A selective phosphatase of regenerating liver phosphatase inhibitor suppresses tumor cell anchorage-independent growth by a novel mechanism involving p130Cas cleavage.

The phosphatase of regenerating liver (PRL) family, a unique class of oncogenic phosphatases, consists of three members: PRL-1, PRL-2, and PRL-3. Aberrant overexpression of PRL-3 has been found in multiple solid tumor types. Ectopic expression of PRLs in cells induces transformation, increases mobility and invasiveness, and forms experimental metastases in mice. We have now shown that small interfering RNA-mediated depletion of PRL expression in cancer cells results in the down-regulation of p130Cas phosphorylation and expression and prevents tumor cell anchorage-independent growth in soft agar. We have also identified a small molecule, 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (thienopyridone), which potently and selectively inhibits all three PRLs but not other phosphatases in vitro. The thienopyridone showed significant inhibition of tumor cell anchorage-independent growth in soft agar, induction of the p130Cas cleavage, and anoikis, a type of apoptosis that can be induced by anticancer agents via disruption of cell-matrix interaction. Unlike etoposide, thienopyridone-induced p130Cas cleavage and apoptosis were not associated with increased levels of p53 and phospho-p53 (Ser(15)), a hallmark of genotoxic drug-induced p53 pathway activation. This is the first report of a potent selective PRL inhibitor that suppresses tumor cell three-dimensional growth by a novel mechanism involving p130Cas cleavage. This study reveals a new insight into the role of PRL-3 in priming tumor progression and shows that PRL may represent an attractive target for therapeutic intervention in cancer.

High-throughput methods for measuring heparanase activity and screening potential antimetastatic and anti-inflammatory agents.

Heparanase plays an important role in the degradation of the extracellular matrix. It is implicated in inflammation, tumor angiogenesis and metastasis. We have developed two high-throughput methods for measuring heparanase activity and screening potential inhibitors. The first method involves coating fibroblast growth factor (FGF) on microtiter plates and capturing fluorescein isothiocyanate (FITC)-labeled heparin sulfate (HS), which is used as a substrate for heparanase digestion. Labeled HS fragments are released into the medium and quantitated by fluorescence intensity measurement. We have implemented this assay method into a Zeiss uHTS system and screened compound libraries for heparanase inhibitors. The second method involves labeling HS with biotin followed by FITC to generate a dual-labeled HS. The labeled material is bound to streptavidin-coated plates and used as a substrate for heparanase digestion. Both methods are sensitive and easily applicable to robotic systems. In addition, we have labeled both HS and biotin-HS with Eu-chelate, a fluorophore that exhibits long decay fluorescence. Assays using Eu-labeled HS and Eu-labeled biotin-HS have been developed and show higher sensitivity than those using FITC-labeled material. Furthermore, assays using Eu-chelate HS (or biotin-HS) should eliminate the interference of fluorescence compounds.

A generic time-resolved fluorescence assay for serine/threonine kinase activity: application to Cdc7/Dbf4.

The serine/threonine protein kinase family is a large and diverse group of enzymes that are involved in the regulation of multiple cellular pathways. Elevated kinase activity has been implicated in many diseases and frequently targeted for the development of pharmacological inhibitors. Therefore, non-radioactive antibody-based kinase assays that allow high throughput screening of compound libraries have been developed. However, they require a generation of antibodies against the phosphorylated form of a specific substrate. We report here a time-resolved fluorescence assay platform that utilizes a commercially-available generic anti-phospho-threonine antibody and permits assaying kinases that are able to phosporylate threonin residues on protein substrates. Using this approach, we developed an assay for Cdc7/Dbf4 kinase activity, determined the K(m) for ATP, and identified rottlerin as a non-ATP competitive inhibitor of this enzyme.

Crystal structure of human cytosolic phosphoenolpyruvate carboxykinase reveals a new GTP-binding site.

We report crystal structures of the human enzyme phosphoenolpyruvate carboxykinase (PEPCK) with and without bound substrates. These structures are the first to be determined for a GTP-dependent PEPCK, and provide the first view of a novel GTP-binding site unique to the GTP-dependent PEPCK family. Three phenylalanine residues form the walls of the guanine-binding pocket on the enzyme's surface and, most surprisingly, one of the phenylalanine side-chains contributes to the enzyme's specificity for GTP. PEPCK catalyzes the rate-limiting step in the metabolic pathway that produces glucose from lactate and other precursors derived from the citric acid cycle. Because the gluconeogenic pathway contributes to the fasting hyperglycemia of type II diabetes, inhibitors of PEPCK may be useful in the treatment of diabetes.