A novel high-throughput screening strategy for targeting alpha-synuclein and other long-lived proteins.

Small-molecule high-throughput screening (HTS) campaigns have frequently been used to identify lead molecules that can alter expression of disease-relevant proteins in cell-based assays. However, most cell-based HTS assays require short compound exposure periods to avoid toxicity and ensure that compounds are stable in media for the duration of the exposure. This limits the ability of HTS assays to detect inhibitors of the synthesis of target proteins with long half-lives, which can often exceed the exposure times utilized in most HTS campaigns.

Cereblon Modulators Target ZBTB16 and Its Oncogenic Fusion Partners for Degradation via Distinct Structural Degrons.

There is a growing interest in using targeted protein degradation as a therapeutic modality in view of its potential to expand the druggable proteome. One avenue to using this modality is via molecular glue based Cereblon E3 Ligase Modulating Drug compounds. Here, we report the identification of the transcription factor ZBTB16 as a Cereblon neosubstrate.

Metabolic Enzyme Sulfotransferase 1A1 Is the Trigger for N-Benzyl Indole Carbinol Tumor Growth Suppression.

In an attempt to identify novel therapeutics and mechanisms to differentially kill tumor cells using phenotypic screening, we identified N-benzyl indole carbinols (N-BICs), synthetic analogs of the natural product indole-3-carbinol (I3C). To understand the mode of action for the molecules we employed Cancer Cell Line Encyclopedia viability profiling and correlative informatics analysis to identify and ultimately confirm the phase II metabolic enzyme sulfotransferase 1A1 (SULT1A1) as the essential factor for compound selectivity. Further studies demonstrate that SULT1A1 activates the N-BICs by rendering the compounds strong electrophiles which can alkylate cellular proteins and thereby induce cell death.

TMEM203 Is a Novel Regulator of Intracellular Calcium Homeostasis and Is Required for Spermatogenesis.

Intracellular calcium signaling is critical for initiating and sustaining diverse cellular functions including transcription, synaptic signaling, muscle contraction, apoptosis and fertilization. Trans-membrane 203 (TMEM203) was identified here in cDNA overexpression screens for proteins capable of modulating intracellular calcium levels using activation of a calcium/calcineurin regulated transcription factor as an indicator. Overexpression of TMEM203 resulted in a reduction of Endoplasmic Reticulum (ER) calcium stores and elevation in basal cytoplasmic calcium levels.

Selective VPS34 inhibitor blocks autophagy and uncovers a role for NCOA4 in ferritin degradation and iron homeostasis in vivo.

Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα.

PIKfyve, a class III PI kinase, is the target of the small molecular IL-12/IL-23 inhibitor apilimod and a player in Toll-like receptor signaling.

Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool.

A hypomorphic lsd1 allele results in heart development defects in mice.

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown.

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling.

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown.

Defective heart development in hypomorphic LSD1 mice.

Lysine-specific demethylase 1 (LSD1/AOF2/KDM1A), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. LSD1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that LSD1-interacting proteins regulate the activity and specificity of LSD1, the significance and roles of such interactions in developmental processes remain largely unknown.

Protein kinase D3 sensitizes RAF inhibitor RAF265 in melanoma cells by preventing reactivation of MAPK signaling.

RAS mutations occur in more than 30% of all human cancers but efforts to directly target mutant RAS signaling as a cancer therapy have yet to succeed. As alternative strategies, RAF and MEK inhibitors have been developed to block oncogenic signaling downstream of RAS. As might be expected, studies of these inhibitors have indicated that tumors with RAS or BRAF mutations display resistance RAF or MEK inhibitors.

The tyrosine kinase BMX is an essential mediator of inflammatory arthritis in a kinase-independent manner.

Inflammatory cytokines like TNF play a central role in autoimmune disorders such as rheumatoid arthritis. We identified the tyrosine kinase bone marrow kinase on chromosome X (BMX) as an essential component of a shared inflammatory signaling pathway. Transient depletion of BMX strongly reduced secretion of IL-8 in cell lines and primary human cells stimulated by TNF, IL-1β, or TLR agonists.

KMT1E mediated H3K9 methylation is required for the maintenance of embryonic stem cells by repressing trophectoderm differentiation.

Dynamic regulation of histone methylation by methyltransferases and demethylases plays a central role in regulating the fate of embryonic stem (ES) cells. The histone H3K9 methyltransferase KMT1E, formerly known as ESET or Setdb1, is essential to embryonic development as the ablation of the Setdb1 gene results in peri-implantation lethality and prevents the propagation of ES cells. However, Setdb1-null blastocysts do not display global changes in H3K9 methylation or DNA methylation, arguing against a genome-wide defect.

Class III phosphatidylinositol 4-kinase alpha and beta are novel host factor regulators of hepatitis C virus replication.

Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase.

Cholesterol biosynthesis modulation regulates dengue viral replication.

We performed a focused siRNA screen in an A549 dengue type 2 New Guinea C subgenomic replicon cell line (Rluc-replicon) that contains a Renilla luciferase cassette. We found that siRNA mediated knock down of mevalonate diphospho decarboxylase (MVD) inhibited viral replication of the Rluc-replicon and DEN-2 NGC live virus replication in A549 cells. When the Rluc-replicon A459 cells were grown in delipidated media the replicon expression was suppressed and MVD knock down could further sensitize Renilla expression.

Identification of novel genes and pathways regulating SREBP transcriptional activity.

Background: Lipid metabolism in mammals is orchestrated by a family of transcription factors called sterol regulatory element-binding proteins (SREBPs) that control the expression of genes required for the uptake and synthesis of cholesterol, fatty acids, and triglycerides. SREBPs are thus essential for insulin-induced lipogenesis and for cellular membrane homeostasis and biogenesis. Although multiple players have been identified that control the expression and activation of SREBPs, gaps remain in our understanding of how SREBPs are coordinated with other physiological pathways.

Role for Med12 in regulation of Nanog and Nanog target genes.

Nanog, Oct4, and Sox2 form the core of a transcription factor network that maintains embryonic stem cells in the pluripotent state in both humans and mice. These critical factors have been implicated as both positive and negative regulators of transcription, varying by promoter and differentiation state of the cell. The Mediator complex, a ubiquitous conserved complex of approximately 30 subunits, facilitates transcription by coordinating RNA polymerase II binding to target promoters via gene-specific activators and can be divided into several functional subcomplexes.

A Smac mimetic rescue screen reveals roles for inhibitor of apoptosis proteins in tumor necrosis factor-alpha signaling.

Smac mimetic compounds targeting the inhibitor of apoptosis proteins (IAP) baculoviral IAP repeat-3 domain are presumed to reduce the threshold for apoptotic cell death by alleviating caspase-9 repression. We explored this tenet in an unbiased manner by searching for small interfering RNAs that are able to confer resistance to the Smac mimetic compound LBW242. Among the screening hits were multiple components of the tumor necrosis factor alpha (TNFalpha) signaling pathway as well as X-linked inhibitor of apoptosis (XIAP) itself.

Integrating high-content screening and ligand-target prediction to identify mechanism of action.

High-content screening is transforming drug discovery by enabling simultaneous measurement of multiple features of cellular phenotype that are relevant to therapeutic and toxic activities of compounds. High-content screening studies typically generate immense datasets of image-based phenotypic information, and how best to mine relevant phenotypic data is an unsolved challenge. Here, we introduce factor analysis as a data-driven tool for defining cell phenotypes and profiling compound activities.

Optimization procedure for small interfering RNA transfection in a 384-well format.

High-throughput screening of RNAi libraries has become an essential part of functional analysis in academic and industrial settings. The transition of a cell-based RNAi assay into a 384-well format requires several optimization steps to ensure the phenotype being screened is appropriately measured and that the signal-to-background ratio is above a certain quantifiable threshold. Methods currently used to assess small interfering RNA (siRNA) efficacy after transfection, including quantitative PCR or branch DNA analysis, face several technical limitations preventing the accurate measurement of mRNA levels in a 384-well format.

Genome-wide functional analysis of human cell-cycle regulators.

Human cells have evolved complex signaling networks to coordinate the cell cycle. A detailed understanding of the global regulation of this fundamental process requires comprehensive identification of the genes and pathways involved in the various stages of cell-cycle progression. To this end, we report a genome-wide analysis of the human cell cycle, cell size, and proliferation by targeting >95% of the protein-coding genes in the human genome using small interfering RNAs (siRNAs).

Transducer of regulated CREB-binding proteins (TORCs) induce PGC-1alpha transcription and mitochondrial biogenesis in muscle cells.

PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator 1alpha) is a master regulator of mitochondrial biogenesis and plays an important role in several other aspects of energy metabolism. To identify upstream regulators of PGC-1alpha gene transcription, 10,000 human full-length cDNAs were screened for induction of the PGC-1alpha promoter. A number of activators of PGC-1alpha transcription were found; the most potent activator was the transducer of regulated CREB (cAMP response element-binding protein) binding protein (TORC) 1, a coactivator of CREB.

The application of systems biology to drug discovery.

Recent advances in the 'omics' technologies, scientific computing and mathematical modeling of biological processes have started to fundamentally impact the way we approach drug discovery. Recent years have witnessed the development of genome-scale functional screens, large collections of reagents, protein microarrays, databases and algorithms for data and text mining. Taken together, they enable the unprecedented descriptions of complex biological systems, which are testable by mathematical modeling and simulation.

A quantitative high-throughput endothelial cell migration assay.

By combining the use of BD Biosciences FluoroBlok membrane-based Boyden chambers with the Cellomics HCS ArrayScan, a more sensitive method for measuring cell migration has been developed. This assay is based on counting nuclei of migrated cells on the bottom of the filter rather than conventional approaches, which use measurement of total well fluorescence. This cell migration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor-mediated endothelial cell migration.

Design of a genome-wide siRNA library using an artificial neural network.

The largest gene knock-down experiments performed to date have used multiple short interfering/short hairpin (si/sh)RNAs per gene. To overcome this burden for design of a genome-wide siRNA library, we used the Stuttgart Neural Net Simulator to train algorithms on a data set of 2,182 randomly selected siRNAs targeted to 34 mRNA species, assayed through a high-throughput fluorescent reporter gene system. The algorithm, (BIOPREDsi), reliably predicted activity of 249 siRNAs of an independent test set (Pearson coefficient r = 0.