Lanthanide Luminescence Assay Development

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With appropriate incorporation of chelating and UV-absorbing side chains, peptides can coordinate lanthanide metal ions and sensitize their fluorescence/luminescence. Phosphorylation can enhance both chelation affinity and sensitization. Inspired by work from the Zondlo, Imperiali and Franz groups, we developed a time-resolved lanthanide luminescence assay using our Syk substrate (SAStide). We then expanded the design strategy to use known substrate preference information to develop a peptide and assay for ALK and a “pipeline” (KINATEST-ID) to develop lanthanide-sensitizing substrates for almost any kinase.

 

This project has been funded by the Purdue Center for Cancer Research and the NIH (grants R00CA127161 and R01CA182543).

KINATEST-ID pipeline for lanthanide-sensitizing substrate design

KINATEST-ID workflow

The KINATEST-ID “pipeline” is comprised of a series of Excel spreadsheets (available for download as supplemental files at the JACS website) that narrow down the sequence space (and thus peptides to make and test empirically) for a potential substrate for a given kinase or kinase family. It works by taking known substrate sequence preference information from online databases and the literature, as well as positional scanning peptide library data (from Benjamin Turk, Yale University), to define the positional scoring matrices for a panel of kinases. This information is also used to define the “site selectivity,” i.e. how much each kinase “cares” about the specific amino acid identity that is present in a given position from -4 to +4 relative to the central phosphorylated residue. These matrices are used to identify preference motifs, which are then expanded to produce all the possible permutations of amino acids in the motif for a given kinase (using the “Generator” tool–noted as Filter #1 above). The list of potential substrate sequences from the motif permutations is cross-referenced against “non-desired” kinases using the “Screener” tool (noted as Filter #2 above). This narrows down the potential sequence list to only those with a bioinformatically-predicted likelihood of being a selective substrate for the targeted kinase. The resulting list of potential sequences is then aligned and scored against a lanthanide binding motif using the “Aligner” tool (indicated as Filter #3 above). This further limits the candidate substrate sequences to those that should be compatible with a Tb-based lanthanide luminescence assay. The final steps in the process are to synthesize a selection of top-scoring sequences from “Aligner,” taking into account any potential clashes with positions that showed high site selectivity and may be less favorable to incorporating the Tb-chelating residues in those positions, and testing them empirically for both kinase substrate performance (efficiency and selectivity in vitro) and phosphorylation-dependent Tb-sensitizing performance in a time-resolved luminescence measurement.

A key aspect of the KINATEST-ID platform is that the “Aligner” tool could be used to align sequences with any given target motif–for example, if a different lanthanide or other metal ion binding was desired, or if some alternative detection strategy that depended on a different type of biomolecular interaction was desired, Aligner could be used to refine the target sequences for those properties.

The KINATEST-ID platform is currently licensed through the Purdue Research Foundation Office of Technology Commercialization to KinaSense LLC, a start-up out of the Parker Laboratory led by former graduate student Dr. Steven Ouellette. Dr. Laurie Parker and former graduate student Dr. Andrew Lipchik are co-founders, holding equity and serving on the Scientific Advisory Board for KinaSense.