Liquid-phase synthesis of a pegylated adenosine-oligoarginine conjugate,cell-permeable inhibitor of cAMP-dependent protein kinase

An adenosine-oligoarginine conjugate (ARC) was assembled in a stepwise manner on a poly(ethylene glycol) carrier. The pegylated conjugate inhibited cAMP-dependent protein kinase with IC(50)=460 nM and the cellular uptake of its BODIPY FL derivative was demonstrated and compared to that of free ARC with fluorescence microscopy.     

A protein kinase inhibitor as an antimycobacterial agent

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.     

A new chitosan-thymine conjugate: synthesis, characterization and biological activity

Conjugation of chitosan with nucleobases is expected to expand its not only antimicrobial activity but also anti-cancer activity. Here, we report the synthesis of a novel chitosan-thymine conjugate by the reaction between chitosan and thymine-1-yl-acetic acid followed by acylation. The synthesized conjugate was characterized by FTIR, XRD, (1)H NMR, TGA and SEM. The microbiological screening results demonstrated the antimicrobial activity of the conjugate against bacteria viz., Escherichia coli, Staphylococcus aureus, and fungi viz., Aspergillus niger. The chitosan-thymine conjugate also inhibited (p<0.05) the proliferation of human liver cancer cells (HepG2) in a dose-dependent manner but had no cellular toxicity in non-cancerous mouse embryonal fibroblast cells (NIH 3T3). Thus, the chitosan-nucleobase conjugate may open a new perspective in biomedical applications.     

Design, synthesis, and characterization of new embelin derivatives as potent inhibitors of X-linked inhibitor of apoptosis protein

X-Linked inhibitor of apoptosis protein (XIAP) is a promising molecular target for the design of new anticancer drugs aiming at promoting apoptosis in cancer cells. We have previously identified embelin as an inhibitor of XIAP through computational structure-based database screening. Herein, we report the design, synthesis, and evaluation of new embelin analogues as inhibitors of XIAP. Our efforts led to the identification of new and more potent inhibitors. For example, compound 6g has a Ki value of 180 nM binding to XIAP BIR3, in a competitive binding assay and represents a promising lead compound for further optimization.     

Amino acid sequence and characterization of a protein inhibitor of protein kinase C

The complete primary structure has been determined for an inhibitor protein of protein kinase C. The bovine brain-derived inhibitor has a pI of 6 and its N-terminal alanine residue is blocked by acetylation. Fragments obtained by chemical and enzymatic cleavage of the purified inhibitor were analyzed by Edman degradation, fast atom bombardment mass spectrometry, and tandem mass spectrometry. The results establish that the protein has a calculated average molecular mass of 13,690 daltons and contains 125 amino acid residues with the following sequence: (sequence: see text) The inhibitor does not show significant homology with any other known protein. Circular dichroism of the freshly prepared apoprotein indicated a secondary structural content of 23% alpha-helix, 31% beta-sheet, and 11% beta-turn. Immobilization on nitrocellulose followed by exposure to a 65Zn2(+)-containing overlay solution showed that, like protein kinase C itself, the inhibitor is a zinc-binding protein, although the sequence does not reveal a "zinc finger" structure. Competition with 10-fold molar excess Ca2+ or Mg2+ did not reduce the zinc-binding specificity of this inhibitor.     

Design and synthesis of a novel tyrosine kinase inhibitor template

We report the design and synthesis of an insulin receptor kinase family-targeted inhibitor template using the inhibitor conformation observed in an IGF1R/inhibitor co-crystal complex by application of a novel molecular design approach that we have recently published. The synthesis of the template involves a one pot Opatz cyclization reaction that provides a versatile indole ester in good yields. We also developed the required chemistry to elaborate this template with additional substituents and have used this chemistry to prepare some initial compounds that show selective inhibition of anaplastic lymphoma kinase (ALK).     

Partial purification and characterization of a protein inhibitor of phosphoenolpyruvate carboxylase kinase

The activity of phosphoenolpyruvate carboxylase (PEPCase) kinase in leaf extracts increased markedly on dilution. This was shown to be caused by the presence of a protein that inhibits the kinase. The inhibitor protein was separated from the kinase and purified partially. It inhibited the kinase reversibly, presumably by a direct interaction; it was neither a protease nor a protein phosphatase. The amounts of kinase and inhibitor in leaves were estimated following separation by hydrophobic chromatography. The amount of inhibitor in the crassulacean acid metabolism plant Kalanchoe fedtschenkoi Hamet et Perrier was sufficient to inhibit the basal level of kinase activity present during the light period and the early stages of the dark period. Similarly, the amount of inhibitor in the C4 plant Zea mays L. was sufficient to inhibit the low amount of kinase activity present in the dark and at moderate light intensity. Analogous to the role of the protein inhibitor of mammalian cyclic AMP-dependent protein kinase, the function of the PEPCase kinase inhibitor may be to inhibit the basal level of kinase present in conditions under which rapid flux through PEPCase is not required.     

Design and characterization of a traceable protein kinase Calpha

Protein kinase Calpha (PKCalpha) is a critical component of pathways that govern cancer-related phenotypes such as invasion and proliferation. Proteins that serve as immediate substrates for PKCalpha offer potential targets for anticancer drug design. To identify specific substrates, a mutant of PKCalpha (M417A) was constructed at the ATP binding site such that it could bind a sterically large ATP analogue derivatized through the N6 amino group of adenosine ([gamma-32P]-N6-phenyl-ATP). Because this analogue could be utilized by the mutant kinase but not by wild-type PKCalpha (or presumably other protein kinase) to phosphorylate peptide or protein substrates, 32P-labeled products were the direct result of the mutant PKCalpha. Kinetic analysis with [gamma-32P]-N6-phenyl-ATP revealed that the mutant retained undiminished affinity for the peptide substrate (Km = 12.4 microM) and a Vmax value (10.3 pmol/min) that was only 3-fold lower than that exhibited by the wild-type enzyme with natural ATP. However, with [gamma-32P]ATP, the mutant had a somewhat lower affinity (Km = 82.8 microM) than the wild-type enzyme (Km = 9.3 microM) in vitro but was competent in causing aggressive motility in nonmotile MCF-10A human breast cells (with endogenous ATP), as previously described for wild-type PKCalpha. The FLAG-tagged PKCalpha mutant was expressed in MCF-10A cells and used to co-immunoprecipitate high-affinity substrates from lysates. Immunopellets were reacted with [gamma-32P]-N6-phenyl-ATP, and radiolabeled products were analyzed by SDS-PAGE and autoradiography. Mass spectrometry of selected bands identified several known substrates of PKC, thereby validating the methods used in these studies. These findings provide a foundation for future applications of this traceable PKCalpha mutant.     

Design, synthesis and characterization of "clickable" 4-anilinoquinazoline kinase inhibitors

Immobilized kinase inhibitors have emerged as powerful reagents for the determination of kinase inhibitor selectivity and for the enrichment of protein targets from cellular lysates. Here, we report the design and synthesis of a set of "clickable" 4-anilinoquinazoline kinase inhibitors. We demonstrate that the attachment of a flexible tether that contains a bio-orthogonal azide functionality does not adversely affect the potency or selectivity of these inhibitors. Furthermore, we demonstrate the utility of these inhibitors through the generation of an affinity matrix for the enrichment of interacting proteins from cellular lysates.     

Design,synthesis, functional and structural characterization of an inhibitor of N-acetylneuraminate-9-phosphate phosphatase: Observation of extensive dynamics in an enzyme/inhibitor complex

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH₂ group, inhibits HDHD4 with a binding affinity (IC₅₀ 11 μM) in the range of the native substrate Neu5Ac-9-P (compound 1, K_m 47 μM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg²⁺. In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca²⁺ and Mg²⁺ are indicative of a highly dynamic process in the active site for the HDHD4/Mg²⁺/3 complex. Possible explanations for this observation are discussed.     

Isolation and characterization of an endogenous inhibitor of protein synthesis in Escherichia coli K-12.

A low-molecular-weight factor was isolated from cell extracts of Escherichia coli K-12. The concentration of the factor in cells was dependent upon nutritional conditions, the concentration being higher in faster growing cells. Treatment of cells with colicin K caused an increase in concentration of the factor. The factor inhibited protein synthesis in E. coli. This inhibition was reversible, apparently because of metabolism of the factor. The inhibition of synthesis of beta-galactosidase lasted longer than the inhibition of protein synthesis; cyclic AMP eliminated this difference. The factor inhibited the synthesis of beta-galactosidase from preformed lac mRNA, indicating an inhibition of translation. Kinetic studies of the onset of inhibition of beta-galactosidase synthesis by the factor suggested that the factor may inhibit protein synthesis at the initiation of translation.     

Design,synthesis, and biological evaluation of 4-phenoxyquinoline derivatives as potent c-Met kinase inhibitor

A series of novel 4-phenoxyquinoline derivatives containing 3-oxo-3,4-dihydro-quinoxaline moiety were synthesized and evaluated for their antiproliferative activity against five human cancer cell lines (A549, H460, HT-29, MKN-45 and U87MG) in vitro. Most of the tested compounds exhibited more potent inhibitory activities than the positive control foretinib. Compound 1b, 1s and 1t were further examined for their inhibitory activity against c-Met kinase. The most promising compound 1s (with c-Met IC₅₀ value of 1.42 nM) showed remarkable cytotoxicity against A549, H460, HT-29, MKN45 and U87MG cell lines with IC₅₀ values of 0.39 μM, 0.18 μM, 0.38 μM, 0.81 μM, respectively. Their preliminary structure-activity relationships (SARs) study indicated that the replacement of the aromatic ring with the cyclohexane improved their antiproliferative activity.     

Hyaluronic acid-paclitaxel conjugate micelles: synthesis, characterization, and antitumor activity

Chemical conjugates of paclitaxel and hyaluronic acid (HA) were synthesized by utilizing a novel HA solubilization method in a single organic phase. Hydrophilic HA was completely dissolved in anhydrous DMSO with addition of poly(ethylene glycol) (PEG) by forming nanocomplexes. Paclitaxel was then chemically conjugated to HA in the DMSO phase via an ester linkage without modifying extremely hydrophilic HA. A series of HA-paclitaxel conjugates with different conjugation percentages were synthesized and characterized. HA-paclitaxel conjugates self-assembled in aqueous solution to form nanosized micellar aggregates, as characterized by dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM). An intact form of paclitaxel was regenerated from HA-paclitaxel conjugate micelles at acidic pH conditions. HA-paclitaxel conjugate micelles exhibited more pronounced cytotoxic effect for HA receptor overexpressing cancer cells than for HA receptor deficient cells, suggesting that they can be potentially utilized as tumor-specific nanoparticulate therapeutic agents.     

Salubrinal, an inhibitor of protein synthesis, promotes deep slow wave sleep

Previous work showed that sleep is associated with increased brain protein synthesis and that arrest of protein synthesis facilitates sleep. Arrest of protein synthesis is induced during the endoplasmic reticulum (ER) stress response, through phosphorylation of eukaryotic initiation factor 2alpha (p-eIF2alpha). We tested a hypothesis that elevation of p-eIF2alpha would facilitate sleep. We studied the effects of intracerebroventricular infusion of salubrinal (Salub), which increases p-eIF2alpha by inhibiting its dephosphorylation. Salub increased deep slow wave sleep by 255%, while reducing active waking by 49%. Delta power within non-rapid eye movement (NREM) sleep was increased, while power in the sigma, beta, and gamma bands during NREM was reduced. We found that Salub increased expression of p-eIF2alpha in the basal forebrain (BF) area, a sleep-wake regulatory brain region. Therefore, we quantified the p-eIF2alpha-immunolabeled neurons in the BF area; Salub administration increased the number of p-eIF2alpha-expressing noncholinergic neurons in the caudal BF. In addition, Salub also increased the intensity of p-eIF2alpha expression in both cholinergic and noncholinergic neurons, but this was more widespread among the noncholinergic neurons. Our findings support a hypothesis that sleep is facilitated by signals associated with the ER stress response.     

Prevention of lithocholate--induced cholestasis by cycloheximide, an inhibitor of protein synthesis

Our previous investigations have shown that lithocholic acid (LCA)-induced cholestasis is associated with an increased synthesis of microsomal cholesterol which is transported with LCA and incorporated in the bile canalicular membrane. As the significance of these changes remains unknown the effect of interference with microsomal protein synthesis and/or with the cellular transport of cholesterol was studied. Male Wistar rats were injected i.p. with cycloheximide at the dose of 15 micrograms/100 g BW 3 times over a 24-hour period. After cannulating the common bile duct and collecting bile for one hour, the animals were either injected i.v. with 12 mumoles C14-LCA/100 g BW or with a 7.5% albumin solution and bile was collected for another hour. LCA injection in untreated animals reduced bile flow by more than 90% of control values. In contrast, bile flow in the group treated with cycloheximide and LCA was normal and did not differ from that of animals given cycloheximide alone. Bile salt secretion rate was increased in the cycloheximide-LCA group over the control groups. This was mainly due to the secretion of more than 80% of the injected LCA and was confirmed by the distribution of the radioactivity. By electron microscopy, the liver in the cycloheximide-LCA group did not show any of the well defined changes associated with LCA-induced cholestasis. These data suggest that microsomes play an important role in the pathogenesis of LCA cholestasis and that inhibition of microsomal protein synthesis can prevent its development.     

Purification and characterization of a protein synthesis inhibitor associated with vaccinia virus

A protein synthesis inhibitor, solubilized from vaccinia virus (Ben-Hamida, F., Person, A., and Beaud, G. (1983) J. Virol. 45, 452-455), has been purified to homogeneity, yielding a basic protein with molecular mass of 11 kDa. This purified protein migrates as a single spot in two-dimensional gel analysis (isoelectric point above 8.6). It is phosphorylated by the vaccinia-associated protein kinase, and it aggregates in the absence of reducing agents. This 11-kDa protein inhibits protein synthesis when added to a reticulocyte lysate at a stoichiometric ratio of approximately one protein molecule/ribosome, and it associates with the ribosome fraction after incubation in reticulocyte lysates or in Ehrlich ascites tumor cell lysates. As previously described for the inhibitor associated with vaccinia cores, the purified inhibitor inhibits the formation of the 40 S ribosomal subunit X Met-tRNAi ribosomal initiation complex. It has no detectable effect on the formation of the ternary complex (Met-tRNAi X GTP X eucaryotic initiation factor 2). This inhibitor associated with vaccinia virus particles may be involved in the shutoff of host protein synthesis and may also be responsible for the absence of virus replication in some cell-virus systems.