Argosomes A Potential Vehicle for the Spread of Morphogens through Epithelia

AbstractThe formation of morphogen gradients is essential for tissue patterning. Morphogens are released from producing cells and spread through adjacent tissue; paradoxically, however, many morphogens, including Wingless, associate tightly with the cell membrane. Here, we describe a novel cell biological mechanism that disperses membrane fragments over large distances through the Drosophila imaginal disc epithelium. We call these membrane exovesicles argosomes. Argosomes are derived from basolateral membranes and are produced by many different regions of the disc. They travel through adjacent tissue where they are found predominantly in endosomes. Wingless protein colocalizes with argosomes derived from Wingless-producing cells. The properties of argosomes are consistent with their being a vehicle for the spread of Wingless protein

Drug Screen Targeted at Plasmodium Liver Stages Identifies a Potent Multistage Antimalarial Drug

Plasmodium parasites undergo a clinically silent and obligatory developmental phase in the host’s liver cells before they are able to infect erythrocytes and cause malaria symptoms. To overcome the scarcity of compounds targeting the liver stage of malaria, we screened a library of 1037 existing drugs for their ability to inhibit Plasmodium hepatic development. Decoquinate emerged as the strongest inhibitor of Plasmodium liver stages, both in vitro and in vivo. Furthermore, decoquinate kills the parasite’s replicative blood stages and is active against developing gametocytes, the forms responsible for transmission. The drug acts by selectively and specifically inhibiting the parasite’s mitochondrial $bc_1$ complex, with little cross-resistance with the antimalarial drug atovaquone. Oral administration of a single dose of decoquinate effectively prevents the appearance of disease, warranting its exploitation as a potent antimalarial compound

Drug Screen Targeted at Plasmodium Liver Stages Identifies a Potent Multistage Antimalarial Drug

Plasmodium parasites undergo a clinically silent and obligatory developmental phase in the host's liver cells before they are able to infect erythrocytes and cause malaria symptoms. To overcome the scarcity of compounds targeting the liver stage of malaria, we screened a library of 1037 existing drugs for their ability to inhibit Plasmodium hepatic development. Decoquinate emerged as the strongest inhibitor of Plasmodium liver stages, both in vitro and in vivo. Furthermore, decoquinate kills the parasite's replicative blood stages and is active against developing gametocytes, the forms responsible for transmission. The drug acts by selectively and specifically inhibiting the parasite's mitochondrial bc1 complex, with little cross-resistance with the antimalarial drug atovaquone. Oral administration of a single dose of decoquinate effectively prevents the appearance of disease, warranting its exploitation as a potent antimalarial compoun

siRNA screen identifies QPCT as a druggable target for Huntington's disease.

Huntington's disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified new modifiers of mutant HTT toxicity by performing a large-scale 'druggable genome' siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen and also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone αB-crystallin and reduced the aggregation of diverse proteins. We generated new QPCT inhibitors using in silico methods followed by in vitro screening, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a new HD druggable target affecting mutant HTT aggregation and provide proof of principle for a discovery pipeline from druggable genome screen to drug development

RNAi screen for NRF2 inducers identifies targets that rescue primary lung epithelial cells from cigarette smoke induced radical stress

Chronic Obstructive Pulmonary Disease (COPD) is a highly prevalent condition characterized by inflammation and progressive obstruction of the airways. At present, there is no treatment that suppresses the chronic inflammation of the disease, and COPD patients often succumb to the condition. Excessive oxidative stress caused by smoke inhalation is a major driving force of the disease. The transcription factor NRF2 is a critical player in the battle against oxidative stress and its function is impaired in COPD. Increasing NRF2 activity may therefore be a viable therapeutic option for COPD treatment. We show that down regulation of KEAP1, a NRF2 inhibitor, protects primary human lung epithelial cells from cigarette-smoke-extract (CSE) induced cell death in an established in vitro model of radical stress. To identify new potential drug targets with a similar effect, we performed a siRNA screen of the 'druggable' genome using a NRF2 transcriptional reporter cell line. This screen identified multiple genes that when down regulated increased NRF2 transcriptional activity and provided a survival benefit in the in vitro model. Our results suggest that inhibiting components of the ubiquitin-proteasome system will have the strongest effects on NRF2 transcriptional activity by increasing NRF2 levels. We also find that down regulation of the small GTPase Rab28 or the Estrogen Receptor ESRRA provide a survival benefit. Rab28 knockdown increased NRF2 protein levels, indicating that Rab28 may regulate NRF2 proteolysis. Conversely ESRRA down regulation increased NRF2 transcriptional activity without affecting NRF2 levels, suggesting a proteasome-independent mechanism

siPools: highly complex but accurately defined siRNA pools eliminate off-target effects

Short interfering RNAs (siRNAs) are widely used as tool for gene inactivation in basic research and therapeutic applications. One of the major shortcomings of siRNA experiments are sequence-specific off-target effects. Such effects are largely unpredictable because siRNAs can affect partially complementary sequences and function like microRNAs (miRNAs), which inhibit gene expression on mRNA stability or translational levels. Here we demonstrate that novel, enzymatically generated siRNA pools-referred to as siPools-containing up to 60 accurately defined siRNAs eliminate off-target effects. This is achieved by the low concentration of each individual siRNA diluting sequence-specific off-target effects below detection limits. In fact, whole transcriptome analyses reveal that single siRNA transfections can severely affect global gene expression. However, when complex siRNA pools are transfected, almost no transcriptome alterations are observed. Taken together, we present enzymatically produced complex but accurately defined siRNA pools with potent on-target silencing but without detectable off-target effects

Evaluation of activity and combination strategies with the microtubule-targeting drug sagopilone in breast cancer cell lines

Sagopilone, a fully synthetic epothilone, is a microtubule-stabilizing agent optimized for high in vitro and in vivo activity against a broad range of tumor models, including those resistant to paclitaxel and other systemic treatments. Sagopilone development is accompanied by translational research studies to evaluate the molecular mode of action, to recognize mechanisms leading to resistance, to identify predictive response biomarkers, and to establish a rationale for combination with different therapies. Here, we profiled sagopilone activity in breast cancer cell lines. To analyze the mechanisms of mitotic arrest and apoptosis and to identify additional targets and biomarkers, an siRNA-based RNAi drug modifier screen interrogating 300 genes was performed in four cancer cell lines. Defects of the spindle assembly checkpoint (SAC) were identified to cause resistance against sagopilone-induced mitotic arrest and apoptosis. Potential biomarkers for resistance could therefore be functional defects like polymorphisms or mutations in the SAC, particularly in the central SAC kinase BUB1B. Moreover, chromosomal heterogeneity and polyploidy are also potential biomarkers of sagopilone resistance since they imply an increased tolerance for aberrant mitosis. RNAi screening further demonstrated that the sagopilone-induced mitotic arrest can be enhanced by concomitant inhibition of mitotic kinesins, thus suggesting a potential combination therapy of sagopilone with a KIF2C (MCAK) kinesin inhibitor. However, the combination of sagopilone and inhibition of the prophase kinesin KIF11 (EG5) is antagonistic, indicating that the kinesin inhibitor has to be highly specific to bring about the required therapeutic benefit

Confirmational screen with 219 genes using 3 individual siRNAs against each gene.

<p>Enrichment for luciferase-inducing genes. Frequency distribution of luciferase induction for all siRNA pools screened in pass 1 (dashed line) and the 657 individual siRNAs used in pass 2 (solid line).</p