Development of self-reporting fluorescent chemosensor peptide substrates as molecular sensors
In vitro assays of protein kinase activity have been routinely performed by monitoring the transfer of the radioactive 32P-phosphate from [γ-32P]ATP to specific protein and peptide substrates catalysed by protein kinases.
Owing to the need to use radioactivity and their cumbersome nature, the assays can only be performed in research laboratories. That is, they are not amenable for routine assays of diseased tissues in pathology laboratories and high throughput screening for potential therapeutic inhibitors of disease-related protein kinases. In light of this, specific self-reporting fluorescent chemosensor peptide substrates of protein kinases are valuable tools for high-throughput non-radioactive assays of protein kinases.
This project aims to design and synthesise self-reporting fluorescent chemosensor peptide substrates of protein kinases. We have successfully produced the first generation of such substrates for protein tyrosine kinases (Figure 1). For applications (see below), we will use these peptide substrates to develop convenient non-radioactive assays for protein kinases governing platelet activation and those directing cancer formation and progression.
Application 1: The aggregometry-based assays of platelet function are routinely performed to diagnose congenital bleeding disorders and to ensure normal platelet function in patients prior to surgery. This assay monitors the aggregation of platelets by light transmission upon stimulation by agonists such as ADP and thrombin. The assay is insensitive and labour-intensive and its use results in unnecessary delays in surgery and treatment of the patients.
Activation of Src-family protein kinases (SFKs) is a key step in platelet activation. Upon activation, SFKs control platelet cell adhesion and blood clot formation and retraction. In this project, we aim to develop self-reporting fluorescent chemosensor peptide substrates to monitor SFK activity in platelets. The peptide substrates will be used to develop sensitive and reliable assays of platelet function to complement or replace the aggregometry-based assay widely used in many pathological laboratories.
Application 2: Protein tyrosine kinases such as SFKs and Bruton's tyrosine kinase (Btk) are involved in the formation and progression of solid tumours, lymphomas and leukaemias.
For example, the aberrant increase in activity and expression of SFKs contributes to the formation and progression of both colon cancer and leukaemia. Btk, an essential non-receptor tyrosine kinase in the B-cell receptor signaling pathway is abnormally activated in several types of B cell lymphoma.
Synthetic inhibitors of SFKs such as Dasatinib and the irreversible inhibitor of Btk PCI-32765 are effective in treating several types of cancer. This project aims to develop self-reporting fluorescent chemosensor peptide substrates to monitor the activity of the pathogenic tyrosine kinases for use in clinical diagnostic laboratories.
Figure 1: Development of the first generation of self-reporting chemosensor peptide substrate of Src-family kinases. A. A schematic presentation of the mechanism of increase in fluorescence intensity due to phosphorylation. Phosphorylation reduces the quenching effect of the tyrosine on the fluorophore in the chemosensor peptide. B. Fluorescence spectra of the phosphorylated and unphosphorylated forms of the pyrene-based chemosensor peptide substrate of Src-family kinases.
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