Identification Of Novel Activators Research Articles

Identification of novel activators of the metal responsive transcription factor (MTF-1) using a gene expression biomarker in a microarray compendium.

Environmental exposure to metals is known to cause a number of human toxicities including cancer. Metal-responsive transcription factor 1 (MTF-1) is an important component of metal regulation systems in mammalian cells. Here, we describe a novel method to identify chemicals that activate MTF-1 based on microarray profiling data. MTF-1 biomarker genes were identified that exhibited consistent, robust expression across 10 microarray comparisons examining the effects of metals (zinc, nickel, lead, arsenic, mercury, and silver) on gene expression in human cells. A subset of the resulting 81 biomarker genes was shown to be altered by knockdown of the MTF1 gene including metallothionein family members and a zinc transporter. The ability to correctly identify treatment conditions that activate MTF-1 was determined by comparing the biomarker to microarray comparisons from cells exposed to reference metal activators of MTF-1 using the rank-based Running Fisher algorithm. The balanced accuracy for prediction was 93%. The biomarker was then used to identify organic chemicals that activate MTF-1 from a compendium of 11 725 human gene expression comparisons representing 2582 chemicals. There were 700 chemicals identified that included those known to interact with cellular metals, such as clioquinol and disulfiram, as well as a set of novel chemicals. All nine of the novel chemicals selected for validation were confirmed to activate MTF-1 biomarker genes in MCF-7 cells and to lesser extents in MTF1-null cells by qPCR and targeted RNA-Seq. Overall, our work demonstrates that the biomarker for MTF-1 coupled with the Running Fisher test is a reliable strategy to identify novel chemical modulators of metal homeostasis using gene expression profiling.

2017 Scientific Sessions Sol Sherry Distinguished Lecture in Thrombosis: Factor XI as a Target for New Anticoagulants.

The goal of anticoagulant therapy is to attenuate thrombosis without compromising hemostasis. Although the direct oral anticoagulants are associated with less intracranial hemorrhage than vitamin K antagonists, bleeding remains their major side effect. Factor XI has emerged as a promising target for anticoagulants that may be safer than those currently available. The focus on factor XI stems from epidemiological evidence of its role in thrombosis, the observation of attenuated thrombosis in factor XI-deficient mice, identification of novel activators, and the fact that factor XI deficiency is associated with only a mild bleeding diathesis. Proof-of-concept comes from the demonstration that compared with enoxaparin, factor XI knockdown reduces venous thromboembolism without increasing bleeding after elective knee arthroplasty. This article rationalizes the selection of factor XI as a target for new anticoagulants, reviews the agents under development, and outlines a potential path forward for their development.

Assessing the Effectiveness of Functional Genetic Screens for the Identification of Bioactive Metabolites

A common limitation for the identification of novel activities from functional (meta) genomic screens is the low number of active clones detected relative to the number of clones screened. Here we demonstrate that constructing libraries with strains known to produce bioactives can greatly enhance the screening efficiency, by increasing the “hit-rate” and unmasking multiple activities from the same bacterial source.

Identification of novel activators of constitutive androstane receptor from FDA-approved drugs by integrated computational and biological approaches.

The constitutive androstane receptor (CAR, NR1I3) is a xenobiotic sensor governing the transcription of numerous hepatic genes associated with drug metabolism and clearance. Recent evidence suggests that CAR also modulates energy homeostasis and cancer development. Thus, identification of novel human (h) CAR activators is of both clinical importance and scientific interest. Docking and ligand-based structure-activity models were used for virtual screening of a database containing over 2000 FDA-approved drugs. Identified lead compounds were evaluated in cell-based reporter assays to determine hCAR activation. Potential activators were further tested in human primary hepatocytes (HPHs) for the expression of the prototypical hCAR target gene CYP2B6. Nineteen lead compounds with optimal modeling parameters were selected for biological evaluation. Seven of the 19 leads exhibited moderate to potent activation of hCAR. Five out of the seven compounds translocated hCAR from the cytoplasm to the nucleus of HPHs in a concentration-dependent manner. These compounds also induce the expression of CYP2B6 in HPHs with rank-order of efficacies closely resembling that of hCAR activation. These results indicate that our strategically integrated approaches are effective in the identification of novel hCAR modulators, which may function as valuable research tools or potential therapeutic molecules.

Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements

The human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) share a unique mechanism of colonization that results from the concerted action of effector proteins translocated into the host cell by a type III secretion system (T3SS). EPEC and EHEC not only induce characteristic attaching and effacing (A/E) lesions, but also subvert multiple host cell signalling pathways during infection. Our understanding of the mechanisms by which A/E pathogens hijack host cell signalling has advanced dramatically in recent months with the identification of novel activities for many effectors. In addition to further characterization of established effectors (Tir, EspH and Map), new effectors have emerged as important mediators of virulence through activities such as mimicry of Rho guanine nucleotide exchange factors (Map and EspM), inhibition of apoptosis (NleH and NleD), interference with inflammatory signalling pathways (NleB, NleC, NleE and NleH) and phagocytosis (EspF, EspH and EspJ). The findings have highlighted the multifunctional nature of the effectors and their ability to participate in redundant, synergistic or antagonistic relationships, acting in a co-ordinated spatial and temporal manner on different host organelles and cellular pathways during infection.

Cre-lox univector acceptor vectors for functional screening in protoplasts: analysis of Arabidopsis donor cDNAs encoding ABSCISIC ACID INSENSITIVE1-like protein phosphatases.

The 14,200 available full length Arabidopsis thaliana cDNAs in the universal plasmid system (UPS) donor vector pUNI51 should be applied broadly and efficiently to leverage a "functional map-space" of homologous plant genes. We have engineered Cre-lox UPS host acceptor vectors (pCR701- 705) with N-terminal epitope tags in frame with the loxH site and downstream from the maize Ubiquitin promoter for use in transient protoplast expression assays and particle bombardment transformation of monocots. As an example of the utility of these vectors, we recombined them with several Arabidopsis cDNAs encoding Ser/Thr protein phosphatase type 2C (PP2Cs) known from genetic studies or predicted by hierarchical clustering meta-analysis to be involved in ABA and stress responses. Our functional results in Zea mays mesophyll protoplasts on ABA-inducible expression effects on the Late Embryogenesis Abundant promoter ProEm:GUS reporter were consistent with predictions and resulted in identification of novel activities of some PP2Cs. Deployment of these vectors can facilitate functional genomics and proteomics and identification of novel gene activities.

Identification of Novel Activities of Immunomodulatory Drugs: Cytoskeleton Reorganization through Modulation of Rho GTPases

Immunomodulatory drugs including the IMiDs®, lenalidomide, and pomalidomide represent a novel class of compounds that have both anti-cancer and anti-inflammatory properties. While many studies have demonstrated that IMiDs have broad in vitro and in vivo biological activities including antiangiogenesis, inhibition of TNFa expression, enhancement of antitumor immunity, and induction of IL-2 in T cells, the molecular mechanism through which these drugs exert their effects is largely undefined. In primary human monocytes, IMID1 selectively activated RhoA and Rac1, but not Cdc42 or Ras. Importantly, the activation of these GTPases occurred immediately following treatment with IMID1 in the absence of any costimulation. Consistent with the activation of Rho GTPases, we found that IMID1enhanced F-actin formation, stabilized microtubules, and increased monocyte cell migration, all of which were blocked by selective inhibitors of ROCK1, a downstream effector of RhoA or Rac1. Finally, we demonstrated that IMID1 was able to regulate the activity of Rho GTPases and formation of F-actin in primary human T cells similarly as it did in monocytes, and showed that the activation of RhoA was essential for IMID1-induced IL-2 expression in T cells. In conclusion, these studies demonstrate a novel and acute molecular activity from IMiDs mediated via Rho GTPases. Activation of Rho by IMiDs and the resulting effect on cytoskeletal reorganization may represent a critical mechanism by which IMiDs function as therapeutic immunomodulatory agents.

Functional identification of novel activities: Activity-based selection of proteins from complete proteomes

The identification of proteins with desired activities, especially from complex samples such as plasma and whole blood, is a continual challenge. We have developed a technology platform called Functional Identification of Novel Activities (FIoNA) to discover desired protein activities from complex biological samples. FIoNA uses immobilized libraries of combinatorial peptide ligands to purify and concentrate essentially all of the components of a complex mixture on ligands synthesized on individual beads. No depletion or prefractionation of the starting material is performed before it is incubated with the library, and no a priori knowledge of the active protein or of the ligand to which it binds is required. Instead, the protein-loaded beads are individually evaluated en masse in disease- relevant assays to identify proteins possessing a desired function. Beads associated with the activity are selected, and the ligand is sequenced and resynthesized in bulk on the original backbone for purification and characterization of the active component. Here we illustrate the use of FIoNA in a cell proliferation assay to detect a growth factor present in conditioned cell medium at nanogram/milliliter concentrations. We also have selected beads associated with hydrolysis of nerve agent analogs in assays performed in 100,000-well microtiter plates.

Identification of novel Ah receptor agonists using a high-throughput green fluorescent protein-based recombinant cell bioassay.

The Ah receptor is a ligand-dependent transcription factor that mediates the biological and toxic effects of polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin). Recent evidence also suggests a role for the AhR in normal physiology and development. Although a variety of structurally diverse chemicals are reported to bind to and activate the AhR, the full spectrum of structural chemical classes that can interact with the AhR remains to be elucidated. Large-scale analysis of the ligand binding specificity of the AhR requires the use of a high-throughput AhR bioassay system for chemical screening. We have utilized a recombinant mouse hepatoma cell line (H1G1.1c3) containing a stably integrated TCDD- and AhR-responsive enhanced green fluorescent protein (EGFP) reporter gene to screen a 1,5-dialkylamino-2,4-dinitrobenzene combinatorial chemical library consisting of 155 parental amines and up to 12 090 combinatorial products in less than 7 days for novel AhR agonists. These analyses have identified numerous parental amines as relatively potent inducers of EGFP (with EC(50)s between 8 and 1000 microM) and also have revealed several novel products of the combinatorial chemical library synthesis with EC(50)s between 10 and 100 microM. Overall, these results have not only allowed the identification of novel activators of the AhR but also demonstrate the utility of the recombinant H1G1.1c3 cell bioassay for high-throughput chemical screening.