Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial ... more Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial growth factor receptor tyrosine kinases, has demonstrated promising preclinical single-agent and synergistic anti-leukemic activity in combination with cytarabine. In this phase 1, multicenter, open-label, dose-escalation study, 45 adults with relapsed/refractory acute myeloid leukemia (AML) received linifanib alone in arm A (n = 29) and linifanib plus intermediate-dose cytarabine in arm B (n = 16). Median treatment duration was 21 days (range 5-110). Linifanib was well tolerated overall. The most common grade 3/4 events were fatigue (arm A) and febrile neutropenia (arm B). The recommended phase 2 dose was 15 mg (alone), and 10 mg (with cytarabine). Evidence of on-target kinase inhibition in patients with FLT3-mutant and wild-type AML was seen. Decreased phosphorylated FLT3 was seen in 3/3 patients with FLT3-internal tandem duplication (ITD) with peripheral blast reductions and in 8/24 (...
The manuscript by Yamada et al. "Depsipeptide-resistant ... more The manuscript by Yamada et al. "Depsipeptide-resistant KU812 cells show reversible P-glycoprotein expression, hyperacetylated histones, and modulated gene expression profile," describes not only development of the major resistance mechanism, P-glycoprotein expression, but how histone deacetylase inhibitors can modulate the gene expression profile to promote survival and resistance to depsipeptide.
Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial ... more Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial growth factor receptor tyrosine kinases, has demonstrated promising preclinical single-agent and synergistic anti-leukemic activity in combination with cytarabine. In this phase 1, multicenter, open-label, dose-escalation study, 45 adults with relapsed/refractory acute myeloid leukemia (AML) received linifanib alone in arm A (n = 29) and linifanib plus intermediate-dose cytarabine in arm B (n = 16). Median treatment duration was 21 days (range 5-110). Linifanib was well tolerated overall. The most common grade 3/4 events were fatigue (arm A) and febrile neutropenia (arm B). The recommended phase 2 dose was 15 mg (alone), and 10 mg (with cytarabine). Evidence of on-target kinase inhibition in patients with FLT3-mutant and wild-type AML was seen. Decreased phosphorylated FLT3 was seen in 3/3 patients with FLT3-internal tandem duplication (ITD) with peripheral blast reductions and in 8/24 (33%) patients with wild-type, D835 or unknown FLT3 mutation. Eight/29 (28%) patients had decreased phosphorylated extracellular signal-regulated kinase (ERK).
α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhi... more α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The α-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.
The protein tyrosine kinase (PTK) inhibitor genistein has been demonstrated to inhibit platelet-a... more The protein tyrosine kinase (PTK) inhibitor genistein has been demonstrated to inhibit platelet-activating factor-stimulated prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-primed P388D1 macrophage-like cells (Glaser et al., J Biol Chem 265: 8658-8664, 1990). Therefore, the role of PTK in eicosanoid biosynthesis was investigated in murine resident peritoneal macrophages using genistein and tyrphostin-25, selective PTK inhibitors. Genistein, a competitive inhibitor of ATP binding on PTK, inhibited PGE2 production (IC50 = 20 microM) in response to zymosan, calcium ionophore A23187, and phorbol myristate acetate stimulation. Genistein also inhibited leukotriene C4 (LTC4) production in response to zymosan and calcium ionophore A23187 (IC50 = 10 and 15 microM, respectively) stimulation. Tyrphostin-25, a competitive inhibitor of substrate binding on PTK, inhibited zymosan-stimulated PGE2 and LTC4 production, IC50 = 20 and 7 microM, respectively. Neither genistein nor tyrophostin-25 had any effect on human synovial fluid phospholipase A2 (PLA2) activity in vitro or on cyclooxygenase activity in the intact macrophage; however, tyrphostin-25 did affect 5-lipoxygenase activity (determined from the metabolism of exogenously applied arachidonic acid). These results suggest PTK-mediated phosphorylation as a common event in the signal transduction mechanisms of different stimuli which activate PLA2 for arachidonic acid release and subsequent eicosanoid biosynthesis. Immunoblot analyses of zymosan-stimulated peritoneal exudate cells with the phosphotyrosine monoclonal antibody clone 4G10 demonstrated an increase in protein phosphotyrosine levels in eight major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: p59, 71, 76, 90, 100, 112, 125 and 150. Maximal phosphorylation of these protein substrates occurred after 1-2 min stimulation. Zymosan and LPS stimulation of peritoneal exudate cells produced similar patterns of protein tyrosine phosphorylation. Zymosan-stimulated tyrosine phosphorylation was inhibited by tyrphostin-25 in a concentration-dependent manner between 10 and 60 microM, demonstrating a similar concentration response between effects on tyrosine phosphorylation and eicosanoid biosynthesis in the murine peritoneal macrophage. The use of selective PTK inhibitors suggests a common role for PTK and tyrosine phosphorylation in eicosanoid biosynthesis in the murine peritoneal macrophage.
In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for ... more In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.
The monocytic cell line THP-1 can be induced to express and release tumor necrosis factor α (TNFα... more The monocytic cell line THP-1 can be induced to express and release tumor necrosis factor α (TNFα) and both TNFα receptors (p55 and p75) upon exposure to bacterial lipopolysaccharide (LPS). The broad-spectrum matrix metalloprotease (MMP) inhibitors [4-(N-hydroxyamino)-2R-isobutyl-3S-(phenylthiomethyl)succinyl]-l-phenylalanine-N-methylamide (GI-129471) and marimastat [2S-[N4(R∗),2R∗,3S∗]]-N4-[2,2-dimethyl-1-[(methylamino)carbonyl]propyl]-N1,2-dihydroxy-3-(2-methylpropyl)butanediamide (BB-2516) were effective inhibitors of LPS-induced TNFα (soluble) release with ic50 values of 0.2 and 4.0 μM, respectively. Upon
Transforming growth factor beta (TGF-beta) is a multifunctional protein that has been shown to po... more Transforming growth factor beta (TGF-beta) is a multifunctional protein that has been shown to possess potent growth-inhibitory activity. To identify small molecular weight compounds with TGF-beta-like activities, high throughput screening was performed using mink lung epithelial cells stably transfected with a TGF-beta-responsive plasminogen activator inhibitor 1 promoter/luciferase construct. Biaryl hydroxamate compounds were identified that demonstrated TGF-beta-like activities. 7-[4-(4-cyanophenyl)phenoxy]-heptanohydroxamic acid (A-161906) demonstrated complete TGF-beta-like agonist activity in the plasminogen activator inhibitor 1/luciferase construct. A-161906 inhibited the proliferation of multiple cell lines in a concentration-dependent manner. Cells were growth arrested at the G1-S checkpoint similar to TGF-beta. Consistent with the G1-S arrest, A-161906 induced the expression of the cyclin-dependent kinase inhibitor p21waf1/cip1. A-161906 produced many cellular effects sim...
Manoalide is a potent antiinflammatory marine natural product and a direct inactivator of venom p... more Manoalide is a potent antiinflammatory marine natural product and a direct inactivator of venom phospholipase A2 (PLA2; EC 3.1.1.4). Manoalide has been shown to irreversibly inhibit PLA2, with the corresponding modification of a selective number of lysine residues. The mechanism of inactivation has not yet been elucidated and structure-activity relationship studies were, therefore, performed in order to determine the contributions of the various functional groups incorporated in the gamma-hydroxybutenolide, alpha-hydroxydihydropyran, and trimethylcyclohexenyl ring systems to the efficacy (irreversibility) and potency of this series of inhibitors. These studies indicate that 1) the presence of the hemiacetal in the alpha-hydroxydihydropyran ring is required for irreversible binding of manoalide, 2) the gamma-hydroxybutenolide ring is involved in the initial interaction of manoalide with PLA2, and 3) the hydrophobic nature of the trimethylcyclohexenyl ring system allows nonbonded inte...
The Journal of pharmacology and experimental therapeutics, 1996
The purpose of this investigation was to pharmacologically probe the signaling pathways thought t... more The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100...
Histone deacetylases (HDACs) are a family of enzymes involved in transcription regulation. HDACs ... more Histone deacetylases (HDACs) are a family of enzymes involved in transcription regulation. HDACs are known to play key roles in the regulation of cell proliferation; consequently, inhibition of HDACs has become an interesting approach for anti-cancer therapy. However, expression of mammalian HDACs has proven to be difficult. All attempts to express these HDACs in E.coli, Pichia and baculovirus systems were unsuccessful. Here we present the stable expression of human recombinant His-tagged HDAC1 and HDAC3 in mammalian cells. Full-length human genes for HDAC1 and HDAC3 were cloned into the pcDNA 3.1 vector containing a N-terminal His-tag with an enterokinase cleavage site. Recombinant HDAC enzyme activity was only detected after nickel affinity purification due to high activity of endogenous HDACs; and removal of the His-tag increased activity 2-4 fold. Western blots demonstrated the nickel affinity purified rhHDAC1 preparation also contained endogenous HDAC2 and HDAC3; likewise, rhHD...
Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial ... more Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial growth factor receptor tyrosine kinases, has demonstrated promising preclinical single-agent and synergistic anti-leukemic activity in combination with cytarabine. In this phase 1, multicenter, open-label, dose-escalation study, 45 adults with relapsed/refractory acute myeloid leukemia (AML) received linifanib alone in arm A (n = 29) and linifanib plus intermediate-dose cytarabine in arm B (n = 16). Median treatment duration was 21 days (range 5-110). Linifanib was well tolerated overall. The most common grade 3/4 events were fatigue (arm A) and febrile neutropenia (arm B). The recommended phase 2 dose was 15 mg (alone), and 10 mg (with cytarabine). Evidence of on-target kinase inhibition in patients with FLT3-mutant and wild-type AML was seen. Decreased phosphorylated FLT3 was seen in 3/3 patients with FLT3-internal tandem duplication (ITD) with peripheral blast reductions and in 8/24 (...
The manuscript by Yamada et al. &amp;amp;amp;amp;amp;amp;amp;amp;quot;Depsipeptide-resistant ... more The manuscript by Yamada et al. &amp;amp;amp;amp;amp;amp;amp;amp;quot;Depsipeptide-resistant KU812 cells show reversible P-glycoprotein expression, hyperacetylated histones, and modulated gene expression profile,&amp;amp;amp;amp;amp;amp;amp;amp;quot; describes not only development of the major resistance mechanism, P-glycoprotein expression, but how histone deacetylase inhibitors can modulate the gene expression profile to promote survival and resistance to depsipeptide.
Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial ... more Linifanib, a potent oral inhibitor of fms-like tyrosine kinase 3 (FLT3) and vascular endothelial growth factor receptor tyrosine kinases, has demonstrated promising preclinical single-agent and synergistic anti-leukemic activity in combination with cytarabine. In this phase 1, multicenter, open-label, dose-escalation study, 45 adults with relapsed/refractory acute myeloid leukemia (AML) received linifanib alone in arm A (n = 29) and linifanib plus intermediate-dose cytarabine in arm B (n = 16). Median treatment duration was 21 days (range 5-110). Linifanib was well tolerated overall. The most common grade 3/4 events were fatigue (arm A) and febrile neutropenia (arm B). The recommended phase 2 dose was 15 mg (alone), and 10 mg (with cytarabine). Evidence of on-target kinase inhibition in patients with FLT3-mutant and wild-type AML was seen. Decreased phosphorylated FLT3 was seen in 3/3 patients with FLT3-internal tandem duplication (ITD) with peripheral blast reductions and in 8/24 (33%) patients with wild-type, D835 or unknown FLT3 mutation. Eight/29 (28%) patients had decreased phosphorylated extracellular signal-regulated kinase (ERK).
α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhi... more α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The α-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.α-Keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.
The protein tyrosine kinase (PTK) inhibitor genistein has been demonstrated to inhibit platelet-a... more The protein tyrosine kinase (PTK) inhibitor genistein has been demonstrated to inhibit platelet-activating factor-stimulated prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-primed P388D1 macrophage-like cells (Glaser et al., J Biol Chem 265: 8658-8664, 1990). Therefore, the role of PTK in eicosanoid biosynthesis was investigated in murine resident peritoneal macrophages using genistein and tyrphostin-25, selective PTK inhibitors. Genistein, a competitive inhibitor of ATP binding on PTK, inhibited PGE2 production (IC50 = 20 microM) in response to zymosan, calcium ionophore A23187, and phorbol myristate acetate stimulation. Genistein also inhibited leukotriene C4 (LTC4) production in response to zymosan and calcium ionophore A23187 (IC50 = 10 and 15 microM, respectively) stimulation. Tyrphostin-25, a competitive inhibitor of substrate binding on PTK, inhibited zymosan-stimulated PGE2 and LTC4 production, IC50 = 20 and 7 microM, respectively. Neither genistein nor tyrophostin-25 had any effect on human synovial fluid phospholipase A2 (PLA2) activity in vitro or on cyclooxygenase activity in the intact macrophage; however, tyrphostin-25 did affect 5-lipoxygenase activity (determined from the metabolism of exogenously applied arachidonic acid). These results suggest PTK-mediated phosphorylation as a common event in the signal transduction mechanisms of different stimuli which activate PLA2 for arachidonic acid release and subsequent eicosanoid biosynthesis. Immunoblot analyses of zymosan-stimulated peritoneal exudate cells with the phosphotyrosine monoclonal antibody clone 4G10 demonstrated an increase in protein phosphotyrosine levels in eight major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: p59, 71, 76, 90, 100, 112, 125 and 150. Maximal phosphorylation of these protein substrates occurred after 1-2 min stimulation. Zymosan and LPS stimulation of peritoneal exudate cells produced similar patterns of protein tyrosine phosphorylation. Zymosan-stimulated tyrosine phosphorylation was inhibited by tyrphostin-25 in a concentration-dependent manner between 10 and 60 microM, demonstrating a similar concentration response between effects on tyrosine phosphorylation and eicosanoid biosynthesis in the murine peritoneal macrophage. The use of selective PTK inhibitors suggests a common role for PTK and tyrosine phosphorylation in eicosanoid biosynthesis in the murine peritoneal macrophage.
In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for ... more In order to ascertain the role of phospholipase A2 (PLA2) in the release of arachidonic acid for eicosanoid biosynthesis, we have characterized a Ca2+-dependent PLA2 from P388D1 cells, evaluated inhibitors of its activity, and correlated the effects of these inhibitors on prostaglandin (PG) E2 production in the intact cell. The Ca2+-dependent PLA2 has little preference for the polar head group or sn-2 fatty acid of phospholipids, and we have now found that it will hydrolyze 1-alkyl,2-acyl phospholipids, but it does not show a preference for this substrate over other phospholipids. Inhibitor studies with the Ca2+-dependent PLA2 have shown that arachidonic acid is an effective inhibitor. The analogs of natural fatty acids, eicosatetraynoic acid and octadecyleicosaynoic acid, were ineffective as inhibitors of the P388D1 PLA2. However, 7,7-dimethyl-5,8-eicosadienoic acid was as effective an inhibitor (IC50 = 16 microM) as arachidonic acid. Manoalide and its analog, manoalogue, were found to be good inhibitors of the P388D1 PLA2 (IC50 = 16 and 26 microM, respectively). The irreversible inhibitor of the extracellular PLA2, p-bromophenacyl bromide, was a very poor inhibitor of the P388D1 PLA2, apparent IC50 = 500-600 microM. Quinacrine was also ineffective as an inhibitor as was the cyclooxygenase inhibitor indomethacin. On the cellular level, the P388D1 cells respond to various stimuli to produce PGD2 and PGE2 as the major cyclooxygenase products with minor production of PGI2 and thromboxane A2. Similar arachidonic acid metabolite profiles were seen for calcium ionophore A23187, melittin, and platelet-activating factor. Manoalide, manoalogue, and 7,7-dimethyl-5,8-eicosadienoic acid, effective inhibitors of the isolated PLA2, inhibited PGE2 production in intact P388D1 cells 40-85% in the concentration range studied. In contrast, p-bromophenacyl bromide, which is ineffective as an inhibitor of the P388D1 PLA2, did not significantly effect PGE2 production in the concentration ranges used. These results demonstrate that there may be important differences between the intracellular P388D1 PLA2 and the more commonly studied extracellular forms of PLA2. These differences are also observed in the intact cell studies and emphasize the need for the evaluation of inhibitors both in vitro and in vivo using the isolated enzyme and intact cell. This is the first example of studies aimed at correlating the inhibition of a purified intracellular PLA2 with inhibition of prostaglandin production in the intact cell from which it is derived.
The monocytic cell line THP-1 can be induced to express and release tumor necrosis factor α (TNFα... more The monocytic cell line THP-1 can be induced to express and release tumor necrosis factor α (TNFα) and both TNFα receptors (p55 and p75) upon exposure to bacterial lipopolysaccharide (LPS). The broad-spectrum matrix metalloprotease (MMP) inhibitors [4-(N-hydroxyamino)-2R-isobutyl-3S-(phenylthiomethyl)succinyl]-l-phenylalanine-N-methylamide (GI-129471) and marimastat [2S-[N4(R∗),2R∗,3S∗]]-N4-[2,2-dimethyl-1-[(methylamino)carbonyl]propyl]-N1,2-dihydroxy-3-(2-methylpropyl)butanediamide (BB-2516) were effective inhibitors of LPS-induced TNFα (soluble) release with ic50 values of 0.2 and 4.0 μM, respectively. Upon
Transforming growth factor beta (TGF-beta) is a multifunctional protein that has been shown to po... more Transforming growth factor beta (TGF-beta) is a multifunctional protein that has been shown to possess potent growth-inhibitory activity. To identify small molecular weight compounds with TGF-beta-like activities, high throughput screening was performed using mink lung epithelial cells stably transfected with a TGF-beta-responsive plasminogen activator inhibitor 1 promoter/luciferase construct. Biaryl hydroxamate compounds were identified that demonstrated TGF-beta-like activities. 7-[4-(4-cyanophenyl)phenoxy]-heptanohydroxamic acid (A-161906) demonstrated complete TGF-beta-like agonist activity in the plasminogen activator inhibitor 1/luciferase construct. A-161906 inhibited the proliferation of multiple cell lines in a concentration-dependent manner. Cells were growth arrested at the G1-S checkpoint similar to TGF-beta. Consistent with the G1-S arrest, A-161906 induced the expression of the cyclin-dependent kinase inhibitor p21waf1/cip1. A-161906 produced many cellular effects sim...
Manoalide is a potent antiinflammatory marine natural product and a direct inactivator of venom p... more Manoalide is a potent antiinflammatory marine natural product and a direct inactivator of venom phospholipase A2 (PLA2; EC 3.1.1.4). Manoalide has been shown to irreversibly inhibit PLA2, with the corresponding modification of a selective number of lysine residues. The mechanism of inactivation has not yet been elucidated and structure-activity relationship studies were, therefore, performed in order to determine the contributions of the various functional groups incorporated in the gamma-hydroxybutenolide, alpha-hydroxydihydropyran, and trimethylcyclohexenyl ring systems to the efficacy (irreversibility) and potency of this series of inhibitors. These studies indicate that 1) the presence of the hemiacetal in the alpha-hydroxydihydropyran ring is required for irreversible binding of manoalide, 2) the gamma-hydroxybutenolide ring is involved in the initial interaction of manoalide with PLA2, and 3) the hydrophobic nature of the trimethylcyclohexenyl ring system allows nonbonded inte...
The Journal of pharmacology and experimental therapeutics, 1996
The purpose of this investigation was to pharmacologically probe the signaling pathways thought t... more The purpose of this investigation was to pharmacologically probe the signaling pathways thought to be involved in protein kinase C (PKC)-stimulated superoxide anion (O2-) generation in all-trans retinoic acid-treated human promyelocytic HL-60 cell line (HL-60), targeting PKC, mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), protein serine-threonine phosphatase(s) (PSP), protein tyrosine kinase(s) (PTK) and phosphatase(s) (PTP), secretory phospholipase A2, cyclooxygenase (CO) and 5-lipoxygenase with selected inhibitors. The following agents inhibited phorbol 12-myristate 13-acetate-stimulated O2- generation significantly in the all-trans retinoic acid-treated HL-60 cells (expressed as percentage of control, P < .05): 1) PKC inhibitors: staurosporine (100 nM, 3 +/- 1%); Ro 31-8220 (1 microM, 3 +/- 2%); sphingosine (100 microM, 15 +/- 7%); 2) PSP 1 and 2a inhibitors, okadaic acid (10 microM, 35 +/- 1%); calyculin A (10 microM, 73 +/- 1%); 3) MAPK inhibitor: SB-203580 (100...
Histone deacetylases (HDACs) are a family of enzymes involved in transcription regulation. HDACs ... more Histone deacetylases (HDACs) are a family of enzymes involved in transcription regulation. HDACs are known to play key roles in the regulation of cell proliferation; consequently, inhibition of HDACs has become an interesting approach for anti-cancer therapy. However, expression of mammalian HDACs has proven to be difficult. All attempts to express these HDACs in E.coli, Pichia and baculovirus systems were unsuccessful. Here we present the stable expression of human recombinant His-tagged HDAC1 and HDAC3 in mammalian cells. Full-length human genes for HDAC1 and HDAC3 were cloned into the pcDNA 3.1 vector containing a N-terminal His-tag with an enterokinase cleavage site. Recombinant HDAC enzyme activity was only detected after nickel affinity purification due to high activity of endogenous HDACs; and removal of the His-tag increased activity 2-4 fold. Western blots demonstrated the nickel affinity purified rhHDAC1 preparation also contained endogenous HDAC2 and HDAC3; likewise, rhHD...
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Papers by Keith Glaser