Association of tenofovir diphosphate and lamivudine triphosphate concentrations with HIV and hepatitis B virus viral suppression.

OBJECTIVE: Concentrations of tenofovir diphosphate (TFV-DP) and lamivudine triphosphate (3TC-TP) in cells are correlates of medication adherence and antiviral activity. However, studies have yet to characterize the simultaneous relationship between TFV-DP and 3TC-TP concentrations with HIV and hepatitis B virus (HBV) suppression. METHODS: Individuals with HIV/HBV coinfection on tenofovir disoproxil fumarate (TDF)-containing antiretroviral therapy (ART) were enrolled. Peripheral blood mononuclear cells (PBMCs) and dried blood spots (DBS) samples were collected and steady-state TFV-DP and 3TC-TP concentrations quantified using validated methods. The relationship between patient factors, TFV-DP, and 3TC-TP concentrations in PBMCs and DBS with HBV and HIV viral suppression were examined. RESULTS: Of 138 participants on TDF-containing ART for a median duration (range) of 6 (0.75-15) years, the median age was 43 years and 64% were women. Overall, 128 (92.8%) and 129 (93.5%) had suppressed HIV and HBV viral loads, respectively. Of the 128 participants with suppressed HIV, 122 (95.3%) had suppressed HBV. Self-reported ART adherence, recent change to dolutegravir-based ART, TFV-DP, and 3TC-TP concentrations in PBMCs and DBS were associated with HIV RNA suppression, while HBe antigen positivity, HIV suppression, and TFV-DP concentrations in DBS were associated with HBV DNA suppression (including six persons with HBV nonsuppression and HIV suppression). CONCLUSION: Long-term TDF/3TC-conatining ART was highly efficacious in individuals with HIV/HBV coinfection. Higher TFV-DP concentrations were predictive of suppression for both viruses. Persistent HBV viremia on TDF/3TC-containg ART requires additional research, but may represent poor adherence and the need for adherence interventions or novel antivirals.

Distinct Antiretroviral Mechanisms Elicited by a Viral Mutagen.

5-aza-cytidine (5-aza-C) has been shown to be a potent human immunodeficiency virus type 1 (HIV-1) mutagen that induces G-to-C hypermutagenesis by incorporation of the reduced form (i.e., 5-aza-dC, 5-aza-dCTP). Evidence to date suggests that this lethal mutagenesis is the primary antiretroviral mechanism for 5-aza-C. To investigate the breadth of application of 5-aza-C as an antiretroviral mutagen, we have conducted a comparative, parallel analysis of the antiviral mechanism of 5-aza-C between HIV-1 and gammaretroviruses - i.e., murine leukemia virus (MuLV) and feline leukemia virus (FeLV). Intriguingly, in contrast to the hallmark G-to-C hypermutagenesis observed with HIV-1, MuLV and FeLV did not reveal the presence of a significant increase in mutational burden, particularly that of G-to-C transversion mutations. The effect of 5-aza-dCTP on DNA synthesis revealed that while HIV-1 RT was not inhibited by 5-aza-dCTP even at 100 µM, 5-aza-dCTP was incorporated and significantly inhibited MuLV RT, generating pause sites and reducing the fully extended product. 5-aza-dCTP was found to be incorporated into DNA by MuLV RT or HIV-1 RT, but only acted as a non-obligate chain terminator for MuLV RT. This biochemical data provides an independent line of experimental evidence in support of the conclusion that HIV-1 and MuLV have distinct primary mechanisms of antiretroviral action with 5-aza-C. Taken together, our data provides striking evidence that an antiretroviral mutagen can have strong potency via distinct mechanisms of action among closely related viruses, unlinking antiviral activity from antiviral mechanism of action.

Resistance to excision determines efficiency of hepatitis C virus RNA-dependent RNA polymerase inhibition by nucleotide analogs.

NS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the hepatitis C virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs mericitabine and sofosbuvir, which get metabolized to 2'-fluoro-2'C-methylcytidine-5'-triphosphate and 2'-fluoro-2'C-methyluridine-5'-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, it has been shown that NS5B can efficiently remove chain terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study, we use transient-state kinetics to understand the efficiency of inhibition for five nucleoside analogs. We show that CTP analogs are readily incorporated into a growing primer by NS5B but are also efficiently excised. In contrast, although UMP analogs are more slowly incorporated, the excision of UMP is slow and inefficient, and modifications to the 2'-carbon of the UTP ribose ring further decreased rates of excision to an undetectable level. Taken together, these data suggest that the clinical effectiveness of sofosbuvir is largely a function of being intractable to nucleotide-mediated excision compared with similar nucleoside analogs.

Depot Medroxyprogesterone Acetate and the Vaginal Microbiome as Modifiers of Tenofovir Diphosphate and Lamivudine Triphosphate Concentrations in the Female Genital Tract of Ugandan Women: Implications for Tenofovir Disoproxil Fumarate/Lamivudine in Preexposure Prophylaxis.

BACKGROUND: Effective concentrations of antiretrovirals in the female genital tract (FGT) are critical for suppression of viral shedding or effective preexposure prophylaxis. The disposition of tenofovir diphosphate (TFV-DP) and emtricitabine triphosphate (FTC-TP) in the FGT have been previously described. Despite widespread use, however, lamivudine triphosphate (3TC-TP) exposure in the FGT is unknown. Depot medroxyprogesterone acetate (DMPA) and vaginal dysbiosis have been implicated in increased risk of human immunodeficiency virus (HIV) acquisition, but whether they alter TFV-DP or 3TC-TP exposure, and therefore compromise prevention efficacy, is unknown. METHODS: Fifty premenopausal women living with HIV in Kampala, Uganda, and receiving daily tenofovir disoproxil fumarate/lamivudine were recruited. Ectocervical biopsies were obtained for quantification of TFV-DP and 3TC-TP using liquid chromatography-mass spectrometry. 16S ribosomal RNA gene sequencing was performed on DNA extracted from vaginal swabs. Wilcoxon rank-sum was used to test for differences between contraceptive groups. RESULTS: 3TC-TP concentrations were on average 17-fold greater than TFV-DP concentrations in cervical tissues. TFV-DP concentrations in cervical biopsies were 76% greater in DMPA users compared with women using nonhormonal contraception (n = 23 per group). Abundance of Lactobacillus in vaginal swabs was correlated with 3TC-TP concentrations in cervical tissues. CONCLUSIONS: We found that TFV-DP concentrations were significantly greater in DMPA users compared with women using nonhormonal contraception, suggesting that prevention efficacy is unlikely to be compromised by DMPA use. Similar to reports of FTC-TP, 3TC-TP exposure was significantly greater than TFV-DP in cervical tissue and was correlated with abundance of Lactobacillus. These data support lamivudine as an option for preexposure prophylaxis. CLINICAL TRIALS REGISTRATION: NCT03377608.

Enzymatic synthesis of base-modified RNA by T7 RNA polymerase. A systematic study and comparison of 5-substituted pyrimidine and 7-substituted 7-deazapurine nucleoside triphosphates as substrates.

We synthesized a small library of eighteen 5-substituted pyrimidine or 7-substituted 7-deazapurine nucleoside triphosphates bearing methyl, ethynyl, phenyl, benzofuryl or dibenzofuryl groups through cross-coupling reactions of nucleosides followed by triphosphorylation or through direct cross-coupling reactions of halogenated nucleoside triphosphates. We systematically studied the influence of the modification on the efficiency of T7 RNA polymerase catalyzed synthesis of modified RNA and found that modified ATP, UTP and CTP analogues bearing smaller modifications were good substrates and building blocks for the RNA synthesis even in difficult sequences incorporating multiple modified nucleotides. Bulky dibenzofuryl derivatives of ATP and GTP were not substrates for the RNA polymerase. In the case of modified GTP analogues, a modified procedure using a special promoter and GMP as initiator needed to be used to obtain efficient RNA synthesis. The T7 RNA polymerase synthesis of modified RNA can be very efficiently used for synthesis of modified RNA but the method has constraints in the sequence of the first three nucleotides of the transcript, which must contain a non-modified G in the +1 position.

Biophysical Analysis of Bacterial CTP Synthase Filaments Formed in the Presence of the Chemotherapeutic Metabolite Gemcitabine-5'-triphosphate.

While enzyme activity is often regulated by a combination of substrate/effector availability and quaternary structure, many cytosolic enzymes may be further regulated through oligomerization into filaments. Cytidine-5'-triphosphate (CTP) synthase (CTPS) forms such filaments-a process that is promoted by the product CTP. The CTP analog and active chemotherapeutic metabolite gemcitabine-5'-triphosphate (dF-dCTP) is a potent inhibitor of CTPS; however, its effect on the enzyme's ability to form filaments is unknown. Alongside electron microscopy studies, dynamic light scattering showed that dF-dCTP induces Escherichia coli CTPS (EcCTPS) to form filaments in solution with lengths ≥30 nm in the presence of CTP or dF-dCTP. The substrate UTP blocks formation of filaments and effects their disassembly. EcCTPS variants were constructed to investigate the role of CTP-binding determinants in CTP- and dF-dCTP-dependent filament formation. Substitution of Glu 149 (i.e., E149D), which interacts with the ribose of CTP, caused reduced affinity for both CTP and dF-dCTP, and obviated filament formation. Phe 227 appears to interact with CTP through an edge-on interaction with the cytosine ring, yet the F227A and F227L variants bound CTP and dF-dCTP. F227A EcCTPS did not form filaments, while F227L EcCTPS formed shorter filaments in the presence of CTP or dF-dCTP. Hence, Phe 227 plays a role in filament formation, although replacement by a bulky hydrophobic amino acid is sufficient for limited filament formation. That dF-dCTP can induce filament formation highlights the fact that nucleotide analogs employed as chemotherapeutic agents may affect the filamentous states of enzymes and potentially alter their regulation in vivo.

Intracellular pharmacokinetics of gemcitabine, its deaminated metabolite 2',2'-difluorodeoxyuridine and their nucleotides.

AIMS: Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) is a prodrug that has to be phosphorylated within the tumour cell to become active. Intracellularly formed gemcitabine diphosphate (dFdCDP) and triphosphate (dFdCTP) are considered responsible for the antineoplastic effects of gemcitabine. However, a major part of gemcitabine is converted into 2',2'-difluoro-2'-deoxyuridine (dFdU) by deamination. In the cell, dFdU can also be phosphorylated to its monophosphate (dFdUMP), diphosphate (dFdUDP) and triphosphate (dFdUTP). In vitro data suggest that these dFdU nucleotides might also contribute to the antitumour effects, although little is known about their intracellular pharmacokinetics (PK). Therefore, the objective of the present study was to gain insight into the intracellular PK of all dFdC and dFdU nucleotides formed during gemcitabine treatment. METHODS: Peripheral blood mononuclear cell (PBMC) samples were collected from 38 patients receiving gemcitabine, at multiple time points after infusion. Gemcitabine, dFdU and their nucleotides were quantified in PBMCs. In addition, gemcitabine and dFdU plasma concentrations were monitored. The individual PK parameters in plasma and in PBMCs were determined. RESULTS: Both in plasma and in PBMCs, dFdU was present in higher concentrations than gemcitabine [mean intracellular area under the concentration-time curve from time zero to 24 h (AUC0-24 h ) 1650 vs. 95 µM*h]. However, the dFdUMP, dFdUDP and dFdUTP concentrations in PBMCs were much lower than the dFdCDP and dFdCTP concentrations. The mean AUC0-24 h for dFdUTP was 312 µM*h vs. 2640 µM*h for dFdCTP. CONCLUSIONS: The study provides the first complete picture of all nucleotides that are formed intracellularly during gemcitabine treatment. Low intracellular dFdU nucleotide concentrations were found, which calls into question the relevance of these nucleotides for the cytotoxic effects of gemcitabine.

Fibroblast drug scavenging increases intratumoural gemcitabine accumulation in murine pancreas cancer.

OBJECTIVE: Desmoplasia and hypovascularity are thought to impede drug delivery in pancreatic ductal adenocarcinoma (PDAC). However, stromal depletion approaches have failed to show clinical responses in patients. Here, we aimed to revisit the role of the tumour microenvironment as a physical barrier for gemcitabine delivery. DESIGN: Gemcitabine metabolites were analysed in LSL-KrasG12D/+ ; LSL-Trp53R172H/+ ; Pdx-1-Cre (KPC) murine tumours and matched liver metastases, primary tumour cell lines, cancer-associated fibroblasts (CAFs) and pancreatic stellate cells (PSCs) by liquid chromatography-mass spectrometry/mass spectrometry. Functional and preclinical experiments, as well as expression analysis of stromal markers and gemcitabine metabolism pathways were performed in murine and human specimen to investigate the preclinical implications and the mechanism of gemcitabine accumulation. RESULTS: Gemcitabine accumulation was significantly enhanced in fibroblast-rich tumours compared with liver metastases and normal liver. In vitro, significantly increased concentrations of activated 2',2'-difluorodeoxycytidine-5'-triphosphate (dFdCTP) and greatly reduced amounts of the inactive gemcitabine metabolite 2',2'-difluorodeoxyuridine were detected in PSCs and CAFs. Mechanistically, key metabolic enzymes involved in gemcitabine inactivation such as hydrolytic cytosolic 5'-nucleotidases (Nt5c1A, Nt5c3) were expressed at low levels in CAFs in vitro and in vivo, and recombinant expression of Nt5c1A resulted in decreased intracellular dFdCTP concentrations in vitro. Moreover, gemcitabine treatment in KPC mice reduced the number of liver metastases by >50%. CONCLUSIONS: Our findings suggest that fibroblast drug scavenging may contribute to the clinical failure of gemcitabine in desmoplastic PDAC. Metabolic targeting of CAFs may thus be a promising strategy to enhance the antiproliferative effects of gemcitabine.

Pharmacological factors affecting accumulation of gemcitabine's active metabolite, gemcitabine triphosphate.

Gemcitabine is an anticancer agent acting against several solid tumors. It requires nucleoside transporters for cellular uptake and deoxycytidine kinase for activation into active gemcitabine-triphosphate, which is incorporated into the DNA and RNA. However, it can also be deaminated in the plasma. The intracellular level of gemcitabine-triphosphate is affected by scheduling or by combination with other chemotherapeutic regimens. Moreover, higher concentrations of gemcitabine-triphosphate may affect the toxicity, and possibly the clinical efficacy. As a consequence, different nucleoside analogs have been synthetized with the aim to increase the concentration of gemcitabine-triphosphate into cells. In this review, we summarize currently published evidence on pharmacological factors affecting the intracellular level of gemcitabine-triphosphate to guide future trials on the use of new nucleoside analogs.

Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol.

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+ and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).

Global DNA methylation profiling uncovers distinct methylation patterns of protocadherin alpha4 in metastatic and non-metastatic rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS), which can be classified as embryonal RMS (ERMS) and alveolar RMS (ARMS), represents the most frequent soft tissue sarcoma in the pediatric population; the latter shows greater aggressiveness and metastatic potential with respect to the former. Epigenetic alterations in cancer include DNA methylation changes and histone modifications that influence overall gene expression patterns. Different tumor subtypes are characterized by distinct methylation signatures that could facilitate early disease detection and greater prognostic accuracy. METHODS: A genome-wide approach was used to examine methylation patterns associated with different prognoses, and DNA methylome analysis was carried out using the Agilent Human DNA Methylation platform. The results were validated using bisulfite sequencing and 5-aza-2'deoxycytidine treatment in RMS cell lines. Some in vitro functional studies were also performed to explore the involvement of a target gene in RMS tumor cells. RESULTS: In accordance with the Intergroup Rhabdomyosarcoma Study (IRS) grouping, study results showed that distinct methylation patterns distinguish RMS subgroups and that a cluster of protocadherin genes are hypermethylated in metastatic RMS. Among these, PCDHA4, whose expression was decreased by DNA methylation, emerged as a down-regulated gene in the metastatic samples. As PCDHA4-silenced cells have a significantly higher cell proliferation rate paralleled by higher cell invasiveness, PCDHA4 seems to behave as a tumor suppressor in metastatic RMS. CONCLUSION: Study results demonstrated that DNA methylation patterns distinguish between metastatic and non-metastatic RMS and suggest that epigenetic regulation of specific genes could represent a novel therapeutic target that could enhance the efficiency of RMS treatments.

Exploring the Potent Inhibition of CTP Synthase by Gemcitabine-5'-Triphosphate.

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [Ki =(3.0±0.1) µm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.

Biochemical Effect of Resistance Mutations against Synergistic Inhibitors of RSV RNA Polymerase.

ALS-8112 is the parent molecule of ALS-8176, a first-in-class nucleoside analog prodrug effective in the clinic against respiratory syncytial virus (RSV) infection. The antiviral activity of ALS-8112 is mediated by its 5'-triphosphate metabolite (ALS-8112-TP, or 2'F-4'ClCH2-cytidine triphosphate) inhibiting the RNA polymerase activity of the RSV L-P protein complex through RNA chain termination. Four amino acid mutations in the RNA-dependent RNA polymerase (RdRp) domain of L (QUAD: M628L, A789V, L795I, and I796V) confer in vitro resistance to ALS-8112-TP by increasing its discrimination relative to natural CTP. In this study, we show that the QUAD mutations specifically recognize the ClCH2 group of ALS-8112-TP. Among the four mutations, A789V conferred the greatest resistance phenotype, which was consistent with its putative position in the active site of the RdRp domain. AZ-27, a non-nucleoside inhibitor of RSV, also inhibited the RdRp activity, with decreased inhibition potency in the presence of the Y1631H mutation. The QUAD mutations had no effect on the antiviral activity of AZ-27, and the Y1631H mutation did not significantly increase the discrimination of ALS-8112-TP. Combining ALS-8112 with AZ-27 in vitro resulted in significant synergistic inhibition of RSV replication. Overall, this is the first mechanistic study showing a lack of cross-resistance between mutations selected by different classes of RSV polymerase inhibitors acting in synergy, opening the door to future potential combination therapies targeting different regions of the L protein.

5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses.

Pharmacokinetic Modeling of Lamivudine and Zidovudine Triphosphates Predicts Differential Pharmacokinetics in Seminal Mononuclear Cells and Peripheral Blood Mononuclear Cells.

The male genital tract is a potential site of viral persistence. Therefore, adequate concentrations of antiretrovirals are required to eliminate HIV replication in the genital tract. Despite higher zidovudine (ZDV) and lamivudine (3TC) concentrations in seminal plasma (SP) than in blood plasma (BP) (SP/BP drug concentration ratios of 2.3 and 6.7, respectively), we have previously reported lower relative intracellular concentrations of their active metabolites, zidovudine triphosphate (ZDV-TP) and lamivudine triphosphate (3TC-TP), in seminal mononuclear cells (SMCs) than in peripheral blood mononuclear cells (PBMCs) (SMC/PBMC drug concentration ratios of 0.36 and 1.0, respectively). Here, we use population pharmacokinetic (PK) modeling-based methods to simultaneously describe parent and intracellular metabolite PK in blood, semen, and PBMCs and SMCs. From this model, the time to steady state in each matrix was estimated, and the results indicate that the PK of 3TC-TP and ZDV-TP in PBMCs are different from the PK of the two in SMCs and different for the two triphosphates. We found that steady-state conditions in PBMCs were achieved within 2 days for ZDV-TP and 3 days for 3TC-TP. However, steady-state conditions in SMCs were achieved within 2 days for ZDV-TP and 2 weeks for 3TC-TP. Despite this, or perhaps because of it, ZDV-TP in SMCs does not achieve the surrogate 50% inhibitory concentration (IC50) (as established for PBMCs, assuming SMC IC50 = PBMC IC50) at the standard 300-mg twice-daily dosing. Mechanistic studies are needed to understand these differences and to explore intracellular metabolite behavior in SMCs for other nucleoside analogues used in HIV prevention, treatment, and cure.

Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH2 groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.

Deregulation of DNMT1, DNMT3B and miR-29s in Burkitt lymphoma suggests novel contribution for disease pathogenesis.

Methylation of CpG islands in promoter gene regions is frequently observed in lymphomas. DNA methylation is established by DNA methyltransferases (DNMTs). DNMT1 maintains methylation patterns, while DNMT3A and DNMT3B are critical for de novo DNA methylation. Little is known about the expression of DNMTs in lymphomas. DNMT3A and 3B genes can be regulated post-transcriptionally by miR-29 family. Here, we demonstrated for the first time the overexpression of DNMT1 and DNMT3B in Burkitt lymphoma (BL) tumor samples (69% and 86%, respectively). Specifically, the treatment of two BL cell lines with the DNMT inhibitor 5-aza-dC decreased DNMT1 and DNMT3B protein levels and inhibited cell growth. Additionally, miR-29a, miR-29b and miR-29c levels were significantly decreased in the BL tumor samples. Besides, the ectopic expression of miR-29a, miR-29b and miR-29c reduced the DNMT3B expression and miR-29a and miR-29b lead to increase of p16(INK4a) mRNA expression. Altogether, our data suggest that deregulation of DNMT1, DNMT3B and miR29 may be involved in BL pathogenesis.

RASSF5A, a candidate tumor suppressor, is epigenetically inactivated in esophageal squamous cell carcinoma.

As a result of alternative splicing and differential promoter usage, RASSF5 exists in at least three isoforms (RASSF5A-RASSF5C), which may play different roles in tumorigenesis. The present study was to detect the role of RASSF5A, B and C in esophageal squamous cell carcinoma (ESCC) and clarify the critical CpG sites of RASSF5A, in order to clarify more information on the role of RASSF5 with regard to the pathogenesis of ESCC. Frequent silencing of RASSF5A but not RASSF5B and RASSF5C were found in esophageal cancer cell lines and the silencing of RASSF5A may be reversed by 5-Aza-dC or TSA treatment. The aberrant CpG island 1 methylation of RASSF5A induces silencing of its expression in TE13 cell line. Decreased mRNA and protein expression of RASSF5A was observed in ESCC tumor tissues and was associated with RASSF5A CpG island 1 methylation status. Unlike RASSF5A, expression variation of RASSF5B and RASSF5C was not found in ESCC tissues. Aberrant promoter methylation of RASSF5C was also not found in ESCC. RASSF5A methylation and protein expression were independently associated with ESCC patients' survival. These data indicated that the inactivation of RASSF5A through CpG island 1 methylation may play an important role in ESCC carcinogenesis, RASSF5A may be a functional tumor suppressor and may serve as a prognostic biomarker for ESCC.

Site-specific fluorescence labelling of RNA using bio-orthogonal reaction of trans-cyclooctene and tetrazine.

This communication describes a general approach for site-specific fluorescence labelling of RNA using a cytidine triphosphate (CTP) analogue derivatized with a trans-cyclooctene group. The analogue was efficiently incorporated into a model RNA strand using in vitro transcription. Bio-orthogonal reaction with fluorescein-labelled tetrazine was utilized to fluorescently tag the synthetic RNA strand.

The epigenetically regulated effects of Wnt antagonists on the expression of genes in the apoptosis pathway in human bladder cancer cell line (T24).

The epigenetic suppression of Wnt antagonists (sFRPs, DKKs, and WIF-1) causes the activation of both ß-catenin and target genes, which play an important role in cell proliferation, metastasis, and angiogenesis. This study is aimed to investigate, on transcriptional and protein levels, the synergic effects of unaccompanied and/or combined use of 5-aza-2'-deoxycytidine (DAC, 5-aza-dC), trichostatin A (TSA), and gemcitabine+cisplatin chemotherapeutic agents on the apoptotic pathway of human bladder cancer cell line T24. The anti-tumor effects of gemcitabine (0-500 nM), cisplatin (0-10 µM), DAC (10 µM), and TSA (300 nM) alone and/or together on T24 cells were determined by WST-1. ELISA method was used to analyze the effects of unaccompanied and combined use of gemcitabine+cisplatin, DAC, and TSA on cell proliferation and determine the cytotoxic and apoptotic dosages at the level of H3 histone acetylation. Methylation-specific PCR was used to evaluate methylation profiles of Wnt antagonist gene (WIF-1). In the case of unaccompanied and/or combined use of specified drugs, the variations in the expression levels of CTNNB1, GSK3ß, c-MYC, CCND1, CASP-3, CASP-8, CASP-9, BCL2L1, and WIF-1 genes were determined by quantitative real-time PCR. Our results indicate that through inhibition of DNA methylation, expression of ß-catenin and Wnt antagonist re-activation and expressions of canonical Wnt/ß-catenin pathway target genes, c-myc and cyclin D1 (CCND1), have decreased. In addition, DAC, TSA, and gemcitabine+cisplatin combination caused an increase in GSK3ß mRNA levels, which in turn significantly decreased CCND1 mRNA levels. Moreover, BCL2L1, an anti-apoptotic gene, was downregulated significantly. Meanwhile, both CASP-3 mRNA and active caspase-3 protein levels increased with respect to control (p<0.01). The results revealed that use of quadruplicate gemcitabine+cisplatin+DAC+TSA combination led to a reduced inhibition of canonical Wnt/ß-catenin pathway and reduced cell proliferation. Our findings may offer a new approach to consider in the treatment of bladder cancer.