphostins of the tyrosine kinase activity of the epidermal growth factor receptor and analysis by a new computer program.

The kinetics of inhibition of the epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase (TK) activity by erbstatin, tyrphostins, and lavendustin derivatives were studied in a system that employs poly(Glu6Ala3Tyr) (GAT) and ATP as substrates, after preactivation with EGF. All data were analyzed for computer best-fit curves by a program that was written for this purpose and is available upon request to those interested. The inhibition kinetics followed a sequential, Bi-Bi, rapid equilibrium, random mechanism, the mechanism of the EGFR-TK. Erbstatin and a few tyrphostins that contain a 3,4-dihydroxy-(cis)-cinnamonitrile [1-(3',4'-dihydroxyphenyl)-2-nitriloethene] group were found to be pure competitive inhibitors with respect to both substrates of the kinase reaction, i.e., GAT and ATP. Two tyrphostins, each containing an additional dihydroxyphenyl group in the alpha-position, were found to be pure competitive inhibitors with respect to GAT and noncompetitive (or mixed-competitive) inhibitors with respect to ATP. A lavendustin derivative with a 2,5-dihydroxyphenyl ring and a lavendustin derivative with a 3,4-dihydroyphenyl ring were also found to be competitive inhibitors with respect to both ATP and GAT. Various possible modes of binding at the EGFR-TK active center for the tyrphostins studied are proposed and the significance of the present findings, as well as the interpretations of computer analyses of kinetic data, is discussed.

Catechols↗

PubMed1993

Tyrphostin-induced inhibition of p210bcr-abl tyrosine kinase activity induces K562 to differentiate.

We report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.

3T3 Cells↗

PubMed1993

Tyrphostins. 3. Structure-activity relationship studies of alpha-substituted benzylidenemalononitrile 5-S-aryltyrphostins.

In this study we describe an extension of our previous studies on cis-benzylidenemalononitrile tyrphostins. We have introduced S-aryl substituents in the 5 position (meta vis-a-vis the malononitrile moiety). We find that these compounds are potent blockers of EGFR kinase and its homolog HER-2 kinase. Interestingly, we find that certain S-aryltryphostins discriminate between EGFR and HER-2 kinase in favor of the HER-2 kinase domain by almost 2 orders of magnitude. When examined in intact cells it was found that these selective S-aryltrphostins are equipotent in inhibiting EGF dependent proliferation of NIH 3T3 harboring either the EGF receptor or the chimera EGF/neu (HER1-2). These findings suggest that the antiproliferative activity of these tyrphostins is mainly due to the inhibition of a mitogenic signaling element downstream to the growth receptor kinase.

3T3 Cells↗

PubMed1993

Non-amine based analogues of lavendustin A as protein-tyrosine kinase inhibitors.

The fermentation product lavendustin A (1) is a protein-tyrosine kinase (PTK) inhibitor whose active pharmacophore has previously been shown to reside in the more simplified salicyl-containing benzylamine 2. Amine 2 bears some structural resemblance to two other natural product PTK inhibitors, erbstatin (3) and piceatannol (4). Non-amine containing analogues of 2 were therefore synthesized which incorporated additional aspects of either erbstatin or piceatannol. Examination of these inhibitors in immunoprecipitated p56lck, epidermal growth factor receptor (EGFR), and c-erb B-2/HER 2/neu PTK preparations showed that compound 12 (IC50 = 60 nM) was one of the most potent p56lck inhibitors reported to date. These results demonstrate that nitrogen is not an essential component of the lavendustin A pharmacophore 2 and that 1,2-diarylethanes and -ethenes bearing a salicyl moiety appear to be valuable structural motifs for the construction of extremely potent PTK inhibitors.

ErbB Receptors↗

PubMed1993

Tyrphostin-induced differentiation of mouse erythroleukemia cells.

Inhibitors of protein-tyrosine kinases (TPKs) from the tyrphostins family induce terminal erythroid differentiation of mouse erythroleukemia (MEL) cells. The most potent tyrphostin was found to be AG-568 which was therefore investigated in more detail. Just prior to differentiation the inhibition of tyrosine phosphorylation of a pp97 protein band was noted. We also found that AG-568 treatment induces the appearance of a putative differentiation factor which could induce tyrphostin-independent differentiation in untreated cells. Our study suggests that the inhibition of tyrosine phosphorylation by AG-568 leads to the production of differentiating factor(s) which induce the MEL cells to differentiate.

Animals↗

PubMed1993

Cloning of the STE5 gene of Saccharomyces cerevisiae as a suppressor of the mating defect of cdc25 temperature-sensitive mutants.

The STE5 gene of Saccharomyces cerevisiae was cloned using a screening procedure designed to isolate genes of the S. cerevisiae pheromone response pathway. We screened a yeast genomic high-copy-number plasmid library for genes that allow mating of cdc25ts mutants at the restrictive temperature without affecting the cell-cycle-arrest phenotype. One of the genes cloned was identified by genetic analysis as STE5. STE5 encodes a predicted open reading frame of 916 amino acids and exhibits significant homology to Far1 protein. RNA blot analysis reveals that STE5 gene transcription is regulated by the mating type of the cell and depends on an intact pheromone-response pathway.

Adaptor Proteins, Signal Transducing↗

PubMed1993

Residues crucial for Ras interaction with GDP-GTP exchangers.

Cdc25 is essential for Ras-mediated activation of adenylyl cyclase in the yeast Saccharomyces cerevisiae. This protein acts by catalyzing GDP-GTP exchange on yeast Ras. Harvey (Ha) ras expressed in S. cerevisiae is also recognized by both Cdc25 and Sdc25, a yeast homolog of Cdc25. Thus it is feasible to examine molecular aspects of mammalian Ras modulation by Cdc25 using the RAS/cAMP pathway in yeast as a model system. Here, we describe mutational analysis of Ha-ras for the identification of residues critical for the ability of Ras to interact with Cdc25 and related guanine nucleotide-release proteins. Mutations within codons 97-108 impaired Ras-mediated activation of adenylyl cyclase in the presence but not in the absence of mammalian GTPase-activating protein. Such mutations, therefore, affected the ability of Ras to undergo GDP-GTP exchange catalyzed by the guanine nucleotide exchanger without preventing Ras activation of the effector. Similar mutations were previously shown to impair the ability of c-ras to transform mammalian cells while having a less drastic effect on v-ras.

Adenylyl Cyclases↗

PubMed1993

Selective inhibition of the epidermal growth factor and HER2/neu receptors by tyrphostins.

The HER2 (neu/erb-B2) proto-oncogene codes for a transmembrane receptor with tyrosine kinase activity and with high homology to the EGF receptor (HER1). The high incidence of HER2 overexpression in breast and ovary carcinomas prompted us to synthesize protein tyrosine kinase inhibitors (tyrphostins) which selectively inhibit the HER2 kinase activity. Two groups of tyrphostins were developed: one highly selective in inhibiting HER1 as opposed to HER2, the other highly selective in inhibiting HER2. Both the HER1 and the HER2 selective blockers were competitive with ATP binding. This suggests that even though the kinase domains of the respective receptors show an 80% degree of homology it is possible to design small molecules capable of discriminating between them. These results also show that the two kinases differ in their ATP binding sites. Mitogenic signaling induced by EGF in NIH3T3 cells overexpressing either HER1 or HER1-2 (possessing the HER2 kinase domain) was blocked identically by the agents that discriminate between the two in vitro. This paradox was further explored and elucidated. We propose that high intracellular ATP levels prevent inhibitor binding to the receptor. The antiproliferative action of the two distinct selective tyrphostins observed may result from the inhibition of a downstream element, presumably a tyrosine kinase, which mediates mitogenic signaling.

3T3 Cells↗

PubMed1993

The tyrosine kinase inhibitor tyrphostin blocks the cellular actions of nerve growth factor.

A series of the synthetic protein kinase inhibitors known as tyrphostins were examined for their effects on the tyrosine autophosphorylation of the pp140c-trk, nerve growth factor (NGF) receptor. One of the tyrphostins, AG879, inhibited NGF-dependent pp140c-trk tyrosine phosphorylation, but did not affect tyrosine phosphorylation of epidermal growth factor or platelet-derived growth factor receptors. In addition, the tyrosine phosphorylation of the receptor-associated protein pp38 was also attenuated by the tyrphostin. This effect was time- and dose-dependent, although inhibition of pp38 phosphorylation occurred earlier and at lower concentrations of the compound. AG879 also inhibited NGF-induced PLC-gamma 1 phosphorylation, phosphatidylinositol-3 (PI3) kinase activation, the association of the tyrosine-phosphorylated proteins pp100 and pp110 with the p85 subunit of PI-3 kinase, mitogen activated protein and raf-1 kinases, and c-fos induction. In addition, AG879 inhibited NGF-induced neurite outgrowth in PC12 cells. These data indicate that tyrosine kinase activity of the pp140c-trk NGF receptor is essential for the cellular actions of this growth factor.

Animals↗

PubMed1993

A Candida albicans homolog of CDC25 is functional in Saccharomyces cerevisiae.

We have cloned, by functional complementation of the cdc25-2 mutation of Saccharomyces cerevisiae, a homolog of CDC25 from the pathogenic yeast Candida albicans. The new gene, named CSC25 codes for a 1333-amino-acid protein. The full length gene, as well as a truncated form coding for 795 amino acids, suppresses the thermosensitive phenotype of cdc25ts mutants. Biochemical analysis has shown that Csc25 activates the Ras/adenylyl cyclase pathway in S. cerevisiae at a rate two to three times faster than Cdc25, under the same conditions. The C-terminal domain of Csc25 is highly similar to the C-terminal domain of Cdc25, to almost the same extent as the C-terminus of the endogenous Cdc25 homolog Sdc25. We show that polyclonal anti-Cdc25 antibodies interact with Csc25 expressed in S. cerevisiae. In addition to the full length protein (approximately 150 kDa), we have found a approximately 50-kDa polypeptide which seems to include the C-terminus of the CSC25 gene product.

Amino Acid Sequence↗

PubMed1993

The signal transduction between beta-receptors and adenylyl cyclase.

The beta-adrenergic receptor-dependent adenylyl cyclase system is the most extensively studied G-protein-coupled system. Studies of the coupling between the receptor and effector can provide an insight into the nature of all these systems in general. In the activation of adenylyl cyclase by the receptor, the binding of an agonist to the stimulatory receptor (Rs) and the binding of GTP to the G-protein (Gs) are both required to activate the catalytic moiety (C). The active state decays as GTP is hydrolysed to GDP and inorganic phosphate (Pi), but reactivation occurs as GTP is replenished. The receptor acts as a catalyst, i.e. one agonist-bound receptor can activate numerous adenylyl cyclase molecules. Kinetic studies led to the formulation of the 'collision coupling' model of receptor activation and show that Gs protein does not shuttle between the receptor and cyclase. The Gs protein appears to undergo conformational changes between an 'open' state in which it can bind with GTP, and a 'closed' state unable to achieve this binding. This mechanism of activation does not involve the dissociation of Gs or of Gi. A model which fits the experimental data suggests that Gi*GTP affects cyclase only in its Gs-activated state via the G alpha 1 subunit, but that the oligomeric state of Gi is required for inhibition. The site on C which interacts with Gi is formed only when C is activated by Gs.

Adenylyl Cyclases↗

PubMed1993

Tyrphostins inhibit follicle-stimulating hormone-mediated functions in cultured rat ovarian granulosa cells.

FSH induces the expression of cholesterol side-chain cleavage cytochrome P450 (P450scc) in rat ovarian granulosa cells. The present study reveals that the tyrphostin AG18, a member of novel protein tyrosine kinase inhibitors, can arrest the FSH-induced synthesis of P450scc with an apparent IC50 of 30 microM. Total inhibition of P450scc expression was achieved at 80 microM AG18. AG18-mediated inhibition of P450scc was also observed when the enzyme was induced by prostaglandin E2, forskolin, or 8-bromo-cAMP. Studies examining functional LH receptors showed that the tyrphostin inhibits the expression of FSH-induced LH receptors. The drug did not affect FSH-induced cAMP accumulation, suggesting that it may interfere with the flow of FSH signal transduction at a site distal intracellular accumulation of cAMP. Control experiments demonstrated that the inhibitory action of AG18 was reversible, did not hamper total protein synthesis in the cells, and did not change the adenine nucleotide (ATP:ADP:AMP) ratio or their levels in the treated cells. A cell-free assay of cAMP-dependent protein kinase showed that the tyrphostin AG18 does not affect this enzyme activity up to concentrations above 200 microM. These results suggest that a putative tyrosine kinase activity is involved in the gonadotropin signal transduction pathway leading to expression of functional genes in ovarian cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.

Adenylyl Cyclases↗

PubMed1992

Kinetic model of the epidermal growth factor (EGF) receptor tyrosine kinase and a possible mechanism of its activation by EGF.

The tyrosine kinase activity of the epidermal growth factor receptor (EGFR-TK) was determined at varying poly-Glu6Ala3Tyr1 (GAT) or [Val5]-angiotensin II (AT) and constant ATP concentrations and vice versa. With GAT as substrate, double reciprocal plots intersected practically on the abscissa following EGFR-TK pre-activation with EGF, but below the abscissa without EGF pre-activation. The EGFR-TK inhibitors App(NH)p (5'-adenylyl-beta, gamma-imidodiphosphate) and ADP were competitive with ATP and noncompetitive with GAT. Four families of 1/v vs. 1/[ATP] plots, constructed at different fixed concentrations of ADP and a different constant concentration of GAT for each family, yielded Slope1/ATP replots which intersected to the left of the ordinate and below the abscissa. GAT and AT, as cosubstrates, were competitive with each other and noncompetitive with ATP; 1/v vs. 1/[GAT] or 1/[AT] plots were hyperbolic and reached horizontal asymptotes when v was expressed as the rate of common product formation. All data were subjected to computer best-fit analysis by a program written especially for this purpose. We conclude that (i) the EGFR-TK reaction follows a Sequential Bi-Bi Rapid Equilibrium Random mechanism, and (ii) EGF induces conformational changes in the EGFR-TK active center which lead to marked decreases in the apparent dissociation constants of both substrates of the kinase reaction and a concomitant increase in initial velocities and Vmax (apparent).

Carcinoma, Squamous Cell↗