A Conditionally Fluorescent Peptide Reporter of Secondary Structure Modulation

Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E‐BP1 is an example of an intrinsically disordered protein that forms an α‐helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding‐induced structural changes are low‐throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E‐BP1 peptide that reports structural changes of its helix in high‐throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α‐helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high‐throughput screening to discover ligands that alter peptide secondary structure.PET lights up peptide dynamics: Photoinduced electron transfer (PET) quenching of fluorescence by thioamides presents an elegant method for monitoring changes in macromolecular conformation. Here we apply this approach to monitor peptide dynamics in a 384‐well plate format. Using a fluorescein‐conjugated, 4E‐BP1‐based peptide containing an embedded thioamide, we probe its transition from disorder to a short α‐helix.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/146852/1/cbic201800377.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146852/2/cbic201800377-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/146852/3/cbic201800377_am.pd

An updated synthesis of N1′‐([11C]methyl)naltrindole for positron emission tomography imaging of the delta opioid receptor

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167550/1/jlcr3898.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167550/2/jlcr3898_am.pd

Expansion of cat-ELCCA for the Discovery of Small Molecule Inhibitors of the Pre-let-7–Lin28 RNA–Protein Interaction

Dysregulation of microRNA (miRNA) expression has been linked to many human diseases; however, because of the challenges associated with RNA-targeted drug discovery, additional approaches are needed for probing miRNA biology. The emerging regulatory role of miRNA-binding proteins in miRNA maturation presents such an alternative strategy. Exploiting our laboratory’s click chemistry-based high-throughput screening (HTS) technology, catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a modular method by which to discover new chemical tools for manipulating pre-miRNA–miRNA–binding protein interactions. Using the pre-let-7d–Lin28 interaction as proof-of-concept, the results presented demonstrate how HTS using cat-ELCCA can enable the discovery of small molecules targeting RNA–protein interactions

Radiosynthesis and In Vivo Evaluation of Four Positron Emission Tomography Tracer Candidates for Imaging of Melatonin Receptors

Melatonin is a neurohormone that modulates several physiological functions in mammals through the activation of melatonin receptor type 1 and 2 (MT1 and MT2). The melatonergic system is an emerging therapeutic target for new pharmacological interventions in the treatment of sleep and mood disorders; thus, imaging tools to further investigate its role in the brain are highly sought-after. We aimed to develop selective radiotracers for in vivo imaging of both MT1 and MT2 by positron emission tomography (PET). We identified four previously reported MT ligands with picomolar affinities to the target based on different scaffolds which were also amenable for radiolabeling with either carbon-11 or fluorine-18. [11C]UCM765, [11C]UCM1014, [18F]3-fluoroagomelatine ([18F]3FAGM), and [18F]fluoroacetamidoagomelatine ([18F]FAAGM) have been synthesized in high radiochemical purity and evaluated in wild-type rats. All four tracers showed moderate to high brain permeability in rats with maximum standardized uptake values (SUVmax of 2.53, 1.75, 3.25, and 4.47, respectively) achieved 1-2 min after tracer administration, followed by a rapid washout from the brain. Several melatonin ligands failed to block the binding of any of the PET tracer candidates, while in some cases, homologous blocking surprisingly resulted in increased brain retention. Two 18F-labeled agomelatine derivatives were brought forward to PET scans in non-human primates and autoradiography on human brain tissues. No specific binding has been detected in blocking studies. To further investigate pharmacokinetic properties of the putative tracers, microsomal stability, plasma protein binding, log D, and membrane bidirectional permeability assays have been conducted. Based on the results, we conclude that the fast first pass metabolism by the enzymes in liver microsomes is the likely reason of the failure of our PET tracer candidates. Nevertheless, we showed that PET imaging can serve as a valuable tool to investigate the brain permeability of new therapeutic compounds targeting the melatonergic system