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Molecular Cancer Therapeutics Jan 2016Increased ploidy is common in tumors but treatments for tumors with excess chromosome sets are not available. Here, we characterize high-ploidy breast cancers and...
Increased ploidy is common in tumors but treatments for tumors with excess chromosome sets are not available. Here, we characterize high-ploidy breast cancers and identify potential anticancer compounds selective for the high-ploidy state. Among 354 human breast cancers, 10% have mean chromosome copy number exceeding 3, and this is most common in triple-negative and HER2-positive types. Women with high-ploidy breast cancers have higher risk of recurrence and death in two patient cohorts, demonstrating that it represents an important group for improved treatment. Because high-ploidy cancers are aneuploid, rather than triploid or tetraploid, we devised a two-step screen to identify selective compounds. The screen was designed to assure both external validity on diverse karyotypic backgrounds and specificity for high-ploidy cell types. This screen identified novel therapies specific to high-ploidy cells. First, we discovered 8-azaguanine, an antimetabolite that is activated by hypoxanthine phosphoribosyltransferase 1 (HPRT1), suggesting an elevated gene-dosage of HPRT1 in high-ploidy tumors can control sensitivity to this drug. Second, we discovered a novel compound, 2,3-diphenylbenzo[g]quinoxaline-5,10-dione (DPBQ). DPBQ activates p53 and triggers apoptosis in a polyploid-specific manner, but does not inhibit topoisomerase or bind DNA. Mechanistic analysis demonstrates that DPBQ elicits a hypoxia gene signature and its effect is replicated, in part, by enhancing oxidative stress. Structure-function analysis defines the core benzo[g]quinoxaline-5,10 dione as being necessary for the polyploid-specific effects of DPBQ. We conclude that polyploid breast cancers represent a high-risk subgroup and that DPBQ provides a functional core to develop polyploid-selective therapy. Mol Cancer Ther; 15(1); 48-59. ©2015 AACR.
Topics: Antineoplastic Agents; Apoptosis; Benzoquinones; Biomarkers, Tumor; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Drug Discovery; Female; Gene Expression Regulation, Neoplastic; Humans; In Situ Hybridization, Fluorescence; Kaplan-Meier Estimate; Karyotype; Polyploidy; Prognosis; Proline; Signal Transduction; Tumor Suppressor Protein p53
PubMed: 26586723
DOI: 10.1158/1535-7163.MCT-15-0527 -
Bioorganic & Medicinal Chemistry Letters Oct 2015A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic...
A one step synthesis of fluorescent 8-aryl-(7-deazaguanines) has been accomplished. Probes exhibit blue to green high quantum yield fluorescence in a variety of organic and aqueous solutions, high extinction coefficients, and large Stokes shifts often above 100 nm. The probes are highly cell permeable, and exhibit stable bright fluorescence once intracellular; therefore are suited to the design of biosensors.
Topics: Azaguanine; Cell Line, Tumor; Cell Membrane Permeability; Fluorescence; Fluorescent Dyes; Humans; KB Cells; Microscopy, Confocal; Molecular Structure
PubMed: 26320620
DOI: 10.1016/j.bmcl.2015.08.054 -
International Journal of Systematic and... Dec 2014A group of strains representing species of the genus Leptospira, isolated from patients with leptospirosis in Mayotte (Indian Ocean), were previously found to be...
A group of strains representing species of the genus Leptospira, isolated from patients with leptospirosis in Mayotte (Indian Ocean), were previously found to be considerably divergent from other known species of the genus Leptospira. This was inferred from sequence analysis of rrs (16S rRNA) and other genetic loci and suggests that they belong to a novel species. Two strains from each serogroup currently identified within this novel species were studied. Spirochaete, aerobic, motile, helix-shaped strains grew well at 30-37 °C, but not at 13 °C or in the presence of 8-azaguanine. Draft genomes of the strains were also analysed to study the DNA relatedness with other species of the genus Leptospira. The new isolates formed a distinct clade, which was most closely related to Leptospira borgpetersenii, in multilocus sequence analysis using concatenated sequences of the genes rpoB, recA, fusA, gyrB, leuS and sucA. Analysis of average nucleotide identity and genome-to-genome distances, which have recently been proposed as reliable substitutes for classical DNA-DNA hybridization, further confirmed that these isolates should be classified as representatives of a novel species. The G+C content of the genomic DNA was 39.5 mol%. These isolates are considered to represent a novel species, for which the name Leptospira mayottensis sp. nov. is proposed, with 200901116(T) ( = CIP 110703(T) = DSM 28999(T)) as the type strain.
Topics: Bacterial Typing Techniques; Base Composition; Comoros; DNA, Bacterial; Genes, Bacterial; Humans; Leptospira; Leptospirosis; Molecular Sequence Data; Multilocus Sequence Typing; Nucleic Acid Hybridization; Phylogeny; RNA, Ribosomal, 16S; Sequence Analysis, DNA
PubMed: 25249563
DOI: 10.1099/ijs.0.066597-0 -
The Journal of Biological Chemistry Dec 2013The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate...
The evolutionarily broad family nucleobase-cation symporter-2 (NCS2) encompasses transporters that are conserved in binding site architecture but diverse in substrate selectivity. Putative purine transporters of this family fall into one of two homology clusters: COG2233, represented by well studied xanthine and/or uric acid permeases, and COG2252, consisting of transporters for adenine, guanine, and/or hypoxanthine that remain unknown with respect to structure-function relationships. We analyzed the COG2252 genes of Escherichia coli K-12 with homology modeling, functional overexpression, and mutagenesis and showed that they encode high affinity permeases for the uptake of adenine (PurP and YicO) or guanine and hypoxanthine (YjcD and YgfQ). The two pairs of paralogs differ clearly in their substrate and ligand preferences. Of 25 putative inhibitors tested, PurP and YicO recognize with low micromolar affinity N(6)-benzoyladenine, 2,6-diaminopurine, and purine, whereas YjcD and YgfQ recognize 1-methylguanine, 8-azaguanine, 6-thioguanine, and 6-mercaptopurine and do not recognize any of the PurP ligands. Furthermore, the permeases PurP and YjcD were subjected to site-directed mutagenesis at highly conserved sites of transmembrane segments 1, 3, 8, 9, and 10, which have been studied also in COG2233 homologs. Residues irreplaceable for uptake activity or crucial for substrate selectivity were found at positions occupied by similar role amino acids in the Escherichia coli xanthine- and uric acid-transporting homologs (XanQ and UacT, respectively) and predicted to be at or around the binding site. Our results support the contention that the distantly related transporters of COG2233 and COG2252 use topologically similar side chain determinants to dictate their function and the distinct purine selectivity profiles.
Topics: Escherichia coli K12; Escherichia coli Proteins; Ligands; Membrane Transport Proteins; Models, Molecular; Mutagenesis, Site-Directed; Nucleoside Transport Proteins; Protein Structure, Tertiary; Structural Homology, Protein; Structure-Activity Relationship
PubMed: 24214977
DOI: 10.1074/jbc.M113.523340 -
Asian Pacific Journal of Cancer... 2013Prostate cancer caused by the abnormal disorderly growth of prostatic acinar cells is the most prevalent cancer of men in western countries. We aimed to screen out...
PURPOSE
Prostate cancer caused by the abnormal disorderly growth of prostatic acinar cells is the most prevalent cancer of men in western countries. We aimed to screen out differentially expressed genes (DEGs) and explore small molecule drugs for prostate cancer.
MATERIALS AND METHODS
The GSE3824 gene expression profile of prostate cancer was downloaded from Gene Expression Omnibus database which including 21 normal samples and 18 prostate cancer cells. The DEGs were identified by Limma package in R language and gene ontology and pathway enrichment analyses were performed. In addition, potential regulatory microRNAs and the target sites of the transcription factors were screened out based on the molecular signature database. In addition, the DEGs were mapped to the connectivity map database to identify potential small molecule drugs.
RESULTS
A total of 6,588 genes were filtered as DEGs between normal and prostate cancer samples. Examples such as ITGB6, ITGB3, ITGAV and ITGA2 may induce prostate cancer through actions on the focal adhesion pathway. Furthermore, the transcription factor, SP1, and its target genes ARHGAP26 and USF1 were identified. The most significant microRNA, MIR-506, was screened and found to regulate genes including ITGB1 and ITGB3. Additionally, small molecules MS-275, 8-azaguanine and pyrvinium were discovered to have the potential to repair the disordered metabolic pathways, abd furthermore to remedy prostate cancer.
CONCLUSIONS
The results of our analysis bear on the mechanism of prostate cancer and allow screening for small molecular drugs for this cancer. The findings have the potential for future use in the clinic for treatment of prostate cancer.
Topics: Computational Biology; Databases, Genetic; Drug Discovery; Humans; Male; MicroRNAs; Pharmaceutical Preparations; Prostatic Neoplasms; Small Molecule Libraries; Transcriptome
PubMed: 24175814
DOI: 10.7314/apjcp.2013.14.9.5281 -
Molecules (Basel, Switzerland) Oct 2013Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the...
Various forms of purine-nucleoside phosphorylase (PNP) were used as catalysts of enzymatic ribosylation of selected fluorescent 8-azapurines. It was found that the recombinant calf PNP catalyzes ribosylation of 2,6-diamino-8-azapurine in a phosphate-free medium, with ribose-1-phosphate as ribose donor, but the ribosylation site is predominantly N7 and N8, with the proportion of N8/N7 ribosylated products markedly dependent on the reaction conditions. Both products are fluorescent. Application of the E. coli PNP gave a mixture of N8 and N9-substituted ribosides. Fluorescence of the ribosylated 2,6-diamino-8-azapurine has been briefly characterized. The highest quantum yield, ~0.9, was obtained for N9-β-d-riboside (λmax 365 nm), while for N8-β-d-riboside, emitting at ~430 nm, the fluorescence quantum yield was found to be close to 0.4. Ribosylation of 8-azaguanine with calf PNP as a catalyst goes exclusively to N9. By contrast, the E. coli PNP ribosylates 8-azaGua predominantly at N9, with minor, but highly fluorescent products ribosylated at N8/N7.
Topics: Animals; Azaguanine; Biocatalysis; Cattle; Escherichia coli Proteins; Fluorescent Dyes; Glycosylation; Kinetics; Purine-Nucleoside Phosphorylase; Recombinant Proteins; Ribosemonophosphates
PubMed: 24126376
DOI: 10.3390/molecules181012587 -
Nucleic Acids Research Nov 2013Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the...
Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.
Topics: Anti-HIV Agents; Azaguanine; CD4-Positive T-Lymphocytes; Cell Line; HIV-1; HeLa Cells; Humans; Quinolines; RNA Splicing; RNA, Viral; Thiophenes; Viral Structural Proteins; Virus Replication; rev Gene Products, Human Immunodeficiency Virus
PubMed: 23945945
DOI: 10.1093/nar/gkt727 -
Nitric Oxide : Biology and Chemistry Nov 2013Nitric oxide (NO) is a very effective radiosensitizer of hypoxic mammalian cells, at least as efficient as oxygen in enhancing cell death in vitro. NO may induce cell...
Nitric oxide (NO) is a very effective radiosensitizer of hypoxic mammalian cells, at least as efficient as oxygen in enhancing cell death in vitro. NO may induce cell death through the formation of base lesions which are difficult to repair, and if they occur within complex clustered damage common to ionizing radiation, they may lead to replication-induced DNA strand breaks. It has previously been shown that 8-azaguanine and xanthine result from the reaction of guanine radicals with nitric oxide. We have now shown that adenine radicals also react with NO to form hypoxanthine and 8-azaadenine. Cells irradiated in exponential growth in the presence of NO are twice as radiosensitive compared to those irradiated in anoxia alone, whereas confluent cells are less radiosensitive to (•)NO. In addition, the numbers of DNA double strand breaks observed as γH2AX staining following radiosensitization by NO, are higher in exponential cells than in confluent cells. DNA damage, detected as 53BP1 foci, is also higher in HF-19 cells expressing Cyclin A, a marker for cells in S and G2 phases of the cell cycle, following radiosensitization by NO. RAD51 foci are highest in V79-4 cells irradiated in the presence of NO compared to in anoxia, 24h after radiolysis. This work presents evidence that radiosensitization of cells by NO is in part through the formation of specific DNA damage, difficult to repair, which in dividing cells may induce the formation of stalled replication forks and as a consequence replication-induced DNA strand breaks which may lead to cell death.
Topics: Adenine; Animals; Cell Cycle; Cell Line; Cell Survival; Cricetinae; DNA; DNA Damage; DNA Replication; Humans; Intracellular Signaling Peptides and Proteins; Nitric Oxide; Rad51 Recombinase; Radiation, Ionizing; Radiation-Sensitizing Agents
PubMed: 23623927
DOI: 10.1016/j.niox.2013.04.005 -
Biochemistry May 2013NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural...
NE0047 from Nitrosomonas europaea has been annotated as a zinc-dependent deaminase; however, the substrate specificity is unknown because of the low level of structural similarity and sequence identity compared to other family members. In this study, the function of NE0047 was established as a guanine deaminase (catalytic efficiency of 1.2 × 10(5) M(-1) s(-1)), exhibiting secondary activity towards ammeline. The structure of NE0047 in the presence of the substrate analogue 8-azaguanine was also determined to a resolution of 1.9 Å. NE0047 crystallized as a homodimer in an asymmetric unit. It was found that the extreme nine-amino acid C-terminal loop forms an active site flap; in one monomer, the flap is in the closed conformation and in the other in the open conformation with this loop region exposed to the solvent. Calorimetric data obtained using the full-length version of the enzyme fit to a sequential binding model, thus supporting a cooperative mode of ligand occupancy. In contrast, the mutant form of the enzyme (ΔC) with the deletion of the extreme nine amino acids follows an independent model of ligand occupancy. In addition, the ΔC mutant also does not exhibit any enzyme activity. Therefore, we propose that the progress of the reaction is communicated via changes in the conformation of the C-terminal flap and the closed form of the enzyme is the catalytically active form, while the open form allows for product release. The catalytic mechanism of deamination was also investigated, and we found that the mutagenesis of the highly conserved active site residues Glu79 and Glu143 resulted in a complete loss of activity and concluded that they facilitate the reaction by serving as proton shuttles.
Topics: Bacterial Proteins; Catalysis; Catalytic Domain; Guanine Deaminase; Ligands; Models, Molecular; Nitrosomonas europaea; Protein Conformation; Substrate Specificity
PubMed: 23557066
DOI: 10.1021/bi400068g -
Journal of Biomolecular Structure &... 2014Analogues of purine bases are highly relevant in the biological context and have been implicated as drug molecules for therapy against a number of diseases....
Analogues of purine bases are highly relevant in the biological context and have been implicated as drug molecules for therapy against a number of diseases. Additionally, these molecules have been implicated to have a role in the prebiotic RNA world. However, experimental data on the structures of these molecules in aqueous solution is lacking. In this work, we report the ultraviolet resonance Raman spectra of 6-chloroguanine, 8-azaguanine and allopurinol, obtained with 260 nm excitation. The reported spectra have been assigned to normal modes computed from density functional theory (B3LYP/6-31G (d,p)) calculations. This work has been useful in identifying the solution-state structures of these molecules at neutral pH. We find that the guanine analogues 6-chloroguanine and 8-azaguanine exist as keto-N9H and keto-N7H tautomers in solution, respectively. On the other hand, the hypoxanthine analogue allopurinol exists as a mixture of keto-N9H and keto-N8H tautomers in solution. We predict that this work would be particularly useful in future vibrational studies where these molecules are present in complexes with their target proteins.
Topics: Allopurinol; Azaguanine; Guanine; Hydrogen-Ion Concentration; Molecular Structure; Spectrum Analysis, Raman
PubMed: 23384120
DOI: 10.1080/07391102.2012.745821