Biomedical subjects

Frosty Loechel

Publications and source records attributed to Frosty Loechel.

6 recordsLinked to original sources

PubMed2007

High content translocation assays for pathway profiling.

This chapter describes the design and development of cell-based assays, in which quantitation of the intracellular translocation of a target protein--rather than binding or catalytic activity--provides the primary assay readout. These are inherently high content assays, and they provide feedback on cellular response at the systems level, rather than data on activities of individual, purified molecules. Multiple protein translocation assays can be used to profile cellular signaling pathways and they can play a key role in determination of mechanism of action for novel classes of compounds with therapeutic potential. This assay technology has developed from laboratory curiosity into main stream industrial research over the past decade, and its promise is beginning to be realized as data acquisition and analysis technology evolve to take advantage of the rich window into systems biology provided by translocation assays.

Animals↗

PubMed2006

Identification of RAS-mitogen-activated protein kinase signaling pathway modulators in an ERF1 redistribution screen.

The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, high-throughput screen using a novel ERF1 Redistribution assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC(50)=< 5 microM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant.

Animals↗

PubMed2005

Identification of Akt pathway inhibitors using redistribution screening on the FLIPR and the IN Cell 3000 analyzer.

The PI3-kinase/Akt pathway is an important cell survival pathway that is deregulated in the majority of human cancers. Despite the apparent druggability of several kinases in the pathway, no specific catalytic inhibitors have been reported in the literature. The authors describe the development of a fluorometric imaging plate reader (FLIPR)-based Akt1 translocation assay to discover inhibitors of Akt1 activation. Screening of a diverse chemical library of 45,000 compounds resulted in identification of several classes of Akt1 translocation inhibitors. Using a combination of classical in vitro assays and translocation assays directed at different steps of the Akt pathway, the mechanisms of action of 2 selected chemical classes were further defined. Protein translocation assays emerge as powerful tools for hit identification and characterization.

Animals↗

PubMed2005

Non-anti-coagulant heparins: a promising approach for prevention of tumor metastasis (review).

The metastatic spread of solid tumors is responsible for most cancer-related deaths, and unfortunately no specific anti-metastatic therapy exists. Heparin is widely used in the clinic today as an anti-coagulant therapy for cancer patients at risk for venous thrombo-embolism. Recent clinical trials with low molecular weight heparin and meta-analyses of earlier clinical trials with unfractionated heparin indicate that heparin has an anti-metastatic activity. Animal studies using non-anti-coagulant heparin (NAC heparin) suggest that it is possible to separate the anti-metastatic and anti-coagulant activities of heparin. The use of heparin as an anti-metastatic agent is limited due to its potent anti-coagulant activity. NAC heparins have clinical potential because they could be administered at a higher dose, thereby fully exploiting the anti-metastatic component of heparin activity, and because they could be used with cancer patients with bleeding complications, where the use of heparin is currently precluded. The mechanism by which NAC heparin inhibits metastasis is not yet fully understood. Possible mechanisms include the inhibition of selectin-mediated cell-cell interactions, heparanase and angiogenesis, and stimulation of tissue factor pathway inhibitor release. The promising results from clinical trials and animal studies clearly warrant further investigation of NAC heparin as a therapeutic for the prevention of tumor metastasis.

Animals↗

PubMed2004

Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen.

Redistribution (BioImage) A/S, Søborg, Denmark) is a novel high-throughput screening technology that monitors translocation of specific protein components of intracellular signaling pathways within intact mammalian cells, using green fluorescent protein as a tag. A single Redistribution assay can be used to identify multiple classes of compounds that act at, or upstream of, the level of the protein target used in the primary screening assay. Such compounds may include both conventional and allosteric enzyme inhibitors, as well as protein-protein interaction modulators. We have developed a series of Redistribution assays to discover and characterize compounds that inhibit tumor necrosis factor-alpha biosynthesis via modulation of the p38 mitogen-activated protein kinase (MAPK) pathway. A primary assay was designed to identify low-molecular-weight compounds that inhibit the activation-dependent nuclear export of the p38 kinase substrate MAPK-activated protein kinase 2 (MK2). Hits from the primary screen were categorized, using secondary assays, either as direct inhibitors of MK2 nuclear export, or as inhibitors of the upstream p38 MAPK pathway. Activity profiles are presented for a nuclear export inhibitor, and a compound that structurally and functionally resembles a known p38 kinase inhibitor. These results demonstrate the utility of Redistribution technology as a pathway screening method for the identification of diverse and novel compounds that are active within therapeutically important signaling pathways.

Active Transport, Cell Nucleus↗

PubMed2002

ADAM 12 protease induces adipogenesis in transgenic mice.

ADAM 12 (meltrin-alpha) is a member of the ADAM (a disintegrin and metalloprotease) family. ADAM 12 functions as an active metalloprotease, supports cell adhesion, and has been implicated in myoblast differentiation and fusion. Human ADAM 12 exists in two forms: the prototype membrane-anchored protein, ADAM 12-L, and a shorter secreted form, ADAM 12-S. Here we report the occurrence of adipocytes in the skeletal muscle of transgenic mice in which overexpression of either form is driven by the muscle creatine kinase promoter. Cells expressing a marker of early adipogenesis were apparent in the perivascular space in muscle tissue of 1- to 2-week-old transgenic mice whereas mature lipid-laden adipocytes were seen at 3 to 4 weeks. Moreover, female transgenics expressing ADAM 12-S exhibited increases in body weight, total body fat mass, abdominal fat mass, and herniation, but were normoglycemic and did not exhibit increased serum insulin, cholesterol, or triglycerides. Male transgenics were slightly overweight and also developed herniation but did not become obese. Transgenic mice expressing a truncated form of ADAM 12-S lacking the prodomain and the metalloprotease domain did not develop this adipogenic phenotype, suggesting a requirement for ADAM 12 protease activity. This is the first in vivo demonstration that an ADAM protease is involved in adipogenesis.

ADAM Proteins↗