In vitro and in vivo irreversible plasma protein binding of beclobric acid enantiomers

Acyl glucuronides are known to be labile conjugates, which undergo hydrolysis and bind irreversibly to proteins. The lipid-regulating agent (±)-beclobrate is immediately converted to the free acid after oral administration. Further metabolism leads to formation of the corresponding diastereomeric acyl glucuronides. Beclobric acid glucuronides were quantified by indirect measurement with an HPLC method based on chiral fluorescent derivatization of the carboxylic acid and subsequent normal-phase chromatography. The renal clearance of unchanged drug is low, with almost all drug excreted into urine as glucuronic acid conjugates. Beclobric acid glucuronide is also detectable in plasma. In vitro degradation studies with beclobric acid glucuronide (at a concentration of 5 μM in 150 mM phosphate buffer pH 7.4) exhibited a minor tendency for acyl migration and hydrolysis, i.e., a higher stability than has been observed for the acyl glucuronides of most other drugs. The in vitro degradation half-lives of the two beclobric acid β-1-O-acyl glucuronides were 22.7 and 25.7 h. After incubation with pooled plasma and human serum albumin in buffer pH 7.4 irreversible binding was measured in vitro. No significant difference between the two enantiomers was detected with respect to the magnitude of in vitro irreversible binding. In 3 healthy male volunteers the extent of irreversible binding of both beclobric acid enantiomers to plasma proteins was investigated after single and multiple oral doses of racemic beclobrate (100 mg once daily). Irreversible binding of both enantiomers was observed in all volunteers. The adduct densities for (?)- and (+)-beclobric acid after single 100 mg beclobrate doses were 0.147 × 10^?4 and 0.177 × 10^?4 mol/mol protein. Multipie dosing increased irreversible binding 3- to 4-fold. © 1993 Wiley-Liss, Inc.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Down-regulation of protein L-isoaspartyl methyltransferase in human epileptic hippocampus contributes to generation of damaged tubulin

Protein L-isoaspartyl methyltransferase (PIMT) repairs the damaged proteins which have accumulated abnormal aspartyl residues during cell aging. Gene targeting has elucidated a physiological role for PIMT by showing that mice lacking PIMT died prematurely from fatal epileptic seizures. Here we investigated the role of PIMT in human mesial temporal lobe epilepsy. Using surgical specimens of hippocampus and neocortex from controls and epileptic patients, we showed that PIMT activity and expression were 50% lower in epileptic hippocampus than in controls but were unchanged in neocortex. Although the protein was down-regulated, PIMT mRNA expression was unchanged in epileptic hippocampus, suggesting post-translational regulation of the PIMT level. Moreover, several proteins with abnormal aspartyl residues accumulate in epileptic hippocampus. Microtubules component beta-tubulin, one of the major PIMT substrates, had an increased amount (two-fold) of L-isoaspartyl residues in the epileptic hippocampus. These results demonstrate that the down-regulation of PIMT in epileptic hippocampus leads to a significant accumulation of damaged tubulin that could contribute to neuron dysfunction in human mesial temporal lobe epilepsy.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

丙戊酸与缺血性脑卒中相关研究进展

动脉粥样硬化和缺血性脑损伤是防治缺血性脑卒中所面临的两大难题,而细胞炎症损伤是它们的共同诱因。丙戊酸作为组蛋白去乙酰化酶抑制剂,具有抑制细胞炎症因子释放及保护神经的作用,所以丙戊酸可能是防治缺血性脑卒中的潜在治疗药物。本文从组蛋白去乙酰化酶对缺血性脑卒中的影响以及丙戊酸的抗炎机制两个方面进行综述。     

Identification of a novel miRNA that increases transient protein expression in combination with valproic acid

Transient gene expression in mammalian cells is an efficient process to produce recombinant proteins for various research applications and large molecule therapeutics development. For the first time, we report a screen to identify human microRNAs (miRNAs) that increase titers after polyethylenimine (PEI) mediated transient transfection of a HEK293 cell line. From a library of 875 miRNAs, we identified 2 miRNAs, miR‐26a‐5p and miR‐337‐5p, that increased human IgG1 (huIgG1) yields by 50 and 25%, respectively. The titer increase was achievable by expressing miR‐26a‐5p from oligonucleotides or a plasmid. Furthermore, combining miR‐26a‐5p with valproic acid (VPA) treatment doubled huIgG1 titers. Assessment of miR‐26a‐5p and VPA treatment across a panel of 32 human and murine antibodies demonstrates that the level of yield enhancement was molecule‐dependent, with most exhibiting a range of 50–100% titer increase. These findings exemplify that combining genetic and chemical manipulation can be an effective strategy to enhance transient transfection productivity. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1139–1145, 2017     

Preliminary studies about novel strategies to reverse chemoresistance to adriamycin regarding glutathione metabolism,peroxisomal and extraperoxisomal hydroperoxide and valproic acid metabolic pathways

Summary— The present work was aimed at defining novel strategies to reverse chemoresistance to anticancer drugs, especially by interfering with cellular glutathione metabolism, peroxisomal and/or extraperoxisomal hydroperoxide metabolic pathways. Preliminary results are presented about molecules we demonstrated to be capable of interfering with hydrogen peroxide metabolism in cells. Prior to describing these molecules, a short overview of glutathione and free radical metabolic pathways is presented as well as a rapid presentation of the characteristics of chemo-sensitivity and -resistance towards the anticancer drug adriamycin, with special emphasis on hydrogen peroxide metabolism. The strategies currently developed to reverse chemoresistance are further presented. in subsequent sections, our own strategy to achieve inhibition of hydrogen peroxide breakdown and stimulation of peroxisomal hydrogen peroxide production is illustrated on the basis of molecular modelling studies and biochemical investigations on extraperoxisomal and peroxisomal metabolic pathways. Preliminary studies on cultured cells have been initiated. The perspective for future studies is presented as well as other possible models of chemoresistance as target for the design of hydrogen peroxide metabolism-interfering pharmacomolecules.     

The action of cytochalasin A on the in vitro polymerization of brain tubulin and muscle G-actin

The presence of cytochalasin A inhibits the self-assembly of beef brain tubulin and rabbit muscle G-actin in vitro and also decreases the colchicine binding of tubulin. Prior reaction of cytochalasin A with 2-mercaptoethanol destroys its inhibitory effects. It is shown that cytochalasin A exerts its actions by reacting with sulfhydryl groups, possibly causing irreversible structural changes in the proteins. Cytochalasin B does not affect the tubulin assembly reaction.     

Regulation by thyroid hormone of microtubule assembly and neuronal differentiation

In this review we examine successively: 1) the major effects of thyroid hormone deficiency seen during brain development with special emphasis on the changes in neuronal morphology and migration occurring postnatally in the cerebellum. 2) The effects of this hormone on microtubule assembly during neurite outgrowth and acquisition of neuronal polarity. 3) The changes in expression of the different tubulin isoforms occurring during development in the normal and hypothyroid rat brain. 4) The regulation by thyroid hormone of the transition occurring during development between the juvenile and adult microtubule-associated protein Tau.Special issue dedicated to Dr. Louis Sokoloff.     

Modulation of angiogenesis-related protein synthesis by valproic acid

Recent studies have attested to the antiangiogenic effects of HDAC inhibitors on solid human tumors. The HDAC inhibitor butyrate has been reported to impair tumor-cell-induced angiogenesis. However, due to its poor bioavailability in vivo, the therapeutic use of butyrate is limited. On the other hand, valproic acid has inhibitory effects on carcinoma cells, is known to be well tolerated, and has an excellent bioavailability. We therefore set out to investigate whether the HDAC inhibitor valproic acid also impairs angiogenesis. Our findings indicate that valproic acid represses the relevant angiogenic factors VEGF and FGF in Caco-2 cells. Both, protein expression as well as mRNA levels of VEGF, were reduced to a similar degree. Suppression of ubiquitin-proteasome activity could be a possible reason for valproic acid effects on regulatory angiogenesis proteins. These results suggest that the HDAC inhibitor valproic acid could become a valuable new addition in the attempt to develop alternative therapeutic approaches in the treatment of colon carcinomas.     

Inhibition of ribulose bisphosphate carboxylase assembly by antibody to a binding protein

We have developed an assay to monitor in vitro the posttranslational assembly of the chloroplast protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Most of the newly synthesized 55-kD catalytic ("large") subunits of this enzyme occur in a 29S complex together with 60- and 61-kD "binding" proteins. When the 29S complex is incubated with ATP and MgCl2 it dissociates into subunits, and the formerly bound large subunits now sediment at 7S (still faster than expected for a monomer). Upon incubation at 24 degrees C, these large subunits assemble into RuBisCO. The minority of newly made large subunits which are not bound to the 29S complex also sediment at 7S. When endogenous ATP was removed by addition of hexokinase and glucose, the dissociation of the 29S complex was inhibited. Nevertheless, the 7S large subunits assembled into RuBisCO, and did so to a greater extent than in controls retaining endogenous ATP. Thus the 7S large subunits are also assembly competent, at least when ATP is removed. Apparently, in chloroplast extracts, ATP can have a dual effect on the assembly of RuBisCO: on the one hand, even at low concentrations it can inhibit incorporation of 7S large subunits RuBisCO; on the other hand, at higher concentrations it can lead to substantial buildup of the 7S large subunit pool by causing dissociation of the 29S complex, and stimulate overall assembly. At both high and zero concentrations of ATP, however, antibody to the binding protein inhibited the assembly of endogenous large subunits into RuBisCO. Thus it appears that all assembly-competent large subunits are associated with the binding protein, either in the 7S complex or in the 29S complex. The involvement of the binding protein in RuBisCO assembly may represent the first example of non-autonomous protein assembly in higher plants and may pose problems for the genetic engineering of RuBisCO from these organisms.     

Inhibition of tumor-stromal interaction through HGF/Met signaling by valproic acid

Hepatocyte growth factor (HGF), which is produced by surrounding stromal cells, including fibroblasts and endothelial cells, has been shown to be a significant factor responsible for cancer cell invasion mediated by tumor-stromal interactions. We found in this study that the anti-tumor agent valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, strongly inhibited tumor-stromal interaction. VPA inhibited HGF production in fibroblasts induced by epidermal growth factor (EGF), platelet-derived growth factor, basic fibroblast growth factor, phorbol 12-myristate 13-acetate (PMA) and prostaglandin E₂ without any appreciable cytotoxic effect. Other HDAC inhibitors, including butyric acid and trichostatin A (TSA), showed similar inhibitory effects on HGF production stimulated by various inducers. Up-regulations of HGF gene expression induced by PMA and EGF were also suppressed by VPA and TSA. Furthermore, VPA significantly inhibited HGF-induced invasion of HepG2 hepatocellular carcinoma cells. VPA, however, did not affect the increases in phosphorylation of MAPK and Akt in HGF-treated HepG2 cells. These results demonstrated that VPA inhibited two critical processes of tumor-stromal interaction, induction of fibroblastic HGF production and HGF-induced invasion of HepG2 cells, and suggest that those activities serve for other anti-tumor mechanisms of VPA besides causing proliferation arrest, differentiation, and/or apoptosis of tumor cells.     

Cloning of rat olfactory bulb tubulin tyrosine ligase cDNA: a dominant negative mutant and an antisense cDNA increase the proliferation rate of cells in culture

In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.     

Structure-activity study of cytotoxicity and microtubule assembly in vitro by taxol and related taxanes

Fractionation of bovine brain cytosol by DEAE cellulose chromatography revealed the presence of a calcium-dependent protein kinase. This soluble neuronal protein kinase selectively phosphorylated several endogenous substrates. The most prominent substrate was a polypeptide with an apparent M_r of 45,000 which was stimulated 20-fold by addition of both calcium and calmodulin. Activation was dose-dependent, with half-maximal phosphorylation occurring at 0.9 μM free Ca²⁺ and 60nM calmodulin. The effect of calmodulin was competitively inhibited by a variety of calmodulin inhibitors, in a manner characteristic of most calmodulin-dependent enzymes. This calcium- and calmodulin-dependent protein kinase is distinct from any previously described protein kinase.     

Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.     

Inhibition of medium-chain fatty acid beta-oxidation in vitro by valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid

Valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid, were found to inhibit strongly the metabolism of decanoic acid in homogenates of rat liver. Reductions in decanoate consumption in response to inhibitors were paralleled by decreases in the formation of octanoic and hexanoic acids, two products of decanoate beta-oxidation. In contrast, 4-pentenoic acid, an established inhibitor of long-chain fatty acid beta-oxidation, had little effect on the metabolism of decanoate. It is concluded that the title compounds are potent, broad-spectrum inhibitors of fatty acid beta-oxidation, a property which may be of key toxicological importance in the pathology of valproate-induced liver injury.     

Teratology study of derivatives of tetramethylcyclopropyl amide analogues of valproic acid in mice

BACKGROUND: Although valproic acid (VPA) is used extensively for treating various kinds of epilepsies, it is well known that it causes neural tube and skeletal defects in both humans and animals. The amide and urea derivatives of the tetramethylcylcopropyl VPA analogue, N-methoxy-2,2,3,3-tetramethylcyclopropanecarboxamide (N-methoxy-TMCD) and 2,2,3,3-tetramethylcyclopropanecarbonylurea (TMC-urea), were synthesized and shown to have a more potent anticonvulsant activity than VPA. The objective of this study was to investigate the teratogenic effects of these compounds in NMRI mice. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of either VPA, N-methoxy-TMCD, or TMC-urea at 1.8 and 3.6 mmol/kg on gestation day (GD) 8. Cesarean section was performed on GD 18. First, the live fetuses were examined to detect any external malformations, then their skeletons were double-stained for bone and cartilage and subsequently examined. RESULTS: Significant increases in fetal losses and neural tube defects were observed with administration of VPA at 3.6 mmol/kg when compared to the vehicle control. In contrast, upon cesarean section, there were no significant differences between either N-methoxy-TMCD or TMC-urea and the control groups for any parameter. Skeletal examination revealed that a number of the abnormalities were induced by VPA dose-dependently at high rates of incidence. These abnormalities were mainly at the axial skeletal level. However, lower frequencies of skeletal abnormality were observed with N-methoxy-TMCD and TMC-urea than with VPA. CONCLUSIONS: In addition to their more potent antiepileptic activity, these findings clearly indicate that N-methoxy-TMCD and TMC-urea are distinctly less teratogenic than VPA in NMRI mice.     

Molecular dissection of valproic acid effects in acute myeloid leukemia identifies predictive networks

Histone deacetylase inhibitors (HDACIs) like valproic acid (VPA) display activity in leukemia models and induce tumor-selective cytotoxicity against acute myeloid leukemia (AML) blasts. As there are limited data on HDACIs effects, we aimed to dissect VPA effects in vitro using myeloid cell lines with the idea to integrate findings with in vivo data from AML patients treated with VPA additionally to intensive chemotherapy (n = 12). By gene expression profiling we identified an in vitro VPA response signature enriched for genes/pathways known to be implicated in cell cycle arrest, apoptosis, and DNA repair. Following VPA treatment in vivo, gene expression changes in AML patients showed concordant results with the in vitro VPA response despite concomitant intensive chemotherapy. Comparative miRNA profiling revealed VPA-associated miRNA expression changes likely contributing to a VPA-induced reversion of deregulated gene expression. In addition, we were able to define markers predicting VPA response in vivo such as CXCR4 and LBH. These could be validated in an independent cohort of VPA and intensive chemotherapy treated AML patients (n = 114) in which they were inversely correlated with relapse-free survival. In summary, our data provide new insights into the molecular mechanisms of VPA in myeloid blasts, which might be useful in further advancing HDAC inhibition based treatment approaches in AML.