Ratiometric Metabolite Sensors for the Early Detection of Disease - The Lemon Group at Montana State University works at the interface of synthetic chemistry and protein biochemistry to address fundamental biomedical questions regarding the diagnosis and treatment of disease. The objective of our research program is to enhance our molecular understanding of various pathologies and improve patient outcomes. Our team of seven graduate students and six undergraduates is actively developing red fluorescent dyes and proteins for biomedical sensing and imaging applications. We are designing novel fluorescent proteins by synthesizing red fluorescent cofactors, such as corroles and boron dipyrrins (BODIPYs), and incorporating them into heme proteins. These constructs will then be paired with a receptor to quantify disease-relevant metabolites using intravital microscopy. The Lemon lab is also studying bacterial HemW proteins, which are closely related to RSAD1, a neuron-associated protein that is upregulated in Alzheimer’s disease. Since little is known about RSAD1, we are leveraging bacterial HemW proteins as tractable model systems to uncover the structure, cofactor binding, and biochemistry of this underexplored family of proteins. Finally, the Lemon lab is synthesizing bio-inspired catalysts that mimic the active sites of enzymes that hydroxylate substrates with strong C–H bonds. Our alkane oxidation catalysts incorporate a secondary coordination sphere, which we hypothesize will overcome the limitations of previous biomimetic catalysts. Knowledge of tumor metabolism has significantly advanced since the initial observation of increased aerobic glycolysis by Otto Warburg and has led to the characterization of “oncometabolites”. Metabolic dysregulation is a hallmark of disease, and characteristic shifts in metabolism represent an opportunity to diagnose pathologies at an early stage. There is a critical need for new diagnostic agents that enable early intervention, particularly in diseases that lack clinically-validated biomarkers. To address this demand, we will develop optical sensors that use fluorescent proteins as a compact, biocompatible platform to quantify metabolic shifts associated with disease phenotypes. Our sensors incorporate two key design features that render these constructs amenable to in vivo biological applications: red or near-infrared (NIR) emission and ratiometric analyte detection. These attributes will yield sensors that quantify metabolic changes in cells and tumor models with high resolution. Complementary projects in the Lemon Group will quantify metabolic dysregulation to diagnose disease and monitor the biodistribution of pharmaceuticals to improve drug delivery. Sensors developed in the Lemon Group will be able to monitor real-time, dynamic changes in analyte concentrations. Fluorescence imaging affords high spatiotemporal resolution to map analyte gradients and heterogeneity in vivo. This will allow us to determine concentration ranges of key metabolites that are uniquely definitive to diagnose a particular pathology. Once we have accurately determined the relevant concentrations of critical analytes, we will then utilize this knowledge to develop sensors that exploit other imaging modalities, such as MRI, to diagnose diseases in human patients.
