Phosphatases inhibitors

Several phosphatase inhibitors are available and have been useful in many aspects of smooth muscle research. Okadaic acid was the first inhibitor to be widely used and inhibition of phosphatase activity by this compound was demonstrated using smooth muscle (Hartshorne et al., 1989). Calyculin A was also used in several earlier studies. Since 1990 other compounds have been described as phosphatase inhibitors. These are remarkable in that despite considerable differences in structure, they are relatively specific as phosphatase inhibitors. These inhibitors include tau-tomycin, as antifungal antibiotic produced by Strep-tomyces (MacKintosh and Klumpp, 1990 Hori et al., 

1991) the cyclic peptide hepatoxins, microcystins, and nodularin (Eriksson et al., 1990 MacKintosh et al., 

1990) produced by cyanobacteria and the cantharidin derivatives (Li and Casida, 1992 Honkanen, 1993), which are widely used in agriculture as herbicides or insecticides. Cantharidin is the active ingredient of blister beetle extract. [This is only a partial listing for a more complete review, see Fujiki and Suganuma 

All of these compounds are effective inhibitors of PPl and PP2A when used in vitro. Okadaic acid shows a marked difference between the two classes with a much more potent inhibition of type 2A. Using phosphorylated LC20 as substrate, K, values for 

Of these inhibitors only tautomycin is more potent for PPl. K values for cantharidin are 1.11 and 0.19 jlM for PPl and PP2A, respectively (Honkanen, 1993), using phosphohistone as substrate. 

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Recently, two examples of the separation of enantiomers using CCC have been published (Fig. 1-2). The complete enantiomeric separation of commercial d,l-kynurenine (2) with bovine serum albumin (BSA) as a chiral selector in an aqueous-aqueous polymer phase system was achieved within 3.5 h [128]. Moreover, the chiral resolution of 100 mg of an estrogen receptor partial agonist (7-DMO, 3) was performed using a sulfated (3-cyclodextrin [129, 130], while previous attempts with unsubstituted cyclodextrin were not successful [124]. The same authors described the partial resolution of a glucose-6-phosphatase inhibitor (4) with a Whelk-0 derivative as chiral selector (5) [129]. 

An illustrative example of an alternative strategy (cf Fig. 11c) involving the use of a novel traceless linker is found in the multistep synthesis of 6-epi-dysidiolide (363) and several dysidiolide-derived phosphatase inhibitors by Waldmann and coworkers [153], outlined in Scheme 70. During the synthesis, the growing skeleton of 363 remained attached to a robust dienic linker. After completion of intermediate 362, the terminal olefin in 363 was liberated from the solid support by the final metathesis process with concomitant formation of a polymer-bound cyclopentene 364. Notably, during the synthesis it turned out that polymer-bound intermediate 365a, in contrast to soluble benzoate 365b, produced diene 367 only in low yield. After introduction of an additional linker (cf intermediate 366), diene 367 was released in distinctly improved yield by RCM. 

A straightforward application of an Ugi reaction in natural product synthesis has been elucidated by Bauer and Armstrong [53]. These authors prepared the intermediate 9-68 in the synthesis of the complex protein phosphatase inhibitor motuporin (9-69), by using an U-4CR process starting from the acid 9-64, the aldehyde 9-65, methylamine, and the isocyanide 9-66 via 9-67. 

Surprisingly few studies have been performed with purified toxins. When added externally to the water, toxins of various origins were tested on the cope-pod Tigriopus californicus. The protein phosphatase inhibitor okadaic acid (17) from red tide dinoflagellates [22] and the neuronal depolarizing agent do-moic acid (10) from diatoms [40, 41] had different effects on the herbivores (Scheme 3). Micromolar concentrations of okadaic acid (17) acted both as toxin... 

Useful serine/threonine protein phosphatase inhibitors include microcystin-LR (which inhibits protein phosphatases 1, 2A, and 2C, and related enzymes) and /1-glycerophosphate. Sodium fluoride may also be employed. Sodium orthovanadate inhibits protein tyrosine phosphatases. 

Protein phosphatase 1 is regulated by protein phosphatase inhibitor proteins 401... 

Whereas inhibitors 1 and 2 and NIPP1 appear to be widely distributed in mammalian tissues, including brain, DARPP-32 shows a much more restricted distribution. The protein is enriched in discrete populations of neurons in the brain, most prominently those that express Dl-dopamine receptors (see Chs 12,46 and 54). Some neuronal cell types thus appear to contain unique species of phosphatase inhibitor proteins. The critical role played by these proteins in neuronal function is illustrated below. 

Protease inhibitors must be present throughout the protein isolation procedure, it may be also of interest to add specific phosphatase inhibitors to the cocktail of protease inhibitors, especially when aiming at the detection of the phosphorylated form of the protein of interest. 

The serine/threonine phosphatase inhibitor okadaic acid (incubation time and concentration vary from 10 nM, 30 minutes to 1 pM, 1 hour at 37°C) leads to selective stimulation of caveolae uptake (18,64). 

In a total synthesis of cdc25A protein phosphatase inhibitor dysidiolide (46) [37], substitution on an sp carbon center by vinyl cuprate was used to accom-... 

Phosphorylase phosphatase inhibitor Glutamate receptor agonist Unknown Na+, Ca++ channel activators... 

Scheme 10.7 Protein tyrosine phosphatase inhibitor assay.

Umezawa K, Kawakami M, Watanabe T. Molecular design and biological activities of protein-tyrosine phosphatase inhibitors. Pharmacol Ther 2003 99 15-24. 

Agrawal GK, Jwa NS, Rakwal R, A novel rice Oryza sativa L.) acidic PRl gene highly responsive to cut, phytohormones, and protein phosphatase inhibitors, Biochem Biophys Res Commun 274 157—165, 2000. 

Chambers TC, Zheng B, Kuo JF (1992) Regulation by phorbol ester and protein kinase C inhibitors, and by a protein phosphatase inhibitor (okadaic acid) of P-glycoprotein phosphorylation and relationship to drug accumulation in multidrug-resistant human KB cells. Mol Pharmacol 41 1008-1015... 

Sakurada K, Zheng B, Kuo JF (1992) Comparative effects of protein phosphatase inhibitors (okadaic acid and calyculin A) on human leukemia HL60, HL60/ADR and K562 cells. Biochem Biophys Res Commun 187 488-492... 

Already in 1982, it was suggested that the intermediate chromium(V) state is involved in the carcinogenic process.9 Reactive Cr(V) and Cr(IV) intermediates may be harmful in many ways acting as tyrosine phosphatase inhibitors, or by forming organic radicals upon reaction with cellular reductants, which in turn can react with O2 and lead to reactive oxygen species.10 Reaction of chromium(VI) with... 

Gunasekera, S.P. McCarthy, P.J. Kelly-Borges, M. Lobkovsky, E. Clardy, J. (1996A) Dysidiolide a novel protein phosphatase inhibitor from the Caribbean sponge Dysidea etheria. J. Am. Chem. Soc., 118, 8759-60. 

In the search for novel orally active hypoglycemic agents, a team from Hoffmann-LaRoche used the readily available (see Section 10.20.9.2.3) chloromethylpyrimido[4,3-( ]-l,2,4-triazine 86 as a starting material for the synthesis of the protein tyrosine phosphatase inhibitors 87 (individual yields were not detailed), as shown in Equation (12) <2003BML2895>. 

The piperazine-substituted pyrimido[5,4-< ][l,2,4]triazine 103 undergoes selective reaction with benzylic halides to provide the benzylic piperazinyl analogues 104 <2003BML2895> as shown in Equation (15). The products are protein tyrosine phosphatase inhibitors. 

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