Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson's disease pathogenesis.

Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.

Oxidative stress is not a major contributor to somatic mitochondrial DNA mutations.

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (∼10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G∶C to T∶A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2'-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations.

Effects of repeated bouts of long-duration endurance exercise on muscle and urinary levels of 8-hydroxy-2'-deoxyguanosine in moderately trained cyclists.

The purpose of this study was to determine the effects of repeated bouts of long-duration endurance exercise on both muscle and urinary levels of oxidative DNA damage in moderately trained individuals. Seven moderately trained male cyclists participated in this study. All participants repeated two sessions consisting of a 5-h cycling period (equivalent to approximately 52%[Formula: see text]O2peak) followed by a 15-h rest, then a 40-km time trial. During the sessions, participants were instructed to take water ad libitum and to consume a standard sports drink consisting of 0.12 g·kg(-1) body weight·hr(-1) of carbohydrates. For each session, 24 h urine output was collected on the day before the 5-h exercise, and also between the 5-h exercise and 40-km time trial, in addition to between days 1-5 post-exercise. Subsequently, muscle and urinary levels of 8-hydroxy-2'- deoxyguanosine (8-OHdG) were determined using high performance liquid chromatography with electrochemical detection. No significant alterations were observed between two sessions at the muscle or urinary levels of 8-OHdG. These results suggest that repeated bouts of exercise with a 7-day washout period may not lead to an accumulation of DNA damage products after a second 5-h stationary cycling bout.

Absence of P-glycoprotein transport in the pharmacokinetics and toxicity of the herbicide paraquat.

Genetic variation in the multidrug resistance gene ABCB1, which encodes the efflux transporter P-glycoprotein (P-gp), has been associated with Parkinson disease. Our goal was to investigate P-gp transport of paraquat, a Parkinson-associated neurotoxicant. We used in vitro transport models of ATPase activity, xenobiotic-induced cytotoxicity, transepithelial permeability, and rhodamine-123 inhibition. We also measured paraquat pharmacokinetics and brain distribution in Friend leukemia virus B-type (FVB) wild-type and P-gp-deficient (mdr1a(-/-)/mdr1b(-/-)) mice following 10, 25, 50, and 100 mg/kg oral doses. In vitro data showed that: 1) paraquat failed to stimulate ATPase activity; 2) resistance to paraquat-induced cytotoxicity was unchanged in P-gp-expressing cells in the absence or presence of P-gp inhibitors GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] and verapamil-37.0 [95% confidence interval (CI): 33.2-41.4], 46.2 (42.5-50.2), and 34.1 µM (31.2-37.2)-respectively; 3) transepithelial permeability ratios of paraquat were the same in P-gp-expressing and nonexpressing cells (1.55 ± 0.39 and 1.39 ± 0.43, respectively); and 4) paraquat did not inhibit rhodamine-123 transport. Population pharmacokinetic modeling revealed minor differences between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice: clearances of 0.47 [95% confidence interval (CI): 0.42-0.52] and 0.78 l/h (0.58-0.98), respectively, and volume of distributions of 1.77 (95% CI: 1.50-2.04) and 3.36 liters (2.39-4.33), respectively; however, the change in clearance was in the opposite direction of what would be expected. It is noteworthy that paraquat brain-to-plasma partitioning ratios and total brain accumulation were the same across doses between FVB wild-type and mdr1a(-/-)/mdr1b(-/-) mice. These studies indicate that paraquat is not a P-gp substrate. Therefore, the association between ABCB1 pharmacogenomics and Parkinson disease is not attributed to alterations in paraquat transport.

Mitochondrial complex I inhibitors, acetogenins, induce HepG2 cell death through the induction of the complete apoptotic mitochondrial pathway.

The development of new anti-neoplastic drugs is a key issue for cancer chemotherapy due to the reality that, most likely, certain cancer cells are resistant to current chemotherapy. The past two decades have witnessed tremendous advances in our understanding of the pathogenesis of cancer. These advances have allowed identification new targets as oncogenes, tumor supressor genes and the possible implication of the mitochondria (Fulda et al. Nat Rev Drug Discov 9:447-464, 2010). Annonaceous Acetogenins (ACGs) have been described as the most potent inhibitors of the respiratory chain because of their interaction with mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116-120, 1994; Zafra-Polo et al. Phytochemistry 42:253-271, 1996; Miyoshi et al. Biochim Biophys Acta 1365:443-452, 1998; Tormo et al. Arch Biochem Biophys 369:119-126, 1999; Motoyama et al. Bioorg Med Chem Lett 12:2089-2092, 2002). To explore a possible application of natural products from Annonaceous plants to cancer treatment, we have selected four bis-tetrahydrofuranic ACGs, three from Annona cherimolia (cherimolin-1, motrilin and laherradurin) and one from Rollinia mucosa (rollinianstatin-1) in order to fully describe their mechanisms responsible within the cell (Fig. 1). In this study, using a hepato-carcinoma cell line (HepG2) as a model, we showed that the bis-THF ACGs caused cell death through the induction of the apoptotic mitochondrial pathway. Their potency and behavior were compared with the classical mitochondrial respiratory chain Complex I inhibitor rotenone in every apoptotic pathway step.

Analysis of cytotoxic activity at short incubation times reveals profound differences among Annonaceus acetogenins, inhibitors of mitochondrial Complex I.

Annonaceous acetogenins are potent cytotoxic agents against tumor cell lines as well as potent inhibitors of mitochondrial Complex I (Degli Esposti and Ghelli Biochim Biophys Acta 1187:116-120, 1994; Degli Esposti et al. Biochem J 301(Pt 1):161-167, 1994; Tormo et al. Arch Biochem Biophys 369:119-126, 1999). Eighteen different ACGs belonging to seven structural sub-families were tested against six tumor and two non tumor cell lines in a MTT cytotoxicity assay to evaluate the correlation between mitochondrial Complex I inhibition and cytotoxic activity potency and selectivity. The results showed a substantial heterogeneity in potency and selectivity among the different compounds tested, although no clear overall structure-activity relationships could be established. To further characterize the biological activity of these compounds, four ACGs were selected based on their inhibition binding sites to Complex I, to evaluate their cytotoxic activity over a 15-minute to 48-hour period using a more sensitive time-course LDH cytotoxicity assay. Our results indicate that, although all of the ACGs were highly cytotoxic in HepG2 cell lines at 24 h, each sub-class behaves rather differently at shorter times. Perhaps other aspects related to how these compounds reach or bind to their target sites, or differences in their ability to cross the cell and/or the mitochondrial membranes, could help explain their different activities. This different behavior between ACGs may provide new clues for a better understanding of their potential antitumor properties.

Platensimycin is a selective FabF inhibitor with potent antibiotic properties.

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of beta-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Role of the serotonergic system in reduced pulmonary function after exposure to methamphetamine.

Although use of methamphetamine (MA) by smoking is the fastest growing method of administration, very limited data are available describing the effects of smoked MA. Using a murine inhalation exposure system, we explored the pulmonary effects of low-dose acute inhalation exposure to MA vapor (smoke). Inhalation of MA vapor resulted in transiently reduced pulmonary function, as measured by transpulmonary resistance, dynamic compliance, and whole-body plethysmography compared with unexposed control animals. These changes were associated with an approximately 34% reduction in serotonin (5-hydroxytryptamine [5-HT]) metabolism/inactivation to 5-hydroxyindolacetic acid, and a nearly 40% reduction in monoamine oxidase (MAO)-A activity in the lung. Pretreatment of mice with a selective 5-HT reuptake inhibitor completely ablated the MA-induced changes in pulmonary function, confirming a key role for the 5-HT transporter (serotonin transporter [SERT]) and the serotonergic system in this effect. Immunofluorescent staining of mouse lung tissue confirmed high expression of SERT in airway epithelial cells. Using mouse airway epithelial cell line, LA-4, and purified human MAO-A, it was demonstrated that MA impedes 5-HT metabolism through direct inhibition of MAO-A activity in vitro. Together, these data demonstrate that low-dose exposure to MA results in reduced pulmonary function mediated via SERT and subsequent perturbation of 5-HT metabolism in the lung. This supports a role for the serotonergic system in MA-mediated pulmonary effects.

Can overmarking be considered as a means of chemical mate guarding in a wild callitrichid?

Mate guarding is a male strategy to monopolize matings and thus to ensure paternity. Since in callitrichids female reproductive status is advertised by scent marks, one may expect mate guarding by chemical means. We addressed this question during an episode of consortship observed in a polyandrous trio of wild saddleback tamarins (Saguinus fuscicollis). During consortship, the consort male was the only one to allomark the female. Scent marking frequency decreased for all individuals, although the consort male marked more than the other male during consortship, while there was no difference in the previous period. During consortship, almost 50% of female scents were overmarked by the consort, and more than 56% of the consort's scent marks were employed to overmark the female's scents. Therefore, the other male had limited access to female scent marks. Mate guarding may thus have a chemical component in tamarins, and olfactory communication may play an important role in mating competition.

Alzheimer's disease (AD)-like pathology in aged monkeys after infantile exposure to environmental metal lead (Pb): evidence for a developmental origin and environmental link for AD.

The sporadic nature of Alzheimer's disease (AD) argues for an environmental link that may drive AD pathogenesis; however, the triggering factors and the period of their action are unknown. Recent studies in rodents have shown that exposure to lead (Pb) during brain development predetermined the expression and regulation of the amyloid precursor protein (APP) and its amyloidogenic beta-amyloid (Abeta) product in old age. Here, we report that the expression of AD-related genes [APP, BACE1 (beta-site APP cleaving enzyme 1)] as well as their transcriptional regulator (Sp1) were elevated in aged (23-year-old) monkeys exposed to Pb as infants. Furthermore, developmental exposure to Pb altered the levels, characteristics, and intracellular distribution of Abeta staining and amyloid plaques in the frontal association cortex. These latent effects were accompanied by a decrease in DNA methyltransferase activity and higher levels of oxidative damage to DNA, indicating that epigenetic imprinting in early life influenced the expression of AD-related genes and promoted DNA damage and pathogenesis. These data suggest that AD pathogenesis is influenced by early life exposures and argue for both an environmental trigger and a developmental origin of AD.

Folate deficiency induces neurodegeneration and brain dysfunction in mice lacking uracil DNA glycosylase.

Folate deficiency and resultant increased homocysteine levels have been linked experimentally and epidemiologically with neurodegenerative conditions like stroke and dementia. Moreover, folate deficiency has been implicated in the pathogenesis of psychiatric disorders, most notably depression. We hypothesized that the pathogenic mechanisms include uracil misincorporation and, therefore, analyzed the effects of folate deficiency in mice lacking uracil DNA glycosylase (Ung-/-) versus wild-type controls. Folate depletion increased nuclear mutation rates in Ung-/- embryonic fibroblasts, and conferred death of cultured Ung-/- hippocampal neurons. Feeding animals a folate-deficient diet (FD) for 3 months induced degeneration of CA3 pyramidal neurons in Ung-/- but not Ung+/+ mice along with decreased hippocampal expression of brain-derived neurotrophic factor protein and decreased brain levels of antioxidant glutathione. Furthermore, FD induced cognitive deficits and mood alterations such as anxious and despair-like behaviors that were aggravated in Ung-/- mice. Independent of Ung genotype, FD increased plasma homocysteine levels, altered brain monoamine metabolism, and inhibited adult hippocampal neurogenesis. These results indicate that impaired uracil repair is involved in neurodegeneration and neuropsychiatric dysfunction induced by experimental folate deficiency.

PAP inhibitor with in vivo efficacy identified by Candida albicans genetic profiling of natural products.

Natural products provide an unparalleled source of chemical scaffolds with diverse biological activities and have profoundly impacted antimicrobial drug discovery. To further explore the full potential of their chemical diversity, we survey natural products for antifungal, target-specific inhibitors by using a chemical-genetic approach adapted to the human fungal pathogen Candida albicans and demonstrate that natural-product fermentation extracts can be mechanistically annotated according to heterozygote strain responses. Applying this approach, we report the discovery and characterization of a natural product, parnafungin, which we demonstrate, by both biochemical and genetic means, to inhibit poly(A) polymerase. Parnafungin displays potent and broad spectrum activity against diverse, clinically relevant fungal pathogens and reduces fungal burden in a murine model of disseminated candidiasis. Thus, mechanism-of-action determination of crude fermentation extracts by chemical-genetic profiling brings a powerful strategy to natural-product-based drug discovery.

Antisense-guided isolation and structure elucidation of pannomycin, a substituted cis-decalin from Geomyces pannorum.

Antisense-based screening strategies can be used to sensitize a microorganism and selectively detect inhibitors against a particular cellular target of interest. A strain of Staphylococcus aureus that generates an antisense RNA against SecA,a central member of the protein secretion machinery, has been used to screen for novel antibacterials. Possible inhibitors of the SecA ATP-ase were selected with a high-throughput, two-plate agar-based whole cell differential sensitivity screen. After screening a library of over 115 000 natural products extracts with the SecA antisense strain, an extract of Geomyces pannorum was identified as providing increased activity against the sensitized strain as compared with the wild-type control. Bioassay-guided isolation of the active component from this fungal extract provided a new cis-decalin secondary metabolite, which we have named pannomycin.

Reconciliation in captive cotton-top tamarins (Saguinus oedipus), a cooperative breeding primate.

Reconciliation has been demonstrated in all primate species in which the phenomenon has been studied. However, reconciliation has been studied in only two species of callitrichids, and conclusions remain controversial. The first aim of this study has been to find out whether captive cotton-top tamarins (Saguinus oedipus) reconcile, since this is the first such study on this species. We examined 227 conflicts in three family groups (N=19). Instances in which individuals remained together in t=0 (29; 12.8%) were not analyzed. The cotton-top tamarins showed heightened affiliation between opponents in the postconflict periods (PC) compared with matched control (MC) periods (39.88+/-5.12% and 3.18+/-1.27%, respectively), with a corrected conciliatory tendency of 37.17+/-5.37%, and a "time window" that included the first 180 sec of the PC period. Former opponents were the most likely recipient of affiliative behaviors during the PC periods: 39.83+/-4.26% vs. 11.36+/-5.33% during MC periods. The proportion of attracted pairs (47.13+/-6.25%) was significantly higher than those of dispersed pairs for male-male conflicts (3.79+/-1.79), but not for male-female conflicts (27.31+/-9.32 and 4.82+/-2.9, respectively). In cooperative-breeding species, specific sex-class dyads might differ in how they resolve conflicts.

Distribution of the antifungal agents sordarins across filamentous fungi.

Sordarins are a class of natural antifungal agents which act by specifically inhibiting fungal protein synthesis through their interaction with the elongation factor 2, EF2. A number of natural sordarins produced by diverse fungi of different classes have been reported in the literature. We have run an exhaustive search of sordarin-producing fungi using two different approaches consecutively, the first one being a differential sensitivity screen using a sordarin-resistant mutant yeast strain run in parallel with a wild type strain, and the second one an empiric screen against Candida albicans followed by early detection of sordarins by LC-MS analysis. Using these two strategies we have detected as many as 22 new strains producing a number of different sordarin analogues, either known (sordarin, xylarin, zofimarin) or novel (isozofimarin and 4'-O-demethyl sordarin). Sordarin and xylarin were the most frequently found compounds in the class. The producing strains were subjected to sequencing of the ITS region to determine their phylogenetic affinities. All the strains were shown to belong to the Xylariales, being distributed across three families in this order, the Xylariaceae, the Amphisphaeriaceae, and the Diatrypaceae. Despite being screened in large numbers, we did not find sordarin production in any other fungal group, including those orders where sordarin producing fungi are known to exist (i.e., Sordariales, Eurotiales, and Microascales), suggesting that the production of sordarin is a trait more frequently associated to members of the Xylariales than to any other fungal order.

Discovery of the parnafungins, antifungal metabolites that inhibit mRNA polyadenylation, from the Fusarium larvarum complex and other Hypocrealean fungi.

Evaluation of fungal fermentation extracts with whole cell Candida albicans activity resulted in the identification of a novel class of isoxazolidinone-containing metabolites named parnafungins. Chemical-genetic profiling with the C. albicans fitness test identified the biochemical target as inhibition of polyadenosine polymerase, a component of the mRNA cleavage and polyadenylation complex. Parnafungins were discovered from fermentation extracts of fungi resembling F. larvarum isolated from plants, plant litter and lichens. Furthermore authentic strains of F. larvarum var. larvarum and F. larvarum var. rubrum could be induced to produce parnafungins and their degradation products in low titers. Relationships among strains of the F. larvarum complex (FLC), including parnafungin-producing strains, were examined by cladistic analyses of rDNA, mitochondrial rDNA, and two protein-coding genes, comparisons of antifungal activity and antifungal metabolite profiles, and morphological phenotypes. Integrated analyses of these data led to the conclusion that the diversity within the FLC exceeded the one-to-one correspondence between F. larvarum and its teleomorph Cosmospora aurantiicola. Based on multiple gene sequence analyses, strains of the FLC formed a monophyletic clade inclusive of the parnafungin-producing strains. The FLC, including newly discovered parnafungin-producing strains, could be resolved into at least six different lineages, possibly representing cryptic' species, of which one was not fully resolved from F. larvarum var. rubrum. Fusarium larvarum var. rubrum represents a species distinct from var. larvarum. Finally we report that two other species from the Hypocreales, Trichonectria rectipila and Cladobotryum pinarense, are able to produce parnafungins and their open-ring forms.

Cell cycle activation linked to neuronal cell death initiated by DNA damage.

Increasing evidence indicates that neurodegeneration involves the activation of the cell cycle machinery in postmitotic neurons. However, the purpose of these cell cycle-associated events in neuronal apoptosis remains unknown. Here we tested the hypothesis that cell cycle activation is a critical component of the DNA damage response in postmitotic neurons. Different genotoxic compounds (etoposide, methotrexate, and homocysteine) induced apoptosis accompanied by cell cycle reentry of terminally differentiated cortical neurons. In contrast, apoptosis initiated by stimuli that do not target DNA (staurosporine and colchicine) did not initiate cell cycle activation. Suppression of the function of ataxia telangiectasia mutated (ATM), a proximal component of DNA damage-induced cell cycle checkpoint pathways, attenuated both apoptosis and cell cycle reentry triggered by DNA damage but did not change the fate of neurons exposed to staurosporine and colchicine. Our data suggest that cell cycle activation is a critical element of the DNA damage response of postmitotic neurons leading to apoptosis.

Isolation and structure elucidation of parnafungins, antifungal natural products that inhibit mRNA polyadenylation.

The Candida albicans Fitness Test, a whole-cell screening platform, was used to profile crude fermentation extracts for novel antifungal natural products with interesting mechanisms of action. An extract with intrinsic antifungal activity from the fungus Fusarium larvarum displayed a Fitness Test profile that strongly implicated mRNA processing as the molecular target responsible for inhibition of fungal growth. Isolation of the active components from this sample identified a novel class of isoxazolidinone-containing natural products, which we have named parnafungins. These natural products were isolated as an interconverting mixture of four structural- and stereoisomers. The isomerization of the parnafungins was due to a retro-Michael ring-opening and subsequent reformation of a xanthone ring system. This interconversion was blocked by methylation of an enol moiety. Structure elucidation of purified parnafungin derivatives was accomplished by X-ray crystallography and NMR analysis. The biochemical target of these natural products has been identified as the fungal polyadenosine polymerase. Parnafungins demonstrated broad spectrum antifungal activity with no observed activity against gram-positive or gram-negative bacteria. The intact isoxazolidinone ring was required for antifungal activity. In addition, the natural products were efficacious in a mouse model of disseminated candidiasis.

Development of an HTS assay for the search of anti-influenza agents targeting the interaction of viral RNA with the NS1 protein.

The NS1 protein is a nonstructural protein encoded by the influenza A virus. It is responsible for many alterations produced in the cellular metabolism upon infection by the virus and for modulation of virus virulence. The NS1 protein is able to perform a large variety of functions due to its ability to bind various types of RNA molecules, from both viral and nonviral origin, and to interact with several cell factors. With the aim of exploring whether the binding of NS1 protein to viral RNA (vRNA) could constitute a novel target for the search of anti-influenza drugs, a filter-binding assay measuring the specific interaction between the recombinant His-NS1 protein from influenza A virus and a radiolabeled model vRNA ( 32P-vNSZ) was adapted to a format suitable for screening and easy automation. Flashplate technology (PerkinElmer, Waltham, MA), either in 96- or 384-well plates, was used. The Flashplate wells were precoated with the recombinant His-NS1 protein, and the binding of His-NS1 to a 35S-vNSZ probe was measured. A pilot screening of a collection of 27,520 mixtures of synthetic chemical compounds was run for inhibitors of NS1 binding to vRNA. We found 3 compounds in which the inhibition of NS1 binding to vRNA, observed at submicromolar concentrations, was correlated with a reduction of the cytopathic effect during the infection of cell cultures with influenza virus. These results support the hypothesis that the binding of NS1 to vRNA could be a novel target for the development of anti-influenza drugs.