G-Quadruplex-Based Supramolecular Assemblies as Activatable Contrast Agents for In Vitro and In Vivo Fluorescence Imaging of Estrogen Receptor

PROJECT SUMMARY/ABSTRACT One in four women undergoing lumpectomy require a repeat surgery because of positive margins. One of the underlying challenges in breast tumor margin assessment is a lack of fast, sensitive and reliable intraoperative tools. Fluorescence-guided surgery (FGS) using activatable contrast agents (ACAs) is emerging as an intraoperative tool for margin assessment during lumpectomy. Current design approaches to ACAs used in the clinic have been limited to targeting extracellular or cell surface proteins and they rely on covalently attaching a fluorophore and a quencher to a peptide substrate. Protease cleavage of this substrate separates the fluorophore quencher pair thereby resulting in activation of fluorescence. A major disadvantage of this approach is that cleavage of the peptide substrate occurs indiscriminately in both normal and malignant tissues giving rise to false positive signals. Studies have shown higher false positive rates with protease-targeting ACAs than with standard- of-care pathology. One could potentially circumvent this disadvantage by targeting alternative breast cancer- specific proteins. However, in the absence of a suitable design approach for non-protease ACA targets, it is not currently feasible to target non-enzymatic molecules. Here, we propose a new design paradigm that could be applied to detect any intracellular ligand-binding protein. We demonstrate our new approach with the estrogen receptor (ER) – a nuclear hormone receptor – which is upregulated in >70% of invasive breast cancer cases and is expressed at 8-10 times higher levels in tumor cells compared to normal cells. In our approach to developing ER-targeting ACAs, we propose to use supramolecular assemblies constructed from G-quadruplexes as hosts and bifunctional estrogens as the fluorescently activated moiety. The latter consist of a G-Quadruplex-binding fluorophore linked to an ER-targeting ligand. When bound to the G-quadruplex, emission of the fluorophore is quenched by guanines. In the presence of ER, binding of the ligand moiety displaces the fluorophore from the G-quadruplex supramolecular assembly thereby turning the emission “on”. In aim 1, we propose to optimize and characterize a panel of G-quadruplex supramolecular assemblies targeting the ER. In aim 2, we propose to validate the efficacy of the G-quadruplex assemblies compared to free probes for the specific detection of ER in vitro and in vivo. Our new design approach has broad applicability to any cancer-specific protein regardless of cellular location. With successful completion of aims 1 and 2 we will achieve our overall objective of using G-quadruplex assemblies as fluorescent ACAs to detect ER+ breast tumors. The establishment of a new design paradigm in general and the availability of ER-targeting ACAs will diversify the pool of targeted biomarkers for FGS to enhance the sensitivity and specificity of intraoperative margin assessment and ultimately improve patient outcomes.