The effect of corticosteroids on human physiology is complex and their use in tuberculosis patients remains controversial. In a high-throughput screening approach designed to discover virulence inhibitors, several corticosteroids were found to prevent cytolysis of fibroblasts infected with mycobacteria. Further experiments with Mycobacterium tuberculosis showed anti-cytolytic activity in the 10 nM range, but no effect on bacterial growth or survival in the absence of host cells at 20 mu M. The results from a panel of corticosteroids with various affinities to the glucocorticoid- and mineralocorticoid receptors indicate that the inhibition of cytolysis most likely is mediated through the glucocorticoid receptor. Using live-imaging of M. tuberculosis-infected human monocyte-derived macrophages, we also show that corticosteroids to some extent control intracellular bacteria. In vitro systems with reduced complexity are to further study and understand the interactions between bacterial infection, immune defense and cell signaling. (C) 2019 The Authors. Published by Elsevier Inc.

Antibiotics are becoming less effective in treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa. Antimicrobial therapies based on the inhibition of specific virulence-related traits, as opposed to growth inhibitors, constitute an innovative and appealing approach to tackle the threat of P. aeruginosa infections. The twin-arginine translocation (Tat) pathway plays an important role in the pathogenesis of P. aeruginosa, and constitutes a promising target for the development of anti-pseudomonal drugs. In this study we developed and optimized a whole-cell, one-well assay, based on native phospholipase C activity, to identify compounds active against the Tat system. Statistical robustness, sensitivity and consequently suitability for high-throughput screening (HTS) were confirmed by a dry run/pre-screening test scoring a Z' of 0.82 and a signal-to-noise ratio of 49. Using this assay, we evaluated ca. 40,000 molecules and identified 59 initial hits as possible Tat inhibitors. Since phospholipase C is exported into the periplasm by Tat, and subsequently translocated across the outer membrane by the type II secretion system (T2SS), our assay could also identify T2SS inhibitors. To validate our hits and discriminate between compounds that inhibited either Tat or T2SS, two separate counter assays were developed and optimized. Finally, three Tat inhibitors and one T2SS inhibitor were confirmed by means of dose-response analysis and additional counter and confirming assays. Although none of the identified inhibitors was suitable as a lead compound for drug development, this study validates our assay as a simple, efficient, and HTS compatible method for the identification of Tat and T2SS inhibitors.

PARP3 has been shown to be a key driver of TGF beta-induced epithelial-to-mesenchymal transition (EMT) and sternness in breast cancer cells, emerging as an attractive therapeutic target. Nevertheless, the therapeutic value of PARP3 inhibition has not yet been assessed. Here we investigated the impact of the absence of PARP3 or its inhibition on the tumorigenicity of BRCA1-proficient versus BRCA1-deficient breast cancer cell lines, focusing on the triple-negative breast cancer subtype (TNBC). We show that PARP3 knockdown exacerbates centrosome amplification and genome instability and reduces survival of BRCA1-deficient TNBC cells. Furthermore, we engineered PARP3(-/- )BRCA1-deficient or BRCA1-proficient TNBC cell lines using the CRISPR/nCas9(D10A) gene editing technology and demonstrate that the absence of PARP3 selectively suppresses the growth, survival and in vivo tumorigenicity of BRCA1-deficient TNBC cells, mechanistically via effects associated with an altered Rictor/mTORC2 signaling complex resulting from enhanced ubiquitination of Rictor. Accordingly, PARP3 interacts with and ADP-ribosylates GSK3 beta, a positive regulator of Rictor ubiquitination and degradation. Importantly, these phenotypes were rescued by re-expression of a wild-type PARP3 but not by a catalytic mutant, demonstrating the importance of PARP3's catalytic activity. Accordingly, reduced survival and compromised Rictor/mTORC2 signaling were also observed using a cell-permeable PARP3-specific inhibitor. We conclude that PARP3 and BRCA1 are synthetic lethal and that targeting PARP3's catalytic activity is a promising therapeutic strategy for BRCA1-associated cancers via the Rictor/mTORC2 signaling pathway.

Human adenoviruses (HAdV) are the most common cause of ocular infections. Species B human adenovirus type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause epidemic keratoconjunctivitis (EKC). Recently, HAdV-D53, -D54, and -D56 emerged as new EKC-causing agents. HAdV-E4 is associated with both PCF and EKC. We have previously demonstrated that HAdV-D37 uses sialic acid (SA)-containing glycans as cellular receptors on human corneal epithelial (HCE) cells, and the virus interaction with SA is mediated by the knob domain of the viral fiber protein. Here, by means of cell-based assays and using neuraminidase (a SA-cleaving enzyme), we investigated whether ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells. We found that HAdV-E4 and -D56 infect HCE cells independent of SAs, whereas HAdV-D53 and -D64 use SAs as cellular receptors. HAdV-D8 and -D54 fiber knobs also bound to cell-surface SAs. Surprisingly, HCE cells were found resistant to HAdV-B3 infection. We also demonstrated that the SA-based molecule i.e., ME0462, designed to bind to SA-binding sites on the HAdV-D37 fiber knob, efficiently prevents binding and infection of several EKC-causing HAdVs. Surface plasmon resonance analysis confirmed a direct interaction between ME0462 and fiber knobs. Altogether, we demonstrate that SA-containing glycans serve as receptors for multiple EKC-causing HAdVs, and, that SA-based compound function as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs.

The identification of chemical compounds that prevent and combat bacterial diseases is fundamental for crop production. Bacterial virulence inhibitors are a promising alternative to classical control treatments, because they have a low environmental impact and are less likely to generate bacterial resistance. The major virulence determinant of most animal and plant bacterial pathogens is the type III secretion system (T3SS). In this work, we screened nine plant extracts and 12 isolated compounds—including molecules effective against human pathogens—for their capacity to inhibit the T3SS of plant pathogens and for their applicability as virulence inhibitors for crop protection. The screen was performed using a luminescent reporter system developed in the model pathogenic bacterium Ralstonia solanacearum. Five synthetic molecules, one natural product and two plant extracts were found to down‐regulate T3SS transcription, most through the inhibition of the regulator hrpB. In addition, for three of the molecules, corresponding to salicylidene acylhydrazide derivatives, the inhibitory effect caused a dramatic decrease in the secretion capacity, which was translated into impaired plant responses. These candidate virulence inhibitors were then tested for their ability to protect plants. We demonstrated that salicylidene acylhydrazides can limit R. solanacearum multiplication in planta and protect tomato plants from bacterial speck caused by Pseudomonas syringae pv. tomato. Our work validates the efficiency of transcription reporters to discover compounds or natural product extracts that can be potentially applied to prevent bacterial plant diseases.

Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3 beta: ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.

Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use.

We demonstrate the versatile use of the cyclopropylmethyl group to protect phenols through the total synthesis of two benzofuran-based natural products, that is, anigopreissin A and the resveratrol-piceatannol hybrid. This protecting group is a good alternative to the conventional methyl group, owing to the feasibility of introduction, stability under a variety of conditions, and its relative ease of removal under different acidic conditions.

Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay. Several mono-ADP-ribosylation-dependent interactions were identified, and they were found to be in the micromolar affinity range using surface plasmon resonance (SPR). We then focused on the interaction between PARP14 macrodomain-2 and the mono-ADP-ribosylated PARP10 catalytic domain, and probed a similar to 1500-compound diverse library for inhibitors of this interaction using AlphaScreen. Initial hit compounds were verified by concentration-response experiments using AlphaScreen and SPR, and they were tested against PARP14 macrodomain-2 and -3. Two initial hit compounds and one chemical analog each were further characterized using SPR and microscale thermophoresis. In conclusion, our results reveal novel macrodomain interactions and establish protocols for identification of inhibitors of such interactions.

A natural product inspired library was synthesized based on 2,3-diarylbenzofuran and 2,3-diaryl-2,3-dihydrobenzofuran scaffolds. The library of forty-eight compounds was prepared by utilizing Pd-catalyzed one-pot multicomponent reactions and ruthenium-catalyzed intramolecular carbenoid C-H insertions. The compounds were evaluated for antibacterial activity in a panel of test systems including phenotypic, biochemical and image-based screening assays. We identified several potent inhibitors that block intracellular replication of pathogenic Chlamydia trachomatis with IC50 ≤ 3 μM. These new C. trachomatis inhibitors can serve as starting points for the development of specific treatments that reduces the global burden of C. trachomatis infections.