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International Immunopharmacology Feb 2019This study identified 8-azaguanine (8-AG) as a novel immunomodulatory drug (IMiD) through a high-throughput screen of the Preswick Chemical Library in a model of human...
This study identified 8-azaguanine (8-AG) as a novel immunomodulatory drug (IMiD) through a high-throughput screen of the Preswick Chemical Library in a model of human NK cell cytotoxicity against blood cancer cells. 8-AG, originally developed as an antineoplastic agent, significantly increased the cytotoxicity of NK cells and was superior in this activity to previously known IMiDs, such as fluoxetine and amphotericin B, identified from the same library. IFN-γ expression was also slightly increased by 8-AG. Mechanistically, 8-AG increased conjugate formation between NK and target cells and subsequent cytolytic granule polarization, but not calcium mobilization, regulation of activating receptors, or expression of perforin or granzyme B. Thus, the antineoplastic activity of 8-AG should be re-evaluated in light of this novel potentiating effect on NK cells.
Topics: Amphotericin B; Antineoplastic Agents; Azaguanine; Cells, Cultured; Cytoplasmic Granules; Cytotoxicity, Immunologic; Drug Screening Assays, Antitumor; Fluoxetine; Granzymes; Hematologic Neoplasms; Humans; Immunologic Factors; Killer Cells, Natural; Lymphocyte Activation; Perforin; Small Molecule Libraries
PubMed: 30551032
DOI: 10.1016/j.intimp.2018.12.020 -
Bioconjugate Chemistry Jul 2018The α-anomers of 8-aza-2'-deoxyguanosine (αG*) and 2'-deoxyguanosine (αG) were site-specifically incorporated in 12-mer duplexes opposite to the four canonical DNA...
The α-anomers of 8-aza-2'-deoxyguanosine (αG*) and 2'-deoxyguanosine (αG) were site-specifically incorporated in 12-mer duplexes opposite to the four canonical DNA constituents dA, dG, dT, and dC. Oligodeoxyribonucleotides containing αG* display significant fluorescence at slightly elevated pH (8.0). Oligodeoxyribonucleotides incorporating β-anomeric 8-aza-2'-deoxyguanosine (G*) and canonical dG were studied for comparison. For αG* synthesis, an efficient purification of anomeric 8-azaguanine nucleosides was developed on the basis of protected intermediates, and a new αG* phosphoramidite was prepared. Differences were observed for sugar conformations ( N vs S) and p K values of anomeric nucleosides. Duplex stability and mismatch discrimination were studied employing UV-dependent melting and fluorescence quenching. A gradual fluorescence change takes place in duplex DNA when the α-nucleoside αG* was positioned opposite to the four canonical β-nucleosides. The strongest fluorescence decrease appeared in duplexes incorporating αG*-C base pair matches. Decreasing fluorescence corresponds to increasing T values. For mismatch discrimination, the α-anomers αG* and αG are more efficient than the corresponding β-nucleosides. Duplexes with single "purine-purine" αG*-αG* or αG-αG base pairs are significantly more stable than those displaying β-d configuration. CD spectra indicate that single mutations by α-anomeric nucleosides do not affect the global structure of B-DNA.
Topics: Azaguanine; Base Pair Mismatch; Base Pairing; DNA, B-Form; Fluorescence; Guanine; Nucleic Acid Conformation; Oligodeoxyribonucleotides; Transition Temperature
PubMed: 29771499
DOI: 10.1021/acs.bioconjchem.8b00261 -
Antimicrobial Agents and Chemotherapy Apr 2018The high acquisition rate of drug resistance by necessitates the ongoing search for new drugs to be incorporated in the tuberculosis (TB) regimen. Compounds used for...
The high acquisition rate of drug resistance by necessitates the ongoing search for new drugs to be incorporated in the tuberculosis (TB) regimen. Compounds used for the treatment of other diseases have the potential to be repurposed for the treatment of TB. In this study, a high-throughput screening of compounds against thiol-deficient strains and subsequent validation with thiol-deficient strains revealed that and mutants had increased susceptibility to azaguanine (Aza) and sulfaguanidine (Su); and mutants had increased susceptibility to bacitracin (Ba); and , , and mutants had increased susceptibility to fusaric acid (Fu). Further analyses revealed that some of these compounds were able to modulate the levels of thiols and oxidative stress in This study reports the activities of Aza, Su, Fu, and Ba against and provides a rationale for further investigations.
Topics: Antitubercular Agents; Azaguanine; Mutation; Mycobacterium tuberculosis; Oxidative Stress; Sulfaguanidine; Sulfhydryl Compounds
PubMed: 29437626
DOI: 10.1128/AAC.02236-17 -
FEMS Microbiology Letters Apr 2018Two nucleobase transporters encoded in the genome of the Honey bee bacterial pathogen Paenibacillus larvae belong to the azaguanine-like transporters and are referred to...
Two nucleobase transporters encoded in the genome of the Honey bee bacterial pathogen Paenibacillus larvae belong to the azaguanine-like transporters and are referred to as PlAzg1 and PlAzg2. PlAzg1 and 2 display significant amino acid sequence similarity, and share predicted secondary structures and functional sequence motifs with two Escherichia coli nucleobase cation symporter 2 (NCS2) members: adenine permease (EcAdeP) and guanine-hypoxanthine permease EcGhxP. However, similarity does not define function. Heterologous complementation and functional analysis using nucleobase transporter-deficient Saccharomyces cerevisiae strains revealed that PlAzg1 transports adenine, hypoxanthine, xanthine and uracil, while PlAzg2 transports adenine, guanine, hypoxanthine, xanthine, cytosine and uracil. Both PlAzg1 and 2 display high affinity for adenine with Km of 2.95 ± 0.22 and 1.92 ± 0.22 μM, respectively. These broad nucleobase transport profiles are in stark contrast to the narrow transport range observed for EcAdeP (adenine) and EcGhxP (guanine and hypoxanthine). PlAzg1 and 2 are similar to eukaryotic Azg-like transporters in that they share a broad solute transport profile, particularly the fungal Aspergillus nidulans AzgA (that transports adenine, guanine and hypoxanthine) and plant AzgA transporters from Arabidopsis thaliana and Zea mays (that collectively move adenine, guanine, hypoxanthine, xanthine, cytosine and uracil).
Topics: Adenine; Amino Acid Sequence; Animals; Aspergillus nidulans; Bacterial Proteins; Bees; Biological Transport; Guanine; Membrane Transport Proteins; Paenibacillus larvae; Sequence Alignment; Substrate Specificity; Uracil; Xanthine
PubMed: 29385571
DOI: 10.1093/femsle/fny018 -
Chembiochem : a European Journal of... Dec 2017A series of nucleotide analogues, with a hypoxanthine base moiety (8-aminohypoxanthine, 1-methyl-8-aminohypoxanthine, and 8-oxohypoxanthine), together with...
A series of nucleotide analogues, with a hypoxanthine base moiety (8-aminohypoxanthine, 1-methyl-8-aminohypoxanthine, and 8-oxohypoxanthine), together with 5-methylisocytosine were tested as potential pairing partners of N -glycosylated nucleotides with an 8-azaguanine or 8-aza-9-deazaguanine base moiety by using DNA polymerases (incorporation studies). The best results were obtained with the 5-methylisocytosine nucleotide followed by the 1-methyl-8-aminohypoxanthine nucleotide. The experiments demonstrated that small differences in the structure (8-azaguanine versus 8-aza-9-deazaguanine) might lead to significant differences in recognition efficiency and selectivity, base pairing by Hoogsteen recognition at the polymerase level is possible, 8-aza-9-deazaguanine represents a self-complementary base pair, and a correlation exists between in vitro incorporation studies and in vivo recognition by natural bases in Escherichia coli, but this recognition is not absolute (exceptions were observed).
Topics: 5-Methylcytosine; Azaguanine; Base Pairing; DNA; DNA-Directed DNA Polymerase; Escherichia coli; Hypoxanthine; Purine Nucleotides
PubMed: 29024251
DOI: 10.1002/cbic.201700393 -
PLoS Neglected Tropical Diseases Dec 2016The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of...
Emerging Infectious Disease Implications of Invasive Mammalian Species: The Greater White-Toothed Shrew (Crocidura russula) Is Associated With a Novel Serovar of Pathogenic Leptospira in Ireland.
The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira.
Topics: Animals; Azaguanine; Bacterial Outer Membrane Proteins; Bacterial Typing Techniques; China; Communicable Diseases, Emerging; Cricetinae; Disease Vectors; Humans; Introduced Species; Ireland; Japan; Leptospira; Leptospirosis; Lipoproteins; Malaysia; Polymerase Chain Reaction; Prohibitins; RNA, Ribosomal, 16S; Serogroup; Shrews; Zoonoses
PubMed: 27935961
DOI: 10.1371/journal.pntd.0005174 -
Molecular Microbiology Jan 2017NCS1 proteins are H or Na symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are...
NCS1 proteins are H or Na symporters responsible for the uptake of purines, pyrimidines or related metabolites in bacteria, fungi and some plants. Fungal NCS1 are classified into two evolutionary and structurally distinct subfamilies, known as Fur- and Fcy-like transporters. These subfamilies have expanded and functionally diversified by gene duplications. The Fur subfamily of the model fungus Aspergillus nidulans includes both major and cryptic transporters specific for uracil, 5-fluorouracil, allantoin or/and uric acid. Here we functionally analyse all four A. nidulans Fcy transporters (FcyA, FcyC, FcyD and FcyE) with previously unknown function. Our analysis shows that FcyD is moderate-affinity, low-capacity, highly specific adenine transporter, whereas FcyE contributes to 8-azaguanine uptake. Mutational analysis of FcyD, supported by homology modelling and substrate docking, shows that two variably conserved residues (Leu356 and Ser359) in transmembrane segment 8 (TMS8) are critical for transport kinetics and specificity differences among Fcy transporters, while two conserved residues (Phe167 and Ser171) in TMS3 are also important for function. Importantly, mutation S359N converts FcyD to a promiscuous nucleobase transporter capable of recognizing adenine, xanthine and several nucleobase analogues. Our results reveal the importance of specific residues in the functional evolution of NCS1 transporters.
Topics: Amino Acid Sequence; Aspergillus nidulans; Biological Evolution; Biological Transport; Conserved Sequence; Fungal Proteins; Gene Duplication; Nucleobase Transport Proteins; Phylogeny; Protein Structure, Tertiary; Purines; Sequence Homology, Amino Acid; Substrate Specificity
PubMed: 27741561
DOI: 10.1111/mmi.13559 -
Breast Cancer Research : BCR Jan 2016Triple-negative breast cancer (TNBC), an aggressive disease comprising several subtypes including basal-like and claudin-low, involves frequent deletions or point...
BACKGROUND
Triple-negative breast cancer (TNBC), an aggressive disease comprising several subtypes including basal-like and claudin-low, involves frequent deletions or point mutations in TP53, as well as loss of PTEN. We previously showed that combined deletion of both tumor suppressors in the mouse mammary epithelium invariably induced claudin-low-like TNBC. The effect of p53 mutation plus Pten deletion on mammary tumorigenesis and whether this combination can induce basal-like TNBC in the mouse are unknown.
METHODS
WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) composite mice were generated in which Pten is deleted and a p53-R270H mutation in the DNA-binding domain is induced upon expression of Cre-recombinase in pregnancy-identified alveolar progenitors. Tumors were characterized by histology, marker analysis, transcriptional profiling [GEO-GSE75989], bioinformatics, high-throughput (HTP) FDA drug screen as well as orthotopic injection to quantify tumor-initiating cells (TICs) and tail vein injection to identify lung metastasis.
RESULTS
Combined Pten deletion plus induction of p53-R270H mutation accelerated formation of four distinct mammary tumors including poorly differentiated adenocarcinoma (PDA) and spindle/mesenchymal-like lesions. Transplantation assays revealed highest frequency of TICs in PDA and spindle tumors compared with other subtypes. Hierarchical clustering demonstrated that the PDA and spindle tumors grouped closely with human as well as mouse models of basal and claudin-low subtypes, respectively. HTP screens of primary Pten(∆):p53(∆) vs. Pten(∆):p53(R270H) spindle tumor cells with 1120 FDA-approved drugs identified 8-azaguanine as most potent for both tumor types, but found no allele-specific inhibitor. A gene set enrichment analysis revealed increased expression of a metastasis pathway in Pten(∆):p53(R270H) vs. Pten(∆):p53(∆) spindle tumors. Accordingly, following tail vein injection, both Pten(∆):p53(R270H) spindle and PDA tumor cells induced lung metastases and morbidity significantly faster than Pten(∆):p53(∆) double-deletion cells, and this was associated with the ability of Pten(∆):p53(R270H) tumor cells to upregulate E-cadherin expression in lung metastases.
CONCLUSIONS
Our results demonstrate that WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice represent a tractable model to study basal-like breast cancer because unlike p53 deletion, p53(R270H) mutation in the mouse does not skew tumors toward the claudin-low subtype. The WAP-Cre:Pten(f/f):p53(lox.stop.lox_R270H) mice develop basal-like breast cancer that is enriched in TICs, can readily form lung metastasis, and provides a preclinical model to study both basal-like and claudin-low TNBC in immune-competent mice.
Topics: Animals; Cadherins; Claudins; Epithelium; Female; Gene Expression Regulation, Neoplastic; Humans; Mammary Glands, Animal; Mammary Glands, Human; Mammary Neoplasms, Animal; Mice; Neoplasms, Basal Cell; Neoplastic Stem Cells; PTEN Phosphohydrolase; Pregnancy; Sequence Deletion; Triple Negative Breast Neoplasms; Tumor Suppressor Protein p53
PubMed: 26781438
DOI: 10.1186/s13058-015-0668-y -
Plant Physiology and Biochemistry : PPB Mar 2016As part of an evolution-function analysis, two nucleobase cation symporter 1 (NCS1) from the moss Physcomitrella patens (PpNCS1A and PpNCS1B) are examined--the first...
As part of an evolution-function analysis, two nucleobase cation symporter 1 (NCS1) from the moss Physcomitrella patens (PpNCS1A and PpNCS1B) are examined--the first such analysis of nucleobase transporters from early land plants. The solute specificity profiles for the moss NCS1 were determined through heterologous expression, growth and radiolabeled uptake experiments in NCS1-deficient Saccharomyces cerevisiae. Both PpNCS1A and 1B, share the same profiles as high affinity transporters of adenine and transport uracil, guanine, 8-azaguanine, 8-azaadenine, cytosine, 5-fluorocytosine, hypoxanthine, and xanthine. Despite sharing the same solute specificity profile, PpNCS1A and PpNCS1B move nucleobase compounds with different efficiencies. The broad nucleobase transport profile of PpNCS1A and 1B differs from the recently-characterized Viridiplantae NCS1 in breadth, revealing a flexibility in solute interactions with NCS1 across plant evolution.
Topics: Bryopsida; Genetic Complementation Test; Nucleobase Transport Proteins; Plant Proteins; Saccharomyces cerevisiae
PubMed: 26773540
DOI: 10.1016/j.plaphy.2015.12.014 -
Molecules (Basel, Switzerland) Dec 2015Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst,...
Enzymatic ribosylation of fluorescent 8-azapurine derivatives, like 8-azaguanine and 2,6-diamino-8-azapurine, with purine-nucleoside phosphorylase (PNP) as a catalyst, leads to N9, N8, and N7-ribosides. The final proportion of the products may be modulated by point mutations in the enzyme active site. As an example, ribosylation of the latter substrate by wild-type calf PNP gives N7- and N8-ribosides, while the N243D mutant directs the ribosyl substitution at N9- and N7-positions. The same mutant allows synthesis of the fluorescent N7-β-d-ribosyl-8-azaguanine. The mutated form of the E. coli PNP, D204N, can be utilized to obtain non-typical ribosides of 8-azaadenine and 2,6-diamino-8-azapurine as well. The N7- and N8-ribosides of the 8-azapurines can be analytically useful, as illustrated by N7-β-d-ribosyl-2,6-diamino-8-azapurine, which is a good fluorogenic substrate for mammalian forms of PNP, including human blood PNP, while the N8-riboside is selective to the E. coli enzyme.
Topics: Azaguanine; Catalysis; Catalytic Domain; Fluorescent Dyes; Humans; Molecular Structure; Point Mutation; Purine-Nucleoside Phosphorylase
PubMed: 26729076
DOI: 10.3390/molecules21010044