Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development and progression by regulating genes that are vital for proliferation, glycolysis, angiogenesis, and metastasis. To identify strategies of targeting the HIF-1 pathway, we screened a siRNA library against the entire druggable genome and a small-molecule library consisting of 691,200 compounds using a HIF-1 reporter cell line. Although the siRNA library screen failed to reveal any druggable targets, the small-molecule library screen identified a class of alkyliminophenylacetate compounds that inhibit hypoxia-induced HIF-1 reporter activity at single-digit nanomolar concentrations. These compounds were found to inhibit hypoxia but not deferoxamine-induced HIF-1␣ protein stabilization. Further analysis indicated that the alkyliminophenylacetate compounds likely inhibit the HIF-1 pathway through blocking the hypoxia-induced mitochondrial reactive oxygen species (ROS) production. Strikingly, all of the nonalkyliminophenylacetate HIF-1 inhibitors identified from the small-molecule library screen were also found to target mitochondria like the alkyliminophenylacetate compounds. The exclusive enrichment of mitochondria inhibitors from a library of >600,000 diverse compounds by using the HIF-1 reporter assay highlights the essential role of mitochondria in HIF-1 regulation. These results also suggest that targeting mitochondrial ROS production might be a highly effective way of blocking HIF-1 activity in tumors.hypoxia-inducible factor ͉ hypoxia ͉ reactive oxygen species ͉ siRNA ͉ RNAi H ypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular responses to low oxygen (1-3). It consists of a constitutively expressed ␤ subunit and an oxygen-regulated ␣ subunit. The proline hydroxylase domain-containing proteins (PHDs) were originally thought to be the main oxygen sensor that mediates oxygen-dependent HIF-1␣ degradation. Under aerobic conditions, PHDs hydroxylate HIF-1␣ at key proline residues by using oxygen as a substrate, which ultimately causes polyubiquitination and degradation of HIF-1␣ through an E3 ubiquitin ligase complex containing the von Hippel-Lindau protein (4, 5). Because oxygen is required for PHD-mediated hydroxylation, the low oxygen level under hypoxic conditions prevents HIF-1␣ hydroxylation, which subsequently leads to the stabilization of HIF-1␣. However, several recent studies demonstrate that the hypoxia-induced production of reactive oxygen species (ROS) in mitochondria is both necessary and sufficient for hypoxia-dependent HIF-1␣ accumulation, suggesting that mitochondria may act as an oxygen sensor for HIF-1␣ regulation by generating ROS under hypoxic conditions (6-9).HIF-1 has emerged as an attractive target for cancer therapy in the last several years. The requirement of HIF-1 for tumor growth has been examined by abrogating the HIF-1 pathway in tumors by using RNAi, small molecule inhibitors, or genetic alterations. Although the degree of tumor responses to HIF-1 inhibition varies among studies, the major...

The authors report the development of a high-throughput screen for inhibitors of Streptococcus pneumoniae transcription and translation (TT) using a luciferase reporter, and the secondary assays used to determine the biochemical spectrum of activity and bacterial specificity. More than 220,000 compounds were screened in mixtures of 10 compounds per well, with 10,000 picks selected for further study. False-positive hits from inhibition of luciferase activity were an extremely common artifact. After filtering luciferase inhibitors and several known classes of antibiotics, approximately 50 hits remained. These compounds were examined for their ability to inhibit Escherichia coli TT, uncoupled S. pneumoniae translation or transcription, rabbit reticulocyte translation, and in vitro toxicity in human and bacterial cells. One of these compounds had the desired profile of broad-spectrum biochemical activity in bacteria and selectivity versus mammalian biochemical and whole-cell assays.

We report the discovery and characterization of a novel ribosome inhibitor (NRI) class that exhibits selective and broad-spectrum antibacterial activity. Compounds in this class inhibit growth of many gram-positive and gram-negative bacteria, including the common respiratory pathogens Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, and Moraxella catarrhalis, and are nontoxic to human cell lines. The first NRI was discovered in a high-throughput screen designed to identify inhibitors of cell-free translation in extracts from S. pneumoniae. The chemical structure of the NRI class is related to antibacterial quinolones, but, interestingly, the differences in structure are sufficient to completely alter the biochemical and intracellular mechanisms of action. Expression array studies and analysis of NRI-resistant mutants confirm this difference in intracellular mechanism and provide evidence that the NRIs inhibit bacterial protein synthesis by inhibiting ribosomes. Furthermore, compounds in the NRI series appear to inhibit bacterial ribosomes by a new mechanism, because NRI-resistant strains are not cross-resistant to other ribosome inhibitors, such as macrolides, chloramphenicol, tetracycline, aminoglycosides, or oxazolidinones. The NRIs are a promising new antibacterial class with activity against all major drug-resistant respiratory pathogens.Respiratory tract infections are the number 1 killer worldwide, responsible for over 50 million deaths each year. Although antibacterial therapy has successfully stemmed the tide against infection since the middle of the last century, antibacterial resistance of Streptococcus pneumoniae, the most commonly identified pathogen associated with community-acquired pneumonia, is on the rise (2,4,8,22). A recent worldwide study documents that a significant fraction of S. pneumoniae isolates have reduced susceptibility to penicillin (36%) and macrolides (31%) (5). Although overall rates of resistance to fluoroquinolones are low, these rates were found to be increasing rapidly in Canada (1). In general, pathogenic bacteria continuously evolve mechanisms of resistance to currently used antibacterial agents. The discovery of novel antibacterial classes would be the most powerful way to generate new therapy against these resistant pathogens. Unfortunately, novel antibacterial classes have been difficult to discover, with the oxazolidinones, identified in 1980, being the last example to successfully reach the clinic.The bacterial ribosome is a proven target for antibacterial chemotherapy (6,17,20,21). Since the 1940s, small-molecule ribosome inhibitors such as chloramphenicol, tetracyclines, macrolides, aminoglycosides, and, more recently, oxazolidinones have been used to combat bacterial infections in humans (11). These diverse chemical classes of ribosome inhibitors each bind to a different site on the ribosome, which is not surprising given its large size and complexity. Accordingly, drug resistance to each class generally develops separately, such that resista...

A novel high-throughput strand transfer assay has been developed, using Microarray Compound Screening (μARCS) technology, to identify inhibitors of human immunodeficiency virus (HIV) integrase. This technology utilizes agarose matrices to introduce a majority of the reagents throughout the assay. Integration of biotinylated donor DNA with fluorescein isothiocyanate (FITC)-labeled target DNA occurs on a SAM membrane in the presence of integrase. An anti-FITC antibody conjugated to alkaline phosphatase (AP) was used to do an enzyme-linked immunosorbent assay with the SAM. An agarose gel containing AttoPhos, a substrate of AP, was used for detection of the integrase reactions on the SAM. For detection, the AttoPhos gel was separated from the SAM after incubation and then the gel was imaged using an Eagle Eye II closed-circuit device camera system. Potential integrase inhibitors appear as dark spots on the gel image. A library of approximately 250,000 compounds was screened using this HIV integrase strand transfer assay in μARCS format. Compounds from different structural classes were identified in this assay as novel integrase inhibitors.

To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the QuantiGene nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-1alpha (IL-1alpha) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.

A novel high-throughput strand transfer assay has been developed, using Microarray Compound Screening (microARCS) technology, to identify inhibitors of human immunodeficiency virus (HIV) integrase. This technology utilizes agarose matrices to introduce a majority of the reagents throughout the assay. Integration of biotinylated donor DNA with fluorescein isothiocyanate (FITC)-labeled target DNA occurs on a SAM membrane in the presence of integrase. An anti-FITC antibody conjugated to alkaline phosphatase (AP) was used to do an enzyme-linked immunosorbent assay with the SAM. An agarose gel containing AttoPhos, a substrate of AP, was used for detection of the integrase reactions on the SAM. For detection, the AttoPhos gel was separated from the SAM after incubation and then the gel was imaged using an Eagle Eye II closed-circuit device camera system. Potential integrase inhibitors appear as dark spots on the gel image. A library of approximately 250,000 compounds was screened using this HIV integrase strand transfer assay in microARCS format. Compounds from different structural classes were identified in this assay as novel integrase inhibitors.

Microarrayed compound screening format (µARCS) is a novel high-throughput screening technology that uses agarose matrices to integrate various biochemical or biological reagents in the assay. To evaluate the feasibility of using the µARCS technology for nucleic acid polymerization assays, the authors developed HIV reverse transcription (RT) and E1-dependent human papillomavirus (HPV) replication assays in this format. HIV RT is an RNA-dependent DNA polymerase, whereas HPV E1 is a DNA helicase. To ensure the efficient capture of the nucleic acid polymerization reaction and to minimize the nonspecific binding, the authors used a SAM 2® biotin capture membrane in the assay. In both studies, the nucleic acid substrate was biotinylated on one end and was bound to the SAM 2® membrane. A low melting-point agarose gel containing the rest of the reaction components was first placed on a polystyrene sheet spotted with compounds to allow passive diffusion of the compounds into the gel. The gel was removed from the compound sheet and applied to the SAM 2® membrane with the immobilized nucleic acid template to initiate the polymerization. After the incubation, the membrane was washed with a high-salt buffer and exposed for imaging. Potential inhibitors can be seen as white spots on a dark background. The sensitivity for the known inhibitors appears to be comparable in µARCS as in a traditional 96-well plate assay. The methodology described in this paper further expands the applications of µARCS technology. (Journal of Biomolecular Screening 2003:273-282)

Protein tyrosine kinases play critical roles in cell signaling and are considered attractive targets for drug discovery. The authors have applied microARCS (microarrayed compound screening) technology to develop a high-throughput screen for finding inhibitors of the p56lck tyrosine kinase. Initial assay development was performed in a homogeneous time-resolved (LANCE) format in 96-well microplates and then converted into the gel-based microARCS format. The microARCS methodology is a well-less screening format in which 8640 compounds are arrayed on a microplate-sized piece of polystyene and subsequently assayed by placing reagents cast in agarose gels in contact with these compound sheets. A blotting paper soaked with adenosine triphosphate is applied on the gel to initiate the kinase reaction in the gel. Using this screening methodology, 300,000 compounds were screened in less than 40 h. Substantial reagent reduction was achieved by converting this tyrosine kinase assay from a 96-well plate assay to microARCS, resulting in significant cost savings.