Lung tumor microphysiological system with 3D endothelium to evaluate modulators of T-cell migration.

Lung cancer is a leading cause of death worldwide, with only a fraction of patients responding to immunotherapy. The correlation between increased T-cell infiltration and positive patient outcomes has motivated the search for therapeutics promoting T-cell infiltration. While transwell and spheroid platforms have been employed, these models lack flow and endothelial barriers, and cannot faithfully model T-cell adhesion, extravasation, and migration through 3D tissue.

A Comprehensive Phenotypic Screening Strategy to Identify Modulators of Cargo Translocation by the Bacterial Type IVB Secretion System.

Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.

The HIV protease inhibitor, ritonavir, corrects diverse brain phenotypes across development in mouse model of DYT-TOR1A dystonia.

Dystonias are a group of chronic movement-disabling disorders for which highly effective oral medications or disease-modifying therapies are lacking. The most effective treatments require invasive procedures such as deep brain stimulation. In this study, we used a high-throughput assay based on a monogenic form of dystonia, DYT1 (DYT-TOR1A), to screen a library of compounds approved for use in humans, the NCATS Pharmaceutical Collection (NPC; 2816 compounds), and identify drugs able to correct mislocalization of the disease-causing protein variant, ∆E302/3 hTorsinA.

Compound screening in cell-based models of tau inclusion formation: Comparison of primary neuron and HEK293 cell assays.

The hallmark pathological features of Alzheimer's disease (AD) brains are senile plaques, comprising β-amyloid (Aβ) peptides, and neuronal inclusions formed from tau protein. These plaques form 10-20 years before AD symptom onset, whereas robust tau pathology is more closely associated with symptoms and correlates with cognitive status. This temporal sequence of AD pathology development, coupled with repeated clinical failures of Aβ-directed drugs, suggests that molecules that reduce tau inclusions have therapeutic potential.

A cross-species whole genome siRNA screen in suspension-cultured Chinese hamster ovary cells identifies novel engineering targets.

High-throughput siRNA screens were only recently applied to cell factories to identify novel engineering targets which are able to boost cells towards desired phenotypes. While siRNA libraries exist for model organisms such as mice, no CHO-specific library is publicly available, hindering the application of this technique to CHO cells. The optimization of these cells is of special interest, as they are the main host for the production of therapeutic proteins.

High-Throughput Zika Viral Titer Assay for Rapid Screening of Antiviral Drugs.

Zika virus has recently emerged as a worldwide pathogen and public health burden due to its rapid spread and identification as a causative agent for multiple neurological defects, including congenital microcephaly. While there has been a flurry of recent research to address this emerging pathogen, there are currently no approved drug treatments for ZIKV infection. The gold standard for testing antiviral activity is to quantify infectious virion production.

Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space.

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays.

A high-throughput imaging and nuclear segmentation analysis protocol for cleared 3D culture models.

Imaging and subsequent segmentation analysis in three-dimensional (3D) culture models are complicated by the light scattering that occurs when collecting fluorescent signal through multiple cell and extracellular matrix layers. For 3D cell culture models to be usable for drug discovery, effective and efficient imaging and analysis protocols need to be developed that enable high-throughput data acquisition and quantitative analysis of fluorescent signal. Here we report the first high-throughput protocol for optical clearing of spheroids, fluorescent high-content confocal imaging, 3D nuclear segmentation, and post-segmentation analysis.

Correction: A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity.

[This corrects the article DOI: 10.1371/journal.pone.

Connecting Neuronal Cell Protective Pathways and Drug Combinations in a Huntington's Disease Model through the Application of Quantitative Systems Pharmacology.

Quantitative Systems Pharmacology (QSP) is a drug discovery approach that integrates computational and experimental methods in an iterative way to gain a comprehensive, unbiased understanding of disease processes to inform effective therapeutic strategies. We report the implementation of QSP to Huntington's Disease, with the application of a chemogenomics platform to identify strategies to protect neuronal cells from mutant huntingtin induced death. Using the STHdh cell model, we investigated the protective effects of small molecule probes having diverse canonical modes-of-action to infer pathways of neuronal cell protection connected to drug mechanism.

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons.

Assessing inhibitors of mutant isocitrate dehydrogenase using a suite of pre-clinical discovery assays.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that are mutated in a variety of cancers to confer a gain-of-function activity resulting in the accumulation of an oncometabolite, D-2-hydroxyglutarate (2-HG). Accumulation of 2-HG can result in epigenetic dysregulation and a block in cellular differentiation, suggesting these mutations play a role in neoplasia. Based on its potential as a cancer target, a number of small molecule inhibitors have been developed to specifically inhibit mutant forms of IDH (mIDH1 and mIDH2).

Exploring Drug Dosing Regimens In Vitro Using Real-Time 3D Spheroid Tumor Growth Assays.

Two-dimensional monolayer cell proliferation assays for cancer drug discovery have made the implementation of large-scale screens feasible but only seem to reflect a simplified view that oncogenes or tumor suppressor genes are the genetic drivers of cancer cell proliferation. However, there is now increased evidence that the cellular and physiological context in which these oncogenic events occur play a key role in how they drive tumor growth in vivo and, therefore, in how tumors respond to drug treatments. In vitro 3D spheroid tumor models are being developed to better mimic the physiology of tumors in vivo, in an attempt to improve the predictability and efficiency of drug discovery for the treatment of cancer.

Cellular and molecular defects in a patient with Hermansky-Pudlak syndrome type 5.

Hermansky-Pudlak syndrome (HPS) is a heterogeneous group of genetic disorders typically manifesting with tyrosinase-positive oculocutaneous albinism, bleeding diathesis, and pulmonary fibrosis, in some subtypes. Most HPS subtypes are associated with defects in Biogenesis of Lysosome-related Organelle Complexes (BLOCs), which are groups of proteins that function together in the formation and/or trafficking of lysosomal-related endosomal compartments. BLOC-2, for example, consists of the proteins HPS3, HPS5, and HPS6.

RNAi High-Throughput Screening of Single- and Multi-Cell-Type Tumor Spheroids: A Comprehensive Analysis in Two and Three Dimensions.

The widespread use of two-dimensional (2D) monolayer cultures for high-throughput screening (HTS) to identify targets in drug discovery has led to attrition in the number of drug targets being validated. Solid tumors are complex, aberrantly growing microenvironments that harness structural components from stroma, nutrients fed through vasculature, and immunosuppressive factors. Increasing evidence of stromally-derived signaling broadens the complexity of our understanding of the tumor microenvironment while stressing the importance of developing better models that reflect these interactions.

A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity.

Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay.

High-throughput fluorescence imaging approaches for drug discovery using in vitro and in vivo three-dimensional models.

Introduction: High-resolution microscopy using fluorescent probes is a powerful tool to investigate individual cell structure and function, cell subpopulations and mechanisms underlying cellular responses to drugs. Additionally, responses to drugs more closely resemble those seen in vivo when cells are physically connected in three-dimensional (3D) systems (either 3D cell cultures or whole organisms), as opposed to traditional monolayer cultures. Combined, the use of imaging-based 3D models in the early stages of drug development has the potential to generate biologically relevant data that will increase the likelihood of success for drug candidates in human studies.

Mammalian mitochondrial and cytosolic folylpolyglutamate synthetase maintain the subcellular compartmentalization of folates.

Folylpoly-γ-glutamate synthetase (FPGS) catalyze the addition of multiple glutamates to tetrahydrofolate derivatives. Two mRNAs for the fpgs gene direct isoforms of FPGS to the cytosol and to mitochondria in mouse and human tissues. We sought to clarify the functions of these two compartmentalized isoforms.

High-Throughput Multiplexed Quantitation of Protein Aggregation and Cytotoxicity in a Huntington's Disease Model.

A hallmark of Huntington's disease is the presence of a large polyglutamine expansion in the first exon of the Huntingtin protein and the propensity of protein aggregation by the mutant proteins. Aberrant protein aggregation also occurs in other polyglutamine expansion disorders, as well as in other neurodegenerative diseases including Parkinson's, Alzheimer's, and prion diseases. However, the pathophysiological role of these aggregates in the cell death that characterizes the diseases remains unclear.

Identification of quaternary ammonium compounds as potent inhibitors of hERG potassium channels.

The human ether-a-go-go-related gene (hERG) channel, a member of a family of voltage-gated potassium (K(+)) channels, plays a critical role in the repolarization of the cardiac action potential. The reduction of hERG channel activity as a result of adverse drug effects or genetic mutations may cause QT interval prolongation and potentially leads to acquired long QT syndrome. Thus, screening for hERG channel activity is important in drug development.

Automated high-content screening for compounds that disassemble the perinucleolar compartment.

All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells.

A new homogeneous high-throughput screening assay for profiling compound activity on the human ether-a-go-go-related gene channel.

Long QT syndrome, either inherited or acquired from drug treatments, can result in ventricular arrhythmia (torsade de pointes) and sudden death. Human ether-a-go-go-related gene (hERG) channel inhibition by drugs is now recognized as a common reason for the acquired form of long QT syndrome. It has been reported that more than 100 known drugs inhibit the activity of the hERG channel.

Exploration and optimization of substituted triazolothiadiazines and triazolopyridazines as PDE4 inhibitors.

An expansion of structure-activity studies on a series of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine PDE4 inhibitors and the introduction of a related [1,2,4]triazolo[4,3-b]pyridazine based inhibitor of PDE4 is presented. The development of SAR included strategic incorporation of known substituents on the critical catachol diether moiety of the 6-phenyl appendage on each heterocyclic core. From these studies, (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (10) and (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-[1,2,4]triazolo[4,3-b]pyridazine (18) were identified as highly potent PDE4A inhibitors.

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening.

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease.

Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.

The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame.