Antibacterial activity of griseofulvin analogues as an example of drug repurposing.

Griseofulvin is a well-known antifungal drug that was launched in 1962 by Merck & Co. for the treatment of dermatophyte infections. However, according to predictions using the Way2Drug computational drug repurposing platform, it may also have antibacterial activity. As no confirmation of this prediction was found in the published literature, this study estimated in-silico antibacterial activity for 42 griseofulvin derivatives. Antibacterial activity was predicted for 33 of the 42 compounds, which led to the conclusion that this activity might be considered as typical for this chemical series. Therefore, experimental testing of antibacterial activity was performed on a panel of Gram-positive and Gram-negative micro-organisms. Antibacterial activity was evaluated using the microdilution method detecting the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). The tested compounds exhibited potent antibacterial activity against all the studied bacteria, with MIC and MBC values ranging from 0.0037 to 0.04 mg/mL and from 0.01 to 0.16 mg/mL, respectively. Activity was 2.5-12 times greater than that of ampicillin and 2-8 times greater than that of streptomycin, which were used as the reference drugs. Similarity analysis for all 42 compounds with the (approximately) 470,000 drug-like compounds indexed in the Clarivate Analytics Integrity database confirmed the significant novelty of the antibacterial activity for the compounds from this chemical class. Therefore, this study demonstrated that by using computer-aided prediction of biological activity spectra for a particular chemical series, it is possible to identify typical biological activities which may be used for discovery of new applications (e.g. drug repurposing).

Modified chitosan coated mesoporous strontium hydroxyapatite nanorods as drug carriers.

Mesoporous strontium hydroxyapatite (SrHAp) nanorods (NRs) have been successfully synthesized using a simple and efficient chemical route, i.e. the hydrothermal method. Structural and morphological characterization of the as-synthesized SrHAp NRs have been performed by transmission electron microscopy (TEM) and high angle annular dark field scanning transmission electron microscopy (HAADF-STEM). TEM and HAADF-STEM measurements of the NRs reveal the coexistence of longer and shorter particles with the length ranging from 50 nm to 400 nm and a diameter of about 20-40 nm. Electron tomography measurements of the NRs allow us to better visualize the mesopores and their facets. Two model drugs, hydrophobic risperidone and hydrophilic pramipexole, were loaded into the SrHAp NRs. These nanorods were coated using a modified chitosan (CS) with poly(2-hydroxyethyl methacrylate) (PHEMA), in order to encapsulate the drug-loaded SrHAp nanoparticles and reduce the cytotoxicity of the loaded materials. The drug release from neat and encapsulated SrHAp NRs mainly depends on the drug hydrophilicity. Importantly, although neat SrHAp nanorods exhibit some cytotoxicity against Caco-2 cells, the Cs-g-PHEMA-SrHAp drug-loaded nanorods show an acceptable cytocompatibility.

Pharmacokinetic interaction between losartan and Rhodiola rosea in rabbits.

AIM: The study investigates the potential interaction of the herbal medicinal product of Rhodiola rosea on the pharmacokinetics of losartan and its active metabolite EXP3174 after concurrent oral administration to rabbits. MATERIALS AND METHODS: We conducted a randomized, single-dose, two-treatment, two-period, two-sequence, cross-over pharmacokinetic study on 6 healthy female New Zealand rabbits, after concurrent oral administration of losartan (5 mg/kg) and the herbal medicinal product of R. rosea (50 mg/kg). Quantification of losartan and its main active metabolite EXP3174 was achieved using a validated HPCL/UV method. Pharmacokinetic and statistical analysis was performed using the EquivTest/PK software. OBSERVATIONS: Administration of the herbal medicinal product of R. rosea resulted in a statistically significant increase of the following pharmacokinetic parameters for losartan: the maximum plasma concentration (C(max)), the area under the curve (AUC) and the apparent total body clearance (CL/F). An almost 2-fold increase in the AUC of losartan was observed after concurrent administration of the herbal medicinal product of R. rosea. No statistically significant alteration was observed in the pharmacokinetic parameters of the active metabolite of losartan EXP3174. CONCLUSION: The data of this study suggest that R. rosea significantly alters the pharmacokinetic properties of losartan after concurrent oral administration to rabbits. A study in humans should be conducted to assess the clinical significance of a possible herb-drug interaction between the herbal medicinal products of R. rosea and drugs such as losartan, which are substrates of both CYPs and P-gp.

MeSHy: Mining unanticipated PubMed information using frequencies of occurrences and concurrences of MeSH terms.

MOTIVATION: PubMed is the most widely used database of biomedical literature. To the detriment of the user though, the ranking of the documents retrieved for a query is not content-based, and important semantic information in the form of assigned Medical Subject Headings (MeSH) terms is not readily presented or productively utilized. The motivation behind this work was the discovery of unanticipated information through the appropriate ranking of MeSH term pairs and, indirectly, documents. Such information can be useful in guiding novel research and following promising trends. METHODS: A web-based tool, called MeSHy, was developed implementing a mainly statistical algorithm. The algorithm takes into account the frequencies of occurrences, concurrences, and the semantic similarities of MeSH terms in retrieved PubMed documents to create MeSH term pairs. These are then scored and ranked, focusing on their unexpectedly frequent or infrequent occurrences. RESULTS: MeSHy presents results through an online interactive interface facilitating further manipulation through filtering and sorting. The results themselves include the MeSH term pairs, along with MeSH categories, the score, and document IDs, all of which are hyperlinked for convenience. To highlight the applicability of the tool, we report the findings of an expert in the pharmacology field on querying the molecularly-targeted drug imatinib and nutrition-related flavonoids. To the best of our knowledge, MeSHy is the first publicly available tool able to directly provide such a different perspective on the complex nature of published work. IMPLEMENTATION AND AVAILABILITY: Implemented in Perl and served by Apache2 at http://bat.ina.certh.gr/tools/meshy/ with all major browsers supported.

On the intracellular trafficking of mouse S5 ribosomal protein from cytoplasm to nucleoli.

The non-ribosomal functions of mammalian ribosomal proteins have recently attracted worldwide attention. The mouse ribosomal protein S5 (rpS5) derived from ribosomal material is an assembled non-phosphorylated protein. The free form of rpS5 protein, however, undergoes phosphorylation. In this study, we have (a) investigated the potential role of phosphorylation in rpS5 protein transport into the nucleus and then into nucleoli and (b) determined which of the domains of rpS5 are involved in this intracellular trafficking. In vitro PCR mutagenesis of mouse rpS5 cDNA, complemented by subsequent cloning and expression of rpS5 truncated recombinant forms, produced in fusion with green fluorescent protein, permitted the investigation of rpS5 intracellular trafficking in HeLa cells using confocal microscopy complemented by Western blot analysis. Our results indicate the following: (a) rpS5 protein enters the nucleus via the region 38-50 aa that forms a random coil as revealed by molecular dynamic simulation. (b) Immunoprecipitation of rpS5 with casein kinase II and immobilized metal affinity chromatography analysis complemented by in vitro kinase assay revealed that phosphorylation of rpS5 seems to be indispensable for its transport from nucleus to nucleoli; upon entering the nucleus, Thr-133 phosphorylation triggers Ser-24 phosphorylation by casein kinase II, thus promoting entrance of rpS5 into the nucleoli. Another important role of rpS5 N-terminal region is proposed to be the regulation of protein's cellular level. The repetitively co-appearance of a satellite C-terminal band below the entire rpS5 at the late stationary phase, and not at the early logarithmic phase, of cell growth suggests a specific degradation balancing probably the unassembled ribosomal protein molecules with those that are efficiently assembled to ribosomal subunits. Overall, these data provide new insights on the structural and functional domains within the rpS5 molecule that contribute to its cellular functions.

Cloning, sequencing and expression of a cDNA encoding the mouse L35a ribosomal protein during differentiation of murine erythroleukemia (MEL) cells.

In a previous study we reported that ribosomal protein S5 gene is suppressed in differentiating and not in proliferating or apoptotic murine erythroleukaemia (MEL) cells (Vizirianakis et al., 1999). We wish to report here the isolation, characterisation and expression of the full length cDNA for another ribosomal protein, the L35a (rpL35a), in MEL cells. This cDNA shares significant structural homology in both DNA and protein levels to genes encoding the rat and human L35a ribosomal proteins. Northern blot hybridisation analysis has shown that the steady-state level of rpL35a mRNA is progressively reduced during differentiation of MEL cells along the erythrocytic maturation pathway induced by DMSO or UDP-4, two structurally unrelated inducers of differentiation. However, in cells where differentiation was inhibited by N(6)-methyladenosine, the level of rpL35a RNA transcripts was not affected. In addition, rpL35a gene expression was not altered in apoptotic MEL cells. Furthermore, the suppression of L35a gene was not correlated to any change in DNA methylation at CCGG sites located at the rpL35a gene locus in undifferentiated and differentiated MEL cells, as we observed for the rpS5 gene. Overall, these data suggest that the expression of ribosomal genes, the L35a of 60S ribosomal subunit and the S5 of 40S ribosomal subunit, are regulated by a common mechanism in differentiating MEL cells, leading to the observed decrease in ribosomal function.

Transfection of MCF-7 carcinoma cells with human integrin alpha7 cDNA promotes adhesion to laminin.

The laminin-binding alpha7beta1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin alpha7 subunit contributes to a form of muscular dystrophy in integrin alpha7 null mice, whereas specific mutations in the alpha7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin alpha7 in human-related muscular disorders, we cloned alpha7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin alpha7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human alpha7X2B alternative splice form. Expression of human alpha7 was further confirmed by transfection of chimeric human/mouse alpha7 cDNA constructs. To demonstrate the functionality of expressed human alpha7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human alpha7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human alpha7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human alpha7 cDNA. These results show for the first time the exogenous expression of functional full-length human alpha7 cDNA, as well as the development of monoclonal antibodies against the human alpha7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving alpha7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.

Transcriptional activation of prion protein gene in growth-arrested and differentiated mouse erythroleukemia and human neoplastic cells.

The prion protein (PrP) is a GPI-anchored sialoglycoprotein that has attracted worldwide attention over the years due to its involvement in the pathogenesis of transmissible spongiform encephalopathies in sheep (scrapie), cattle (BSE), and humans (CJD). To understand the precise role of the Prn-p gene in cell growth and differentiation we investigated the expression pattern of the Prn-p gene in proliferating cells and in cells arrested in growth either by confluency or by induction of terminal differentiation. Viral-transformed mouse spleen hematopoietic cells named murine erythroleukemia (MEL) and other types of inducible cells (human neuroectodermal RD/TE-671, myoid RD cells) were employed. Cells grown exponentially, at confluency, or irreversibly arrested in growth at terminal differentiation state were analyzed by fluorescence cell sorting and Northern blot hybridization to estimate the steady-state level of PrP mRNA at different phases of the cell cycle. MEL cells that failed to differentiate from treatment with N(6)-methyladenosine (N(6)mAdo), an inhibitor of differentiation, were also analyzed for PrP mRNA level. Our results indicate the following: (a) growth arrest of cells at G(1) phase by confluency or by induction of terminal differentiation led to increased accumulation of PrP mRNA transcripts, an event observed also in differentiated MEL, RD/TE-671, and RD cells independent of the inducer used; (b) treatment of MEL cells with N(6)mAdo prevented early activation of the Prn-p gene in cells treated with the inducer; and (c) cell-free nuclear run-off studies showed enhanced expression of the Prn-p gene due to transcriptional activation. These findings indicate, for the first time, that the Prn-p gene, which is thought to be a housekeeping gene, is transcriptionally activated in G(1) phase in confluent and terminally differentiated cells. This information may be valuable in understanding the overaccumulation of PrP in some differentiated tissues and may let us repress Prn-p gene activation by novel agents.

Expression of ribosomal protein S5 cloned gene during differentiation and apoptosis in murine erythroleukemia (MEL) cells.

Murine erythroleukemia (MEL) cells have been used as a suitable model system for studying cellular and molecular mechanisms of erythroid differentiation. In an effort to isolate and characterize genes whose expression change during differentiation, we cloned and sequenced a cDNA of 715 bp (rpS5) from a MEL cDNA library. The cloned mouse cDNA showed significant degree of structural homology in both DNA and protein level to known human and rat genes that encode the S5 proteins of 40S ribosomal subunit. The use of 715-bp cDNA as probe revealed the presence of an RNA transcript in the cytoplasm of MEL and human neuroectodermal RD/TE-671 cells. The steady-state accumulation level of this RNA transcript decreased upon induction of differentiation of both cell lines by treatment with DMSO and UDP-4, two structurally different inducers. Blockade of MEL cell differentiation by the inhibitor N6-methyladenosine preserved the constitutive expression of the rpS5 gene. DNA methylation analysis at CCGG sites located at the rpS5 gene locus in undifferentiated and differentiated MEL cells revealed that the suppression of the rpS5 gene during MEL cell differentiation is not related to any change in methylation at these sites. Moreover, the rpS5 gene continued to be expressed in cells undergoing serum-deprived apoptosis, like in control untreated cells. Therefore, we conclude that there may be a disparate pattern of expression of the rpS5 gene in differentiating and apoptotic cells. These data can be valuable in understanding the role of ribosomal proteins during differentiation and cell death (apoptosis) of neoplastic cells, although there is no experimental evidence that the suppression of the rpS5 gene is related mechanistically to the induction of differentiation. It may well be considered as part of the differentiation process.

Induction of murine erythroleukemia cell differentiation is associated with methylation and differential stability of poly(A)+ RNA transcripts.

Murine erythroleukemia (MEL) cells exposed to DMSO were assessed for their ability to methylate poly(A)+ RNA and accumulate RNA transcripts of globin and nonglobin genes (c-myc, beta-actin and MER5). Cells were pulse-labeled with L-[methyl-3H]methionine, cytoplasmic RNA was isolated, selected for poly(A)+ RNA and analyzed by HPLC chromatography for methylated nucleosides. When MEL cells were exposed to inhibitors of RNA methylation (neplanocin A, 3-deazaneplanocin A and cycloleucine) and assessed for their ability to differentiate by DMSO, accumulate RNA transcripts, produce hemoglobin, methylate poly(A)+ and poly(A)- RNA and synthesize S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), we observed the following: (a) MEL cells treated with DMSO underwent hypermethylation in poly(A)+ RNA that preferentially occurred at the 5'-cap structures (7-methylguanosine and 2'-O-methylcytidine and 2'-O-methyluridine); (b) inducer-treated MEL cells exhibited a decrease in the intracellular level of SAH that led to a lower ratio of SAH/SAM, an event that favors methylation; and (c) treatment of MEL cells with inhibitors of RNA methylation suppressed methylation of poly(A)- and poly(A)+ RNA, reversed the ratio SAH/SAM seen in differentiated MEL cells and prevented differentiation to occur. Moreover, we observed that treatment of MEL cells with selective inhibitors of RNA methylation caused fragmentation of beta major globin and c-myc mRNAs, two RNA transcripts coded by developmentally regulated genes, while had no detectable effect on the structural integrity of poly(A)+ RNA transcripts transcribed by two housekeeping genes (beta-actin and MER5). These data indicate that induction of erythroid cell differentiation of MEL cells is associated with changes in methylation of poly(A)+ RNA and selective differential stability of RNA transcripts, two events that might be related to each other.

N6-methyladenosine inhibits murine erythroleukemia cell maturation by blocking methylation of RNA and memory via conversion to S-(N6-methyl)-adenosylhomocysteine.

We have shown earlier that N6-methyladenosine (N6mAdo) and other methylated derivatives block commitment of murine erythroleukemia (MEL) cells to terminal erythroid maturation. In this study, we further investigated the mechanism of this blockade. Treatment of MEL cells with N6mAdo inhibited cell growth, prevented accumulation of committed cells, suppressed methylation of total cytoplasmic RNA, and erased the expression of "memory" response, an event that precedes initiation of commitment. Furthermore, N6mAdo increased the level of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) and altered the SAH/SAM ratio that influences methylation of ribonucleic acid (RNA). Moreover, analysis of the intracellular extracts revealed that N6-mAdo is converted into S-(N6-methyl)-adenosylhomocysteine (N6-SAH) in MEL cells, an active intermediate that affects methylation of RNA. Therefore, we conclude that N6-mAdo prevents induction of MEL cell differentiation by affecting methylation of critical RNA transcripts involved in expression of "memory" and initiation of commitment. It is likely that this inhibition occurs via conversion of N6mAdo into N6-SAH.

Analysis of the inhibition of commitment of murine erythroleukemia (MEL) cells to terminal maturation by N6-methyladenosine.

Treatment of cultured murine erythroleukemia (MEL or Friend) cells with N6-methylated derivatives of adenosine inhibited erythroid cell differentiation induced by various agents. N6-Methyladenosine (N6mAdo) inhibited initiation of commitment to terminal maturation and prevented accumulation of hemoglobin in a concentration-dependent manner. Treatment with N6mAdo slowed cell growth without causing substantial inhibition in the rate of DNA synthesis and a marked decrease in viability and clonogenic potential of MEL cells. Furthermore, N6mAdo decreased the cytoplasmic accumulation of beta(major) globin mRNA and affected its structural integrity in MEL cells. Cells pre-exposed to N6mAdo failed to initiate commitment as early as control cells upon challenge with the inducer dimethyl sulfoxide. N6mAdo-induced inhibition of commitment was not reversed but rather was potentiated by the presence of adenine, L-homocysteine and/or L-methionine, agents involved in the active methylation cycle. To this respect, N6mAdo-induced inhibition of commitment was found to be different from that caused by cordycepin (3'-deoxyadenosine, an inhibitor of RNA methylation and mRNA polyadenylation). The latter inhibition was fully reversed by the addition of L-methionine. These findings indicate that N6-methyladenosine: (a) blocks a central process that is required for initiation of commitment; and (b) decreases accumulation of beta (major) globin mRNA, causes mRNA degradation and prevents hemoglobin synthesis. Due to the differential sensitivity of N6mAdo- and cordycepin-induced blockade of commitment to L-methionine, these agents inhibit commitment by acting via two different mechanisms impinging on the final pathway of MEL erythroid cell maturation.

Synthesis, molecular structure determination, and antitumor activity of platinum(II) and palladium(II) complexes of 2-substituted benzimidazole.

The complexes of 2-aminomethyl benzimidazole, 2-(beta-aminoethyl)benzimidazole, and 2-(alpha-aminoethy-l)benzimidazole with Pt(II) and Pd(II) have been prepared. The molecular structure of the free ligands and their complexes were studied by IR and 1H NMR. It was concluded that the substituted benzimidazole derivatives behave as bidentate ligands, being bound to the metal atoms via the nitrogen of the -N = group and the amino group of the side chain of the benzimidazole ring. The metal complexes were tested for antineoplastic activity both in cultures of neoplastic cells (MEL-745, K-562, Colon 205, IMP-32, SK-N-SH) and in vivo in rodents bearing L-1210 leukemia. The antiproliferative activity of these agents was compared to that of cis-platin.