Development of fluorescence and luminescence-based Src protein kinase chemosensors — ASN Events
  1. Schedule
  2. Session 7 SPPS Automation, Materials & Applications
  3. Development of fluorescence and luminescence-based Src protein kinase chemosensors

Development of fluorescence and luminescence-based Src protein kinase chemosensors (#28)

Denis Scanlon 1 , Hueng-Chin Cheng 2 , Bim Graham 3 , Philip Thompson 3 , Aizuddin Kamaruddin 2
  1. Chemistry and Physics, University of Adelaide, Adelaide, SA, Australia
  2. Biochemistry, The University of Melbourne, Melbourne, Vic, Australia
  3. Chemistry, School of Pharmacy, Melbourne, Vic, Australia

Protein kinases (PKs) are key components  of cellular signalling processes and are responsible for 30% of all protein synthesis within the cell. Aberrant kinase activity has been implicated in numerous cancers and neurological disorders . This has resulted in protein kinases being targeted by the pharmaceutical industry as one of the most important drug targets in the last decade. However, there are only nine kinase inhibitors that have currently been approved and there are only two kinase targets, which are the EGFR kinase and Bcr-Abl kinases. All the small molecules compounds were identified through a high throughput screening (HTS) program. Despite this fact, there are many limitations with the current kinase assay.

The P32 radioactive assay is the gold standard to monitor activity of PKs as it is sensitive and specific. However, the limitations with this assay are the requirement for radioactive materials, and it is a discontinuous process. Recently novel methods have been  published  based on fluorescence ([1], [2]), chelation enhanced fluorescence ( [3] ), or lanthanide(Terbium) based assays ( [4], [5]). Our aim was to develop new, simple and robust peptide based assays for monitoring Src kinase activity by fluorescence or lanthanide-based luminescence which were applicable to HTS applications. We have used the Src optimal peptide sequence AEEEIYGEFEAKKKK as our peptide substrate throughout the study.

We will present a solid-phase peptide synthesis approach to assembling eight separate Src optimal peptides, incorporating “click” chemistry to attach fluorescent moieties in position 9 of the peptide and their utility as chemosensor peptides in a FRET based Src kinase assay.  We will also present the development of a novel MAP peptide construct and its utility in a Terbium based luminescence Src kinase assay.

  1. [1] Ting, A.Y., et.al. (2001) Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells. Proceedings of the Natioal Acadamy of Sciences, 98(26): p.15003
  2. [2] Sharma, V., Wang, Q. and Lawrence D.S. (2008 ) Peptide-based fluorescent sensors of protein kinase activity: design and applications. Biochimica et Biophysica Acta (BBA)-Proteins & Proteomics, 1784(1): p. 94-99.
  3. [3] Shults, M.D., Carrico-Moniz, D. and Imperiali, B. (2006) Optimal Sox-based fluorescent chemosensor design for serine/threonine protein kinases. Analytical Biochemistry, 352(2): p.198-207.
  4. [4]Tremblay, M.S., Lee, M. and Sames, D. (2008) A luminescent sensor for tyrosine phosphorylation.Organic Letters, 10(1): p.5-8.
  5. [5] Akiba,H., Sumaoka, J. and Komiyama, M. (2010) Binuclear Terbium III complex as a probe for Tyrosine phosphorylation Chemistry-A European Journa.l 16(7): p. 5018-5025.