Translational homeostasis via the mRNA cap-binding protein, eIF4E.

Translational control of gene expression plays a key role in many biological processes. Consequently, the activity of the translation apparatus is under tight homeostatic control. eIF4E, the mRNA 5' cap-binding protein, facilitates cap-dependent translation and is a major target for translational control. eIF4E activity is controlled by a family of repressor proteins, termed 4E-binding proteins (4E-BPs). Here, we describe the surprising finding that despite the importance of eIF4E for translation, a drastic knockdown of eIF4E caused only minor reduction in translation. This conundrum can be explained by the finding that 4E-BP1 is degraded in eIF4E-knockdown cells. Hypophosphorylated 4E-BP1, which binds to eIF4E, is degraded, whereas hyperphosphorylated 4E-BP1 is refractory to degradation. We identified the KLHL25-CUL3 complex as the E3 ubiquitin ligase, which targets hypophosphorylated 4E-BP1. Thus, the activity of eIF4E is under homeostatic control via the regulation of the levels of its repressor protein 4E-BP1 through ubiquitination.

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11.

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.

Functional genomic screen for modulators of ciliogenesis and cilium length.

Primary cilia are evolutionarily conserved cellular organelles that organize diverse signalling pathways. Defects in the formation or function of primary cilia are associated with a spectrum of human diseases and developmental abnormalities. Genetic screens in model organisms have discovered core machineries of cilium assembly and maintenance. However, regulatory molecules that coordinate the biogenesis of primary cilia with other cellular processes, including cytoskeletal organization, vesicle trafficking and cell-cell adhesion, remain to be identified. Here we report the results of a functional genomic screen using RNA interference (RNAi) to identify human genes involved in ciliogenesis control. The screen identified 36 positive and 13 negative ciliogenesis modulators, which include molecules involved in actin dynamics and vesicle trafficking. Further investigation demonstrated that blocking actin assembly facilitates ciliogenesis by stabilizing the pericentrosomal preciliary compartment (PPC), a previously uncharacterized compact vesiculotubular structure storing transmembrane proteins destined for cilia during the early phase of ciliogenesis. The PPC was labelled by recycling endosome markers. Moreover, knockdown of modulators that are involved in the endocytic recycling pathway affected the formation of the PPC as well as ciliogenesis. Our results uncover a critical regulatory step that couples actin dynamics and endocytic recycling with ciliogenesis, and also provides potential target molecules for future study.

Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor.

The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ~330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 µM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.

Development and use of a high-throughput screen to identify novel modulators of the corticotropin releasing factor binding protein.

BACKGROUND: Stress responses are believed to involve corticotropin releasing factor (CRF), its two cognate receptors (CRF1 and CRF2), and the CRF-binding protein (CRFBP). Whereas decades of research has focused on CRF1, the role of CRF2 in the central nervous system (CNS) has not been thoroughly investigated. We have previously reported that CRF2, interacting with a C terminal fragment of CRFBP, CRFBP(10kD), may have a role in the modulation of neuronal activity. However, the mechanism by which CRF interacts with CRFBP(10kD) and CRF2 has not been fully elucidated due to the lack of useful chemical tools to probe CRFBP. METHODS: We miniaturized a cell-based assay, where CRFBP(10kD) is fused as a chimera with CRF2, and performed a high-throughput screen (HTS) of 350,000 small molecules to find negative allosteric modulators (NAMs) of the CRFBP(10kD)-CRF2 complex. Hits were confirmed by evaluating activity toward parental HEK293 cells, toward CRF2 in the absence of CRFBP(10kD), and toward CRF1 in vitro. Hits were further characterized in ex vivo electrophysiology assays that target: 1) the CRF1+ neurons in the central nucleus of the amygdala (CeA) of CRF1:GFP mice that express GFP under the CRF1 promoter, and 2) the CRF-induced potentiation of N-methyl-D-aspartic acid receptor (NMDAR)-mediated synaptic transmission in dopamine neurons in the ventral tegmental area (VTA). RESULTS: We found that CRFBP(10kD) potentiates CRF-intracellular Ca2+ release specifically via CRF2, indicating that CRFBP may possess excitatory roles in addition to the inhibitory role established by the N-terminal fragment of CRFBP, CRFBP(27kD). We identified novel small molecule CRFBP-CRF2 NAMs that do not alter the CRF1-mediated effects of exogenous CRF but blunt CRF-induced potentiation of NMDAR-mediated synaptic transmission in dopamine neurons in the VTA, an effect mediated by CRF2 and CRFBP. CONCLUSION: These results provide the first evidence of specific roles for CRF2 and CRFBP(10kD) in the modulation of neuronal activity and suggest that CRFBP(10kD)-CRF2 NAMs can be further developed for the treatment of stress-related disorders including alcohol and substance use disorders.

High-throughput screening based identification of small molecule antagonists of integrin CD11b/CD18 ligand binding.

Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high-throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.

Identification of genes that function in the TNF-alpha-mediated apoptotic pathway using randomized hybrid ribozyme libraries.

Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-alpha (TNF-alpha)-mediated pathway of apoptosis, using RNA helicase-associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-alpha-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-alpha-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.

siRNAs generated by recombinant human Dicer induce specific and significant but target site-independent gene silencing in human cells.

RNA interference has emerged as a powerful tool for the silencing of gene expression in animals and plants. It was reported recently that 21 nt synthetic small interfering RNAs (siRNAs) specifically suppressed the expression of endogenous genes in several lines of mammalian cells. However, the efficacy of siRNAs is dependent on the presence of a specific target site within the target mRNA and it remains very difficult to predict the best or most effective target site. In this study, we demonstrate that siRNAs that have been generated in vitro by recombinant human Dicer (re-hDicer) significantly suppress not only the exogenous expression of a puromycin-resistance gene but also the endogenous expression of H-ras, c-jun and c-fos. In our system, selection of a target site is not necessary in the design of siRNAs. However, it is important to avoid homologous sequences within a target mRNA in a given protein family. Our diced siRNA system should be a powerful tool for the inactivation of genes in mammalian cells.

Identification of genes responsible for cell migration by a library of randomized ribozymes.

Several genes appear to be associated with metastasis, but the underlying mechanisms of metastasis still remain unclear. In this study, we used a library of randomized ribozymes to identify, by inactivation of transcripts, genes involved in cell migration that is an essential aspect of metastasis. Using a chemotaxis assay, the ribozymes that inhibited cell migration were selected from the library. Among such ribozymes, we found two ribozymes that targeted and cleaved ROCK1 mRNA at independent sites. ROCK1 and ROCK2 are Rho kinases, and it has been demonstrated that they regulate the organization of the actin cytoskeleton and are responsible for cell motility and cytokinesis. The two ribozymes that specifically cleaved ROCK1 mRNA inhibited both the migration and invasion of invasive HT1080 fibrosarcoma, but neither had any effect on cell proliferation. Our analysis indicates that the ribozymes toward ROCK1 can block invasive activity but not the proliferation of HT1080 cells without having any effect on expression of ROCK2. Ribozymes identified in this study, including the ribozymes against ROCK1, might be useful in understanding the mechanisms of cell migration and metastasis.

Identification of a caspase 3-independent role of pro-apoptotic factor Bak in TNF-alpha-induced apoptosis.

By using our recently developed gene discovery system, we have identified Bak, a member of the Bcl-2 family, as a pro-apoptotic factor in the tumor necrosis factor (TNF)-alpha-induced apoptotic pathway in caspase 3-deficient cells. Unlike Bcl-2, Bak stimulates several apoptotic pathways, however the molecular mechanism(s) of its action remains unclear. For example, it is unclear whether Bak induces apoptosis in caspase 3-deficient cells. In this study, we examined the effects of overexpression of Bak in MCF-7 cells that lack caspase 3. We found that despite the absence of caspase 3 in MCF-7 cells, they were more sensitive to the cell death effects of Bak as compared to caspase 3-expressing HeLa S3 cells. The targeting of Bak function by ribozymes suggests that Bak is required for the TNF-alpha-induced apoptotic pathway in caspase 3-deficient cells. This study demonstrates the caspase 3-independent function of Bak in the TNF-alpha-induced apoptotic pathway.

A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro.

Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the N-terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.

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. For this assay, cells were incubated with oleic acid to stimulate TAG accumulation prior to adding compounds. We used Nile red as a fluorescent dye to quantify TAG content with a microplate reader. The cell nuclei were counterstained with DAPI nuclear stain to assess cell count and filter cytotoxic compounds. In parallel, we developed an image-based assay in H9c2 cells to measure lipid accumulation levels via high-content analysis, exploiting the dual-emission spectra characteristic of Nile red staining of neutral and phospholipids. Using both approaches, we successfully screened ~227,000 compounds from the National Institutes of Health library. The screening data from the plate reader and IC50 values correlated with that from the Opera QEHS cell imager. The 1536-well plate reader assay is a powerful high-throughout screening platform to identify potent inhibitors of TAG accumulation to better understand the molecular pathways involved in lipid metabolism that lead to lipotoxicity.

Discovery of ML314, a Brain Penetrant Non-Peptidic ß-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 ß-arrestin biased agonist for NTR1. The lead compound, 2-cyclopropyl-6,7-dimethoxy-4-(4-(2-methoxyphenyl)- piperazin-1-yl)quinazoline, 32 (ML314), exhibits full agonist behavior against NTR1 (EC50 = 2.0 µM) in the primary assay and selectivity against NTR2. The effect of 32 is blocked by the NTR1 antagonist SR142948A in a dose dependent manner. Unlike peptide based NTR1 agonists, compound 32 has no significant response in a Ca2+ mobilization assay and is thus a biased agonist that activates the ß-arrestin pathway rather than the traditional G q coupled pathway. This bias has distinct biochemical and functional consequences that may lead to physiological advantages. Compound 32 displays good brain penetration in rodents, and studies examining its in vivo properties are underway.

Discovery of a Plasmodium falciparum glucose-6-phosphate dehydrogenase 6-phosphogluconolactonase inhibitor (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) that reduces parasite growth in vitro.

A high-throughput screen of the NIH's MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum , the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (PfGluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11 (ML276), is a submicromolar inhibitor of PfG6PD (IC(50) = 889 nM). It is completely selective for the enzyme's human isoform, displays micromolar potency (IC(50) = 2.6 µM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting PfG6PD in vivo are in progress.

Identification of Inhibitors of NOD1-Induced Nuclear Factor-κB Activation.

NOD1 (nucleotide-binding oligomerization domain 1) protein is a member of the NLR (NACHT and leucine rich repeat domain containing proteins) protein family, which plays a key role in innate immunity as a sensor of specific microbial components derived from bacterial peptidoglycans and induction of inflammatory responses. Mutations in NOD proteins have been associated with various inflammatory diseases that affect NF-κB (nuclear factor κB) activity, a major signaling pathway involved in apoptosis, inflammation, and immune response. A luciferase-based reporter gene assay was utilized in a high-throughput screening program conducted under the NIH-sponsored Molecular Libraries Probe Production Center Network program to identify the active scaffolds. Herein, we report the chemical synthesis, structure-activity relationship studies, downstream counterscreens, secondary assay data, and pharmacological profiling of the 2-aminobenzimidazole lead (compound 1c, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation.

Identification of MicroRNAs that control lipid droplet formation and growth in hepatocytes via high-content screening.

Hepatic lipid droplets (LDs) are associated with metabolic syndrome, type 2 diabetes, hepatitis C, and both alcoholic and nonalcoholic fatty liver disease. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the level of translation. Approximately 1000 different miRNA species are encoded within the human genome, and many are differentially expressed by healthy and diseased liver. However, few studies have investigated the role of miRNAs in regulating LD expression. Accordingly, a high-content assay (HCA) was performed in which human hepatocytes (Huh-7 cells) were transiently transfected with 327 unique human miRNAs; the cells were then fixed, labeled for nuclei and lipid droplets, and imaged with an automated digital microscopy workstation. LD expression was analyzed on a cell-by-cell basis, using automated image analysis. Eleven miRNAs were identified that altered LDs. MiR-181d was the most efficacious inhibitor, decreasing LDs by about 60%. miRNA-181d was also confirmed to reduce cellular triglycerides and cholesterol ester via biochemical assays. Furthermore, a series of proteins was identified via miRNA target analysis, and siRNAs directed against many of these proteins also modified LDs. Thus, HCA-based screening identified novel miRNA and protein regulators of LDs and cholesterol metabolism that may be relevant to hepatic diseases arising from obesity and alcohol abuse.