Searching for stimulators of the innate antiviral response is an appealing approach to develop novel therapeutics against viral infections. Here, we established a cell-based reporter assay to identify compounds stimulating expression of interferon-inducible antiviral genes. DD264 was selected out of 41,353 compounds for both its immuno-stimulatory and antiviral properties. While searching for its mode of action, we identified DD264 as an inhibitor of pyrimidine biosynthesis pathway. This metabolic pathway was recently identified as a prime target of broad-spectrum antiviral molecules, but our data unraveled a yet unsuspected link with innate immunity. Indeed, we showed that DD264 or brequinar, a well-known inhibitor of pyrimidine biosynthesis pathway, both enhanced the expression of antiviral genes in human cells. Furthermore, antiviral activity of DD264 or brequinar was found strictly dependent on cellular gene transcription, nuclear export machinery, and required IRF1 transcription factor. In conclusion, the antiviral property of pyrimidine biosynthesis inhibitors is not a direct consequence of pyrimidine deprivation on the virus machinery, but rather involves the induction of cellular immune response.

Aminopeptidase N (APN)/CD13 (EC 3.4.11.2) is a transmembrane protease present in a wide variety of human tissues and cell types (endothelial, epithelial, fibroblast, leukocyte). APN/CD13 expression is dysregulated in inflammatory diseases and in cancers (solid and hematologic tumors). APN/CD13 serves as a receptor for coronaviruses. Natural and synthetic inhibitors of APN activity have been characterized. These inhibitors have revealed that APN is able to modulate bioactive peptide responses (pain management, vasopressin release) and to influence immune functions and major biological events (cell proliferation, secretion, invasion, angiogenesis). Therefore, inhibition of APN/CD13 may lead to the development of anti-cancer and anti-inflammatory drugs. This review provides an update on the biological and pharmacological profiles of known natural and synthetic APN inhibitors. Current status on their potential use as therapeutic agents is discussed with regard to toxicity and specificity.

An earlier report suggested that actin and myosin I alpha (MMIalpha), a myosin associated with endosomes and lysosomes, were involved in the delivery of internalized molecules to lysosomes. To determine whether actin and MMIalpha were involved in the movement of lysosomes, we analyzed by time-lapse video microscopy the dynamic of lysosomes in living mouse hepatoma cells (BWTG3 cells), producing green fluorescent protein actin or a nonfunctional domain of MMIalpha. In GFP-actin cells, lysosomes displayed a combination of rapid long-range directional movements dependent on microtubules, short random movements, and pauses, sometimes on actin filaments. We showed that the inhibition of the dynamics of actin filaments by cytochalasin D increased pauses of lysosomes on actin structures, while depolymerization of actin filaments using latrunculin A increased the mobility of lysosomes but impaired the directionality of their long-range movements. The production of a nonfunctional domain of MMIalpha impaired the intracellular distribution of lysosomes and the directionality of their long-range movements. Altogether, our observations indicate for the first time that both actin filaments and MMIalpha contribute to the movement of lysosomes in cooperation with microtubules and their associated molecular motors.

The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have used in vitro splicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction. Importantly, compounds of this family specifically inhibit exonic splicing enhancer (ESE)-dependent splicing, because they interact directly and selectively with members of the serine-arginine-rich protein family. Treatment of cells expressing reporter constructs with ESE sequences demonstrated that selected indole derivatives mediate inhibition of ESE usage in vivo and prevent early splicing events required for HIV replication. This discovery opens the exciting possibility of a causal pharmacological treatment of aberrant splicing in human genetic disorders and development of new antiviral therapeutic approaches.splicing correction ͉ exonic splicing enhancer ͉ small chemicals ͉ pathologic splicing R emoval of introns from newly transcribed RNA polymerase II precursors (pre-mRNA) during splicing not only is an essential step for the expression of most genes in higher eukaryotic cells but also constitutes an important mechanism for generation of protein diversity and regulation of gene expression (1, 2). It is estimated that Ͼ70% of human genes are subjected to alternative splicing, and it is not surprising that many point mutations causing human diseases are associated with aberrant splicing (3, 4).Current models of constitutive and a fortiori alternative splicing suggest that splice site recognition is strongly modulated by the interaction of specific exonic and intronic pre-mRNA sequences with at least two classes of nonspliceosomal nuclear RNA-binding proteins: serine-arginine-rich (SR) proteins (5-7) and heterogeneous nuclear ribonucleoproteins (8-10). These proteins interact with spliceosomal components (5-7) and either activate or prevent the use of degenerate splice sites in their vicinity. Thus, binding of SR proteins to exonic splicing enhancers (ESE) through their RNA-recognition motif (RRM) promotes exon definition by recruiting constitutive factors via protein-protein interactions mediated by their arginine-serine-rich (RS) domain and prevents the action of nearby splicing silencers (4, 6, 11).Mutations causing human diseases may affect splice sites as well as regulatory sequences leading to the production of defective proteins (4, 11). Thus, targeting either the mutated sequences or the factors that bind them may prove to be a valuable strategy to correct aberrant splicing. Recently, antisense strategies targeting ESEdependent mechanisms have been used to induce skipping of exons containing nonsense mutations or, conversely, to restore exon inclusion by synthetic exon-specific effectors (bifunctional antisense peptide molecules or tailed antisense oligonucleotides) or spliceosome-mediat...