Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
2002-05-13
2004-03-30
Leary, Louise N. (Department: 1654)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S004000, C435S968000
Reexamination Certificate
active
06713274
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of sulfotransferase enzymes, and in particular assay methods for identifying agents that modulate sulfotransferase activity.
BACKGROUND OF THE INVENTION
Sulfation of biomolecules is now appreciated as a major regulatory modification that affects interactions in the extracellular space. The sulfotransferases (STs) are a family of enzymes that catalyzes the sulfation of biomolecules. Many STs act on glycoproteins in the Golgi compartment. Dozens of STs have been discovered, and in some cases the epitopes they generate have been linked to disease processes. For example, specific carbohydrate sulfates govern growth factor binding and activation, and leukocyte adhesion during inflammation. Tyrosine sulfation is a prerequisite for HIV-1 binding to host cell co-receptors in addition to other cell adhesion events. These examples have piqued the interests of both biologists and medicinal chemists, as STs are emerging as attractive therapeutic targets.
There has been some progress in the development of relatively efficient assays for individual carbohydrate STs. A traditional architecture involves radiolabel transfer from
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S-labeled 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a carbohydrate substrate bearing a hydrophobic tail for capture on reversed-phase cartridges. While this approach can be adapted to 96-well format, it requires several labor-intensive washing and elution steps in order to purify the sulfated product before quantitation by liquid scintillation. Biotinylated substrates have been used for sulfotransferase assays, wherein capture with immobilized avidin separates the
35
S-labeled products from unreacted
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S-labeled PAPS. Fewer washing steps are required, but microtiter plate-based systems that immobilize avidin are fairly expensive, particularly for high-throughput screens. Wong and co-workers reported an enzyme-coupled continuous spectrophotometric assay that could in principle be applied to any sulfotransferase. While ideal for kinetic assays, the presence of a second enzyme, in this case an aryl sulfotransferase, might complicate ST-targeted inhibitor screens.
Therefore, there is a general need for simple, high-throughput screening strategies for measuring activities in the presence and absence of inhibitors. The present invention addresses this need.
Literature
Armstrong et al. (2000)
Curr. Opin. Drug Disc. Dev
. 3:502-515; Sueyoshi et al. (1998)
FEBS Lett
. 433:211-214; Cook et al. (2000)
J. Am. Chem. Soc
. 122:8612-8622; Hemmerich and Rosen (2000)
Glycobiol
. 10:849-856; Sugahara et al. (1987)
Anal. Biochem
. 166:404-412; Mandon et al. (1994)
J. Biol. Chem
. 269:11729-11733; Hooper et al. (1993)
Anal. Biochem
. 212:128-133; Kehoe et al. (2002)
Bioorg. Med. Chem. Lett
. 12:329-332; Burkart and Wong (1999)
Anal. Biochem
. 274:131-137.
SUMMARY OF THE INVENTION
The instant invention provides methods for identifying agents that modulate an enzymatic activity of a carbohydrate sulfotransferase. The methods generally involve contacting the sulfotransferase, in a reaction solution, with a sulfate donor, a test agent, and a polymeric sulfate acceptor that is readily separated from the reaction solution. Determination of an effect of the test agent on the sulfotransferase is by detecting the amount of sulfate in the polymeric sulfate acceptor that has been separated from the reaction solution. The invention further provides kits for use in carrying out the subject methods.
REFERENCES:
patent: 6365365 (2002-04-01), Bistrup et al.
patent: 2003/0109501 (2003-06-01), Yang et al.
Kusche et al, J. Biological Chemistry, vol. 266, No. 12, p 7400-7409, (Apr. 26, 1991).*
Armstrong, et al. “Sulfotransferases as targets for therapeutic intervention”,Curr. Opin. Drug Disc. Dev., (2000) vol. 3: 502-515.
Burkart, et al. “A continuous assay for the spectrophotometric analysis sulfotransferases using aryl sulfotransferase IV”,Anal. Biochem., (1999) vol. 274: 131-137.
Cook, et al. “Differential carbohydrate recognition of two GIcNAc-6-sulfotransferases with possible roles in L-selectin ligand biosynthesis”,J. Am. Chem. Soc., (2000) vol. 122: 8612-8622.
Hemmerich, et al. “Carbohydrate sulfotransferases in lymphocyte homing”,Glycobiol., (2000) vol. 10(9): 849-856.
Hooper, et al. “Sulfotransferase and glycosyltransferase analyses using a 96-well filtration plate”,Anal. Biochem., (1993) vol. 212: 128-133.
Kehoe, et al. “Tyrosylprotein sulfotransferase inhibitors generated by combinational target-guided ligand assembly”,Bio. Org. Med. Chem. Lett., (2002) vol. 12: 329-332.
Mandon, et al. “A monomeric protein in the golgi membrane catalyzes both N-deacetylation and N-sulfation of heparan sulfate”,J. Biol. Chem., (1994) vol. 269: 11729-11733.
Sueyoshi, et al. “A role of Lys614in the sulfotransferase activity of human heparan sulfate N-deacetylation/N-sulfation”,FEBS Lett., (1998) vol. 433: 211-214.
Sugahara, et al. “Paper disk assay for glycosaminoglycan sulfotransferases”,Anal. Biochem., (1987) vol. 166: 404-412.
Bertozzi Carolyn
Verdugo Dawn
Borden Paula A.
Bozicevic Field & Francis LLP
Leary Louise N.
The Regents of the University of California
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