Biology subjects

Turcatti, G.

Publications and source records attributed to Turcatti, G..

6 recordsLinked to original sources

bioRxiv2025

A human cell-free translation screen identifies the NT-2 mycotoxin as a ribosomal peptidyl transferase inhibitor

Translation inhibitors are invaluable for probing ribosome function and therapeutic applications, but systematic discovery in human systems is limited by the lack of scalable, screening-compatible cell-free platforms. Here, we establish a robust high-throughput screening using human lysates that bypasses cellular cytotoxic effects. After screening [~]28,000 small molecules, we identified known and a novel translation inhibitor, including NT-2, a trichothecene mycotoxin produced by the pathogenic Fusarium sporotrichioides. NT-2 suppressed protein synthesis in human cells and yeast lysates, while sparing translation in bacteria and intact yeast cells. Cryo-EM at 1.76 [A] revealed NT-2 bound at the peptidyl transferase center of the human 60S ribosome, confirming NT-2 as a ribosomal inhibitor associated with ribosomes in an inactive eEF2/SERBP1-associated dormant state. Together, these results expose NT-2 as a previously unrecognized environmental inhibitor of mammalian protein synthesis and demonstrate the power of cell-free translation screening to reveal new inhibitors with unexpected ribosome fates.

biochemistry↗

bioRxiv2024

An all-in-one pipeline for the in vitro discovery and in vivo testing of Plasmodium falciparum malaria transmission blocking drugs

Elimination and eradication of malaria will depend on new drugs with potent activity against Plasmodium falciparum mature stage V gametocytes, the only stages able to infect the mosquito vector for onward parasite transmission. The identification of molecules active against these quiescent stages is difficult due to the specific biology of gametocyte maturation and challenges linked to their cultivation in vitro. Furthermore, the antimalarial drug development pipeline lacks a suitable animal model for evaluating the transmission-blocking potential of promising lead compounds and preclinical and clinical drug candidates in vivo. Here, we established a transmission-blocking drug discovery and development platform based on transgenic P. falciparum parasites engineered to produce large numbers of pure stage V gametocytes expressing a red-shifted firefly luciferase as reporter for cellular viability. This NF54/iGP1_RE9Hulg8 line facilitated the development of a highly efficient and robust in vitro screening assay for the identification of stage V gametocytocidal compounds. Importantly, by infecting humanized NODscidIL2R{gamma}null mice with pure NF54/iGP1_RE9Hulg8 stage V gametocytes, we also established a preclinical P. falciparum in vivo transmission model. Using whole animal bioluminescence imaging and quantification of gametocyte densities over a period of 14 days, we assessed the gametocyte killing and clearance kinetics in vivo of antimalarial reference drugs as well as five clinical drug candidates and identified markedly different pharmacodynamic response profiles. Furthermore, we successfully integrated this mouse model with mosquito feeding assays and thus firmly established a valuable tool for the systematic in vivo evaluation of gametocytocidal and transmission-blocking drug efficacy. One sentence summaryWe applied robust new assays for gametocytocidal drug discovery and in vivo efficacy testing using a humanized mouse model for malaria transmission

microbiology↗

bioRxiv2021

Arrayed multicycle drug screens identify broadly acting chemical inhibitors for repurposing against SARS CoV 2

Coronaviruses (CoVs) circulate in humans and animals, and expand their host range by zoonotic and anthroponotic transmissions. Endemic human CoVs, such as 229E and OC43 cause limited respiratory disease, and elicit short term anti-viral immunity favoring recurrent infections. Yet, severe acute respir-atory syndrome (SARS)-CoV-2 spreads across the globe with unprecedented impact on societies and economics. The world lacks broadly effective and affordable anti-viral agents to fight the pandemic and reduce the death toll. Here, we developed an image-based multicycle replication assay for focus for-mation of -coronavirus hCoV-229E-eGFP infected cells for screening with a chemical library of 5440 compounds arrayed in 384 well format. The library contained about 39% clinically used compounds, 26% in phase I, II or III clinical trials, and 34% in preclinical development. Hits were counter-selected against toxicity, and challenged with hCoV-OC43 and SARS-CoV-2 in tissue culture and human bronchial and nasal epithelial explant cultures from healthy donors. Fifty three compounds inhibited hCoV-229E-GFP, 39 of which at 50% effective concentrations (EC50) < 2M, and were at least 2-fold separated from toxicity. Thirty nine of the 53 compounds inhibited the replication of hCoV-OC43, while SARS-CoV-2 was inhibited by 11 compounds in at least two of four tested cell lines. Six of the 11 compounds are FDA-approved, one of which is used in mouth wash formulations, and five are systemic and orally available. Here, we demonstrate that methylene blue (MB) and mycophenolic acid (MPA), two broadly available low cost compounds, strongly inhibited shedding of infectious SARS-CoV-2 at the apical side of the cultures, in either pre- or post-exposure regimens, with somewhat weaker effects on viral RNA release indicated by RT-qPCR measurements. Our study illustrates the power of full cycle screens in repurposing clinical compounds against SARS-CoV-2. Importantly, both MB and MPA reportedly act as immunosuppressants, making them interesting candidates to counteract the cytokine storms affecting COVID-19 patients.

microbiology↗

bioRxiv2020

The FDA-approved drug Nelfinavir inhibits lytic cell-free transmission of human adenoviruses

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. The human AdV (HAdV) species B and C, such as HAdV-C2, C5 and B14, cause respiratory disease, and constitute a health threat for immuno-compromised individuals. HAdV-Cs are well known for lysing cells, owing to the E3 CR1-{beta}-encoded adenovirus death protein (ADP). We previously reported a high-throughput image-based screening framework and identified an inhibitor of HAdV-C2 multi-round infection, Nelfinavir Mesylate. Nelfinavir is the active ingredient of Viracept, an FDA-approved inhibitor of the human immuno-deficiency virus (HIV) aspartyl protease, and used to treat acquired immunodeficiency syndrome (AIDS). It is not effective against single round HAdV infections. Here, we show that Nelfinavir inhibits the lytic cell-free transmission of HAdV, indicated by the suppression of comet-shaped infection foci in cell culture. Comet-shaped foci occur upon convection-based transmission of cell-free viral particles from an infected cell to neighbouring uninfected cells. HAdV lacking ADP was insensitive to Nelfinavir, but gave rise to comet-shaped foci indicating that ADP enhances but is not required for cell lysis. This was supported by the notion that HAdV-B14 and B14p1 lacking ADP were highly sensitive to Nelfinavir, although HAdV-A31, B3, B7, B11, B16, B21, D8, D30 or D37 were less sensitive. Conspicuously, Nelfinavir uncovered slow-growing round-shaped HAdV-C2 foci, independent of neutralizing antibodies in the medium, indicative of non-lytic cell-to-cell transmission. Our study demonstrates the repurposing potential of Nelfinavir with post-exposure efficacy against different HAdVs, and describes an alternative non-lytic cell-to-cell transmission mode of HAdV. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/098061v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@8aa32corg.highwire.dtl.DTLVardef@1dea943org.highwire.dtl.DTLVardef@13aa2fborg.highwire.dtl.DTLVardef@a23352_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO C_FIG

microbiology↗

bioRxiv2020

A full cycle anti-viral drug screen identifies a clinical compound against adenovirus infection

Human adenoviruses (HAdVs) are fatal to immuno-suppressed people, but no effective anti-HAdV therapy is available. Here, we present a novel image-based high-throughput screening (HTS) platform, which scores the full viral replication cycle from virus entry to dissemination of progeny. We analysed 1,280 small molecular weight compounds of the Prestwick Chemical Library (PCL) for interference with HAdV-C2 infection in a quadruplicate, blinded format, and included robust image analyses, and hit filtering. We present the entire set of the screening data including all the images, image analyses and data processing pipelines. The data are made available at the Image Data Repository (IDR) 1, accession number idr0081. Our screen identified Nelfinavir mesylate as an inhibitor of HAdV-C2 multi-round plaque formation, but not single round infection. Nelfinavir has been FDA-approved for anti-retroviral therapy in humans. Our results underscore the power of image-based full cycle infection assays in identifying viral inhibitors with clinical potential.

microbiology↗

bioRxiv2019

Loss of Bacterial Cell Pole Stabilization in Caulobacter crescentus Sensitizes to Outer Membrane Stress and Peptidoglycan-Directed Antibiotics

Rod-shaped bacteria frequently localise proteins to one or both cell poles in order to regulate processes such as chromosome replication or polar organelle development. However, the role of such polar factors in responses to extracellular stimuli has been generally unexplored. We employed chemical-genetic screening to probe the interaction between one such factor from Caulobacter crescentus, TipN, and extracellular stress and found that TipN is required for normal tolerance of cell envelope-directed antibiotics, including vancomycin that does not normally inhibit growth of Gram-negative bacteria. Forward genetic screening for suppressors of vancomycin sensitivity in the absence of TipN revealed the TonB-dependent receptor ChvT as the mediator of vancomycin tolerance. Loss of ChvT improved resistance to vancomycin and cefixime in the otherwise sensitive {Delta}tipN strain. The activity of the two-component system regulating ChvT (ChvIG) was increased in {Delta}tipN cells relative to wild type under some, but not all, cell wall stress conditions that this strain was sensitised to, in particular cefixime and detergent exposure. Together, these results indicate that the ChvIG two-component system has been co-opted as a sensor of cell wall stress and that TipN can influence cell envelope stability and ChvIG-mediated signaling in addition to its roles in intracellular development.\n\nAuthor summaryMaintenance of an intact cell envelope is essential for free-living bacteria to survive harsh conditions they may encounter in their environment. In the case of rod-shaped bacteria, the poles of the cell are potential weak points in the cell envelope due to the high curvature of the layers and the need to break and re-form parts of the cell envelope at the division plane in order to form new poles as the cells replicate and divide. We have found that TipN, a factor required for correct division and cell pole development in the rod-shaped bacterium, Caulobacter crescentus, is also needed for maintaining normal levels of resistance to cell wall-targeting antibiotics such as vancomycin and cefixime, which interfere with peptidoglycan synthesis. We also identified an outer membrane receptor, ChvT, that was responsible for allowing vancomycin access to the cells and found that the two-component system that negatively regulates ChvT production was activated by various kinds of cell wall stress. Presence or absence of TipN influenced how active this system was in the presence of cefixime or of the membrane-disrupting detergent sodium deoxycholate. Since TipN is normally located at the poles of the cell and at the division plane just before cells complete division, our results suggest that it is involved in stabilisation of these weak points of the cell envelope as well as its other roles inside the cell.

microbiology↗