Genome-wide siRNA screen identifies the retromer as a cellular entry factor for human papillomavirus.

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.

Small-molecule modulators of the NF-kappaB pathway newly identified by a translocation-based cellular assay.

Nuclear factor kappa B (NF-kappaB) is an important transcription factor. Aberrant regulation of the NF-kappaB pathway is frequently observed in a number of major ailments such as cancer and inflammatory diseases. Hence NF-kappaB modulators have been intensely pursued for their potential therapeutic applications. Numerous reviews have described recent progress in the development of these agents. More recently, a variety of structurally and functionally novel small molecules, identified through high-throughput screens conducted within the Molecular Libraries Screening Center Network (MLSCN) of the NIH Roadmap for Medical Research, have been added to the current list of NF-kappaB regulators. This review will discuss the inhibitors and activators newly discovered by Columbia's Molecular Libraries Screening Center (MLSC) using a well-designed and stable cellular assay.

Cell-based assays to probe the ERK MAP kinase pathway in endothelial cells.

To understand signaling pathways in mammalian cells, cell-based assays are relatively new and extremely powerful tools. We have developed a battery of phenotypic assays to study signaling; two of them are described in detail in this chapter. A subset of these assays monitors mitogen-activated protein (MAP) kinase pathways. MAP kinases are principal regulators of fundamental processes in mammalian cells, including growth, cell division, differentiation, stress responses, and neoplastic transformation. Here we describe two cell-based assays querying the function of ERK (extracellular signal regulated kinase), one of the three principal MAP kinases in mammalian cells. We selected human umbilical vein endothelial cells (HUVECs), a primary cell type, because they show a very dynamic response to various activators. Both assays are phenotypic assays and use well-established phosphorylation-specific primary antibodies to study activation. Fluorochrome-coupled secondary antibodies were used to label phosphorylated target proteins; images were captured with the INCell Analyzer 3000 and analyzed with the INCell Analyzer 3000 software. The first of these two assays monitors phosphorylation of ERK1/2, while the second assay monitors activation of the transcription factor CREB (cAMP response element-binding protein). The assays described in this chapter cover major checkpoints of the ERK signaling pathway: (1) MAP kinase activation and (2) subsequent transcription factor activation. Both assays exhibit robust performance and can easily be used for high-throughput screening.

Discovery of novel small molecule cell type-specific enhancers of NF-kappaB nuclear translocation.

An IKKbeta inhibitor reported to block NF-kappaB transcriptional activities in Jurkat T cells, was found to enhance NF-kappaB translocation in HUVEC cells. These studies suggested a noncanonical NF-kappaB signaling pathway independent of IKKbeta in HUVEC cells.

Identification of N-(quinolin-8-yl)benzenesulfonamides as agents capable of down-regulating NFkappaB activity within two separate high-throughput screens of NFkappaB activation.

We describe here a series of N-(quinolin-8-yl)benzenesulfonamides capable of suppressing the NFkappaB pathway identified from two high-throughput screens run at two centers of the NIH Molecular Libraries Initiative. These small molecules were confirmed in both primary and secondary assays of NFkappaB activation and expanded upon through analogue synthesis. The series exhibited potencies in the cell-based assays at as low as 0.6 microM, and several indications suggest that the targeted activity lies within a common region of the NFkappaB pathway.

Cell-based assays using primary endothelial cells to study multiple steps in inflammation.

Cell-based assays are powerful tools for drug discovery and provide insight into complex signal transduction pathways in higher eukaryotic cells. Information gleaned from assays that monitor a cellular phenotype can be used to elucidate the details of a single pathway and to establish patterns of cross talk between pathways. By selecting the appropriate cell model, cell-based assays can be used to understand the function of a specific cell type in a complex disease process such as inflammation. We have used human umbilical vein endothelial cells to establish three cell-based, phenotypic assays that query different stages of a major signaling pathway activated in inflammation. One assay analyzes the tumor necrosis factor alpha (TNFalpha)-induced translocation of the transcription factor NF-kappaB from the cytoplasm into the nucleus 20 min after stimulation with TNFalpha. Two more assays monitor the expression of E-selectin and VCAM-1, 4 and 24 h after stimulation with TNFalpha. Indirect immunofluorescence and high-throughput automated microscopy were used to analyze cells. Imaging was performed with the IN Cell Analyzer 3000. All assays proved to be highly robust. Z' values between 0.7 and 0.8 make each of the three assays well suited for use in high-throughput screening for drug or probe discovery.

Analysis of mouse embryonic patterning and morphogenesis by forward genetics.

Many aspects of the genetic control of mammalian embryogenesis cannot be extrapolated from other animals. Taking a forward genetic approach, we have induced recessive mutations by treatment of mice with ethylnitrosourea and have identified 43 mutations that affect early morphogenesis and patterning, including 38 genes that have not been studied previously. The molecular lesions responsible for 14 mutations were identified, including mutations in nine genes that had not been characterized previously. Some mutations affect vertebrate-specific components of conserved signaling pathways; for example, at least five mutations affect previously uncharacterized regulators of the Sonic hedgehog (Shh) pathway. Approximately half of all of the mutations affect the initial establishment of the body plan, and several of these produce phenotypes that have not been described previously. A large fraction of the genes identified affect cell migration, cellular organization, and cell structure. The findings indicate that phenotype-based genetic screens provide a direct and unbiased method to identify essential regulators of mammalian development.

Mouse Dispatched homolog1 is required for long-range, but not juxtacrine, Hh signaling.

Precise patterning of cell types along the dorsal-ventral axis of the spinal cord is essential to establish functional neural circuits. In order to prove the feasibility of studying a single biological process through random mutagenesis in the mouse, we have identified recessive ENU-induced mutations in six genes that prevent normal specification of ventral cell types in the spinal cord. We positionally cloned the genes responsible for two of the mutant phenotypes, smoothened and dispatched, which are homologs of Drosophila Hh pathway components. The Dispatched homolog1 (Disp1) mutation causes lethality at midgestation and prevents specification of ventral cell types in the neural tube, a phenotype identical to the Smoothened (Smo) null phenotype. As in Drosophila, mouse Disp1 is required to move Shh away from the site of synthesis. Despite the existence of a second mouse disp homolog, Disp1 is essential for long-range signaling by both Shh and Ihh ligands. Our data indicate that Shh signaling is required within the notochord to maintain Shh expression and to prevent notochord degeneration. Disp1, unlike Smo, is not required for this juxtacrine signaling by Shh.