A phosphatase is an enzyme that hydrolyzes phosphoric acid monoesters into a phosphate ion and a molecule with a free hydroxyl group. This action is directly opposite to that of phosphorylases and kinases, which attach phosphate groups to their substrates by using chemical energy conserving molecules like ATP.
Phosphatases can be categorized into two main categories: metalloenzymes (which are dependent on the presence of two or more metal ions in their active sites for activity), and non-metalloenzymes. These categories can then be divided into further sub-categories. The metalloenzymes by far comprise the greatest bulk of phosphatases and contain such enzymes as alkaline phosphatase (three metal ions, only two of which are catalytically active), the serine threonine phosphatases, and inositol monophosphatase. Best known of the non-metalloenzymes are the protein tyrosine phosphatases, which hydrolyze phospho-tyrosine residues.
The presence or absence of a phosphate group on proteins is known to play a regulatory role in many biochemical and particularly signal transduction pathways. Tyrosine residues can be tagged with a phosphate group (phosphorylated) by protein kinases. In its phosphorylated state, the tyrosine residue is referred to as phosphotyrosine. Tyrosine phosphorylation is considered as one of the key steps in signal transduction and regulation of enzymatic activity. Phosphotyrosine can be detected through specific antibodies. Together, specialized kinases and phosphatases regulate the activity of enzymes, receptors and other components of signal transduction pathways, transcription factors, and other functional proteins.
Thus, there are numerous biochemical methods aimed at detecting phosphorylated proteins in a biological sample. For instance, sampled cell material is lysed and the protein fraction is subjected to one-dimensional (e.g., SDS-PAGE) or two-dimensional (e.g., 1st dimension: isoelectric focussing, 2nd dimension: SDS PAGE) separation, and phosphorylated proteins of individual bands or spots are detected by phosphoserine- or phosphotyrosine-specific antibodies. Other more specific detection methods make use of antibodies which specifically bind to particular phosphorylated proteins. Such antibodies are frequently used for histocytochemical detection of phosphorylated target proteins in formalin fixed paraffin embedded tissue sections from biopsy material.
Regardless of the detection method used, the result of the analysis of phosphorylated proteins is desired to reflect the status of phosphorylation at the time point when the experiment is started, that is to say when the cells are lysed or the tissue is fixed and sectioned. More generally, it is desired to conserve the status of protein phosphorylation at a certain point in time. Conservation is achieved by preventing phosphate esters to be hydrolyzed from their target proteins. To this end, the state of the art provides a number of substances capable of inhibiting phosphatase activity. Inhibitors are applied routinely when performing assays for the detection of phosphorylated proteins but also when phosphorylated proteins are to be purified in larger amounts.
Among vanadium-containing inhibitors of phosphatase activity, pervanadate and vanadate are the best known and most widely employed substances. Vanadate is a phosphate analogon which mimics the transition state of phosphate hydrolysis and is therefore considered as a general phosphatase inhibitor. However, vanadate is recognized to be particularly suited to inhibit tyrosine phosphatases (Huyer, G., et al., J. Biol. Chem. 272 (1997) 843-851) and alkaline phosphatases (e.g., Stankiewicz, P. J., et al., in Met. Ions Biol. Syst. 31 (1995) 287-324). But inhibition of other phosphatase like ATPases, glucose-6 phosphatase, acid phosphatase, or fructose-2,6-bisphosphatase had also been reported.
However, a disadvantage is that in order to provide an effective amount, vanadate has to be in a concentration in the micromolar or even millimolar range. In this regard, an “effective amount” is understood as being between 1× (one time) and 50× (fifty times) the concentration of the inhibitor in an aqueous solution which at a 1× concentration reduces the activity of a phosphatase by the factor of 20. In contrast to vanadate, other inhibitors of phosphatase activity are known which are effective at nanomolar concentrations. An example therefor is cantharidin.
It has been reported, however, that the inhibitory effect can be enhanced by way of forming stable vanadate-containing complexes. E.g., potassium bisperoxo (bipyridine) oxovanadate (V) and potassium bisperoxo (1,10 phenanthroline) oxovanadate (V)) both are more potent than orthovanadate. However, complexation of vanadate it not necessarily a prerequisite for improvement of potency. For instance, hydroxylamine or dimethylhydroxylamine spontaneously form complexes with vanadate. The potency of those complexes remain the same or is reduced to a certain extent (Cuncic, C., et al., Biochem. Pharmacol. 58 (1999) 1859-1867). In cell-based assays, it was found that these two compounds increase the uptake of vanadate into the cellular lumen (Nxumalo, F., et al., J. Biol. Inorg. Chem. 3 (1998) 534-542; Cuncic, C., et al., Biochem. Pharmacol. 58 (1999) 1859-1867).
It is further known that in the presence of certain complex-forming reagents like EDTA, the phosphatase inhibiting effect of vanadate is reduced by a factor of about 1,000 (Huyer, G., et al., J. Biol. Chem. 272, (1997) 843-851). This is particularly disadvantageous because EDTA is frequently used in biochemistry as a stabilizer and as an inhibitor of metalloenzymes such as phosphatases and proteases. Another important stabilizer for use in the preparation of cell lysates is dithiothreitol (DTT). The inventors have found that DTT also reduces to a significant extent the inhibitory effect of vanadate on phosphatases.
In view of the disadvantages of the state of the art, it is an object of the present invention to provide alternative compounds which enhance the inhibitory effect of vanadate-containing compounds on enzymes with phosphatase activity. It is another object of the invention to provide compounds which counteract the negative effects of EDTA and DTT on vanadate-containing compounds.
The inventors have surprisingly found that in the presence of a polyol the inhibitory effect of vanadate on phosphatases and particularly on phosphotyrosine-specific phosphatases is potentiated. This effect was observed even with a very simple polyol like glycerol but also with sugar alcohols like mannitol. Even more surprising, the negative effects of EDTA and DTT on vanadate were found to be reduced significantly or even abolished completely.
USPTO Backgrounds
USPTO Backgrounds