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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. (Journal of Biomolecular Screening 2005:20-29)

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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.

Plasminogen contains a unique disulphide bond, Cys558 Cys566, responsible for the cyclic nature of the peptide sequence surrounding the activation site at Arg561-Val562. A recombinant [Ser558, Ser566]-Lys78-plasminogen variant was produced in which the two cysteine residues were replaced by serine residues. The variant was used to study the functional implications of removing the structural restrains imposed to the activation loop by this disulphide bond. Elimination of the Cys558 Cys566 bond attenuated activation by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), but resulted in an increased susceptibility to cleavage by trypsin and plasma kallikrein. Two opposite effects on the interaction of plasminogen with streptokinase were produced by modification of this bond; (a) attenuation of the rate at which the active complex with streptokinase was formed and (b) a 7.5-fold increase in plasminogen activation catalysed by this complex. Activation by tPA in the presence of fibrin, in contrast to activation in its absence, was not attenuated by elimination of this disulphide bond. However, the activation rate as a function of plasminogen concentration followed a different saturation curve, and the fibrin degradation pattern was changed. The results suggest that the Cys558 Cys566 disulphide bond is of importance for the specificity of plasminogen. This applies to its activation and also to its role in subsequent fibrin clot degradation.Keywords : disulphide bond; plasminogen mutant; urokinase-type plasminogen activator ; tissue-type plasminogen activator ; streptokinase.Plasminogen is the zymogen form of the serine protease, necting kringles 2 and 3, may also serve to stabilise the protein in a conformation important for its presentation of fibrinplasmin, consisting of 791 amino acids with a molecular mass of 92 000 Da. The protein contains an N-terminal Glu1-Lys77 binding sites. Plasminogen is activated by cleavage of a peptide bond at Arg561-Val562 located in a short disulphide linked peptide, five homologous kringle domains and a C-terminal serine protease domain. Native Glu1-plasminogen is converted to (Cys558 Cys566) peptide loop, CPGRVVGGC. Activation is Lys78-plasminogen by plasmin which cleaves at the Lys77-mediated by urokinase (uPA), tissue-type plasminogen activator Lys78 peptide bond. Kringle 1 and kringle 4 each contain a ly-(tPA) or a complex of streptokinase and plasminogen/plasmin. sine-binding site and kringle 5 contains an aminohexyl-binding After activation, the heavy and light chains are linked by two site. Several studies have indicated that kringle 1 and kringle 5 interdomain Cys548 Cys670 and Cys558 Cys566 disulphide are involved in fibrin binding via their lysine-binding sites, bonds. Plasminogen activators are remarkably specific proteinwhereas this may not be the case for kringle 4 although it con-ases since the activation loop in plasminogen is their only known tains such a site ([1] and [2] for reviews). Specific functions physiologically relevant subs...

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