Fine‐tuned photochromic sulfonylureas for optical control of beta cell Ca ²⁺ fluxes

We previously developed, synthesized and tested light-activated sulfonylureas for optical control of KATP channels and pancreatic beta cell activity in vitro and in vivo. Such technology relies on installation of azobenzene photoswitches onto the sulfonylurea backbone, affording light-dependent isomerization, alteration in ligand affinity for SUR1 and hence KATP channel conductance. Inspired by molecular dynamics simulations and to further improve photoswitching characteristics, we set out to develop a novel push-pull closed ring azobenzene unit, before installing this on the sulfonylurea glimepiride as a small molecule recipient. Three fine-tuned, light-activated sulfonylureas were synthesized, encompassing azetidine, pyrrolidine and piperidine closed rings. Azetidine-, pyrrolidine- and piperidine-based sulfonylureas all increased beta cell Ca2+ -spiking activity upon continuous blue light illumination, similarly to first generation JB253. Notably, the pyrrolidine-based sulfonylurea showed superior switch OFF performance to JB253. As such, third generation sulfonylureas afford more precise optical control over primary pancreatic beta cells, and showcase the potential of pyrrolidine-azobenzenes as chemical photoswitches across drug classes

Enhanced endosomal signaling and desensitization of GLP-1R versus GIPR in pancreatic beta cells

The incretin receptors, glucagon-like peptide-1 receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide receptor (GIPR), are prime therapeutic targets for the treatment of type 2 diabetes (T2D) and obesity. They are expressed in pancreatic beta cells where they potentiate insulin release in response to food intake. Despite GIP being the main incretin in healthy individuals, GLP-1R has been favored as a therapeutic target due to blunted GIPR responses in T2D patients and conflicting effects of GIPR agonists and antagonists in improving glucose tolerance and preventing weight gain. There is, however, a recently renewed interest in GIPR biology following the realization that GIPR responses can be restored after an initial period of blood glucose normalization and the recent development of dual GLP-1R/GIPR agonists with superior capacity for controlling blood glucose levels and weight. The importance of GLP-1R trafficking and subcellular signaling in the control of receptor outputs is well established, but little is known about the pattern of spatiotemporal signaling from the GIPR in beta cells. Here, we have directly compared surface expression, trafficking and signaling characteristics of both incretin receptors in pancreatic beta cells to identify potential differences that might underlie distinct pharmacological responses associated with each receptor. Our results indicate increased cell surface levels, internalization, degradation, and endosomal versus plasma membrane activity for the GLP-1R, while the GIPR is instead associated with increased plasma membrane recycling, reduced desensitization, and enhanced downstream signal amplification. These differences might have potential implications for the capacity of each incretin receptor to control beta cell function

Enzyme self-label-bound ATTO700 in single-molecule and super-resolution microscopy

Herein, we evaluate near-infrared ATTO700 as an acceptor in SNAP- and Halo-tag protein labelling for Förster Resonance Energy Transfer (FRET) by ensemble and single molecule measurements. Microscopy of cell surface proteins in live cells is perfomed including super-resolution stimulated emission by depletion (STED) nanoscopy

A red-shifted photochromic sulfonylurea for the remote control of pancreatic beta cell function

Azobenzene photoresponsive elements can be installed on sulfonylureas, yielding optical control over pancreatic beta cell function and insulin release. An obstacle to such photopharmacological approaches remains the use of ultraviolet-blue illumination. Herein, we synthesize and test a novel yellow light-activated sulfonylurea based on a heterocyclic azobenzene bearing a push–pull system

A red-shifted photochromic sulfonylurea for the remote control of pancreatic beta cell function

Azobenzene photoresponsive elements can be installed on sulfonylureas, yielding optical control over pancreatic beta cell function and insulin release. An obstacle to such photopharmacological approaches remains the use of ultraviolet-blue illumination. Herein, we synthesize and test a novel yellow light-activated sulfonylurea based on a heterocyclic azobenzene bearing a push–pull system

An expanded LUXendin color palette for GLP1R detection and visualization in vitro and in vivo

The glucagon-like peptide-1 receptor (GLP1R) is expressed in peripheral tissues and the brain, where it exerts pleiotropic actions on metabolic and inflammatory processes. Detection and visualization of GLP1R remains challenging, partly due to a lack of validated reagents. Previously, we generated LUXendins, antagonistic red and far-red fluorescent probes for specific labeling of GLP1R in live and fixed cells/tissue. We now extend this concept to the green and near-infrared color ranges by synthesizing and testing LUXendin492, LUXendin551, LUXendin615 and LUXendin762. All four probes brightly and specifically label GLP1R in cells and pancreatic islets. Further, LUXendin551 acts as chemical beta cell reporter in preclinical rodent models, while LUXendin762 allows non-invasive imaging, highlighting differentially-accessible GLP1R populations. We thus expand the color palette of LUXendins to seven different spectra, opening up a range of experiments using widefield microscopy available in most labs through super-resolution imaging and whole animal imaging. With this, we expect that LUXendins will continue to generate novel and specific insight into GLP1R biology

Signalling, trafficking and glucoregulatory properties of glucagon-like peptide-1 receptor agonists exendin-4 and lixisenatide.

BACKGROUND AND PURPOSE: Amino acid substitutions at the N-termini of glucagon-like peptide-1 receptor agonist (GLP-1RA) peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here we aimed to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1RAs exendin-4 and lixisenatide lead to similar phenomena. EXPERIMENTAL APPROACH: Exendin-4, lixisenatide, and N-terminally substituted analogues with biased signalling characteristics were compared across a range of in vitro trafficking and signalling assays in different cell types. Fluorescent ligands and new time-resolved FRET approaches were developed to study agonist behaviours at the cellular and sub-cellular level. Anti-hyperglycaemic and anorectic effects of each parent ligand, and their biased derivatives, were assessed in mice. KEY RESULTS: Lixisenatide and exendin-4 showed equal binding affinity, but lixisenatide was 5-fold less potent for cAMP signalling. Both peptides induced extensive GLP-1R clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1R recycled more slowly to the cell surface after lixisenatide treatment. These combined deficits resulted in reduced maximal sustained insulin secretion and reduced anti-hyperglycaemic and anorectic effects in mice with lixisenatide. N-terminal substitution of His1 by Phe1 to both ligands had favourable effects on their pharmacology, resulting in improved insulin release and lowering of blood glucose. CONCLUSION AND IMPLICATIONS: Changes to the C-terminus of exendin-4 affect signalling potency and GLP-1R trafficking via mechanisms unrelated to GLP-1R occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant

DFT‐Guided Discovery of Ethynyl‐Triazolyl‐Phosphinates as Modular Electrophiles for Chemoselective Cysteine Bioconjugation and Profiling

We report the density functional theory (DFT) guided discovery of ethynyl‐triazolyl‐phosphinates (ETPs) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using CuI‐catalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer, we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynyl‐phosphinate. ETP‐reagents were used to obtain fluorescent peptide‐conjugates for receptor labelling on live cells and a stable and a biologically active antibody‐drug‐conjugate. Moreover, we were able to incorporate ETP‐electrophiles into an azide‐containing ubiquitin under native conditions and demonstrate their potential in protein–protein conjugation. Finally, we showcase the excellent cysteine‐selectivity of this new class of electrophile in mass spectrometry based, proteome‐wide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.By means of density functional theory calculations, ethynyl‐triazolyl‐phosphinates (ETPs) were discovered as modular and cysteine‐selective electrophiles for bioconjugation. Using CuI‐click chemistry in aqueous buffers, this functional group can be easily introduced into azide‐containing (bio‐)molecules. These reagents can be used for proteome‐wide cysteine profiling and to obtain functional peptide‐ and protein conjugates, as well as protein–protein conjugates . imageDeutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Leibniz-Gemeinschaft http://dx.doi.org/10.13039/501100001664Studienstiftung des Deutschen Volkes http://dx.doi.org/10.13039/501100004350Alexander von Humboldt-Stiftung http://dx.doi.org/10.13039/100005156Institute for Basic Science in KoreaPeer Reviewe

Remote control of glucose homeostasis in vivo using photopharmacology

Photopharmacology describes the use of light to precisely deliver drug activity in space and time. Such approaches promise to improve drug specificity by reducing off-target effects. As a proof-of-concept, we have subjected the fourth generation photoswitchable sulfonylurea JB253 to comprehensive toxicology assessment, including mutagenicity and maximum/repeated tolerated dose studies, as well as in vivo testing in rodents. Here, we show that JB253 is well-tolerated with minimal mutagenicity and can be used to optically-control glucose homeostasis in anesthetized mice following delivery of blue light to the pancreas. These studies provide the first demonstration that photopharmacology may one day be applicable to the light-guided treatment of type 2 diabetes and other metabolic disease states in vivo in humans

DFT‐basierte Entdeckung von Ethynyl‐Triazolyl‐Phosphinaten als modulare Elektrophile für die chemoselektive Cystein‐Biokonjugation und Profilierung

Wir berichten über eine Dichtefunktionaltheorie (DFT)-basierte Entdeckung von Ethinyl-Triazolyl-Phosphinaten (ETP) als eine neue Klasse elektrophiler Verbindungen für die selektive Biokonjugation von Cystein. Mit Hilfe der CuI-katalysierten Azid-Alkin-Cycloaddition (CuAAC) in wässrigem Puffer konnten wir eine Vielzahl funktioneller elektrophiler Bausteine, darunter auch Proteine, aus Diethynylphosphinat herstellen. Wir verwendeten diese ETP-Reagenzien, um fluoreszierende Peptid-Konjugate für die Markierung von Rezeptoren auf lebenden Zellen sowie ein stabiles und biologisch aktives Antikörper-Wirkstoff-Konjugat zu erhalten. Darüber hinaus konnten wir ETP-Elektrophile unter nativen Bedingungen in ein Azid-haltiges Ubiquitin einbauen und ihr Potenzial für die Protein-Protein-Konjugation demonstrieren. Schließlich zeigen wir die exzellente Cystein-Selektivität dieser neuen Klasse von Elektrophilen in Massenspektrometrie basierten, proteomweiten Reaktivitätsstudien und unterstreichen damit die generelle Anwendbarkeit in homogenen Biokonjugationsstrategien zur Verknüpfung zweier komplexer Biomoleküle.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Leibniz-Gemeinschaft http://dx.doi.org/10.13039/501100001664Studienstiftung des Deutschen Volkes http://dx.doi.org/10.13039/501100004350Alexander von Humboldt-Stiftung http://dx.doi.org/10.13039/100005156Institute for Basic Science in KoreaPeer Reviewe