title: Quantitating drug-target engagement in single cells in vitro and in vivo.
abstract: Quantitation of drug target engagement in single cells has proven to be difficult, often leaving unanswered questions in the drug development process. We found that intracellular target engagement of unlabeled new therapeutics can be quantitated using polarized microscopy combined with competitive binding of matched fluorescent companion imaging probes. We quantitated the dynamics of target engagement of covalent BTK inhibitors, as well as reversible PARP inhibitors, in populations of single cells using a single companion imaging probe for each target. We then determined average in vivo tumor concentrations and found marked population heterogeneity following systemic delivery, revealing single cells with low target occupancy at high average target engagement in vivo.
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: J Matthew
authors.initials: JM
authors.lastname: Dubach
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Eunha
authors.initials: E
authors.lastname: Kim
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Yang
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Cuccarese
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Randy J
authors.initials: RJ
authors.lastname: Giedt
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Labros G
authors.initials: LG
authors.lastname: Meimetis
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Claudio
authors.initials: C
authors.lastname: Vinegoni
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.|Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
mesh_terms: D004305:Dose-Response Relationship, Drug; D006801:Humans; D015394:Molecular Structure; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D059010:Single-Cell Analysis; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
chemical_list: D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases