Development of LanthaScreen™ Cellular Assays for Key Components within the PI3K/AKT/mTOR Pathway

Abstract

The PI3K/AKT/mTOR pathway is central to cell growth and survival, cell cycle regulation, and programmed cell death. Aberrant activation of this signaling cascade is linked to several disease states, and thus many components of the pathway are attractive targets for therapeutic intervention. However, the considerable degree of complexity, crosstalk, and feedback regulation that exists within the pathway (especially with respect to the regulation of mTOR and its complexes) underscores the need for a comprehensive set of cell-based assays to properly identify and characterize small-molecule modulators. Here, the development and application of time-resolved Förster resonance energy transfer (TR-FRET)—based assays to enable the phosphoprotein analysis of key pathway components in a cellular format are reported. The LanthaScreen™ cellular assay platform uses FRET between a terbium-labeled phosphorylation site-specific antibody and an expressed green fluorescent protein fusion of particular kinase substrate and provides an assay readout that is ratiometric, robust, and amenable to high-throughput screening applications. Assays specific for 5 different targets within the pathway are highlighted: Ser183 and Thr246 on the proline-rich AKT substrate 40 kDa (PRAS40), Ser457 on programmed cell death protein 4 (PDCD4), and Thr308 and Ser473 on AKT. Each assay was evaluated under various experimental conditions and individually optimized for performance. Known pathway agonists and a small panel of commercially available compounds were also used to complete the assay validation. Taken together, these data demonstrate the utility of a related set of cell-based assays to interrogate PI3K/AKT/mTOR signaling and provide a template for the development of similar assays for other targets. (Journal of Biomolecular Screening 2009:121-132)

Keywords

PI3K AKT mTOR TR-FRET GFP

References

Corradetti MN , Guan K. -L: Upstream of the mammalian target of rapamycin: do all roads pass through mTOR? Oncogene 2006 ;25:6347-6360.

Manning BD , Cantley LC : AKT/PKB signaling: navigating downstream. Cell 2007;129:1261-1274.

Tsang CK , Qi H. , Liu LF , Zheng XJS : Targeting mammalian target of rapamycin (mTOR) for health and diseases . Drug Discov Today 2007;12:112-124.

Horton RA , Strachan EA , Vogel KW , Riddle SM : A substrate for deubiquitinating enzymes based on time-resolved fluorescence resonance energy transfer between terbium and yellow fluorescent protein. Anal Biochem 2007;360: 138-143.

Riddle SM , Vedvik KL , Hanson GT , Vogel KW : Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: applications of terbium-fluorescein and terbium-green fluorescent protein fluorescence resonance energy transfer pairs. Anal Biochem 2006;356:108-116.

Robers MB , Horton RA , Bercher MR , Vogel KW , Machleidt T. : High-throughput cellular assays for regulated post-translational modifications . Anal Biochem 2008;372:189-197.

Robers MB , Machleidt T. , Carlson CB , Bi K. : Cellular LanthaScreen and beta-lactamase reporter assays for high-throughput screening of JAK2 inhibitors. ASSAY Drug Dev Technol 2008;6:519-529.

Binder C. , Lafayette A. , Archibeque I. , Sun Y. , Plewa C. , Sinclair A. , et al: Optimization and utilization of the SureFire phospho-STAT5 assay for a cell-based screening campaign. ASSAY Drug Dev Technol 2008;6:27-37.

Vander Haar E. , Lee S. , Bandhakavi S. , Griffin TJ , Kim D-H. : Insulin signaling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat Cell Biol 2007 ;9:316-323.

10.

Oshiro N. , Takahashi R. , Yoshino K. , Tanimura K. , Nakashima A. , Eguchi S. , et al: The proline-rich AKT substrate of 40 kDa (PRAS40) is a physiological substrate of the mammalian target of rapamycin complex 1. J Biol Chem 2007;282:20329-20339.

11.

Lankat-Buttgereit B. , Göke R. : Programmed cell death protein 4 (pdcd4): a novel target for antineoplastic therapy? Biol Cell 2003;95:515-519.

12.

Sarbassov DD , Guertin DA , Ali SM , Sabatini DM : Phosphorylation and regulation of the AKT/PKB by the rictor-mTOR complex. Science 2005;307:1098-1101.

13.

Zhang JH , Chung TD , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 1999;4:67-73.

14.

Fonesca BD , Smith EM , Lee Vh-Y , MacKintosh C. , Proud CG : PRAS40 is a target for mammalian target of rapamycin complex 1 and is required for signaling downstream of this complex. J Biol Chem 2007;282:24514-24524.

15.

Pagliaro L. , Pr stegaard M. : Transfected cell lines as tools for high throughput screening: a call for standards. J Biomol Screen 2001;6:133-136.

16.

Vlahos CJ , Matter WF , Hui KY , Brown RF : A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 1994;269:5241-5248.

17.

Fan Q-W. , Knight ZA , Goldenberg DD , Yu W. , Mostov KE , Stokoe D. , et al: A dual PI3 kinase / mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 2006;9:341-349.

18.

Knight ZA , Gonzalez B. , Feldman ME , Zunder ER , Goldenberg DD , Williams O. , et al: A pharmacological map of the PI3-K family defines a role for p110a in insulin signaling. Cell 2006;125:733-747.

19.

Chaussade C. , Rewcastle GW , Kendall JD , Denny WA , Cho K. , Grønning LM , et al: Evidence for functional redundancy of class IA PI3K isoforms on insulin signalling . Biochem J 2007;404:449-458.

20.

Kau TR , Schroeder F. , Ramaswamy S. , Wojciechowski CL , Zhao JJ , Roberts TM , et al: A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTENdeficient tumor cells . Cancer Cell 2003;4:463-476.

21.

Lindsley CW , Zhao Z. , Leister WH , Robinson RG , Barnett SF , Defeo-Jones D. , et al: Allosteric Akt (PKB) inhibitors: discovery and SAR of isozyme selective inhibitors . Bioorg Med Chem Lett 2005;15:761-764.

22.

Thimmaiah KN , Easton JB , Germain GS , Morton CL , Kamath S. , Buolamwini JK , et al: Identification of N10-substituted phenoxazines as potent and specific inhibitors of Akt signaling . J Biol Chem 2005; 280:31924-31935.

23.

Yang L. , Dan HC , Sun M. , Liu Q. , Sun X-M. , Feldman RI , et al: Akt/protein kinase B signaling inhibitor-2, a selective small-molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt. Cancer Res 2004;64:4394-4399.

24.

Blum G. , Gazit A. , Levitzki A. : Development of new insulin-like growth factor receptor kinase inhibitors using catechol mimics. J Biol Chem 2003;278:40442-40454.

25.

Hayakawa M. , Kaizawa H. , Moritomo H. , Koizumi T. , Ohishi T. , Okada M. , et al: Synthesis and biological evaluation of 4-morpholino-2-phenylquinazolines and related derivatives as novel PI3 kinase p110α inhibitors. Bioorg Med Chem 2006;14:6847-6858.

26.

Zaman GJR , de Roos JADM , Blomenröhr M. , van Koppen CJ , Oosterom J. : Cryopreserved cells facilitate cell-based drug discovery. Drug Discov Today 2007;12:521-526.