Two-photon Fluorescence Anisotropy Microscopy for Imaging and Direct Measurement of Intracellular Drug Target Engagement.

Small molecule therapeutic drugs must reach their intended cellular targets (pharmacokinetics) and engage them to modulate therapeutic effects (pharmacodynamics). These processes are often difficult to measure in vivo due to their complexities and occurrence within single cells. It has been particularly difficult to directly measure cellular drug target binding. Fluorescence polarization is commonly used in pharmacological screening assays to measure drug-protein or protein-protein interactions. We hypothesized that fluorescence polarization imaging could be adapted and used with fluorescently labeled drugs to measure drug target engagement in vivo. Here we summarize recent results using two photon fluorescence anisotropy microscopy. Our imaging technique offers quantitative pharmacological binding information of diverse molecular interactions at the microscopic level, differentiating between bound and unbound states. Used in combination with other recent advances in the development of novel fluorescently labeled drugs, we expect that the described imaging modality will provide a window into the distribution and efficacy of drugs in real time and in vivo at the cellular and subcellular level.

Optimized Near-IR Fluorescent Agents for in Vivo Imaging of Btk Expression.

Bruton's tyrosine kinase (Btk) is intricately involved in anti-apoptotic signaling pathways in cancer and in regulating innate immune response. A number of Btk inhibitors are in development for use in treating B-cell malignancies and certain immunologic diseases. To develop robust companion imaging diagnostics for in vivo use, we set out to explore the effects of red wavelength fluorochrome modifications of two highly potent irreversible Btk inhibitors, Ibrutinib and AVL-292. Surprisingly, we found that subtle chemical differences in the fluorochrome had considerable effects on target localization. Based on iterative designs, we developed a single optimized version with superb in vivo imaging characteristics enabling single cell Btk imaging in vivo. This agent (Ibrutinib-SiR-COOH) is expected to be a valuable chemical tool in deciphering Btk biology in cancer and host cells in vivo.