Preclinical spheroid models identify BMX as a therapeutic target for metastatic MYCN nonamplified neuroblastoma.

The development of targeted therapies offers new hope for patients affected by incurable cancer. However, multiple challenges persist, notably in controlling tumor cell plasticity in patients with refractory and metastatic illness. Neuroblastoma (NB) is an aggressive pediatric malignancy originating from defective differentiation of neural crest-derived progenitors with oncogenic activity due to genetic and epigenetic alterations and remains a clinical challenge for high-risk patients.

Phosphoproteomics and morphology of stored human red blood cells treated by protein tyrosine phosphatases inhibitor.

The process of protein phosphorylation is involved in numerous cell functions. In particular, phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions, including the cytoskeleton organization. During their storage before transfusion, RBCs suffer from storage lesions that affect their energy metabolism and morphology.

Automated Dual-Mode Cell Monitoring To Simultaneously Explore Calcium Dynamics and Contraction-Relaxation Kinetics within Drug-Treated Stem Cell-Derived Cardiomyocytes.

This manuscript proposes a new dual-mode cell imaging system for studying the relationships between calcium dynamics and the contractility process of cardiomyocytes derived from human-induced pluripotent stem cells. Practically, this dual-mode cell imaging system provides simultaneously both live cell calcium imaging and quantitative phase imaging based on digital holographic microscopy. Specifically, thanks to the development of a robust automated image analysis, simultaneous measurements of both intracellular calcium, a key player of excitation-contraction coupling, and the quantitative phase image-derived dry mass redistribution, reflecting the effective contractility, namely, the contraction and relaxation processes, were achieved.

In vitro-transfusional model for red-blood-cell study: the advantage of lowering hematocrit.

Background: The quality of red blood cells (RBCs) stored in red cell concentrates (RCCs) is influenced by processing, storage and donor characteristics, and can have a clinical impact on transfused patients. To evaluate RBC properties and their potential impact in a transfusion setting, a simple in vitro-transfusional model has been developed.

Materials And Methods: Transfusion was simulated by mixing a washed RBC pool from two male-derived RCCs stored at 4°C with a pool of 15 male-derived fresh frozen plasma (FFP) units, representing the recipient, at a hematocrit (HCT) of 30% ("control" setting) or 5% (alternative model).

High-content, arrayed compound screens with rhinovirus, influenza A virus and herpes simplex virus infections.

Viruses are genetically and structurally diverse, and outnumber cells by orders of magnitude. They can cause acute and chronic infections, suppress, or exacerbate immunity, or dysregulate survival and growth of cells. To identify chemical agents with pro- or antiviral effects we conducted arrayed high-content image-based multi-cycle infection screens of 1,280 mainly FDA-approved compounds with three human viruses, rhinovirus (RV), influenza A virus (IAV), and herpes simplex virus (HSV) differing in genome organization, composition, presence of an envelope, and tropism.

Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale.

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology.

Novel High-Throughput Fluorescence-Based Assay for the Identification of Nematocidal Compounds That Target the Blood-Feeding Pathway.

Hookworm infections cause a neglected tropical disease (NTD) affecting ~740 million people worldwide, principally those living in disadvantaged communities. Infections can cause high morbidity due to their impact on nutrient uptake and their need to feed on host blood, resulting in a loss of iron and protein, which can lead to severe anaemia and impaired cognitive development in children. Currently, only one drug, albendazole is efficient to treat hookworm infection and the scientific community fears the rise of resistant strains.

Identification of New Vulnerabilities in Conjunctival Melanoma Using Image-Based High Content Drug Screening.

Recent evidence suggests that numerous similarities exist between the genomic landscapes of both conjunctival and cutaneous melanoma. Since alterations of several components of the MAP kinases, PI3K/mTOR, and cell cycle pathways have been reported in conjunctival melanoma, we decided to assess the sensitivity of conjunctival melanoma to targeted inhibition mostly of kinase inhibitors. A high content drug screening assay based on automated fluorescence microscopy was performed in three conjunctival melanoma cell lines with different genomic backgrounds with 489 kinase inhibitors and 53 other inhibitors.

Identification of broad anti-coronavirus chemical agents for repurposing against SARS-CoV-2 and variants of concern.

Endemic human coronaviruses (hCoVs) 229E and OC43 cause respiratory disease with recurrent infections, while severe acute respiratory syndrome (SARS)-CoV-2 spreads across the world with impact on health and societies. Here, we report an image-based multicycle infection procedure with α-coronavirus hCoV-229E-eGFP in an arrayed chemical library screen of 5440 clinical and preclinical compounds. Toxicity counter selection and challenge with the β-coronaviruses OC43 and SARS-CoV-2 in tissue culture and human airway epithelial explant cultures (HAEEC) identified four FDA-approved compounds with oral availability.

Highly Potent Host-Specific Small-Molecule Inhibitor of Paramyxovirus and Pneumovirus Replication with High Resistance Barrier.

Multiple enveloped RNA viruses of the family and like measles virus (MeV), Nipah virus (NiV), canine distemper virus (CDV), or respiratory syncytial virus (RSV), are of high clinical relevance. Each year a huge number of lives are lost as a result of these viral infections. Worldwide, MeV infection alone is responsible for over a hundred thousand deaths each year despite available vaccine.

Picomole-Scale Synthesis and Screening of Macrocyclic Compound Libraries by Acoustic Liquid Transfer.

Macrocyclic compounds are an attractive class of therapeutic ligands against challenging targets, such as protein-protein interactions. However, the development of macrocycles as drugs is hindered by the lack of large combinatorial macrocyclic libraries, which are cumbersome, expensive, and time consuming to make, screen, and deconvolute. Here, we established a strategy for synthesizing and screening combinatorial libraries on a picomolar scale by using acoustic droplet ejection to combine building blocks at nanoliter volumes, which reduced the reaction volumes, reagent consumption, and synthesis time.

Macrocycle synthesis strategy based on step-wise "adding and reacting" three components enables screening of large combinatorial libraries.

Macrocycles provide an attractive modality for drug development, but generating ligands for new targets is hampered by the limited availability of large macrocycle libraries. We have established a solution-phase macrocycle synthesis strategy in which three building blocks are coupled sequentially in efficient alkylation reactions that eliminate the need for product purification. We demonstrate the power of the approach by combinatorially reacting 15 bromoacetamide-activated tripeptides, 42 amines, and 6 bis-electrophile cyclization linkers to generate a 3780-compound library with minimal effort.

Image- and Fluorescence-Based Test Shows Oxidant-Dependent Damages in Red Blood Cells and Enables Screening of Potential Protective Molecules.

An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions.

A high-content image-based drug screen of clinical compounds against cell transmission of adenovirus.

Human adenoviruses (HAdVs) are fatal to immuno-suppressed individuals, 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 and second-round infections. 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 performed robust image analyses and hit filtering.

The FDA-Approved Drug Nelfinavir Inhibits Lytic Cell-Free but Not Cell-Associated Nonlytic Transmission of Human Adenovirus.

Adenoviruses (AdVs) are prevalent and give rise to chronic and recurrent disease. Human AdV (HAdV) species B and C, such as HAdV-C2, -C5, and -B14, cause respiratory disease and constitute a health threat for immunocompromised individuals. HAdV-Cs are well known for lysing cells owing to the E3 CR1-β-encoded adenovirus death protein (ADP).

High-throughput automated organoid culture via stem-cell aggregation in microcavity arrays.

Stem-cell-derived epithelial organoids are routinely used for the biological and biomedical modelling of tissues. However, the complexity, lack of standardization and quality control of stem cell culture in solid extracellular matrices hampers the routine use of the organoids at the industrial scale. Here, we report the fabrication of microengineered cell culture devices and scalable and automated methods for suspension culture and real-time analysis of thousands of individual gastrointestinal organoids trapped in microcavity arrays within a polymer-hydrogel substrate.

Image-Based Marker-Free Screening of GABA Agonists, Antagonists, and Modulators.

The ionotropic GABA receptors represent the main target for different groups of widely used drugs having hypnotic and anxiolytic effects. So far, most approaches used to assess GABA activity involve invasive low -throughput electrophysiological techniques or rely on fluorescent dyes, preventing the ability to conduct noninvasive and thus nonperturbing screens. To address this limitation, we have developed an automated marker-free cell imaging method, based on digital holographic microscopy (DHM).

Thiol-to-amine cyclization reaction enables screening of large libraries of macrocyclic compounds and the generation of sub-kilodalton ligands.

Macrocyclic compounds are an attractive modality for drug development, but the limited availability of large, structurally diverse macrocyclic libraries hampers the discovery of leads. Here, we describe the discovery of efficient macrocyclization reactions based on thiol-to-amine ligations using bis-electrophiles, their application to synthesize and screen large libraries of macrocyclic compounds, and the identification of potent small macrocyclic ligands. The thiol-to-amine cyclization reactions showed unexpectedly high yields for a wide substrate range, which obviated product purification and enabled the generation and screening of an 8988 macrocycle library with a comparatively small effort.

A Universal Assay for Aminopeptidase Activity and Its Application for Dipeptidyl Peptidase-4 Drug Discovery.

Aminopeptidases, such as dipeptidyl peptidase-4 (DPP-4, CD26), are potent therapeutic targets for pharmacological interventions because they play key roles in many important pathological pathways. To analyze aminopeptidase activity in vitro (including high-throughput screening [HTS]), in vivo, and ex vivo, we developed a highly sensitive and quantitative bioluminescence-based readout method. We successfully applied this method to screening drugs with potential DPP-4 inhibitory activity.

Quantification of stored red blood cell fluctuations by time-lapse holographic cell imaging.

We propose methods to quantitatively calculate the fluctuation rate of red blood cells with nanometric axial and millisecond temporal sensitivity at the single-cell level by using time-lapse holographic cell imaging. For this quantitative analysis, cell membrane fluctuations (CMFs) were measured for RBCs stored at different storage times. Measurements were taken over the whole membrane for both the ring and dimple sections separately.

Targeting STING with covalent small-molecule inhibitors.

Aberrant activation of innate immune pathways is associated with a variety of diseases. Progress in understanding the molecular mechanisms of innate immune pathways has led to the promise of targeted therapeutic approaches, but the development of drugs that act specifically on molecules of interest remains challenging. Here we report the discovery and characterization of highly potent and selective small-molecule antagonists of the stimulator of interferon genes (STING) protein, which is a central signalling component of the intracellular DNA sensing pathway.