ABSTRACT
A full understanding of biological phenomena involves sensitive and noninvasive detection. Herein, we report the optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized 129 Xe NMR spectroscopy detection. The fluorescence detection part is composed of six residues containing a tetracysteine tag (-CCXXCC-) genetically incorporated into the protein of interest and of a small organic molecule, CrAsH. CrAsH becomes fluorescent if it binds to the tetracysteine tag. The part of the biosensor that enables detection by means of 129 Xe NMR spectroscopy, which is linked to the CrAsH moiety by a spacer, is based on a cryptophane core that is fully suited to reversibly host xenon. Three different peptides, containing the tetracysteine tag and four organic biosensors of different stereochemistry, are benchmarked to propose the best couple that is fully suited for the in vitro detection of proteins.
Subject(s)
Biosensing Techniques , Luminescent Proteins/chemistry , Organometallic Compounds/chemistry , Cysteine/chemistry , Magnetic Resonance Spectroscopy/methods , Spectrometry, Fluorescence/methods , Xenon/chemistry , Red Fluorescent ProteinABSTRACT
Although Non-Small Cell Lung Cancer (NSCLC) is one of the main causes of cancer death, very little improvement has been made in the last decades regarding diagnosis and outcomes. In this study, a bimodal fluorescence/129Xe NMR probe containing a xenon host, a fluorescent moiety and a therapeutic antibody has been designed to target the Epidermal Growth Factor Receptors (EGFR) overexpressed in cancer cells. This biosensor shows high selectivity for the EGFR, and a biological activity similar to that of the antibody. It is detected with high specificity and high sensitivity (sub-nanomolar range) through hyperpolarized 129Xe NMR. This promising system should find important applications for theranostic use.
Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , ErbB Receptors/antagonists & inhibitors , Fluorescent Dyes/pharmacology , Lung Neoplasms/drug therapy , Molecular Imaging , Protein Kinase Inhibitors/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , ErbB Receptors/metabolism , Fluorescence , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Magnetic Resonance Spectroscopy , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured , Xenon IsotopesABSTRACT
We report the synthesis of new water-soluble cryptophane host molecules that can be used for the preparation of (129)Xe NMR-based biosensors. We show that the cryptophane-223 skeleton can be modified to introduce a unique secondary alcohol to the propylenedioxy linker. This chemical functionality can then be exploited to introduce a functional group that is different from the six chemical groups attached to the aromatic rings. In this approach, the generation of a statistical mixture when trying to selectively functionalize a symmetrical host molecule is eliminated, which enables the efficient large-scale production of new cryptophanes that can be used as chemical platforms ready to use for the preparation of xenon biosensors. To illustrate this approach, two molecular platforms have been prepared, and the ability of these new derivatives to bind xenon has been investigated.
