Identification and Characterization of IMD-0354 as a Glutamine Carrier Protein Inhibitor in Melanoma.

A key hallmark of cancer, altered metabolism, is central to cancer pathogenesis and therapy resistance. Robust glutamine metabolism is among cellular processes regulating tumor progression and responsiveness to therapy in a number of cancers, including melanoma and breast cancer. Among mechanisms underlying the increase in glutamine metabolism in tumors is enhanced glutamine uptake mediated by the glutamine transporters, with SLC1A5 (also known as ASCT2) shown to play a predominant role.

ERN1 and ALPK1 inhibit differentiation of bi-potential tumor-initiating cells in human breast cancer.

Cancers are heterogeneous by nature. While traditional oncology screens commonly use a single endpoint of cell viability, altering the phenotype of tumor-initiating cells may reveal alternative targets that regulate cellular growth by processes other than apoptosis or cell division. We evaluated the impact of knocking down expression of 420 kinases in bi-lineage triple-negative breast cancer (TNBC) cells that express characteristics of both myoepithelial and luminal cells.

Protein kinase A can block EphA2 receptor-mediated cell repulsion by increasing EphA2 S897 phosphorylation.

The EphA2 receptor tyrosine kinase plays key roles in tissue homeostasis and disease processes such as cancer, pathological angiogenesis, and inflammation through two distinct signaling mechanisms. EphA2 "canonical" signaling involves ephrin-A ligand binding, tyrosine autophosphorylation, and kinase activity; EphA2 "noncanonical" signaling involves phosphorylation of serine 897 (S897) by AKT and RSK kinases. To identify small molecules counteracting EphA2 canonical signaling, we developed a high-content screening platform measuring inhibition of ephrin-A1-induced PC3 prostate cancer cell retraction.

Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation.

Biogenesis of the primary cilium, a cellular organelle mediating various signaling pathways, is generally coordinated with cell cycle exit/re-entry. Although the dynamic cell cycle-associated profile of the primary cilium has been largely accepted, the mechanism governing the link between ciliogenesis and cell cycle progression has been poorly understood. Using a human genome-wide RNAi screen, we identify genes encoding subunits of the spliceosome and proteasome as novel regulators of ciliogenesis.

Cardiac Glycosides Activate the Tumor Suppressor and Viral Restriction Factor Promyelocytic Leukemia Protein (PML).

Cardiac glycosides (CGs), inhibitors of Na+/K+-ATPase (NKA), used clinically to treat heart failure, have garnered recent attention as potential anti-cancer and anti-viral agents. A high-throughput phenotypic screen designed to identify modulators of promyelocytic leukemia protein (PML) nuclear body (NB) formation revealed the CG gitoxigenin as a potent activator of PML. We demonstrate that multiple structurally distinct CGs activate the formation of PML NBs and induce PML protein SUMOylation in an NKA-dependent fashion.

Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.

Discovery of ML314, a Brain Penetrant Non-Peptidic β-Arrestin Biased Agonist of the Neurotensin NTR1 Receptor.

The neurotensin 1 receptor (NTR1) is an important therapeutic target for a range of disease states including addiction. A high throughput screening campaign, followed by medicinal chemistry optimization, led to the discovery of a non-peptidic β-arrestin biased agonist for NTR1. The lead compound, 2-cyclopropyl-6,7-dimethoxy-4-(4-(2-methoxyphenyl)- piperazin-1-yl)quinazoline, (), exhibits full agonist behavior against NTR1 (EC = 2.

Identification of the GPR55 antagonist binding site using a novel set of high-potency GPR55 selective ligands.

GPR55 is a class A G protein-coupled receptor (GPCR) that has been implicated in inflammatory pain, neuropathic pain, metabolic disorder, bone development, and cancer. Initially deorphanized as a cannabinoid receptor, GPR55 has been shown to be activated by non-cannabinoid ligands such as l-α-lysophosphatidylinositol (LPI). While there is a growing body of evidence of physiological and pathophysiological roles for GPR55, the paucity of specific antagonists has limited its study.

Identification of Inhibitors of triacylglyceride accumulation in muscle cells: comparing HTS results from 1536-well plate-based and high-content platforms.

Excess caloric consumption leads to triacylglyceride (TAG) accumulation in tissues that do not typically store fat, such as skeletal muscle. This ectopic accumulation alters cells, contributing to the pathogenesis of metabolic syndrome, a major health problem worldwide. We developed a 1536-well assay to measure intracellular TAG accumulation in differentiating H9c2 myoblasts.

Inhibition of melanoma growth by small molecules that promote the mitochondrial localization of ATF2.

Purpose: Effective therapy for malignant melanoma, the leading cause of death from skin cancer, remains an area of significant unmet need in oncology. The elevated expression of PKCε in advanced metastatic melanoma results in the increased phosphorylation of the transcription factor ATF2 on threonine 52, which causes its nuclear localization and confers its oncogenic activities. The nuclear-to-mitochondrial translocation of ATF2 following genotoxic stress promotes apoptosis, a function that is largely lost in melanoma cells, due to its confined nuclear localization.

Functional genomic and high-content screening for target discovery and deconvolution.

Introduction: Functional genomic screens apply knowledge gained from the sequencing of the human genome toward rapid methods of identifying genes involved in cellular function based on a specific phenotype. This approach has been made possible through advances in both molecular biology and automation. The utility of this approach has been further enhanced through the application of image-based high-content screening: an automated microscopy and quantitative image analysis platform.

Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy.

Herein we present the outcome of a high throughput screening (HTS) campaign-based strategy for the rapid identification and optimization of selective and general chemotypes for both kappa (κ) opioid receptor (KOR) activation and inhibition. In this program, we have developed potent antagonists (IC(50) < 120 nM) or agonists of high binding affinity (K(i) < 3 nM). In contrast to many important KOR ligands, the compounds presented here are highly modular, readily synthesized and, in most cases, achiral.

Optimization and application of median filter corrections to relieve diverse spatial patterns in microtiter plate data.

The standard (STD) 5 × 5 hybrid median filter (HMF) was previously described as a nonparametric local backestimator of spatially arrayed microtiter plate (MTP) data. As such, the HMF is a useful tool for mitigating global and sporadic systematic error in MTP data arrays. Presented here is the first known HMF correction of a primary screen suffering from systematic error best described as gradient vectors.

A cell-based high-content screening assay reveals activators and inhibitors of cancer cell invasion.

Acquisition of invasive cell behavior underlies tumor progression and metastasis. To further define the molecular mechanisms underlying invasive behavior, we developed a high-throughput screening strategy to quantitate invadopodia, which are actin-rich membrane protrusions of cancer cells that contribute to tissue invasion and matrix remodeling. We tested the LOPAC 1280 collection of pharmacologically active agents in a high-content, image-based assay and identified compounds that inhibited invadopodium formation without overt toxicity, as well as compounds that increased invadopodia number.

Identification of the GPR55 agonist binding site using a novel set of high-potency GPR55 selective ligands.

Marijuana is the most widely abused illegal drug, and its spectrum of effects suggests that several receptors are responsible for the activity. Two cannabinoid receptor subtypes, CB1 and CB2, have been identified, but the complex pharmacological properties of exogenous cannabinoids and endocannabinoids are not fully explained by their signaling. The orphan receptor GPR55 binds a subset of CB1 and CB2 ligands and has been proposed as a cannabinoid receptor.

A high-content screening (HCS) assay for the identification of chemical inducers of PML oncogenic domains (PODs).

PML is a multi-functional protein with roles in tumor suppression and host defense against viruses. When active, PML localizes to subnuclear structures named PML oncogenic domains (PODs) or PML nuclear bodies (PML-NBs), whereas inactive PML is located diffusely throughout the nucleus of cells. The objective of the current study was to develop a high content screening (HCS) assay for the identification of chemical activators of PML.