title: Multicolored, Tb³⁺-Based Antibody-Free Detection of Multiple Tyrosine Kinase Activities.
abstract: Kinase signaling is a major mechanism driving many cancers. While many inhibitors have been developed and are employed in the clinic, resistance due to crosstalk and pathway reprogramming is an emerging problem. High-throughput assays to detect multiple pathway kinases simultaneously could better model these complex relationships and enable drug development to combat this type of resistance. We developed a strategy to take advantage of time-resolved luminescence of Tb(3+)-chelated phosphotyrosine-containing peptides, which facilitated efficient energy transfer to small molecule fluorophores conjugated to the peptides to produce orthogonally colored biosensors for two different kinases. This enabled multiplexed detection with high signal-to-noise in a high-throughput-compatible format. This proof-of-concept study provides a platform that could be applied to other lanthanide metal and fluorophore combinations to achieve even greater multiplexing without the need for phosphospecific antibodies.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and Purdue Center for Cancer Research, Purdue University, 201 S. University Street, West Lafayette, Indiana 47907, United States.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Lipchik
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and Purdue Center for Cancer Research, Purdue University, 201 S. University Street, West Lafayette, Indiana 47907, United States.
authors.forename: Minervo
authors.initials: M
authors.lastname: Perez
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and Purdue Center for Cancer Research, Purdue University, 201 S. University Street, West Lafayette, Indiana 47907, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Cui
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy and Purdue Center for Cancer Research, Purdue University, 201 S. University Street, West Lafayette, Indiana 47907, United States.
authors.forename: Laurie L
authors.initials: LL
authors.lastname: Parker
mesh_terms: D000906:Antibodies / Q000737:chemistry; D015374:Biosensing Techniques; D003116:Color; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013725:Terbium / Q000737:chemistry*
chemical_list: D000906:Antibodies; D013725:Terbium; D011505:Protein-Tyrosine Kinases