There is a need to develop more sensitive, specific and robust strategies capable of imaging and quantitating therapeutic targets In cancers in vivo. To date, many biological targets and processes remain largely unobserved due to a) lack of affinity ligands, b) mismatches between target abundance and the amount of imaging agent required for target visualization, c) delivery barriers, d) unfavorable pharmacokinetics, e) high background signals, f) the unavailability of appropriate affinity ligands, and/or g) the sheer numbers of targets. The goal of this project is to use a recently developed bioorthogonal platform technology (known as BIND) for the rapid development of intracellular imaging agents such as polo-lil<e kinase (PLK1) and poly-ADP-ribose-polymerase 1 (PARP1), which are key intracellular cancer targets. BIND involves a {4+2} inverse Diels-Alder cycloaddition between fraA7s-cyclooctene (TCO) and a tetrazine (Tz), either attached to an imaging reporter or to a small molecule affinity ligand. BIND is biocompatible, has one of the most favorable reaction kinetics among bioorthogonal reactions, does not require catalysts, works well with small-molecule therapeutic drugs and can be combined with efficient amplifications strategies. This reaction can also be easily incorporated into development strategies for the vast number of existing small molecule affinity ligands for kinases and other intracellular targets. Cell permeable, small-molecule binders for PLK1 and PARP will be modified using TCO/Tz and imaged using complementary TCO/Tzimaging reporters. We will first validate the approach using cell based microscopic imaging, before performing in vivo experiments in mouse models of pancreatic ductal adenocarcinoma (PDAC).
The specific aims are: 1) to develop a library of PLK-1 and PARP1-targeted BIND agents and characterize them biochemically, using live cell imaging and intravital microscopy; 2) to perform target-identification and network analysis using in vivo proteomics (SILAC); and 3) to explore the translational potential using ^^F-labeled agents in gemcitabine and PLKHnhibitor/PARPinhibitor treated mice.
The proposed work will likely lead to more sensitive, rapid and robust generic methods for imaging and quantifying therapeutic targets in cancer. Such technologies will enable physicians to stratify patients into appropriate treatment groups and to detect emerging drug resistance.
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