Silver and Copper Complexes with Ibuprofen and Caffeine-Preparation and Evaluation of Their Selected Biological Effects.

Several organometallic complexes based on more than twenty different metals have already been approved for medical applications. The aim of the presented research was to obtain complexes of silver and copper with the non-steroidal anti-inflammatory drugs ibuprofen and xanthine alkaloid caffeine and evaluate selected aspects of their bioactivity and biosafety in terms of their future possible applications. The obtained complexes were characterized by Fourier-transform infrared spectroscopy, thermogravimetry, UV-VIS spectroscopy, conductometry, elemental analysis, and bioassays. Cytotoxicity for normal human cells of the CCD-Co18 cell line was evaluated by determining the IC50 value, with metabolic and morphology assessments. It was observed that complexes containing ibuprofen and caffeine exhibited lower toxicity than those with ibuprofen only. Complexes with copper showed lower toxicity towards healthy human fibroblasts compared to silver-based compounds, with an IC50 above 140 µg mL-1. However, in the silver complexes, the presence of caffeine increased the potency of COX-2 inhibition. Antimicrobial effects against different Gram-positive and Gram-negative bacterial strains were evaluated by MIC determination with values less than 20 µg mL-1.

Yeast Protein Asf1 Possesses Modulating Activity towards Protein Kinase CK2.

Protein kinase CK2 plays an important role in cell survival and protects regulatory proteins from caspase-mediated degradation during apoptosis. The consensus sequence of proteins phosphorylated by CK2 contains a cluster of acidic amino acids around the phosphorylation site. The poly-acidic sequence in yeast protein Asf1 is similar to the acidic loop in CK2ß, which possesses a regulatory function. We observed that the overexpression of Asf1 in yeast cells influences cell growth. Experiments performed in vitro and in vivo indicate that yeast protein Asf1 inhibits protein kinase CK2. Our data suggest that each CK2 isoform might be regulated in a different way. Deletion of the amino or carboxyl end of Asf1 reveals that the acidic cluster close to the C-terminus is responsible for the activation or inhibition of CK2 activity.

CK2 and protein kinases of the CK1 superfamily as targets for neurodegenerative disorders.

Casein kinases are involved in a variety of signaling pathways, and also in inflammation, cancer, and neurological diseases. Therefore, they are regarded as potential therapeutic targets for drug design. Recent studies have highlighted the importance of the casein kinase 1 superfamily as well as protein kinase CK2 in the development of several neurodegenerative pathologies, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. CK1 kinases and their closely related tau tubulin kinases as well as CK2 are found to be overexpressed in the mammalian brain. Numerous substrates have been detected which play crucial roles in neuronal and synaptic network functions and activities. The development of new substances for the treatment of these pathologies is in high demand. The impact of these kinases in the progress of neurodegenerative disorders, their bona fide substrates, and numerous natural and synthetic compounds which are able to inhibit CK1, TTBK, and CK2 are discussed in this review.

Metabolic and Proteomic Analysis of Chlorella sorokiniana, Chloroidium saccharofilum, and Chlorella vulgaris Cells Cultured in Autotrophic, Photoheterotrophic, and Mixotrophic Cultivation Modes.

Chlorella is one of the most well-known microalgal genera, currently comprising approximately a hundred species of single-celled green algae according to the AlgaeBase. Strains of the genus Chlorella have the ability to metabolize both inorganic and organic carbon sources in various trophic modes and synthesize valuable metabolites that are widely used in many industries. The aim of this work was to investigate the impact of three trophic modes on the growth parameters, productivities of individual cell components, and biochemical composition of Chlorella sorokiniana, Chloroidium saccharofilum, and Chlorella vulgaris cells with special consideration of protein profiles detected by SDS-PAGE gel electrophoresis and two-dimensional gel electrophoresis with MALDI-TOF/TOF MS. Mixotrophic conditions with the use of an agro-industrial by-product stimulated the growth of all Chlorella species, which was confirmed by the highest specific growth rates and the shortest biomass doubling times. The mixotrophic cultivation of all Chlorella species yielded a high amount of protein-rich biomass with reduced contents of chlorophyll a, chlorophyll b, carotenoids, and carbohydrates. Additionally, this work provides the first information about the proteome of Chloroidium saccharofilum, Chlorella sorokiniana, and Chlorella vulgaris cells cultured in molasses supplementation conditions. The proteomic analysis of the three Chlorella species growing photoheterotrophically and mixotrophically showed increased accumulation of proteins involved in the cell energy metabolism and carbon uptake, photosynthesis process, and protein synthesis, as well as proteins involved in intracellular movements and chaperone proteins.

Organofluorine Hydrazone Derivatives as Multifunctional Anti-Alzheimer's Agents with CK2 Inhibitory and Antioxidant Features.

A set of novel hydrazone derivatives were synthesized and analyzed for their biological activities. The compounds were tested for their inhibitory effect on the phosphorylating activity of the protein kinase CK2, and their antioxidant activity was also determined in three commonly used assays. The hydrazones were evaluated for their radical scavenging against the DPPH, ABTS and peroxyl radicals. Several compounds have been identified as good antioxidants as well as potent protein kinase CK2 inhibitors. Most hydrazones containing a 4-N(CH3 )2 residue or perfluorinated phenyl rings showed high activity in the radical-scavenging assays and possess nanomolar IC50 values in the kinase assays.

Compounds from Natural Sources as Protein Kinase Inhibitors.

The advantage of natural compounds is their lower number of side-effects when compared to most synthetic substances. Therefore, over the past several decades, the interest in naturally occurring compounds is increasing in the search for new potent drugs. Natural compounds are playing an important role as a starting point when developing new selective compounds against different diseases. Protein kinases play a huge role in several diseases, like cancers, neurodegenerative diseases, microbial infections, or inflammations. In this review, we give a comprehensive view of natural compounds, which are/were the parent compounds in the development of more potent substances using computational analysis and SAR studies.

Powder bed 3D-printing of highly loaded drug delivery devices with hydroxypropyl cellulose as solid binder.

3D-printing is a promising tool to pave the way to the widespread adaption of individualized medicine. Several printing techniques have been investigated and introduced to pharmaceutical research. Until now, only one 3D-printed medicine is approved on the US market. The medicine is manufactured via drop-on-powder deposition, which uses inkjet printing to jet a liquid binder on a powder bed to create 3D objects. However, inkjet processes are prone to nozzle clogging when binders or active pharmaceutical ingredients (APIs) are included in the printing ink. This renders the formulation development of the ink the most challenging step. In this study, different hydroxypropyl cellulose (HPC) grades were investigated as solid binders in the powder formulation on a commercially available DoP printer. The printed ink only consisted of a water/ethanol mixture. Formulations containing 70% caffeine as model API were developed and tablets printed. It was found that the friability of the tablets greatly depends on the particle size of the employed binder, whereas disintegration time and dissolution properties mainly depend on the viscosity of the employed binders. Higher viscous binders led to slower disintegration and dissolution whereas lower viscous binders led to faster disintegration and dissolution. The study demonstrates that HPC is a suitable solid binder for DoP printing and that 3D-DoP printing can be used to print robust dosage forms.

Inhibitory influence of natural flavonoids on human protein kinase CK2 isoforms: effect of the regulatory subunit.

CK2 is a pleiotropic, constitutively active protein kinase responsible for the phosphorylation of more than 300 physiological substrates. Typically, this enzyme is found in tetrameric form consisting of two regulatory subunits CK2ß and two catalytic subunits CK2α or CK2α'. Several natural occurring flavonoids were tested for their ability to inhibit both CK2 holoenzymes, CK2α2ß2 and CK2α'2ß2. We identified few substances selectively inhibiting only the α' subunit. Other compounds showed similar effect towards all four isoforms. In some cases, like chrysoeriol, pedalitin, apigenin, and luteolin, the α2ß2 holoenzyme was at least six times better inhibited than the free α subunit. Otherwise, we have found a luteolin derivative decreased the kinase activity of CK2α' with an IC50 value of 0.8 µM, but the holoenzyme only with 9.5 µM.

Selected flavonoid compounds as promising inhibitors of protein kinase CK2α and CK2α', the catalytic subunits of CK2.

CK2 is a ubiquitous protein kinase involved in many cell functions. During the last years it became an interesting target in cancer research. A series of flavonoid compounds was tested as inhibitors of protein kinase CK2. Several substances were found to be highly active against both catalytic subunits with IC50 values below 1 µM in case of CK2α'. The most promising inhibitor we identified is chrysoeriol with IC50 values of 250 and 34 nM for CK2α and CK2α', respectively.

Dual inhibition of HCV and HIV by ring-expanded nucleosides containing the 5:7-fused imidazo[4,5-e][1,3]diazepine ring system. In vitro results and implications.

Examples of ring-expanded nucleosides (RENs), represented by general structures 1 and 2, exhibited dual anti-HCV and anti-HIV activities in both cell culture systems and the respective target enzyme assays, including HCV NTPase/helicase and human RNA helicase DDX3. Since HCV is a leading co-infection in late stage HIV AIDS patients, often leading to liver cirrhosis and death, the observed dual inhibition of HCV and HIV by the target nucleoside analogues has potentially beneficial implications in treating HIV patients infected with HCV.

UK-1 and structural analogs are potent inhibitors of hepatitis C virus replication.

The bacterial natural product UK-1 and several structural analogs inhibit replication of the hepatitis C virus in the replicon assay, with IC50 values as low as 0.50 µM. The NS3 helicase has been identified as a possible target of inhibition for several of these compounds, while the remaining inhibitors act via an undetermined mechanism. Gel shift assays suggest that helicase inhibition is a direct result of inhibitor-enzyme binding as opposed to direct RNA binding, and the ATPase activity of NS3 is not affected. The syntheses and biological results are presented herein.

The NTPase/helicase domain of hepatitis C virus nonstructural protein 3 inhibits protein kinase C independently of its NTPase activity.

Helicase motif VI is a short arginine-rich motif within the NTPase/helicase domain of the non-structural protein 3 (NS3) of the hepatitis C virus (HCV). We previously demonstrated that it reduces the catalytic activity and intracellular shuttling of protein kinase C (PKC). Thus, NS3-mediated PKC inhibition may be involved in HCV-associated hepatocellular carcinoma (HCC). In this study, we expand on our earlier results, which were obtained in experiments with short fragments of NS3, to show for the first time that the catalytically active, longer C-terminal NTPase/helicase of NS3 acts as a potent PKC inhibitor in vitro. PKC inhibition assays with the NTPase-inactive mutant NS3h-D1316A revealed a mixed type kinetic inhibition pattern. A broad range of 11 PKC isotypes was tested and all of the PKC isotypes were inhibited with IC50-values in the low micromolar range. These findings were confirmed for the wild-type NTPase/helicase domain in a non-radiometric PKC inhibition assay with ATP regeneration to rule out any effect of ATP hydrolysis caused by its NTPase activity. PKCα was inhibited with a micromolar IC50 in this assay, which compares well with our result for NS3h-D1316A (IC50 = 0.7 µM). In summary, these results confirm that catalytically active NS3 NTPase/helicase can act in an analogous manner to shorter NS3 fragments as a pseudosubstrate inhibitor of PKC.

CK2α and CK2α' subunits differ in their sensitivity to 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazole derivatives.

The goal of this study was to test the inhibitory activity of a series of tetrahalogenobenzimidazoles, including a number of novel derivatives, on individual catalytic subunits of human CK2. 4,5,6,7-tetrabromo- and 4,5,6,7-tetraiodo-1H-benzimidazoles and their newly obtained N(1)- and 2-S-carboxyalkyl derivatives showed potent inhibitory activity against both these subunits. CK2α' was up to 6 times more sensitive to the studied compounds than CK2α. The investigated iododerivatives showed, in most cases, stronger inhibitory properties than the respective brominated congeners, but the differences showed considerable dependence on the protein substrate used.

Cloning and purification of protein kinase CK2 recombinant alpha and beta subunits from the Mediterranean fly Ceratitis capitata.

The Mediterranean fruit fly Ceratitis capitata is an insect capable of wreaking extensive damage to a wide range of fruit crops. Protein kinase CK2 is a ubiquitous Ser/Thr kinase that is highly conserved among eukaryotes; it is a heterotetramer composed of two catalytic (α) and a dimer of regulatory (ß) subunits. We present here the construction of the cDNA molecules of the CK2α and CK2ß subunits from the medfly C. capitata by the 5'/3' RACE and RT-PCR methods, respectively. CcCK2α catalytic subunit presents the characteristic and conserved features of a typical protein kinase, similar to the regulatory CcCK2ß subunit, that also possess the conserved features of regulatory CK2ß subunits, as revealed by comparison of their predicted amino acid sequences with other eukaryotic species. The recombinant CcCK2α and CcCK2ß proteins were purified by affinity chromatography to homogeneity, after overexpression in Escherichia coli. CcCK2α is capable to utilize GTP and its activity and is inhibited by polyanions and stimulated by polycations in phosphorylation assays, using purified acidic ribosomal protein P1 as a substrate.

Interactions between subunits of protein kinase CK2 and their protein substrates influences its sensitivity to specific inhibitors.

Five isoforms of CK2 may exist simultaneously in yeast cells: free catalytic subunits CK2α', CK2α and three holoenzymatic structures composed of αα'ßß', α(2)ßß' and α'(2)ßß'. Each isolated and purified form exhibits properties typical for CK2, but they differ in substrate specificity as well as in sensitivity to specific modulators. All five isoforms of protein kinase CK2 from Saccharomyces cerevisiae were examined for their binding capacity with ATP/GTP and two commonly used ATP-competitive inhibitors TBB and TBI. Enzymes were tested with protein substrates differently interacting with CK2 subunits: Elf1, Fip1, Svf1, P2B and synthetic peptide. Obtained results show that K(m) for ATP varies from 2.4-53 µM for Elf1/CK2α' and Svf1/CK2α, respectively. Similar differences can be seen in case when GTP was used as phosphate donor. The inhibitory effect depends on composition of CK2/substrate complexes. Highest sensitivity to TBB shows all complexes containing αα'ßß' isoform with K (i) values between 0.2 and 1.1 µM. The prospect that TBB and TBI could be utilized to discriminate between different molecular forms of CK2 in yeast cells was examined. Both inhibitors, TBB as well as TBI, decreases cell growth to extents devoting interactions with different CK2 isoforms present in the cell but the presence of ß/ß'-dimer has a high importance towards sensitivity. Conceivably, a given inhibitor concentration can inhibit only selected CK2-mediated processes in the cell.

Isolation of a CK2α subunit and the holoenzyme from the mussel Mytilus galloprovincialis and construction of the CK2α and CK2ß cDNAs.

Protein kinase CK2 is a ubiquitous, highly pleiotropic, and constitutively active phosphotransferase that phosphorylates mainly serine and threonine residues. CK2 has been studied and characterized in many organisms, from yeast to mammals. The holoenzyme is generally composed of two catalytic (α and/or α') and two regulatory (ß) subunits, forming a differently assembled tetramer. The free and catalytically active α/α' subunits can be present in cells under some circumstances. We present here the isolation of a putative catalytic CK2α subunit and holoenzyme from gills of the mussel Mytilus galloprovincialis capable of phosphorylating the purified recombinant ribosomal protein rMgP1. For further analysis of M. galloprovincialis protein kinase CK2, the cDNA molecules of CK2α and CK2ß subunits were constructed and cloned into expression vectors, and the recombinant proteins were purified after expression in Escherichia coli. The recombinant MgCK2ß subunit and MgP1 were phosphorylated by the purified recombinant MgCK2α subunit. The mussel enzyme presented features typical for CK2: affinity for GTP, inhibition by both heparin and ATP competitive inhibitors (TBBt, TBBz), and sensitivity towards NaCl. Predicted amino acid sequence comparison showed that the M. galloprovincialis MgCK2α and MgCK2ß subunits have similar features to their mammalian orthologs.

Regulation of the biochemical function of motif VI of HCV NTPase/helicase by the conserved Phe-loop.

In previous works, we demonstrated a potent inhibition of diverse protein kinase C (PKC) functions by a fragment of nonstructural protein 3 (NS3) of hepatitis C virus (HCV), mainly mediated by the Arg-rich amino acid motif HCV(1487-1500). This sequence is localized on the surface of Domain 2 of the NS3 NTPase/helicase in direct vicinity to a flexible loop that is localized between Val1458 and Thr1476. Here, we assessed the regulation of the accessibility of the Arg-rich amino acid motif for PKC by this loop, using two variants of domain 2. The first construct, termed NS3d2Delta, comprises the complete domain, HCV(1361-1503), devoid the loop. The second variant, NS3d2wt corresponds to wild type domain 2. The results indicated an enhanced inhibitory potential of NS3d2Delta towards rat brain PKC and towards the majority of PKC isoforms. This effect and the accompanying change of the mode of inhibition from a mixed mode, exerted by NS3d2wt to a competitive mode, exerted by NS3d2Delta are caused by the deletion of the loop. Accordingly, the presence of the intact loop abolished the binding of domain 2 to the tailed duplex RNA used as helicase substrate, without affecting the binding of dsDNA. Furthermore, a direct competition of dsRNA and PKC for the same binding site HCV(1487-1500), could be documented. The binding of dsRNA to NS3d2Delta previously overlaid with PPKCalpha was reduced to 30% and completely abolished in case of NS3d2Delta overlaid with cAMP-dependent protein kinase A (PKA).

Viral NS3 helicase activity is inhibited by peptides reproducing the Arg-rich conserved motif of the enzyme (motif VI).

The NTPase/helicase of Flaviviridae viruses is one of the essential components of their replication complex. The enzyme is defined by the presence of seven highly conserved amino acid motifs. Random screening of numerous hepatitis C virus (HCV) derived peptides, revealed a basic amino acid stretch corresponding to motif VI of the HCV NTPase/helicase (amino acids 1487-1500 of the HCV polyprotein). This peptide inhibited the unwinding activity of the enzyme with an IC(50)=0.2 microM. Peptides corresponding to motif VI of HCV, West Nile virus (WNV) and Japanese encephalitis virus (JEV) were synthesized and tested as inhibitors of NTPase and unwinding reactions mediated by the viral enzymes. Peptides distinguished in regard to their length and structure. Between the peptides tested HCV(1487-1500) reproducing the sequence of motif VI was the most potent inhibitor of helicase activities of investigated enzymes. Other respective peptides were rather modest inhibitors. The examined peptides inhibited the Flaviviridae helicases in the following order of potency: HCV(1487-1500)>WNV(1959-1572)>JEV(1962-1975). Interestingly, the susceptibility of the helicase activity to the inhibition by the peptides was similar and in the row: HCV>WNV>JEV. The inhibition results from binding and blockade of the active site of the enzyme lyes beyond the NTP-binding and hydrolyzing site. The kinetic analyses indicated that the binding of the peptides do not interfere with the NTPase activity of the enzymes. The peptide may serve as effective and selective tool to reduce the virus propagation.

Tropolone and its derivatives as inhibitors of the helicase activity of hepatitis C virus nucleotide triphosphatase/helicase.

In this report, we demonstrate the interaction of the non-structural protein 3 (NS3) of hepatitis C virus (HCV) with alkaloide tropolone (2-hydroxy-2,4,6-heptatriene-1-one) and its derivatives. The compounds were biochemically screened separately against the ATPase and helicase activities of HCV NS3. In the investigations presented, alkaIoide tropolone and its derivatives significantly inhibited the helicase activity of the viral protein when using a DNA substrate, with 50% inhibitory concentration values within a low micromolar range. The results using the RNA substrate were unexpected--none of the tropolone derivatives excerted any modulating influence towards the unwinding activity. Surprisingly, no influence of the nucleoside triphosphatase (NTPase) turnover was observed. Evidence is presented confirming that these compounds do not act by blocking the NTP-binding site, but by occupying an additional allosteric regulatory site. Further mechanisms of action, particularly of some of the derivatives, are discussed.

Synaptonemal complex protein SYCP3: Conserved polymerization properties among vertebrates.

Synaptonemal complexes (SCs) are meiosis-specific, nuclear structures that are critically involved in synapsis, recombination and segregation of homologous chromosomes. Although the SC structure is conserved in evolution this is not the case for its protein components. To provide information on SC proteins which would be important for our understanding of the conserved SC structure and function, here we compared ortholog SYCP3 proteins of two evolutionary distant vertebrate species, namely rat and medaka fish. To this end we have investigated the polymerization properties of both proteins by immunocytochemistry, electron microscopy and cell fractionation. We found that despite of the sequence differences that have accumulated over the last 450 million years mammalian and fish SYCP3 have similar properties that allow them to co-assemble higher order structures under experimental conditions. We also provide a likely explanation as to how heterozygous mutations in the SYCP3 gene can lead to a defective meiosis.