Nuclear Export Inhibitors and Kinase Inhibitors Identified Using a MAPK-Activated Protein Kinase 2 Redistribution^®Screen

Abstract

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.

Pharmacological Profiling of Chemokine Receptor–Directed Compounds Using High-Content Screening

“…24,25 HCS combines high-resolution fluorescence microscopy with automated image analysis. [26][27][28][29][30][31] HCS provides several advantages over normal high-throughput screening (HTS). Cellular HTS conventionally monitors the mean response of the whole-cell population of a microtiter plate (MTP) well.…”

Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

“…The Rev Redistribution ® assay is used to detect nonspecific inhibition of Crm1-mediated export. 21 Rev continuously shuttles between the nucleus and cytoplasm, and upon inhibition of nuclear export, Rev accumulates in the nucleus. Ratjadone A, a nonspecific inhibitor of Crm1-mediated nuclear export, was used as the positive control in this assay, 22 with an IC 50 value of approximately 4.5 nM (data not shown).…”

“…17,21 The MAPK pathway is a key regulator of cellular proliferation. Several proteins in this pathway are known as relevant targets for cancer therapy, and a number of drugs inhibiting this pathway are already in preclinical or clinical development.…”

“…In the ERF1 Redistribution ® screen, we were able to obtain a low hit rate (0.65%) compared to similar types of screens. 17,21,42 This may be due to the design of the assay. The ERF1 Redistribution ® assay was developed in T24 cells, which harbor an activating H-RAS mutation, causing spontaneous activation of the MAPK pathway.…”

Identification of Akt Pathway Inhibitors Using Redistribution Screening on the FLIPR and the IN Cell 3000 Analyzer

“…One of the most important aspects of protein function is subcellular localization, but this parameter has been exceedingly difficult to measure at high throughput, making it difficult to use as a primary screening output. With the introduction of new highcontent screening (HCS) systems, as well as our pioneering Redistribution drug discovery concept, this goal is now within reach, [1][2][3][4][5] and protein translocation assays with medium to high throughput (25,000 data points per day) have been devised. However, due to the specialized nature and the high acquisition costs of the HCS equipment and the very limited number of truly high-throughput HCS platforms in use within the industry, there is still a need for new methods that allow the formatting of Redistribution assays on more traditional equipment such as fluorometric imaging plate readers (FLIPRs).…”