56 research outputs found
Azobenzene-based inhibitors of human carbonic anhydrase II
Aryl sulfonamides are a widely used drug class for the inhibition of carbonic anhydrases. In the context of our program of photochromic pharmacophores we were interested in the exploration of azobenzene-containing sulfonamides to block the catalytic activity of human carbonic anhydrase II (hCAII). Herein, we report the synthesis and in vitro evaluation of a small library of nine photochromic sulfonamides towards hCAII. All molecules are azobenzene-4-sulfonamides, which are substituted by different functional groups in the 4 '-position and were characterized by X-ray crystallography. We aimed to investigate the influence of electrondonating or electron-withdrawing substituents on the inhibitory constant Ki. With the aid of an hCAII crystal structure bound to one of the synthesized azobenzenes, we found that the electronic structure does not strongly affect inhibition. Taken together, all compounds are strong blockers of hCAII with K-i = 25-65 nM that are potentially photochromic and thus combine studies from chemical synthesis, crystallography and enzyme kinetics
Fineâtuned photochromic sulfonylureas for optical control of beta cell Ca <sup>2+</sup> 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
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 versatile Halo- and SNAP-tagged BMP/TGFÎČ receptor library for quantification of cell surface ligand binding
TGFÎČs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFÎČ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFÎČ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFÎČ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways
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 pushâpull 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 pushâpull system
DFTâGuided Discovery of EthynylâTriazolylâPhosphinates as Modular Electrophiles for Chemoselective Cysteine Bioconjugation and Profiling
We report the density functional theory (DFT) guided discovery of ethynylâtriazolylâphosphinates (ETPs) as a new class of electrophilic warheads for cysteine selective bioconjugation. By using CuIâcatalysed azide alkyne cycloaddition (CuAAC) in aqueous buffer, we were able to access a variety of functional electrophilic building blocks, including proteins, from diethynylâphosphinate. ETPâreagents were used to obtain fluorescent peptideâconjugates for receptor labelling on live cells and a stable and a biologically active antibodyâdrugâconjugate. Moreover, we were able to incorporate ETPâelectrophiles into an azideâcontaining ubiquitin under native conditions and demonstrate their potential in proteinâprotein conjugation. Finally, we showcase the excellent cysteineâselectivity of this new class of electrophile in mass spectrometry based, proteomeâwide cysteine profiling, underscoring the applicability in homogeneous bioconjugation strategies to connect two complex biomolecules.By means of density functional theory calculations, ethynylâtriazolylâphosphinates (ETPs) were discovered as modular and cysteineâselective electrophiles for bioconjugation. Using CuIâclick chemistry in aqueous buffers, this functional group can be easily introduced into azideâcontaining (bioâ)molecules. These reagents can be used for proteomeâwide cysteine profiling and to obtain functional peptideâ and protein conjugates, as well as proteinâprotein 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
DFTâbasierte Entdeckung von EthynylâTriazolylâPhosphinaten als modulare Elektrophile fĂŒr die chemoselektive CysteinâBiokonjugation und Profilierung
Wir berichten ĂŒber eine Dichtefunktionaltheorie (DFT)-basierte Entdeckung von Ethinyl-Triazolyl-Phosphinaten (ETP) als eine neue Klasse elektrophiler Verbindungen fĂŒr die selektive Biokonjugation von Cystein. Mit Hilfe der CuI-katalysierten Azid-Alkin-Cycloaddition (CuAAC) in wĂ€ssrigem Puffer konnten wir eine Vielzahl funktioneller elektrophiler Bausteine, darunter auch Proteine, aus Diethynylphosphinat herstellen. Wir verwendeten diese ETP-Reagenzien, um fluoreszierende Peptid-Konjugate fĂŒr die Markierung von Rezeptoren auf lebenden Zellen sowie ein stabiles und biologisch aktives Antikörper-Wirkstoff-Konjugat zu erhalten. DarĂŒber hinaus konnten wir ETP-Elektrophile unter nativen Bedingungen in ein Azid-haltiges Ubiquitin einbauen und ihr Potenzial fĂŒr die Protein-Protein-Konjugation demonstrieren. SchlieĂlich zeigen wir die exzellente Cystein-SelektivitĂ€t dieser neuen Klasse von Elektrophilen in Massenspektrometrie basierten, proteomweiten ReaktivitĂ€tsstudien und unterstreichen damit die generelle Anwendbarkeit in homogenen Biokonjugationsstrategien zur VerknĂŒpfung zweier komplexer BiomolekĂŒle.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
- âŠ