Development of a Fluorescence Polarization Bead-Based Coupled Assay to Target Different Activity/Conformation States of a Protein Kinase

Abstract

Most of the protein kinase inhibitors being developed are directed toward the adenosine triphosphate (ATP) binding site that is highly conserved in many kinases. A major issue with these inhibitors is the specificity for a given kinase. Structure determination of several kinases has shown that protein kinases adopt distinct conformations in their inactive state, in contrast to their strikingly similar conformations in their active states. Hence, alternative assay formats that can identify compounds targeting the inactive form of a protein kinase are desirable. The authors describe the development and optimization of an Immobilized Metal Assay for Phosphochemicals (IMAP™)-based couple™d assay using PDK1 and inactive Akt-2 enzymes. PDK1 phosphorylates Akt-2 at Thr 309 in the catalytic domain, leading to enzymatic activation. Activation of Akt by PDK1 is measured by quantitating the phosphorylation of Akt-specific substrate peptide using the IMAP assay format. This IMAP-coupled assay has been formatted in a 384-well microplate format with a Z′ of 0.73 suitable for high-throughput screening. This assay was evaluated by screening the biologically active sample set LOPAC™ and validated with the protein kinase C inhibitor staurosporine. The IC₅₀ value generated was comparable to the value obtained by the radioactive 33P-γ-ATP flashplate transfer assay. This coupled assay has the potential to identify compounds that target the inactive form of Akt and prevent its activation by PDK1, in addition to finding inhibitors of PDK1 and activated Akt enzymes.

Keywords

protein kinase inhibitors IMAP high-throughput screening Akt

References

1. Cohen P : Protein kinases—the major drug targets of the twenty-first century? Nat Rev Drug Discovery 2002;1:309-315.

2. Dancey J , Sausville EA : Issues and progress with protein kinase inhibitors for cancer treatment. Nat Rev Drug Discovery 2003;2:296-313.

3. Blume-Jensen P , Hunter T : Oncogenic kinase signalling. Nature 2001;411:355-365.

4. Kumar C , Madison V : Drugs targeted against protein kinases. Expert Opin Emerging Drugs 2001;6:303-315.

5. Manning G , Whyte DB , Martinez R , Hunter T , Sudarsanam S : The protein kinase complement of the human genome. Science 2002;298:1912-1934.

6. Gaudet E , Huang KS , Zhang Y , Huang W , Mark D , Sportsman JR : A homogeneous fluorescence polarization assay adaptable for a range of protein serine/threonine and tyrosine kinases. J Biomol Screen 2003;8:164-175.

7. Turek T , Small EC , Bryant RW , Hill WAG : Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody. Anal Biochem 2001;299:45-53.

8. Turek T , Kober TP , Stafford JM , Bryant RW : Development of a fluorescence polarization Akt serine/threonine kinase assay using an immobilized metal affinity-based technology. Assay Drug Dev Tech 2003;1:1-9.

9. Sportsman J , Lee SK , Diley H , Bukar R : Fluorescence polarization. In Delvin JP (ed), High Throughput Screening: The Discovery of Bioactive Substances. New York: Marcel Dekker, 1997:389-399.

10.

10. Sportsman J , Daijo J , Gaudet EA : Fluorescence polarization assays in signal transduction discovery. Comb Chem High Throughput Screen 2003;6:195-200.

11.

11. Huse M , Kuriyan J : The conformational plasticity of protein kinases. Cell 2002;109:275-282.

12.

12. Nagar B , Bornmann WG , Pellicena P , Schindler T , Veach DR , Miller WT , et al : Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res 2002;62:4236-4243.

13.

13. Schindler T , Bornmann W , Pellicena P , Miller WT , Clarkson B , Kuriyan J : Structural mechanism for STI-571 inhibition of abelson tyrosine kinase. Science 2000;289:1938-1942.

14.

14. Pargellis C , Tong L , Churchill L , Cirillo PF , Gilmore T , Graham AG , et al : Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site. Nat Struct Biol 2002;4:268-272.

15.

15. Druker B : Perspectives on the development of a molecularly targeted agent. Cancer Cell 2002;1:31-36.

16.

16. Capdeville R , Buchdunger E , Zimmermann J , Matter A : Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discovery 2002;1:493-502.

17.

17. Staal SP : Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. Proc Natl Acad Sci USA 1987;84:5034-5037.

18.

18. Vivanco I , Sawyers CL : The phosphatidylinositol 3-kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2:489-501.

19.

19. Brazil D , Hemmings BA : Ten years of protein kinase B signalling: a hard Akt to follow. Trends Biochem Sci 2001;26:657-664.

20.

20. Lawlor M , Alessi DR : PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci 2001;114:2903-2910.

21.

21. Testa J , Bellacosa A : AKT plays a central role in tumorigenesis. Proc Natl Acad Sci USA 2001;98:10983-10985.

22.

22. Scheid M , Woodgett JR : Unravelling the activation mechanism of protein kinase B/Akt. FEBS Lett 2003;27349:1-5.

23.

23. Alessi D , James SR , Downes CP , Holmes AB , Gaffney PR , Reese CB , et al : Characterization of a 3′-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase B-alpha. Curr Biol 1997;7:261-269.

24.

24. Lawlor M , Mora A , Ashby PR , Williams MR , Murray-Tait V , Malone L , et al : Essential role of PDK1 in regulating cell size and development in mice. Embo J 2002;21:3728-3738.

25.

25. Kumar C , Diao R , Yin Z , Liu Y , Samatar AA , Madison V , et al : Expression, purification, characterization and homology modeling of active Akt/PKB, a key enzyme involved in cell survival signaling. Biochim Biophys Acta 2001;1526:257-268.

26.

26. Alessi D , Cohen P : Mechanism of activation and function of protein kinase B. Curr Opin Genet Dev 1998;8:55-62.

27.

27. Zhang J , Chung T , Oldenburg K : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biol Chem 1999;4:67-73.

28.

28. Schindler T , Sicheri F , Pico A , Gazit A , Levitzki A , Kuriyan J : Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor. Mol Cell 1999;5:639-648.

29.

29. Xu W , Harrison SC , Eck MJ : Three-dimensional structure of the tyrosine kinase c-Src. Nature 1999;385:595-602.