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.

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.

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.

Identification of intracellular residues in the dopamine transporter critical for regulation of transporter conformation and cocaine binding.

Recently we showed evidence that mutation of Tyr-335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-264, Asp-345, and Asp-436, the mutation of which to alanine produces a phenotype similar to that of Y335A. Like Y335A, the mutants (K264A, D345A, and D436A) were characterized by low uptake capacity that was potentiated by Zn(2+). Moreover, the mutants displayed lower affinity for cocaine and other inhibitors, suggesting a role for these residues in maintaining the structural integrity of the inhibitor binding crevice. The conformational state of K264A, Y335A, and D345A was investigated by assessing the accessibility to MTSET ([2-(trimethylammonium)ethyl]-methanethiosulfonate) of a cysteine engineered into position 159 (I159C) in transmembrane segment 3 of the MTSET-insensitive "E2C" background (C90A/C306A). Unlike its effect at the corresponding position in the homologous norepinephrine transporter (NET I155C), MTSET did not inhibit uptake mediated by E2C I159C. Furthermore, no inhibition was observed upon treatment with MTSET in the presence of dopamine, cocaine, or Zn(2+). Without Zn(2+), E2C I159C/K264A, E2C I159C/Y335A, and E2C I159C/D345A were also not inactivated by MTSET. In the presence of Zn(2+) (10 microm), however, MTSET (0.5 mm) caused up to approximately 60% inactivation. As in NET I155C, this inactivation was protected by dopamine and enhanced by cocaine. These data are consistent with a Zn(2+)-dependent partial reversal of a constitutively altered conformational equilibrium in the mutant transporters. They also suggest that the conformational equilibrium produced by the mutations resembles that of the NET more than that of the DAT. Moreover, the data provide evidence that the cocaine-bound state of both DAT mutants and of the NET is structurally distinct from the cocaine-bound state of the DAT.

N-terminal truncation of the dopamine transporter abolishes phorbol ester- and substance P receptor-stimulated phosphorylation without impairing transporter internalization.

The structural basis of phosphorylation and its putative role in internalization were investigated in the human dopamine transporter (hDAT). Activation of protein kinase C (PKC) was achieved either directly by treatment with 4-alpha-phorbol 12-myristate 13-acetate (PMA) or by activating the Galpha(q)-coupled human substance P receptor (hNK-1) co-expressed with hDAT in HEK293 cells and in N2A neuroblastoma cells. In both cell lines, activation of the hNK-1 receptor by substance P reduced the V(max) for [(3)H]dopamine uptake to the same degree as did PMA ( approximately 50 and approximately 20% in HEK293 and N2A cells, respectively). In HEK293 cells, the reduction in transport capacity could be accounted for by internalization of the transporter, as assessed by cell surface biotinylation experiments, and by fluorescence microscopy using enhanced green fluorescent protein-tagged hDAT. In HEK293 cells, hNK-1 receptor activation, as well as direct PKC activation by PMA, was accompanied by a marked increase in transporter phosphorylation. However, truncation of the first 22 N-terminal residues almost abolished detectable phosphorylation without affecting the SP- or PMA-induced reduction in transport capacity and internalization. In this background truncation construct, systematic mutation of all the phosphorylation consensus serines and threonines in hDAT, alone and in various combinations, did also not alter the effect of hNK-1 receptor activation or PMA treatment in either HEK293 or N2A cells. Mutation of a dileucine and of two tyrosine-based motifs in hDAT was similarly without effect. We conclude that the major phosphorylation sites in hDAT are within the distal N terminus, which contains several serines. Moreover, the present data strongly suggest that neither this phosphorylation, nor the phosphorylation of any other sites within hDAT, is required for either receptor-mediated or direct PKC-mediated internalization of the hDAT.