N-Substituted pyrazole-3-carboxamides as inhibitors of human 15-lipoxygenase.

High-throughput screening was used to find selective inhibitors of human 15-lipoxygenase-1 (15-LOX-1). One hit, a 1-benzoyl substituted pyrazole-3-carboxanilide (1a), was used as a starting point in a program to develop potent and selective 15-LOX-1 inhibitors.

3-Substituted pyrazoles and 4-substituted triazoles as inhibitors of human 15-lipoxygenase-1.

Investigation of 1N-substituted pyrazole-3-carboxanilides as 15-lipoxygenase-1 (15-LOX-1) inhibitors demonstrated that the 1N-substituent was not essential for activity or selectivity. Additional halogen substituents on the pyrazole ring, however, increased activity. Further development led to triazole-4-carboxanilides and 2-(3-pyrazolyl) benzoxazoles, which are potent and selective 15-LOX-1 inhibitors.

Syntheses and antiproliferative evaluation of oxyphenisatin derivatives.

Syntheses and structure-antiproliferative relationship for oxyphenisatin analogues are described. The cell proliferation data showed that the presence of substituents (especially F, Cl, Me, CF(3), and OMe) in the 6- and 7-position of oxyphenisatin markedly enhanced the potency in the MDA-468 cell line without affecting the MDA-231 cell line. The best compounds from this series showed low nanomolar antiproliferative activity towards the MDA-468 cell line and a 1000-fold selectivity over the MDA-231 cell line.

High content screening for G protein-coupled receptors using cell-based protein translocation assays.

G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide the capability to probe GPCR function at the cellular level with better resolution than has previously been possible, and offer practical strategies for more definitive selectivity evaluation and counter-screening in the early stages of drug discovery. The potential of cell-based translocation assays for GPCR discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells as well as to play a role in metastasis of certain cancer cells.

Emerging classes of protein-protein interaction inhibitors and new tools for their development.

Protein-protein interactions play a key role in the signal transduction pathways that regulate cellular function. Three years ago, few descriptions of small molecule protein-protein interaction inhibitors (SMPPIIs) existed in the literature. Today, the number of examples of both the biology and chemistry of such interaction inhibitors is growing rapidly. This growth occurs at the convergence of medicinal chemistry, signaling biology and novel assay technology for profiling emerging compound classes and modes of action. Protein translocation assays provide a unique new tool for identifying, profiling, and optimizing SMPPIIs. This review summarizes recent work in the field, and outlines future developments we can anticipate.

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.