Abstract:Disruption of regulatory protein phosphorylation can lead to disease, and is particularly prevalent in cancers. Inhibitors that target deregulated kinases are therefore a major focus of chemotherapeutic development. Achieving sensitivity and specificity in high-throughput compatible kinase assays is key to successful inhibitor development. Here we describe the application of time-resolved luminescence detection to the direct sensing of Syk kinase activity and inhibition using a novel peptide substrate. Chelati… Show more
“…ELISA), light scattering based methods using SERS and RLS, and fluorescence-based methods such as chelation enhanced fluorescence (CHEF), FRET and fluorescence quenching. 9-19 In particular, CHEF methods that sensitize lanthanide ions such as terbium (Tb 3+ ) in a phosphorylation-dependent manner 19-22 can enable high sensitivity and analytical reproducibility. Previously, we described the application of a kinase specific peptide substrate (SAStide) for the sensitive detection of spleen tyrosine kinase (Syk) activity in vitro through phosphorylation-dependent enhanced sensitization of Tb 3+ luminescence.…”
Section: Introductionmentioning
confidence: 99%
“…Previously, we described the application of a kinase specific peptide substrate (SAStide) for the sensitive detection of spleen tyrosine kinase (Syk) activity in vitro through phosphorylation-dependent enhanced sensitization of Tb 3+ luminescence. 22 The luminescence signal is generated when phosphorylation of the tyrosine residue results in exclusion of water and completion of the Tb 3+ coordination sphere. Phosphorylation also alters the excitation wavelength of the aromatic side chain, increases the binding affinity for the peptide, and increases the luminescence lifetime, 22 resulting in a large increase in signal to noise (16-fold in the case of SAStide).…”
Non-receptor protein tyrosine kinases (NRTKs) are essential for cellular homeostasis, and thus are a major focus of current drug discovery efforts. Peptide substrates that can enhance lanthanide ion luminescence upon tyrosine phosphorylation enable rapid, sensitive screening of kinase activity, however design of suitable substrates that can distinguish between tyrosine kinase families is a huge challenge. Despite their different substrate preferences, many NRTKs are structurally similar even between families. Furthermore, the development of lanthanide-based kinase assays is hampered by incomplete understanding of how to integrate sequence selectivity with metal ion binding, necessitating laborious iterative substrate optimization. We used curated proteomic data from endogenous kinase substrates and known Tb3+-binding sequences to build a generalizable in silico pipeline with tools to generate, screen, align and select potential phosphorylation-dependent Tb3+-sensitizing substrates that are most likely to be kinase specific. We demonstrated the approach by developing several substrates that are selective within kinase families and amenable to HTS applications. Overall, this strategy represents a pipeline for developing efficient and specific assays for virtually any tyrosine kinase that use high throughput screening-compatible lanthanide-based detection. The tools provided in the pipeline also have the potential to be adapted to identify peptides for other purposes, including other enzyme assays or protein binding ligands.
“…ELISA), light scattering based methods using SERS and RLS, and fluorescence-based methods such as chelation enhanced fluorescence (CHEF), FRET and fluorescence quenching. 9-19 In particular, CHEF methods that sensitize lanthanide ions such as terbium (Tb 3+ ) in a phosphorylation-dependent manner 19-22 can enable high sensitivity and analytical reproducibility. Previously, we described the application of a kinase specific peptide substrate (SAStide) for the sensitive detection of spleen tyrosine kinase (Syk) activity in vitro through phosphorylation-dependent enhanced sensitization of Tb 3+ luminescence.…”
Section: Introductionmentioning
confidence: 99%
“…Previously, we described the application of a kinase specific peptide substrate (SAStide) for the sensitive detection of spleen tyrosine kinase (Syk) activity in vitro through phosphorylation-dependent enhanced sensitization of Tb 3+ luminescence. 22 The luminescence signal is generated when phosphorylation of the tyrosine residue results in exclusion of water and completion of the Tb 3+ coordination sphere. Phosphorylation also alters the excitation wavelength of the aromatic side chain, increases the binding affinity for the peptide, and increases the luminescence lifetime, 22 resulting in a large increase in signal to noise (16-fold in the case of SAStide).…”
Non-receptor protein tyrosine kinases (NRTKs) are essential for cellular homeostasis, and thus are a major focus of current drug discovery efforts. Peptide substrates that can enhance lanthanide ion luminescence upon tyrosine phosphorylation enable rapid, sensitive screening of kinase activity, however design of suitable substrates that can distinguish between tyrosine kinase families is a huge challenge. Despite their different substrate preferences, many NRTKs are structurally similar even between families. Furthermore, the development of lanthanide-based kinase assays is hampered by incomplete understanding of how to integrate sequence selectivity with metal ion binding, necessitating laborious iterative substrate optimization. We used curated proteomic data from endogenous kinase substrates and known Tb3+-binding sequences to build a generalizable in silico pipeline with tools to generate, screen, align and select potential phosphorylation-dependent Tb3+-sensitizing substrates that are most likely to be kinase specific. We demonstrated the approach by developing several substrates that are selective within kinase families and amenable to HTS applications. Overall, this strategy represents a pipeline for developing efficient and specific assays for virtually any tyrosine kinase that use high throughput screening-compatible lanthanide-based detection. The tools provided in the pipeline also have the potential to be adapted to identify peptides for other purposes, including other enzyme assays or protein binding ligands.
“…The binding curve was hyperbolic and reached saturation at 20 µM of Tb 3+ , suggesting one-site binding behavior of the pALAStide (Figure 2d). The calculated K d for this pALAStide-Tb 3+ complex was 0.8 ± 0.2 µM, which is quite high Tb 3+ affinity in the context of other known Tb 3+ binding sequences 15,17,23 .…”
mentioning
confidence: 74%
“…As a strategy to overcome such problems, peptide-based approaches can be integrated into various analytical workflows for developing HTS compatible kinase assays 12–15 . Several examples of detecting tyrosine phosphorylation directly by lanthanide luminescence (rather than the antibody-dependent technologies) have been demonstrated 17–20 , either by using sensitizer-labeled peptides or peptides with inherent lanthanide-sensitizing sequences.…”
mentioning
confidence: 99%
“…We previously employed time-resolved lanthanide luminescence to design a high-throughput screening (HTS) compatible biosensor assay (Scheme 1) for Syk kinase activity 15 . However, lanthanide sensitization by the Syk substrate we developed was fortuitous based on the inherent substrate sequence, and not necessarily applicable to other substrates, which may not have the same advantageous chelating residues.…”
A novel time-resolved luminescence biosensor assay for anaplastic lymphoma kinase (ALK) was developed. We used a straightforward strategy to modify a known ALK substrate into a peptide biosensor that can accommodate terbium luminescence sensitization upon its phosphorylation by ALK. Since this strategy is generalizable, this high-throughput screening compatible assay serves as an example for development of other kinase assays that employ terbium luminescence as a read-out.
in aqueous solution under the visible excitation of up to 488 nm. This nanoprobe exhibits multiple advantages, including long excitation wavelength, high quantum effi ciency, long emission lifetime, narrow emission bands, high photostability, excellent water dispersibility, and good biocompatibility, all of which are very benefi cial for applications in bioimaging. The successful application of nanoprobe in bioimaging with visible excitation has been demonstrated. Thus, the design strategy will be a versatile and convenient way to realize excellent lanthanide(III) complex-based bioprobes for practical biomedical applications. Scheme 1. Design strategy and synthesis procedure of lanthanide(III) complex-based bioporbes sensitized by Ir(III) complexes. 235 wileyonlinelibrary.com
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