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Respiratory syncytial virus (RSV) is a seasonal virus that infects the lungs and airways of 64 million children and adults every year. It is a major cause of acute lower respiratory tract infection and is associated with significant morbidity and mortality. Despite the large medical and economic burden, treatment options for RSV-associated bronchiolitis and pneumonia are limited and mainly consist of supportive care. This publication covers the medicinal chemistry efforts resulting in the identification of JNJ-53718678, an orally bioavailable RSV inhibitor that was shown to be efficacious in a phase 2a challenge study in healthy adult subjects and that is currently being evaluated in hospitalized infants and adults. Cocrystal structures of several new derivatives helped in rationalizing some of the structure-activity relationship (SAR) trends observed.

Respiratory syncytial virus (RSV) is a negative sense single-stranded RNA virus and one of the main causes of severe lower respiratory tract infections in infants and young children. RSV RNA replication/transcription and capping are ensured by the viral Large (L) protein. The L protein contains a polymerase domain associated with a polyribonucleotidyl transferase domain in its N-terminus, and a methyltransferase (MTase) domain followed by the C-terminal domain (CTD) enriched in basic amino acids at its C-terminus. The MTase-CTD of Mononegavirales forms a clamp to accommodate RNA that is subsequently methylated on the cap structure and depending on the virus, on internal positions. These enzymatic activities are essential for efficient viral mRNA translation into proteins, and to prevent the recognition of uncapped viral RNA by innate immunity sensors. In this work, we demonstrated that the MTase-CTD of RSV, as well as the full-length L protein in complex with phosphoprotein (P), catalyzes the N7- and 2’-O-methylation of the cap structure of a short RNA sequence that corresponds to the 5’ end of viral mRNA. Using different experimental systems, we showed that the RSV MTase-CTD methylates the cap structure with a preference for N7-methylation as first reaction. However, we did not observe cap-independent internal methylation, as recently evidenced for the Ebola virus MTase. We also found that at μM concentrations, sinefungin, a S-adenosylmethionine analogue, inhibits the MTase activity of the RSV L protein and of the MTase-CTD domain. Altogether, these results suggest that the RSV MTase domain specifically recognizes viral RNA decorated by a cap structure and catalyzes its methylation, which is required for translation and innate immune system subversion., Author summary Respiratory syncytial virus (RSV) is responsible of infant bronchiolitis and severe lower respiratory tract infections in infants and young children, and the leading cause of hospitalization in children under one year of age. However, we still lack a vaccine and therapeutics against this important pathogen. The main enzymatic activities involved in RSV propagation are embedded in the Large (L) protein that contains the polymerase domain and also all the activities required for RNA cap structure synthesis and methylation. These post-transcriptional RNA modifications play a key role in virus replication because cap N7-methylation is required for viral RNA translation into proteins, and 2’-O-methylation hides viral RNA from innate immunity detection. Viral methyltransferase (MTase) activities are now considered potential antiviral targets because their inhibition might limit the virus production and strengthen early virus detection by innate immunity sensors. In this work, we compared the enzymatic activities of the MTase expressed as a single domain or in the context of the full-length L protein. We demonstrated that the MTase protein catalyzes the specific methylation of the cap structure at both N7- and 2’-O-positions, and we obtained the proof of concept that a S-adenosylmethionine analogue can inhibit the MTase activity of the L protein.

Respiratory syncytial virus (RSV) is a seasonal virus that infects the lungs and airways of 64 million children and adults every year. It is a major cause of acute lower respiratory tract infection and is associated with significant morbidity and mortality. Despite the large medical and economic burden, treatment options for RSV-associated bronchiolitis and pneumonia are limited and mainly consist of supportive care. This publication covers the medicinal chemistry efforts resulting in the identification of JNJ-53718678, an orally bioavailable RSV inhibitor that was shown to be efficacious in a phase 2a challenge study in healthy adult subjects and that is currently being evaluated in hospitalized infants and adults. Cocrystal structures of several new derivatives helped in rationalizing some of the structure-activity relationship (SAR) trends observed.

Summary Numerous interventions are in clinical development for respiratory syncytial virus (RSV) infection, including small molecules that target viral transcription and replication. These processes are catalyzed by a complex comprising the RNA-dependent RNA polymerase (L) and the tetrameric phosphoprotein (P). RSV P recruits multiple proteins to the polymerase complex and, with the exception of its oligomerization domain, is thought to be intrinsically disordered. Despite their critical roles in RSV transcription and replication, structures of L and P have remained elusive. Here, we describe the 3.2-Å cryo-EM structure of RSV L bound to tetrameric P. The structure reveals a striking tentacular arrangement of P, with each of the four monomers adopting a distinct conformation. The structure also rationalizes inhibitor escape mutants and mutations observed in live-attenuated vaccine candidates. These results provide a framework for determining the molecular underpinnings of RSV replication and transcription and should facilitate the design of effective RSV inhibitors., Graphical Abstract, Highlights • Cryo-EM structure of RSV L bound by tetrameric RSV P solved to 3.2 Å • P tetramer adopts an asymmetric tentacular arrangement when bound to L • L priming loop adopts elongation-compatible state without PRNTase-RdRp separation • Structure rationalizes escape from small-molecule antivirals, Respiratory syncytial virus (RSV) remains a leading cause of bronchiolitis and hospitalization, especially of infants. Gilman et al. present a 3.2-Å cryo-EM structure of the RSV L polymerase in complex with the P phosphoprotein—components of the core viral replication machinery that represent an attractive target for the development of therapeutic agents.

Currently, there is an increasing interest to apply pre-fusion (pre-F) protein of respiratory syncytial virus (RSV) as antigen for the development of a subunit vaccine. A pre-F-containing powder would increase the flexibility regarding the route of administration. For instance, a pre-F-containing powder could be incorporated into a single-injection system releasing a primer, and after a lag time, a booster. The most challenging aspect, obtaining the booster after a lag time, may be achieved by incorporating the powder into a core encapsulated by a nonporous poly(dl-lactic-co-glycolic acid) (PLGA) shell. We intended to develop a stable freeze-dried pre-F-containing powder. Furthermore, we investigated whether incorporation of this powder into the core-shell implant was feasible and whether this system would induce a delayed RSV virus-neutralizing antibody (VNA) response in mice. The developed pre-F-containing powder, consisting of pre-F in a matrix of inulin, HEPES, sodium chloride, and Tween 80, was stable during freeze-drying and storage for at least 28 days at 60 &deg, C. Incorporation of this powder into the core-shell implant was feasible and the core-shell production process did not affect the stability of pre-F. An in vitro release study showed that pre-F was incompletely released from the core-shell implant after a lag time of 4 weeks. The incomplete release may be the result of pre-F instability within the core-shell implant during the lag time and requires further research. Mice subcutaneously immunized with a pre-F-containing core-shell implant showed a delayed RSV VNA response that corresponded with pre-F release from the core-shell implant after a lag time of approximately 4 weeks. Moreover, pre-F-containing core-shell implants were able to boost RSV VNA titers of primed mice after a lag time of 4 weeks. These findings could contribute to the development of a single-injection pre-F-based vaccine containing a primer and a booster.

A small molecule that targets influenza Many of us rely on seasonal vaccines for protection against influenza and are only too aware of their limited breadth. Broadly neutralizing antibodies (bnAbs) that target the conserved hemagglutinin (HA) stem of the influenza virus provide hope for the development of universal vaccines and are being evaluated in clinical trials. Van Dongen et al. selected and optimized a small-molecule lead compound that recapitulates key interactions of the bnAb with HA. Like the bnAb, the compound inhibited viral fusion in the endosomes of target cells. The compound protected mice from influenza after oral administration and neutralized virus infection in a 3D cell culture of human bronchial epithelial cells. Science , this issue p. eaar6221

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections in infants and the elderly, and yet there remains no effective treatment or vaccine. The surface of the virion is decorated with the fusion glycoprotein (RSV F) and the attachment glycoprotein (RSV G), which binds to CX3CR1 on human airway epithelial cells to mediate viral attachment and subsequent infection. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region of G have been shown to neutralize both subtypes of RSV and to protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated two human antibodies directed against the central conserved region of RSV G and demonstrated that they neutralize RSV infection of human bronchial epithelial cell cultures in the absence of complement. Moreover, the antibodies protected cotton rats from severe RSV disease. Both antibodies bound with high affinity to a secreted form of RSV G as well as to a peptide corresponding to the unglycosylated central conserved region. High-resolution crystal structures of each antibody in complex with the G peptide revealed two distinct conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with the structure of fractalkine (CX3CL1) alone or in complex with a viral homolog of CX3CR1 (US28) suggests that RSV G would bind to CX3CR1 in a mode that is distinct from that of fractalkine. Collectively, these results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide the development of new vaccines and antibody-based therapies for RSV., Author summary Respiratory syncytial virus (RSV) is a common cause of bronchiolitis and pneumonia, and is a leading cause of infant deaths globally due to infectious disease. Despite decades of research, there is still no vaccine or widespread treatment for RSV. In this study, we isolated two antibodies that bind to the central conserved region of the viral attachment glycoprotein, RSV G. The antibodies effectively neutralize both subtypes of RSV and protect RSV-challenged animals from severe disease. We also determined high-resolution crystal structures of each antibody in complex with the central conserved region of RSV G to gain a better understanding of how these antibodies bind to RSV G and how they neutralize the virus. Because RSV G is a small folded domain bounded by unstructured mucin-like domains, structural elucidation of the central conserved region provides atomic-level information for the complete folded portion of RSV G. The results presented here will help develop effective antibodies for passive prophylaxis as well as guide efforts to design vaccines that elicit broadly neutralizing antibodies against RSV G.

Background Respiratory syncytial virus (RSV) disease has no effective treatment. JNJ-53718678 is a fusion inhibitor with selective activity against RSV. Methods After confirmation of RSV infection or 5 days after inoculation with RSV, participants (n = 69) were randomized to JNJ-53718678 75 mg (n = 15), 200 mg (n = 17), 500 mg (n = 18), or placebo (n = 17) orally once daily for 7 days. Antiviral effects were evaluated by assessing RSV RNA viral load (VL) area under the curve (AUC) from baseline (before the first dose) until discharge, time-to-peak VL, duration of viral shedding, clinical symptoms, and quantity of nasal secretions. Results Mean VL AUC was lower for individuals treated with different doses of JNJ-53718678 versus placebo (203.8-253.8 vs 432.8 log10 PFUe.hour/mL). Also, mean peak VL, time to peak VL, duration of viral shedding, mean overall symptom score, and nasal secretion weight were lower in each JNJ-53718678-treated group versus placebo. No clear exposure-response relationship was observed. Three participants discontinued due to treatment-emergent adverse events of grade 2 and 1 electrocardiogram change (JNJ-53718678 75 mg and 200 mg, respectively) and grade 2 urticaria (placebo). Conclusions JNJ-53718678 at all 3 doses substantially reduced VL and clinical disease severity, thus establishing clinical proof of concept and the compound's potential as a novel RSV treatment. Clinical trials registration ClinicalTrials.gov: NCT02387606; EudraCT number: 2014-005041-41.

Influenza and respiratory syncytial virus (RSV) cause acute infections of the respiratory tract. Since the viruses both cause illnesses with similar symptoms, researchers often try to apply knowledge gleaned from study of one virus to the other virus. This can be an effective and efficient strategy for understanding viral dynamics or developing treatment strategies, but only if we have a full understanding of the similarities and differences between the two viruses. This study used mathematical modeling to quantitatively compare the viral kinetics of in vitro RSV and influenza virus infections. Specifically, we determined the viral kinetics parameters for RSV A2 and three strains of influenza virus, A/WSN/33 (H1N1), A/Puerto Rico/8/1934 (H1N1), and pandemic H1N1 influenza virus. We found that RSV viral titer increases at a slower rate and reaches its peak value later than influenza virus. Our analysis indicated that the slower increase of RSV viral titer is caused by slower spreading of the virus from one cell to another. These results provide estimates of dynamical differences between influenza virus and RSV and help provide insight into the virus-host interactions that cause observed differences in the time courses of the two illnesses in patients.

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure–activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection., Respiratory syncytial virus causes lung infections in children, immunocompromised adults, and in the elderly. Here the authors show that a chemical inhibitor to a viral fusion protein is effective in reducing viral titre and ameliorating infection in rodents and neonatal lambs.

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.

Respiratory syncytial virus (RSV) causes bronchiolitis in young children and common colds in adults. There is no licensed vaccine, and prophylactic treatment with palivizumab is very expensive and limited to high-risk infants. Ribavirin is used as an antiviral treatment in infants and immunosuppressed patients, and its use is limited due to side-effects, toxicity to the recipient and staff, and evidence of marginal clinical efficacy. Therefore, we studied the in vivo kinetics, and the antiviral and protective properties of a novel candidate for RSV disease treatment. The drug is a small molecule (TMC353121) discovered by screening for fusion inhibitory properties against RSV in a cellular infection model. The pharmacokinetics of TMC353121 was studied in BALB/c mice and antiviral effects determined by testing viral loads in lung tissue by quantitative RT-PCR and plaque assay after intranasal RSV infection. At doses of 0.25-10 mg · kg(-1), TMC353121 significantly reduced viral load, bronchoalveolar lavage cell accumulation and the severity of lung histopathological change after infection. Treatment remained effective if started within 48 h of infection, but was ineffective thereafter. Therefore, TMC353121 is a novel potent antiviral drug, in vivo reducing RSV replication and inhibiting consequential lung inflammation, with a great potential for further clinical development.

Cell-based in vitro models are invaluable tools in elucidating the pharmacokinetic profile of a drug candidate during its drug discovery and development process. As biotransformation is one of the key determinants of a drug's disposition in the body, many in vitro models to study drug metabolism have been established, and others are still being developed and validated. This review is aimed at providing the reader with a concise overview of the characteristics and optimal application of established and emerging in vitro cell-based models to study human drug metabolism and induction of drug metabolising enzymes in the liver. The strengths and weaknesses of liver-derived models, such as primary hepatocytes, either freshly isolated or cryopreserved, and from adult or fetal donors, precision-cut liver slices, and cell lines, including immortalised cells, reporter cell lines, hepatocarcinoma-derived cell lines and recombinant cell lines, are discussed. Relevant cell culture configuration aspects as well as other models such as stem cell-derived hepatocyte-like cells and humanised animal models are also reviewed. The status of model development, their acceptance by health authorities and recommendations for the most appropriate use of the models are presented.

Cyclic AMP-dependent differentiation of rat C6 glioma cells into an astrocyte type II is characterized by inhibition of cell growth and induction of glial fibrillary acidic protein (GFAP) synthesis. Activation of the P2Y(12) receptor with 2-methylthioadenosine-5'-diphosphate inhibited beta-adrenergic receptor-induced differentiation. The selective P2Y(12) receptor antagonist N(6)-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene ATP abolished the receptor-mediated effect on differentiation. In contrast non-selective antagonists of P2Y receptors did not revert the inhibiting effect of the P2Y(12) receptor on differentiation. Reactive blue 2 (RB2), a potent P2Y(12) receptor antagonist, completely inhibited the synthesis of GFAP, while the P2Y receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid were less efficient. However, although P2Y receptor antagonists inhibited GFAP synthesis to a different extent they were unable to relieve the growth inhibition that accompanied induction of differentiation, whereas stimulation of the P2Y(12) receptor with 2-methylthioadenosine-5'-diphosphate inhibited GFAP expression and restored cell proliferation. Assay of the activity of phosphatidylinositol 3-kinase (PI 3-K), an enzyme required for GFAP expression [J. Neurochem. 76 (2001) 610], showed that RB2 inhibited this enzyme after cellular uptake, while suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid inhibited PI 3-K to a lesser extent. The intracellular concentration of RB2 increased in time and attained the ic(50) for PI 3-K inhibition (4microM) after 40-min incubation with 50microM RB2. In conclusion, cAMP-induced differentiation in C6 cells is inhibited by activation of the P2Y(12) receptor. In addition, synthesis of GFAP is also inhibited by cellular uptake of non-selective nucleotide receptor antagonists that inhibit PI 3-K, a kinase required for the cAMP-dependent induction of differentiation.

Tumor metastasis is responsible for a high degree of mortality in cancer patients. One of the genes involved in tumor metastasis is NM23. At present, eight human isoforms, transcribed from different NM23 genes, have been detected. The gene products have been identified as nucleoside diphosphate kinases (NDPKs), most of which catalyse the transfer of the γ-phosphate of a (deoxy)nucleoside triphosphate to a (deoxy)nucleoside diphosphate. However, the function of NDPK isoforms involved in tumor metastasis cannot be explained on the basis of their phosphotransferase activity alone. At present, several other properties, like transcriptional regulation and protein kinase activity, have been assigned to these proteins. Moreover, it has also been shown that NDPKs interact with several other proteins, and binding partners of NDPKs are identified at an increasing rate. Accumulating evidence indicates that protein–protein interactions modulate the molecular action of NDPKs. In this review we provide a brief overview of how NDPKs are correlated with cancer, and discuss when and how the activities assigned to NDPKs may affect metastasis, with special emphasis on the role of protein–NDPK interactions in this process.

We have previously shown that an ecto-NPPase modulates the ATP- and ADP-mediated P2YAC-receptor activation in rat C6 glioma. In the present study, 2MeSADP and Ap3A induced no detectable PI turnover and were identified as specific agonists of the P2YAC-receptor with EC50 values of 250 ± 37 pm and 1 ± 0.5 µm, respectively. P2YAC-receptor stimulation increased MAP kinase (ERK1/2) activation that returned to the basal level 4 h after stimulation and was correlated with a gradual desensitization of the P2YAC-purinoceptor. The purinoceptor antagonists DIDS and RB2 blocked MAP kinase activation. An IP3-independent Ca2+-influx was observed after P2YAC-receptor activation. Inhibition of this influx by Ca2+-chelation, did not affect MAP kinase activation. Pertussis toxin, toxin B, selective PKC-inhibitors and a specific MEK-inhibitor inhibited the 2MeSADP- and Ap3A-induced MAP kinase activation. In addition, transfection with dominant negative RhoAAsn19 rendered C6 cells insensitive to P2YAC-receptor-mediated MAP kinase activation whereas dominant negative ras was without effect. Immunoprecipitation experiments indicated a significant increase in the phosphorylation of raf-1 after P2YAC-receptor activation. We may conclude that P2YAC-purinoceptor agonists activate MAP kinase through a Gi-RhoA-PKC-raf-MEK-dependent, but ras- and Ca2+-independent cascade.

Extracellularly added P1,P3-di(adenosine-5′) triphosphate (Ap3A), P1,P4-di(adenosine-5′) tetraphosphate (Ap4A), ATP, ADP, AMP and adenosine are growth inhibitory for rat C6 glioma cells. Analysis of nucleotide hydrolysis and the use of nucleotidase inhibitors demonstrated that the latter inhibition is due to hydrolysis of the nucleotides to adenosine. Agonists of the P2YAC−-receptor enhance the growth of C6 cells if their hydrolysis to adenosine is inhibited by pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS). In these conditions, the potency to stimulate cell growth parallels the ranking of the receptor agonists, i.e. 2-methylthioadenosine-5′-diphosphate (2MeSADP)>Ap3A>Ap4A. ATP and ADP are still hydrolysed in the presence of PPADS and have no proliferative effect on C6 cells. The enhanced growth is due to a P2YAC−-receptor-mediated activation of p42/44 mitogen-activated protein kinase (MAPK) as shown by immunoblotting and protein kinase assays for active MAPK and the use of the MAPK/extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059. The UTP-induced enhancement of the growth of C6 cells is due to activation of MAPK by a PPADS sensitive nucleotide receptor. In conclusion, the effect of nucleotides on the growth of C6 cells is determined by ecto-nucleotidases and by activation of nucleotide receptors. Hydrolysis of nucleotides to adenosine induces growth inhibition while inhibition of the hydrolysis of agonists of the P2YAC−-receptor enhances cell growth by activation of MAPK. Keywords: Ap3A, Ap4A, C6 cells, growth inhibition, MAPK, NPPase, nucleotide receptor, proliferation, purinoceptor, P2YAC−-receptor Introduction Nucleotides, and in particular ATP and adenosine, have been reported to affect the proliferation of different cell types. The effects are not always unequivocal, e.g. ATP stimulates the proliferation of primary cultures of astrocytes and rat aortic smooth muscle cells (Abbracchio et al., 1994; Harper et al., 1998), is ineffective on the growth of transformed mouse fibroblasts (Weisman et al., 1988) and has an inhibitory effect on the growth of several human tumour cell lines (Seetulsingh-Goorah & Stewart, 1998; Rapaport et al., 1983). Adenosine has a growth inhibitory effect on most of the examined cell types (Seetulsingh-Goorah & Stewart, 1998; Fishman et al., 1998), although a growth stimulation is also reported (Lelievre et al., 1998). This apparent discrepancy may be explained by the existence of several mechanisms that take place at the cell surface: firstly, extracellular nucleotides are degraded by a cascade of cell surface-bound enzymes, i.e. ecto-ATPase, ecto-apyrase, ecto-nucleotide pyrophosphatase/phosphodiesterase I (NPPase) and ecto-5′-nucleotidase, hydrolysing ATP into ADP, AMP and adenosine (Zimmermann, 1996). Cellular uptake of the latter by adenosine transporters can induce an adenosine-dependent pyrimidine starvation resulting in inhibition of proliferation (Lasso de la Vega et al., 1994). Secondly, the added nucleotides and their breakdown products affect cell proliferation by activation of nucleotide receptors, coupled to phospolipase C (PLC) or adenylate cyclase (AC). In addition, ATP may activate ecto-protein kinases that modulate the activity of autocrine growth factors and growth inhibitors (Vilgrain & Baird, 1991; Friedberg et al., 1995). The inhibitory effect of ATP and other adenosine phosphates on cell proliferation may be explained by one or a combination of these mechanisms. Two main classes of nucleotide receptors, P1 and P2, have been described (Burnstock, 1978). The P1-receptors are mainly responsive to adenosine. The P1-receptor subtypes A1 and A3 are negatively, and A2 is positively coupled to AC. The P2-receptors, activated by ATP/ADP or UTP/UDP, are subdivided in metabotropic P2Y- and ionotropic P2X- receptors. P2Y-receptors are coupled to a PLC-dependent Ca2+ mobilization and activation of protein kinase C (PKC) or to AC (P2Y11, P2Y12), while activation of the P2X-receptors, ligand gated ion-channels, generate a Ca2+-influx (Fredholm et al., 1994; Communi et al., 1997; Boeynaems et al., 2000; Hollopeter et al., 2001). We investigated the effect of adenosine and its mono- and dinucleotides on the proliferation of rat C6 glioma cells. The latter is a bipotential cell line often used as a model system for astrocytes. Several purinergic receptors are reported to be present on C6 cells: an A2b-receptor, a ‘nucleotide receptor' coupled to PLC and activated by all nucleoside triphosphates, a PLC-coupled P2Y6-receptor activated by UDP, and a P2Y-receptor negatively coupled to AC presumed to be the P2Y12-receptor (Ohkubo et al., 2001; Lin & Chuang, 1994; Nicholas et al., 1996; Boyer et al., 1993; Hollopeter et al., 2001). The agonist potency of the latter AC-coupled receptor is more or less the same as that of the P2Y1-receptor and has hitherto been called the P2Y1-like receptor (Albert et al., 1997; Grobben et al., 2000). However, in contrast to the P2Y1-receptor, this receptor is not coupled to PLC and it is not sensitive to the antagonists PPADS (pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid) and 5′-phosphoadenosine 3′-phosphate (Boyer et al., 1994). Although Webb et al. (1996) concluded that the P2Y1-like- and the P2Y1-receptor are identical but coupled to a different second messenger system, Schachter et al. (1997) disproved this finding. To describe more accurately the pharmacological properties of the P2Y1-like receptor we propose the name P2YAC− for future reference to this receptor. The recently cloned P2Y12-receptor has been proposed to be present on C6 glioma cells (Hollopeter et al., 2001). However, ATP, an antagonist for the P2Y12-receptor, is an agonist for the P2YAC−-receptor (Grobben et al., 2000), suggesting that P2Y12 and P2YAC− are different receptors. When the P2YAC−-receptor is cloned and appears to be a unique receptor, and not the P2Y1-receptor coupled to a different second messenger system, the P2Yn nomenclature can be used. In this study we examined the effect of adenosine nucleotides on the growth of C6 cells. Both growth inhibitory and proliferative effects were observed, depending on whether the added adenosine nucleotides were hydrolysed by ecto-nucleotidases or not. Hydrolysis to adenosine proved to be essential for growth inhibition. We previously studied the hydrolysis of extracellular nucleotides, and identified NPPase as the main ATP hydrolysing ecto-enzyme of C6 cells (Grobben et al., 1999). When nucleotide hydrolysis was inhibited by PPADS, an enhanced proliferation was observed for all potent agonists of the P2YAC−-receptor, except for ADP and to a lesser extent ATP, which are hydrolysed even in the presence of PPADS. The enhanced proliferation is due to a P2YAC−-receptor-mediated activation of p42/44 MAPK.

Many cellular processes have been identified in which phosphatidylinositol 3-kinase has a key regulatory function. As an oncogene, it is also involved in the development of cancer. The transformation and progression of normal cells towards an advanced stage tumor and/or towards metastatic lesions involves a complex series of events, including genetic alterations, leading to aberrant cell cycle progression, altered adhesion and motility characteristics, inhibition of apoptosis and induction of angiogenesis. This review highlights the processes involved in the pathogenesis of cancer in which phosphatidylinositol 3-kinase is involved and provides an overview of the possible mechanisms by which the enzyme exerts its oncogenic action.

Processes like cell proliferation, differentiation, and tumor metastasis require a flexible adaptation of cell shape and cell plasticity. A regulator of cell structure and shape is the centrosome and its associated microtubules. Recently, oncogenes like p53, pRB, and the tumor suppressor BRCA1 have been characterized as members of the centrosome. In this communication, we identified rat Nm23-R1/NDPKbeta, a homologue of the human tumor metastasis suppressor Nm23-H1 and a regulator of cell proliferation and differentiation, as a component of the centrosomal complex. We used confocal laser scanning microscopy on different cell types and biochemical analysis of purified centrosomes to demonstrate that Nm23-R1 is located in the centrosome of dividing and nondividing cells. We also showed that the centrosomal enzyme is catalytically active and able to transfer the gamma-phosphate from a nucleoside triphosphate to a nucleoside diphosphate. In addition, Nm23-R1 coimmunoprecipitated with gamma-tubulin, a core centrosomal protein essential for microtubule nucleation. In addition, human Nm23-R1/-H1 was also shown to be present in the centrosome of different human and rat cell types, demonstrating that the presence of Nm23-H1 homologues in the latter organelle is a general event.

1. The effect of ecto-nucleotide pyrophosphatase (ecto-NPPase; EC 3.6.1. 9) on the ATP- and ADP-mediated receptor activation was studied in rat C6 glioma cells. The P2-purinoceptor antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and reactive blue (RB2) are potent inhibitors (IC(50)=12+/-3 microM) of the latter enzyme. 4,4'-diisothiocyanatostilbene-2,2' disulfonic acid (DIDS), 5'-phosphoadenosine 3'-phosphate (PAP) and suramin were less potent inhibitors with an IC(50) of 22+/-4, 36+/-7 and 72+/-11 microM respectively. 2. P1-purinoceptor antagonists CGS 15943, cyclo-pentyl theophylline (CTP) and theophylline did not affect the activity of the ecto-NPPase. 3. ATP- and ADP-mediated P2Y(1)-like receptor activation inhibited the (-)-isoproterenol-induced increase of intracellular cyclic AMP concentration. PPADS, an ineffective P2Y-antagonist in C6, potentiated the ATP and ADP effect approximately 3 fold due to inhibition of nucleotide hydrolysis by the ecto-NPPase. 4. We conclude that ecto-NPPase has a modulator effect on purinoceptor-mediated signalling in C6 glioma cell cultures.

The presence of a nucleotide pyrophosphatase (EC 3.6.1.9) on the plasma membrane of rat C6 glioma has been demonstrated by analysis of the hydrolysis of ATP labeled in the base and in the alpha- and gamma-phosphates. The enzyme degraded ATP into AMP and PPi and, depending on the ATP concentration, accounted for approximately 50-75% of the extracellular degradation of ATP. The association of the enzyme with the plasma membrane was confirmed by ATP hydrolysis in the presence of a varying concentration of pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), a membrane-impermeable inhibitor of the enzyme. PPADS concentration above 20 microM abolished the degradation of ATP into AMP and PPi. The nucleotide pyrophosphatase has an alkaline pH optimum and a Km for ATP of 17 +/- 5 microM. The enzyme has a broad substrate specificity and hydrolyzes nucleoside triphosphates, nucleoside diphosphates, dinucleoside polyphosphates, and nucleoside monophosphate esters but is inhibited by nucleoside monophosphates, adenosine 3',5'-bisphosphate, and PPADS. The substrate specificity characterizes the enzyme as a nucleotide pyrophosphatase/phosphodiesterase I (PD-I). Immunoblotting and autoadenylylation identified the enzyme as a plasma cell differentiation antigen-related protein. Hydrolysis of ATP terminates the autophosphorylation of a nucleoside diphosphate kinase (NDPK/nm23) detected in the conditioned medium of C6 cultures. A function of the pyrophosphatase/PD-I and NDPK in the purinergic and pyrimidinergic signal transduction in C6 is discussed.

Infection with human respiratory syncytial virus (hRSV) causes a wide spectrum of respiratory disease in infants, young children, and elderly persons. No vaccine is available today and hRSV treatment options are limited. As a consequence, the treatment of hRSV infection remains largely supportive and new therapeutic options are needed to treat severe lower respiratory tract hRSV disease. Several animal models have been developed to study hRSV disease and evaluate novel therapies or preventive measures such as vaccines. However, each of these models reproduces different aspects of hRSV disease, and therefore, an appropriate model should be selected on the basis of the scientific question under investigation. In this chapter, we describe how cotton rats and Balb/c mice are used in our laboratory to test the in vivo efficacy of small-molecule inhibitors against hRSV.

Respiratory syncytial virus (RSV) is a common virus that infects people of all ages and causes cold-like symptoms in most cases. However, more serious infections occur in the younger and older extremities of the population causing severe lung infections such as bronchiolitis and pneumonia. The current standard of care is mostly limited to supportive treatment, although prophylaxis by passive immunization with the humanized monoclonal antibody palivizumab and therapeutic intervention with aerosolized ribavirin are available. Unfortunately, administration of palivizumab is restricted to at-risk infants up to the age of two and is associated with high cost, while ribavirin treatment is hindered by questionable efficacy and safety reasons. Consequently, the development of novel specific RSV antiviral drugs is needed to help decrease RSV-related morbidity and mortality. We describe here a fluorescence-based high-throughput screening assay to discover RSV inhibitors which is based on the infection of HeLa cells with a recombinant RSV strain that contains an enhanced green fluorescent protein coding sequence in its viral genome.

Infection with human respiratory syncytial virus (hRSV) causes a wide spectrum of respiratory disease in infants, young children, and elderly persons. No vaccine is available today and hRSV treatment options are limited. As a consequence, the treatment of hRSV infection remains largely supportive and new therapeutic options are needed to treat severe lower respiratory tract hRSV disease. Several animal models have been developed to study hRSV disease and evaluate novel therapies or preventive measures such as vaccines. However, each of these models reproduces different aspects of hRSV disease, and therefore, an appropriate model should be selected on the basis of the scientific question under investigation. In this chapter, we describe how cotton rats and Balb/c mice are used in our laboratory to test the in vivo efficacy of small-molecule inhibitors against hRSV.

Respiratory syncytial virus (RSV) is a common virus that infects people of all ages and causes cold-like symptoms in most cases. However, more serious infections occur in the younger and older extremities of the population causing severe lung infections such as bronchiolitis and pneumonia. The current standard of care is mostly limited to supportive treatment, although prophylaxis by passive immunization with the humanized monoclonal antibody palivizumab and therapeutic intervention with aerosolized ribavirin are available. Unfortunately, administration of palivizumab is restricted to at-risk infants up to the age of two and is associated with high cost, while ribavirin treatment is hindered by questionable efficacy and safety reasons. Consequently, the development of novel specific RSV antiviral drugs is needed to help decrease RSV-related morbidity and mortality. We describe here a fluorescence-based high-throughput screening assay to discover RSV inhibitors which is based on the infection of HeLa cells with a recombinant RSV strain that contains an enhanced green fluorescent protein coding sequence in its viral genome.

Respiratory syncytial virus (RSV) has emerged since its isolation from infected children in 1957 as an important respiratory pathogen (Falsey et al, 2005; Hall et al, 2009; Nair et al, 2010; Ruuskanen et al, 2011). Generally, infection is restricted to the upper respiratory tract and not associated with long-term pathology, but progression to a more severe lower respiratory tract infection is frequent.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants, young children, elderly persons, and severely immunocompromised patients. Effective postinfection treatments are not widely available, and currently there is no approved vaccine. TMC353121 is a potent RSV fusion inhibitor in vitro , and its ability to reduce viral loads in vivo was demonstrated in cotton rats following prophylactic intravenous administration. Here, the pharmacokinetics of TMC353121 in the cotton rat, which is semipermissive for RSV replication, were further explored to build a pharmacokinetic-pharmacodynamic (PK-PD) model and to estimate the plasma drug levels needed for significant antiviral efficacy. TMC353121 reduced the viral titers in bronchoalveolar lavage fluid in a dose-dependent manner after a single subcutaneous administration and intranasal RSV inoculation 24 h after compound administration. The viral titer reduction and plasma TMC353121 concentration at the time of RSV inoculation were well described using a simple E max model with a maximal viral titer reduction ( E max ) of 1.5 log 10 . The plasma drug level required to achieve 50% of the E max (200 ng/ml) was much higher than the 50% inhibitory concentration observed in vitro in HeLaM cells (0.07 ng/ml). In conclusion, this simple PK-PD approach may be useful in predicting efficacious exposure levels for future RSV inhibitors.

Six-helix bundle (6HB) formation is an essential step for many viruses that rely on a class I fusion protein to enter a target cell and initiate replication. Because the binding modes of small molecule inhibitors of 6HB formation are largely unknown, precisely how they disrupt 6HB formation remains unclear, and structure-based design of improved inhibitors is thus seriously hampered. Here we present the high resolution crystal structure of TMC353121, a potent inhibitor of respiratory syncytial virus (RSV), bound at a hydrophobic pocket of the 6HB formed by amino acid residues from both HR1 and HR2 heptad-repeats. Binding of TMC353121 stabilizes the interaction of HR1 and HR2 in an alternate conformation of the 6HB, in which direct binding interactions are formed between TMC353121 and both HR1 and HR2. Rather than completely preventing 6HB formation, our data indicate that TMC353121 inhibits fusion by causing a local disturbance of the natural 6HB conformation.

Human respiratory syncytial virus (hRSV) is a significant cause of respiratory illness in at-risk pediatric patients, immunocompromised adults and the elderly. No vaccine is currently available for the virus and treatment options are limited to the prophylactic treatment of at-risk infants with the mAb palivizumab (Synagis) and to therapeutic intervention with the nucleoside analog ribavirin (Rebetol). The clinical use of these agents is limited and a need exists for more effective treatment for the at-risk population. The merging of viral and cellular membranes is a crucial event in the hRSV life cycle that enables the virus to enter a host cell. The multistep fusion process is facilitated by the substantial refolding of a trimeric class I fusion protein (F protein), which is the main target of fusion inhibitors. Several small-molecule fusion inhibitors have been discovered, of which some have progressed significantly in the drug development process. BTA-9881 (Biota Holdings Ltd/MedImmune) and TMC-353121 (JohnsonJohnson) are the most advanced of this drug class. In addition, progress has been made in the development of next-generation antibodies such as motavizumab (Numax; MedImmune). This review will discuss the status and latest developments of compounds and antibodies that inhibit hRSV fusion.

Fresh human hepatocytes are still considered as the “gold standard” to screen in vitro for cytochrome P450 (P450) induction. However, sparse availability of good quality human liver tissue for research purposes and the demand for standardized cell populations, together with the need for proper storage of the cells not immediately required, have resulted in the development of cryopreservation techniques that provide adequate viability and plateability of hepatocytes after thawing. This study aimed at validating cryopreserved human hepatocytes as a model to investigate P450 induction. Cryopreserved cells from four different donors were plated and cultured for 48 h, followed by incubation in the presence of typical P450 inducers. During the experiments, quality of the cultured cells was monitored both physiologically and morphologically. Concomitantly, the activity of CYP1A2, 2B6, 2C9, 2E1, and 3A4 was measured together with their mRNA and protein expression. Determination of CYP1A2, 2B6, 2C9, 2E1, and 3A4 activity in control versus prototypical inducer-treated hepatocytes revealed a maximal significant mean 11.6-, 2.8-, 1.9-, 1.5-, and 9.0-fold induction over their basal expression, respectively. Protein expression analysis of these P450s confirmed these results. Moreover, a mean 44.9-, 3.5-, 3.2-, and 13.8-fold induction of CYP1A2, 2B6, 2C9, and 3A4 mRNA was observed. Our data demonstrate that cryopreserved human hepatocytes are a valuable tool to study the induction of CYP1A2, 2B6, 2C9, 2E1, and 3A4.

Inositol 1,4,5-trisphosphate (InsP(3)) 3-kinase catalyzes the phosphorylation of InsP(3) to inositol 1,3,4,5-tetrakisphosphate (InsP(4)). cDNAs encoding three isoenzymes of InsP(3) 3-kinase (3-kinases A, B, and C) have been previously reported; however, a demonstrably full-length cDNA encoding human InsP(3) 3-kinase B was still lacking. Here we report the cloning of a full-length 2841-bp cDNA encoding human InsP(3) 3-kinase B. Northern blot analysis shows the presence of an ubiquitous transcript of approximately 7.2 kb in a large number of human tissues. InsP(3) 3-kinase activity measured in COS-7 cells transfected with InsP(3) 3-kinase B shows an activity that was 8-fold increased upon the addition of Ca(2+)/calmodulin in the assay mixture.

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.

Glial fibrillary acidic protein (GFAP) is expressed upon cAMP-mediated induction of differentiation of glial progenitor cells into type II astrocytes. The protein is regulated by hormones, growth factors and cytokines but the signal transduction pathways involved in the regulation of GFAP expression are largely unknown. Specific protein kinase inhibitors were used to study their effect on the expression of GFAP in rat C6 glioma cells. Herbimycin A, a selective protein tyrosine kinase inhibitor, reduced GFAP mRNA and protein expression upon cAMP analog or beta-adrenergic receptor-mediated induction of differentiation. The latter inhibitor attenuated the elevation of cAMP by adenylate cyclase and abolished the activity of phosphatidylinositol 3-kinase (PI 3-K). These data indicate that GFAP expression is regulated by protein tyrosine phosphorylations, modulating the cAMP concentration and PI 3-K activity in C6 glioma cells.

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKβ and Nm23-R2/NDPKα isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.

Nucleoside diphosphate kinase (NDPK/nm23) ATP/ GDP phosphotransferase activity and serine autophosphoryla- tion is inhibited by M-mbcAMP, 8-ClcAMP and 8-BrcAMP. Inhibition of the enzymatic activity largely depends on the concentration of ATP and becomes significant at ATP concentrations up to 0.5 mM and at effector concentrations measured in C6 cells stimulated with 1 mM cAMP analogue. N6- mbcAMP is a substrate of the enzyme. DbcAMP and O'2- mbcAMP, cAMP analogues with a modified 0'2-ribose, did not affect the NDPK activity. Cyclic AMP is only a moderate inhibitor of NDPK even at low ATP concentrations. Possible inhibitory effects of cAMP and cAMP analogues on reported extra- and intracellular functions of NDPK/nm23 are discussed. 2.3. Serine autophosphorylation of NDPK Autophosphorylation was performed in buffer B (25 mM HEPES (pH 7.3), 140 mM NaCl, 1 mM MgCl2, 0.8 mM CaCl2, 5 mM KC1, 5 mM EDTA) and in the presence of 20 nM fr 32P)ATP (4500 Ci/ mmol). Incubation was at 37°C for 30 min. The reaction was stopped by addition of SDS-PAGE sample buffer to a final concentration of 0.5% (w/v) SDS, 10% (v/v) glycerol, 0.5% (v/v) 2-mercaptoethanol, 6 mM Tris-HCl (pH 6.8). After boiling of the samples for 5 min, they were analyzed by SDS-PAGE on a 20% (w/v) polyacrylamide gel. The gel was Coomassie stained (30 min in 0.1% (w/v) CBB-R250, 7.5% (v/v) acetic acid, 40% (v/v) methanol) and destained for 16 h by successive washes in 10% (v/v) acetic acid, 40% (v/v) methanol. After dehydration in 50% ethanol the gel was dried and autoradiographed.