KINATEST-ID: A Pipeline To Develop Phosphorylation-Dependent Terbium Sensitizing Kinase Assays

Abstract

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.

High-throughput identification of FLT3 wild-type and mutant kinase substrate preferences and application to design of sensitive in vitro kinase assay substrates

“…29,30 We then used the identified substrate preferences to rationally design a panel of candidate peptides incorporating key sequence features predicted to make them favorable for phosphorylation by the FLT3 kinase variants, following our previously reported substrate development pipeline KINATEST-ID. 31 We demonstrated that these substrates enable efficient inhibitor screening for all three forms of FLT3. These peptides could be used in many different types of drug discovery settings to more rapidly and efficiently screen for and validate FLT3 inhibition.…”

“…Frequency, Non-substrate Motifs, and Screener.csv, the scripts "Kinatestpart1.R" and "Kinatestpart2.R" were written to replicate the functionality of the KINATEST-ID workbooks previously described, 31 Table; and 3) the list of predicted substrates ranked according to lowest "off-target" kinase scores using the Screener comparisons. For more information about these functions see the previously published description of KINATEST-ID.…”

“…Overall, we identified more than 10-fold more substrates for FLT3 and the two mutant variants than we had curated for most of the kinases we had evaluated in our previous publication using KINATEST-ID, in which substrate numbers ranged from ~15 to ~170. 31 Using a relatively strict identification quality cut-off of 1% false discovery rate (FDR), we observed 1559 phosphorylated peptides from the kinase reaction with the WT FLT3 treatment, 2010 from the FLT3-D835Y mutant, and 344 from the FLT3-ITD mutant. Of these, 244 overlapped in common from each reaction ( Figure 1).…”

“…Due to its larger molecular weight relative to the WT and D835Y variants, less of this kinase was used in the KALIP reactions, which likely accounts for the lower number of substrates observed for that variant. Nevertheless, compared to the manual curation of the literature-reported substrates and phosphoproteomic databases that was implemented previously, 31 even for the ITD mutant our approach identified many more phosphopeptide sequences than before, which could be used as input for the KINATEST-ID pipeline.…”

Multicolored, Tb3+-Based Antibody-Free Detection of Multiple Tyrosine Kinase Activities

“…21–23 Here we show extension to a multiplexed detection platform for simultaneous monitoring of multiple tyrosine kinase activities (Lyn and Syk) via SFAStide-A and SAStide substrates (sequences given in Table 1). 21, 22 Multi-colored detection was achieved through time-resolved luminescence energy transfer (TR-LRET) by employing the phosphopeptide-Tb 3+ complexes as the energy donors and the conjugated fluorophores cyanine 5 (Cy5) and 5-carboxyfluorescein (5-FAM) respectively, as the energy acceptors (Figure 1A). …”