Arenaviruses are a large family of emerging enveloped negative-strand RNA viruses that include several causative agents of viral hemorrhagic fevers. For cell entry, human-pathogenic arenaviruses use different cellular receptors and endocytic pathways that converge at the level of acidified late endosomes, where the viral envelope glycoprotein mediates membrane fusion. Inhibitors of arenavirus entry hold promise for therapeutic antiviral intervention and the identification of “druggable” targets is of high priority. Using a recombinant vesicular stomatitis virus pseudotype platform, we identified the clotrimazole-derivative TRAM-34, a highly selective antagonist of the calcium-activated potassium channel KCa3.1, as a specific entry inhibitor for arenaviruses. TRAM-34 specifically blocked entry of most arenaviruses, including hemorrhagic fever viruses, but not Lassa virus and other enveloped viruses. Anti-arenaviral activity was likewise observed with the parental compound clotrimazole and the derivative senicapoc, whereas structurally unrelated KCa3.1 inhibitors showed no antiviral effect. Deletion of KCa3.1 by CRISPR/Cas9 technology did not affect the antiarenaviral effect of TRAM-34, indicating that the observed antiviral effect of clotrimazoles was independent of the known pharmacological target. The drug affected neither virus-cell attachment, nor endocytosis, suggesting an effect on later entry steps. Employing a quantitative cell-cell fusion assay that bypasses endocytosis, we demonstrate that TRAM-34 specifically inhibits arenavirus-mediated membrane fusion. In sum, we uncover a novel antiarenaviral action of clotrimazoles that currently undergo in vivo evaluation in the context of other human diseases. Their favorable in vivo toxicity profiles and stability opens the possibility to repurpose clotrimazole derivatives for therapeutic intervention against human-pathogenic arenaviruses. IMPORTANCE Emerging human-pathogenic arenaviruses are causative agents of severe hemorrhagic fevers with high mortality and represent serious public health problems. The current lack of a licensed vaccine and the limited treatment options makes the development of novel antiarenaviral therapeutics an urgent need. Using a recombinant pseudotype platform, we uncovered that clotrimazole drugs, in particular TRAM-34, specifically inhibit cell entry of a range of arenaviruses, including important emerging human pathogens, with the exception of Lassa virus. The antiviral effect was independent of the known pharmacological drug target and involved inhibition of the unusual membrane fusion mechanism of arenaviruses. TRAM-34 and its derivatives currently undergo evaluation against a number of human diseases and show favorable toxicity profiles and high stability in vivo. Our study provides the basis for further evaluation of clotrimazole derivatives as antiviral drug candidates. Their advanced stage of drug development will facilitate repurposing for therapeutic intervention against human-pathogenic arenaviruses.

Antibiotic resistance has become a major health issue. Nosocomial infections and the prevalence of resistant pathogenic bacterial strains are rising steadily. Therefore, there is an urgent need to develop new classes of antibiotics effective on multi-resistant nosocomial pathogenic bacteria. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317−326, induces cancer cell death, inhibits metastatic progression, and sensitizes tumor cells to various anti-cancer treatments in vitro and in vivo. We here report that TAT-RasGAP317−326 also possesses antimicrobial activity. In vitro, TAT-RasGAP317−326, but not mutated or truncated forms of the peptide, efficiently killed a series of bacteria including Escherichia coli, Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa. In vivo experiments revealed that TAT-RasGAP317−326 protects mice from lethal E. coli-induced peritonitis if administrated locally at the onset of infection. However, the protective effect was lost when treatment was delayed, likely due to rapid clearance and inadequate biodistribution of the peptide. Peptide modifications might overcome these shortcomings to increase the in vivo efficacy of the compound in the context of the currently limited antimicrobial options.

Cell-penetrating peptides (CPPs) allow intracellular delivery of bioactive cargo molecules. The mechanisms allowing CPPs to enter cells are ill-defined. Using a CRISPR/Cas9-based screening, we discovered that KCNQ5, KCNN4, and KCNK5 potassium channels positively modulate cationic CPP direct translocation into cells by decreasing the transmembrane potential (Vm). These findings provide the first unbiased genetic validation of the role of Vm in CPP translocation in cells. In silico modeling and live cell experiments indicate that CPPs, by bringing positive charges on the outer surface of the plasma membrane, decrease the Vm to very low values (–150 mV or less), a situation we have coined megapolarization that then triggers formation of water pores used by CPPs to enter cells. Megapolarization lowers the free energy barrier associated with CPP membrane translocation. Using dyes of varying dimensions in CPP co-entry experiments, the diameter of the water pores in living cells was estimated to be 2 (–5) nm, in accordance with the structural characteristics of the pores predicted by in silico modeling. Pharmacological manipulation to lower transmembrane potential boosted CPP cellular internalization in zebrafish and mouse models. Besides identifying the first proteins that regulate CPP translocation, this work characterized key mechanistic steps used by CPPs to cross cellular membranes. This opens the ground for strategies aimed at improving the ability of cells to capture CPP-linked cargos in vitro and in vivo.

We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.

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30Cell-penetrating peptides (CPPs) allow intracellular delivery of cargo molecules. CPPs 31 provide efficient methodology to transfer bioactive molecules in cells, in particular in 32 conditions when transcription or translation of cargo-encoding sequences is not 33 desirable or achievable. The mechanisms allowing CPPs to enter cells are ill-defined 34 and controversial. This work identifies potassium channels as key regulators of cationic 35 CPP translocation. Using a CRISPR/Cas9-based screening, we discovered that 36 KCNQ5, KCNN4, and KCNK5 positively modulate CPP cellular direct translocation by 37 reducing transmembrane potential (Vm). Cationic CPPs further decrease the Vm to 38 megapolarization values (about -150 mV) leading to the formation of ~2 nm-wide water 39 pores used by CPPs to access the cell's cytoplasm. Pharmacological manipulation to 40 lower transmembrane potential boosted CPPs cellular uptake in zebrafish and mouse 41 models. Besides identifying the first genes that regulate CPP translocation, this work 42 characterizes key mechanistic steps used by CPPs to cross cellular membrane. This 43 opens the ground for pharmacological strategies augmenting the susceptibility of cells 44 to capture CPP-linked cargos in vitro and in vivo. 45 46Cell-penetrating peptides (CPPs) are non-toxic molecules of 5-30 amino acids that can 47 translocate into living cells. CPPs can be hooked to a variety of cargos (siRNAs, DNA, 48 polypeptides, liposomes, nanoparticles, etc.) to allow their transport into cells for 49 therapeutic or experimental purposes (1-10). The origin of CPPs is diverse. For 50 example, TAT48-57 is a 10 amino-acid fragment derived from the trans-activator of 51 transcription (TAT) HIV-1 protein (11, 12), penetratin is a 16 amino-acid peptide 52 derived from the Antennapedia Drosophila melanogaster protein (13), and MAP (model 53 amphipatic peptide) is a synthetic alanine/leucine/lysine-rich peptide (14). The vast 54 majority of CPPs are cationic (1, 3, 6, 7). 55How CPPs enter cells is debated and not fully characterized at the molecular level 56 (reviewed in (1-8)). Due to this knowledge gap, it is difficult to ameliorate CPP cellular 57 entry and this slows down development of CPP-based interventions. Two main modes 58 of CPP entry have been described: endocytosis and direct translocation(1-8). 59Endocytosed CPPs gain cytosolic access by escaping endosomes. Direct 60 translocation has been proposed to occur through transient pore formation or 61 membrane destabilization. Endocytosis and direct translocation are not mutually 62 exclusive. Several entry routes can be followed simultaneously by a given CPP in a 63 given cell line (9, 10). 64Here, we used CRISPR/Cas9-screenings to identify proteins required for the cellular 65 uptake of CPPs. This approach identified three potassium channels as mandatory for 66 the direct translocation of CPPs into various cell types. Further, we highlighted the 67 requirement of an appropriate membrane potential to generate a 2 nm-wide water 68 pores through which...

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High-density lipoproteins (HDLs) prevent cell death induced by a variety of cytotoxic drugs. The underlying mechanisms are however still poorly understood. Here we present evidence that HDLs efficiently protect cells against thapsigargin (TG), a sarco/ endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) inhibitor, by extracting the drug from cells. Drug efflux could also be triggered to some extent by low-density lipoproteins and serum. HDLs did not reverse the non-lethal mild ER stress response induced by low TG concentrations or by SERCA knock-down but HDLs inhibited the toxic SERCA-independent effects mediated by high TG concentrations. HDLs could extract other lipophilic compounds, but not hydrophilic substances This work shows that HDLs utilize their capacity of loading themselves with lipophilic compounds, akin to their ability to extract cellular cholesterol, to reduce the cell content of hydrophobic drugs. This can be beneficial if lipophilic xenobiotics are toxic but may be detrimental to the therapeutic benefit of lipophilic drugs such as glibenclamide.