Publications by authors named "Johannes Broichhagen"

47 Publications

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Mol Pharmacol 2021 Jul 27. Epub 2021 Jul 27.

Imperial College London, United Kingdom

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitisation and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cyclic adenosine monophosphate (cAMP). Here we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analogue, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and β-arrestins, endocytic and post-endocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency but exendin-4-C16 showed ~2.5-fold bias towards G protein recruitment and a ~60% reduction in β-arrestin-2 recruitment efficacy compared to exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting towards recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach, and a ~70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology. Acylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. Our work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking and interactions with the plasma membrane, which may be therapeutically important.
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http://dx.doi.org/10.1124/molpharm.121.000270DOI Listing
July 2021

SNAP-tag-enabled super-resolution imaging reveals constitutive and agonist-dependent trafficking of GPR56 in pancreatic β-cells.

Mol Metab 2021 Jul 2;53:101285. Epub 2021 Jul 2.

Department of Diabetes, School of Life Course Sciences, King's College London, Guy's Campus, London SE1 1UL, UK. Electronic address:

Objective: Members of the adhesion G protein-coupled receptor (aGPCR) subfamily are important actors in metabolic processes, with GPR56 (ADGRG1) emerging as a possible target for type 2 diabetes therapy. GPR56 can be activated by collagen III, its endogenous ligand, and by a synthetic seven amino-acid peptide (TYFAVLM; P7) contained within the GPR56 Stachel sequence. However, the mechanisms regulating GPR56 trafficking dynamics and agonist activities are not yet clear.

Methods: Here, we introduced SNAPf-tag into the N-terminal segment of GPR56 to monitor GPR56 cellular activity in situ. Confocal and super-resolution microscopy were used to investigate the trafficking pattern of GPR56 in native MIN6 β-cells and in MIN6 β-cells where GPR56 had been deleted by CRISPR-Cas9 gene editing. Insulin secretion, changes in intracellular calcium, and β-cell apoptosis were determined by radioimmunoassay, single-cell calcium microfluorimetry, and measuring caspase 3/7 activities, respectively, in MIN6 β-cells and human islets.

Results: SNAP-tag labelling indicated that GPR56 predominantly underwent constitutive internalisation in the absence of an exogenous agonist, unlike GLP-1R. Collagen III further stimulated GPR56 internalisation, whereas P7 was without significant effect. The overexpression of GPR56 in MIN6 β-cells did not affect insulin secretion. However, it was associated with reduced β-cell apoptosis, while the deletion of GPR56 made MIN6 β-cells more susceptible to cytokine-induced apoptosis. P7 induced a rapid increase in the intracellular calcium in MIN6 β-cells (in a GPR56-dependent manner) and human islets, and it also caused a sustained and reversible increase in insulin secretion from human islets. Collagen III protected human islets from cytokine-induced apoptosis, while P7 was without significant effect.

Conclusions: These data indicate that GPR56 exhibits both agonist-dependent and -independent trafficking in β-cells and suggest that while GPR56 undergoes constitutive signalling, it can also respond to its ligands when required. We have also identified that constitutive and agonist-dependent GPR56 activation is coupled to protect β-cells against apoptosis, offering a potential therapeutic target to maintain β-cell mass in type 2 diabetes.
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http://dx.doi.org/10.1016/j.molmet.2021.101285DOI Listing
July 2021

Evaluation of efficacy- versus affinity-driven agonism with biased GLP-1R ligands P5 and exendin-F1.

Biochem Pharmacol 2021 Aug 12;190:114656. Epub 2021 Jun 12.

Section of Endocrinology and Investigative Medicine, Imperial College London, London, United Kingdom.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of glucose homeostasis and has been successfully targeted for the treatment of type 2 diabetes. Recently described biased GLP-1R agonists with selective reductions in β-arrestin versus G protein coupling show improved metabolic actions in vivo. However, two prototypical G protein-favouring GLP-1R agonists, P5 and exendin-F1, are reported to show divergent effects on insulin secretion. In this study we aimed to resolve this discrepancy by performing a side-by-side characterisation of these two ligands across a variety of in vitro and in vivo assays. Exendin-F1 showed reduced acute efficacy versus P5 for several readouts, including recruitment of mini-G proteins, G protein-coupled receptor kinases (GRKs) and β-arrestin-2. Maximal responses were also lower for both GLP-1R internalisation and the presence of active GLP-1R-mini-G complexes in early endosomes with exendin-F1 treatment. In contrast, prolonged insulin secretion in vitro and sustained anti-hyperglycaemic efficacy in mice were both greater with exendin-F1 than with P5. We conclude that the particularly low acute efficacy of exendin-F1 and associated reductions in GLP-1R downregulation appear to be more important than preservation of endosomal signalling to allow sustained insulin secretion responses. This has implications for the ongoing development of affinity- versus efficacy-driven biased GLP-1R agonists as treatments for metabolic disease.
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http://dx.doi.org/10.1016/j.bcp.2021.114656DOI Listing
August 2021

Interrogating surface intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates.

Chem Sci 2020 Jul 7;11(30):7871-7883. Epub 2020 Jul 7.

Max Planck Institute for Medical Research, Department of Chemical Biology Jahnstr. 29 69120 Heidelberg Germany.

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labelling sites. Here we describe a novel approach to improve extracellular labelling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation can be applied to any SNAP-tag substrate, improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic superresolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.
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http://dx.doi.org/10.1039/d0sc02794dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8163392PMC
July 2020

Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin.

Sci Rep 2021 May 18;11(1):10536. Epub 2021 May 18.

Department of Biochemistry, Weill Cornell Medical College, New York, NY, 10065, USA.

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wild-type and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.
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http://dx.doi.org/10.1038/s41598-021-90039-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8131606PMC
May 2021

Differences in interactions between transmembrane domains tune the activation of metabotropic glutamate receptors.

Elife 2021 Apr 21;10. Epub 2021 Apr 21.

Physiology, Biophysics and Systems Biology Graduate Program, Weill Cornell Graduate School of Medical Sciences, New York, United States.

The metabotropic glutamate receptors (mGluRs) form a family of neuromodulatory G-protein-coupled receptors that contain both a seven-helix transmembrane domain (TMD) and a large extracellular ligand-binding domain (LBD) which enables stable dimerization. Although numerous studies have revealed variability across subtypes in the initial activation steps at the level of LBD dimers, an understanding of inter-TMD interaction and rearrangement remains limited. Here, we use a combination of single molecule fluorescence, molecular dynamics, functional assays, and conformational sensors to reveal that distinct TMD assembly properties drive differences between mGluR subtypes. We uncover a variable region within transmembrane helix 4 (TM4) that contributes to homo- and heterodimerization in a subtype-specific manner and tunes orthosteric, allosteric, and basal activation. We also confirm a critical role for a conserved inter-TM6 interface in stabilizing the active state during orthosteric or allosteric activation. Together this study shows that inter-TMD assembly and dynamic rearrangement drive mGluR function with distinct properties between subtypes.
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http://dx.doi.org/10.7554/eLife.67027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102066PMC
April 2021

A fine-tuned azobenzene for enhanced photopharmacology in vivo.

Cell Chem Biol 2021 Mar 13. Epub 2021 Mar 13.

Department of Chemical Biology, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany; Department of Chemical Biology, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, 13125 Berlin, Germany. Electronic address:

Despite the power of photopharmacology for interrogating signaling proteins, many photopharmacological systems are limited by their efficiency, speed, or spectral properties. Here, we screen a library of azobenzene photoswitches and identify a urea-substituted "azobenzene-400" core that offers bistable switching between cis and trans with improved kinetics, light sensitivity, and a red-shift. We then focus on the metabotropic glutamate receptors (mGluRs), neuromodulatory receptors that are major pharmacological targets. Synthesis of "BGAG," a photoswitchable orthogonal, remotely tethered ligand (PORTL), enables highly efficient, rapid optical agonism following conjugation to SNAP-tagged mGluR2 and permits robust optical control of mGluR1 and mGluR5 signaling. We then produce fluorophore-conjugated branched PORTLs to enable dual imaging and manipulation of mGluRs and highlight their power in ex vivo slice and in vivo behavioral experiments in the mouse prefrontal cortex. Finally, we demonstrate the generalizability of our strategy by developing an improved soluble, photoswitchable pore blocker for potassium channels.
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http://dx.doi.org/10.1016/j.chembiol.2021.02.020DOI Listing
March 2021

Chemical Biology Tools To Investigate Malaria Parasites.

Chembiochem 2021 Jul 31;22(13):2219-2236. Epub 2021 Mar 31.

Centre for Infectious Diseases, Parasitology, Heidelberg University Hospital, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.

Parasitic diseases like malaria tropica have been shaping human evolution and history since the beginning of mankind. After infection, the response of the human host ranges from asymptomatic to severe and may culminate in death. Therefore, proper examination of the parasite's biology is pivotal to deciphering unique molecular, biochemical and cell biological processes, which in turn ensure the identification of treatment strategies, such as potent drug targets and vaccine candidates. However, implementing molecular biology methods for genetic manipulation proves to be difficult for many parasite model organisms. The development of fast and straightforward applicable alternatives, for instance small-molecule probes from the field of chemical biology, is essential. In this review, we will recapitulate the highlights of previous molecular and chemical biology approaches that have already created insight and understanding of the malaria parasite Plasmodium falciparum. We discuss current developments from the field of chemical biology and explore how their application could advance research into this parasite in the future. We anticipate that the described approaches will help to close knowledge gaps in the biology of P. falciparum and we hope that researchers will be inspired to use these methods to gain knowledge - with the aim of ending this devastating disease.
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http://dx.doi.org/10.1002/cbic.202000882DOI Listing
July 2021

Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells.

Int J Mol Sci 2020 Nov 9;21(21). Epub 2020 Nov 9.

Section of Endocrinology and Investigative Medicine, Imperial College London, London W12 0NN, UK.

The glucagon-like peptide-1 receptor (GLP-1R) is an important regulator of blood glucose homeostasis. Ligand-specific differences in membrane trafficking of the GLP-1R influence its signalling properties and therapeutic potential in type 2 diabetes. Here, we have evaluated how different factors combine to control the post-endocytic trafficking of GLP-1R to recycling versus degradative pathways. Experiments were performed in primary islet cells, INS-1 832/3 clonal beta cells and HEK293 cells, using biorthogonal labelling of GLP-1R to determine its localisation and degradation after treatment with GLP-1, exendin-4 and several further GLP-1R agonist peptides. We also characterised the effect of a rare GLP1R coding variant, T149M, and the role of endosomal peptidase endothelin-converting enzyme-1 (ECE-1), in GLP1R trafficking. Our data reveal how treatment with GLP-1 versus exendin-4 is associated with preferential GLP-1R targeting towards a recycling pathway. GLP-1, but not exendin-4, is a substrate for ECE-1, and the resultant propensity to intra-endosomal degradation, in conjunction with differences in binding affinity, contributes to alterations in GLP-1R trafficking behaviours and degradation. The T149M GLP-1R variant shows reduced signalling and internalisation responses, which is likely to be due to disruption of the cytoplasmic region that couples to intracellular effectors. These observations provide insights into how ligand- and genotype-specific factors can influence GLP-1R trafficking.
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http://dx.doi.org/10.3390/ijms21218404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7664906PMC
November 2020

Photocaged Hoechst Enables Subnuclear Visualization and Cell Selective Staining of DNA in vivo.

Chembiochem 2021 Feb 2;22(3):548-556. Epub 2020 Nov 2.

Department of Chemical Biology, Max Planck Institute for Medical Research, Jahnstr. 29, 69120, Heidelberg, Germany.

Selective targeting of DNA by means of fluorescent labeling has become a mainstay in the life sciences. While genetic engineering serves as a powerful technique and allows the visualization of nucleic acid by using DNA-targeting fluorescent fusion proteins in a cell-type- and subcellular-specific manner, it relies on the introduction of foreign genes. On the other hand, DNA-binding small fluorescent molecules can be used without genetic engineering, but they are not spatially restricted. Herein, we report a photocaged version of the DNA dye Hoechst33342 (pcHoechst), which can be uncaged by using UV to blue light for the selective staining of chromosomal DNA in subnuclear regions of live cells. Expanding its application to a vertebrate model organism, we demonstrate uncaging in epithelial cells and short-term cell tracking in vivo in zebrafish. We envision pcHoechst as a valuable tool for targeting and interrogating DNA with precise spatiotemporal resolution in living cells and wild-type organisms.
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http://dx.doi.org/10.1002/cbic.202000465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894298PMC
February 2021

Photohormones Enable Optical Control of the Peroxisome Proliferator-Activated Receptor γ (PPARγ).

J Med Chem 2020 10 21;63(19):10908-10920. Epub 2020 Sep 21.

Department of Chemistry, New York University, New York, New York 10003, United States.

Photopharmacology aims at the optical control of protein activity using synthetic photoswitches. This approach has been recently expanded to nuclear hormone receptors with the introduction of "photohormones" for the retinoic acid receptor, farnesoid X receptor, and estrogen receptor. Herein, we report the development and profiling of photoswitchable agonists for peroxisome proliferator-activated receptor γ (PPARγ). Based on known PPARγ ligands (MDG548, GW1929, and rosiglitazone), we have designed and synthesized azobenzene derivatives, termed and , which were confirmed to be active in cell-based assays. Subsequent computer-aided optimization of resulted in the photohormone , which bound and activated PPARγ preferentially in its light-activated -configuration.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00654DOI Listing
October 2020

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

Br J Pharmacol 2020 09 19;177(17):3905-3923. Epub 2020 Jun 19.

Section of Endocrinology and Investigative Medicine, Imperial College London, London, UK.

Background And Purpose: Amino acid substitutions at the N-termini of glucagon-like peptide-1 (GLP-1) receptor agonist peptides result in distinct patterns of intracellular signalling, sub-cellular trafficking and efficacy in vivo. Here, we to determine whether sequence differences at the ligand C-termini of clinically approved GLP-1 receptor agonists 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 fivefold less potent for cAMP signalling. Both peptides induced extensive GLP-1 receptor clustering in the plasma membrane and were rapidly endocytosed, but the GLP-1 receptor 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-1 receptor trafficking via mechanisms unrelated to GLP-1 receptor occupancy. These differences were associated with changes in their ability to control blood glucose and therefore may be therapeutically relevant.
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http://dx.doi.org/10.1111/bph.15134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429481PMC
September 2020

The Influence of Peptide Context on Signaling and Trafficking of Glucagon-like Peptide-1 Receptor Biased Agonists.

ACS Pharmacol Transl Sci 2020 Apr 17;3(2):345-360. Epub 2020 Mar 17.

Section of Endocrinology and Investigative Medicine, Imperial College London, London, W12 0NN, United Kingdom.

Signal bias and membrane trafficking have recently emerged as important considerations in the therapeutic targeting of the glucagon-like peptide-1 receptor (GLP-1R) in type 2 diabetes and obesity. In the present study, we have evaluated a peptide series with varying sequence homology between native GLP-1 and exendin-4, the archetypal ligands on which approved GLP-1R agonists are based. We find notable differences in agonist-mediated cyclic AMP signaling, recruitment of β-arrestins, endocytosis, and recycling, dependent both on the introduction of a His → Phe switch at position 1 and the specific midpeptide helical regions and C-termini of the two agonists. These observations were linked to insulin secretion in a beta cell model and provide insights into how ligand factors influence GLP-1R function at the cellular level.
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http://dx.doi.org/10.1021/acsptsci.0c00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155199PMC
April 2020

Super-resolution microscopy compatible fluorescent probes reveal endogenous glucagon-like peptide-1 receptor distribution and dynamics.

Nat Commun 2020 01 24;11(1):467. Epub 2020 Jan 24.

Institute of Metabolism and Systems Research (IMSR), and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK.

The glucagon-like peptide-1 receptor (GLP1R) is a class B G protein-coupled receptor (GPCR) involved in metabolism. Presently, its visualization is limited to genetic manipulation, antibody detection or the use of probes that stimulate receptor activation. Herein, we present LUXendin645, a far-red fluorescent GLP1R antagonistic peptide label. LUXendin645 produces intense and specific membrane labeling throughout live and fixed tissue. GLP1R signaling can additionally be evoked when the receptor is allosterically modulated in the presence of LUXendin645. Using LUXendin645 and LUXendin651, we describe islet, brain and hESC-derived β-like cell GLP1R expression patterns, reveal higher-order GLP1R organization including membrane nanodomains, and track single receptor subpopulations. We furthermore show that the LUXendin backbone can be optimized for intravital two-photon imaging by installing a red fluorophore. Thus, our super-resolution compatible labeling probes allow visualization of endogenous GLP1R, and provide insight into class B GPCR distribution and dynamics both in vitro and in vivo.
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http://dx.doi.org/10.1038/s41467-020-14309-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981144PMC
January 2020

Transformation of Receptor Tyrosine Kinases into Glutamate Receptors and Photoreceptors.

Angew Chem Int Ed Engl 2020 04 4;59(17):6720-6723. Epub 2020 Mar 4.

Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204, Center for Infection and Immunity of Lille (CIIL), 59000, Lille, France.

Receptor tyrosine kinases (RTKs) are key regulators of cellular functions in metazoans. In vertebrates, RTKs are mostly activated by polypeptides but are not naturally sensitive to amino acids or light. Taking inspiration from Venus kinase receptors (VKRs), an atypical family of RTKs found in nature, we have transformed the human insulin (hIR) and hepatocyte growth factor receptor (hMET) into glutamate receptors by replacing their extracellular binding domains with the ligand-binding domain of metabotropic glutamate receptor type 2 (mGluR2). We then imparted light sensitivity through covalent attachment of a synthetic glutamate-based photoswitch via a self-labelling SNAP tag. By employing a Xenopus laevis oocyte kinase activity assay, we demonstrate how these chimeric RTKs, termed light-controlled human insulin receptor (LihIR) and light-controlled human MET receptor (LihMET), can be used to exert optical control over the insulin or MET signaling pathways. Our results outline a potentially general strategy to convert RTKs into photoreceptors.
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http://dx.doi.org/10.1002/anie.201915352DOI Listing
April 2020

Branched Photoswitchable Tethered Ligands Enable Ultra-efficient Optical Control and Detection of G Protein-Coupled Receptors In Vivo.

Neuron 2020 02 26;105(3):446-463.e13. Epub 2019 Nov 26.

Biochemistry, Cell and Molecular Biology Graduate Program, Weill Cornell Medicine, New York, NY 10065, USA; Department of Biochemistry, Weill Cornell Medicine, New York, NY 10065, USA; Tri-Institutional PhD Program in Chemical Biology, New York, NY 10065, USA. Electronic address:

The limitations of classical drugs have spurred the development of covalently tethered photoswitchable ligands to control neuromodulatory receptors. However, a major shortcoming of tethered photopharmacology is the inability to obtain optical control with an efficacy comparable with that of the native ligand. To overcome this, we developed a family of branched photoswitchable compounds to target metabotropic glutamate receptors (mGluRs). These compounds permit photo-agonism of G-coupled group II mGluRs with near-complete efficiency relative to glutamate when attached to receptors via a range of orthogonal, multiplexable modalities. Through a chimeric approach, branched ligands also allow efficient optical control of G-coupled mGluR5, which we use to probe the spatiotemporal properties of receptor-induced calcium oscillations. In addition, we report branched, photoswitch-fluorophore compounds for simultaneous receptor imaging and manipulation. Finally, we demonstrate this approach in vivo in mice, where photoactivation of SNAP-mGluR2 in the medial prefrontal cortex reversibly modulates working memory in normal and disease-associated states.
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http://dx.doi.org/10.1016/j.neuron.2019.10.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216301PMC
February 2020

Genetically Targeted Optical Control of an Endogenous G Protein-Coupled Receptor.

J Am Chem Soc 2019 07 10;141(29):11522-11530. Epub 2019 Jul 10.

Department of Molecular and Cell Biology , University of California , Berkeley , California 94720 , United States.

G protein-coupled receptors (GPCRs) are membrane proteins that play important roles in biology. However, our understanding of their function in complex living systems is limited because we lack tools that can target individual receptors with sufficient precision. State-of-the-art approaches, including DREADDs, optoXRs, and PORTL gated-receptors, control GPCR signaling with molecular, cell type, and temporal specificity. Nonetheless, these tools are based on engineered non-native proteins that may (i) express at nonphysiological levels, (ii) localize and turnover incorrectly, and/or (iii) fail to interact with endogenous partners. Alternatively, membrane-anchored ligands (t-toxins, DARTs) target endogenous receptors with molecular and cell type specificity but cannot be turned on and off. In this study, we used a combination of chemistry, biology, and light to control endogenous metabotropic glutamate receptor 2 (mGluR2), a Family C GPCR, in primary cortical neurons. mGluR2 was rapidly, reversibly, and selectively activated with photoswitchable glutamate tethered to a genetically targeted-plasma membrane anchor (embrane nchored hotoswitchable rthogonal emotely ethered igand; maPORTL). Photoactivation was tuned by adjusting the length of the PORTL as well as the expression level and geometry of the membrane anchor. Our findings provide a template for controlling endogenous GPCRs with cell type specificity and high spatiotemporal precision.
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http://dx.doi.org/10.1021/jacs.9b02895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271769PMC
July 2019

Optical Regulation of Class C GPCRs by Photoswitchable Orthogonal Remotely Tethered Ligands.

Methods Mol Biol 2019 ;1947:103-136

Department of Biochemistry, Weill Cornell Medicine, New York, NY, USA.

G protein-coupled receptors (GPCRs) respond to a wide range of extracellular cues to initiate complex downstream signaling cascades that control myriad aspects of cell function. Despite a long-standing appreciation of their importance to both basic physiology and disease treatment, it remains a major challenge to understand the dynamic activation patterns of GPCRs and the mechanisms by which they modulate biological processes at the molecular, cellular, and tissue levels. Unfortunately, classical methods of pharmacology and genetic knockout are often unable to provide the requisite precision needed to probe such questions. This is an especially pressing challenge for the class C GPCR family which includes receptors for the major excitatory and inhibitory neurotransmitters, glutamate and GABA, which signal in a rapid, spatially-delimited manner and contain many different subtypes whose roles are difficult to disentangle. The desire to manipulate class C GPCRs with spatiotemporal precision, genetic targeting, and subtype specificity has led to the development of a variety of photopharmacological tools. Of particular promise are the photoswitchable orthogonal remotely tethered ligands ("PORTLs") which attach to self-labeling tags that are genetically encoded into full length, wild-type metabotropic glutamate receptors (mGluRs) and allow the receptor to be liganded and un-liganded in response to different wavelengths of illumination. While powerful for studying class C GPCRs, a number of detailed considerations must be made when working with these tools. The protocol included here should provide a basis for the development, characterization, optimization, and application of PORTLs for a wide range of GPCRs.
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http://dx.doi.org/10.1007/978-1-4939-9121-1_6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538505PMC
August 2019

Optical tools for understanding the complexity of β-cell signalling and insulin release.

Nat Rev Endocrinol 2018 12;14(12):721-737

Institute of Metabolism and Systems Research (IMSR) and Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham, Birmingham, UK.

Following stimulation, pancreatic β-cells must orchestrate a plethora of signalling events to ensure the appropriate release of insulin and maintenance of normal glucose homeostasis. Failure at any point in this cascade leads to impaired insulin secretion, elevated blood levels of glucose and eventually type 2 diabetes mellitus. Likewise, β-cell replacement or regeneration strategies for the treatment of both type 1 and type 2 diabetes mellitus might fail if the correct cell signalling phenotype cannot be faithfully recreated. However, current understanding of β-cell function is complicated because of the highly dynamic nature of their intracellular and intercellular signalling as well as insulin release itself. β-Cells must precisely integrate multiple signals stemming from multiple cues, often with differing intensities, frequencies and cellular and subcellular localizations, before converging these signals onto insulin exocytosis. In this respect, optical approaches with high resolution in space and time are extremely useful for properly deciphering the complexity of β-cell signalling. An increased understanding of β-cell signalling might identify new mechanisms underlying insulin release, with relevance for future drug therapy and de novo stem cell engineering of functional islets.
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http://dx.doi.org/10.1038/s41574-018-0105-2DOI Listing
December 2018

SNAP-Tagged Nanobodies Enable Reversible Optical Control of a G Protein-Coupled Receptor via a Remotely Tethered Photoswitchable Ligand.

ACS Chem Biol 2018 09 7;13(9):2682-2688. Epub 2018 Sep 7.

Department of Chemical Biology , Max Planck Institute for Medical Research , Jahnstrasse 29 , 69120 Heidelberg , Germany.

G protein-coupled receptors (GPCRs) mediate the transduction of extracellular signals into complex intracellular responses. Despite their ubiquitous roles in physiological processes and as drug targets for a wide range of disorders, the precise mechanisms of GPCR function at the molecular, cellular, and systems levels remain partially understood. To dissect the function of individual receptor subtypes with high spatiotemporal precision, various optogenetic and photopharmacological approaches have been reported that use the power of light for receptor activation and deactivation. Here, we introduce a novel and, to date, most remote way of applying photoswitchable orthogonally remotely tethered ligands by using a SNAP-tag fused nanobody. Our nanobody-photoswitch conjugates can be used to target a green fluorescent protein-fused metabotropic glutamate receptor by either gene-free application of purified complexes or coexpression of genetically encoded nanobodies to yield robust, reversible control of agonist binding and subsequent downstream activation. By harboring and combining the selectivity and flexibility of both nanobodies and self-labeling proteins (or suicide enzymes), we set the stage for targeting endogenous receptors in vivo.
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http://dx.doi.org/10.1021/acschembio.8b00628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620112PMC
September 2018

A New Fluorogenic Small-Molecule Labeling Tool for Surface Diffusion Analysis and Advanced Fluorescence Imaging of β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Based on Silicone Rhodamine: SiR-BACE1.

J Med Chem 2018 Jul 10;61(14):6121-6139. Epub 2018 Jul 10.

Institute of Physiology and Pathophysiology , Friedrich-Alexander-Universität Erlangen-Nürnberg , Universitaetsstrasse 17 , 91054 Erlangen , Germany.

β-site APP-cleaving enzyme 1 (BACE1) is a major player in the pathogenesis of Alzheimer's disease. Structural and functional fluorescence microscopy offers a powerful approach to learn about the physiology and pathophysiology of this protease. Up to now, however, common labeling techniques require genetic manipulation, use large antibodies, or are not compatible with live cell imaging. Fluorescent small molecules that specifically bind to the protein of interest can overcome these limitations. Herein, we introduce SiR-BACE1, a conjugate of the BACE1 inhibitor S-39 and SiR647, as a novel fluorogenic, tag-free, and antibody-free label for BACE1. We present its chemical development, characterize its photophysical and pharmacologic properties, and evaluate its behavior in solution, in overexpression systems, and in native brain tissue. We demonstrate its applicability in confocal, stimulated emission depletion and dynamic single-molecule microscopy. The first functional studies with SiR-BACE1 on the surface mobility of BACE1 revealed a markedly confined diffusion pattern.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00387DOI Listing
July 2018

Optical control of GPR40 signalling in pancreatic β-cells.

Chem Sci 2017 Nov 30;8(11):7604-7610. Epub 2017 Aug 30.

Department of Chemistry , Center for Integrated Protein Science , Ludwig Maximilians University Munich , Butenandtstraße 5-13 , 81377 Munich , Germany.

Fatty acids activate GPR40 and K channels to modulate β-cell function. Herein, we describe the design and synthesis of , a light-controllable GPR40 agonist based on Gw-9508. is a potent GPR40 agonist in the -configuration and can be inactivated on isomerization to with UV-A light. Irradiation with blue light reverses this effect, allowing activity to be cycled ON and OFF with a high degree of spatiotemporal precision. In dissociated primary mouse β-cells, also inactivates voltage-activated and ATP-sensitive K channels, and allows us to control glucose-stimulated Ca oscillations in whole islets with light. As such, is a useful tool to study the complex effects, with high specificity, which FA-derivatives such as Gw-9508 exert at multiple targets in mouse β-cells.
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http://dx.doi.org/10.1039/c7sc01475aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5848828PMC
November 2017

Author Correction: Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor.

Nat Commun 2018 03 13;9(1):1112. Epub 2018 Mar 13.

Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA.

Kevin J. Cao and Richard H. Kramer, who developed extended release with beta cyclodextrin, were inadvertently omitted from the author list and author contributions section of this Article. These errors have now been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-018-03585-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849763PMC
March 2018

Conditional and Reversible Activation of Class A and B G Protein-Coupled Receptors Using Tethered Pharmacology.

ACS Cent Sci 2018 Feb 16;4(2):166-179. Epub 2018 Jan 16.

Institute of Metabolism and Systems Research (IMSR), University of Birmingham, B15 2TT, Birmingham, United Kingdom.

Understanding the activation and internalization of G protein-coupled receptors (GPCRs) using conditional approaches is paramount to developing new therapeutic strategies. Here, we describe the design, synthesis, and testing of , a benzylguanine-linked peptide agonist of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR required for maintenance of glucose levels in humans. covalently binds to SNAP-tagged GLP-1R-expressing cells, leading to prolonged cAMP generation, Ca rises, and intracellular retention of the receptor. These effects were readily switched OFF following cleavage of the introduced disulfide bridge using the cell-permeable reducing agent -mercaptoethanol (BME). A similar approach could be extended to a class A GPCR using , a benzylguanine-linked peptide agonist of the growth hormone secretagogue receptor 1a (GHS-R1a), which is involved in food intake and growth. Thus, and allow SNAP-tag-directed activation of class A and B GPCRs involved in gut hormone signaling in a reversible manner. This tactic, termed reductively cleavable agONist (RECON), may be useful for understanding GLP-1R and GHS-R1a function both and , with applicability across GPCRs.
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http://dx.doi.org/10.1021/acscentsci.7b00237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5832994PMC
February 2018

A (+)-Larixol Congener with High Affinity and Subtype Selectivity toward TRPC6.

ChemMedChem 2018 05 26;13(10):1028-1035. Epub 2018 Apr 26.

Rudolf Boehm Institute for Pharmacology and Toxicology, Leipzig University, Härtelstr. 16-18, 04107, Leipzig, Germany.

Natural products have many health benefits, and their application can improve the quality of life. Recently, the diterpene (+)-larixol and its acetylated congeners demonstrated selective inhibition of the second-messenger-gated cation channel transient receptor potential canonical 6 (TRPC6) over its close isoforms TRPC3 and TRPC7. Building on this knowledge, we expanded these findings by chemical diversification of (+)-larixol mostly at position C6. Implementing high-throughput Ca FLIPR screening assays and electrophysiological patch-clamp recordings, we showcase larixyl N-methylcarbamate, termed SH045, as a compound with nanomolar affinity and 13-fold subtype selectivity over TRPC3 in stably expressing HEK293 cells. Expanding on this finding, TRPC6 inhibition was also observed in rat pulmonary smooth muscle cells. Furthermore, treatment of isolated perfused lung preparations with SH045 led to a decrease in lung ischemia-reperfusion edema (LIRE), a life-threatening condition associated with TRPC6 that may occur after organ transplantation. Taken together, and given the inexpensive, straightforward, and scalable preparation of SH045, we report a TRPC6 blocker that holds promise for the translational treatment of LIRE.
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http://dx.doi.org/10.1002/cmdc.201800021DOI Listing
May 2018

A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity.

ACS Chem Biol 2018 03 9;13(3):793-800. Epub 2018 Feb 9.

Department of Chemistry , Ludwig-Maximilian-University Munich and Munich Center for Integrated Protein Science (CIPSM) , Butenandtstrasse 5-13 , 83177 Munich , Germany.

Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Herein, we describe an in silico screening approach that is designed to propose a small number of optimal combinations. By applying this computational approach to human carbonic anhydrase and a set of three photochromic tethered ligands, the number of potential site-ligand combinations was narrowed from over 750 down to 6, which we then evaluated experimentally. Two of these six combinations resulted in light-responsive human Carbonic Anhydrases (LihCAs), which were characterized with enzymatic activity assays, mass spectrometry, and X-ray crystallography. Our study also provides insights into the reactivity of cysteines toward maleimides and the hydrolytic stability of the adducts obtained.
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http://dx.doi.org/10.1021/acschembio.7b00862DOI Listing
March 2018

Optical Control of a Biological Reaction-Diffusion System.

Angew Chem Int Ed Engl 2018 02 26;57(9):2362-2366. Epub 2018 Jan 26.

Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152, Martinsried, Germany.

Patterns formed by reaction and diffusion are the foundation for many phenomena in biology. However, the experimental study of reaction-diffusion (R-D) systems has so far been dominated by chemical oscillators, for which many tools are available. In this work, we developed a photoswitch for the Min system of Escherichia coli, a versatile biological in vitro R-D system consisting of the antagonistic proteins MinD and MinE. A MinE-derived peptide of 19 amino acids was covalently modified with a photoisomerizable crosslinker based on azobenzene to externally control peptide-mediated depletion of MinD from the membrane. In addition to providing an on-off switch for pattern formation, we achieve frequency-locked resonance with a precise 2D spatial memory, thus allowing new insights into Min protein action on the membrane. Taken together, we provide a tool to study phenomena in pattern formation using biological agents.
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http://dx.doi.org/10.1002/anie.201712002DOI Listing
February 2018

Restoration of patterned vision with an engineered photoactivatable G protein-coupled receptor.

Nat Commun 2017 11 30;8(1):1862. Epub 2017 Nov 30.

Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA.

Retinitis pigmentosa results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. We used a retinal G protein-coupled receptor, mGluR2, which we chemically engineered to respond to light. In retinal ganglion cells (RGCs) of blind rd1 mice, photoswitch-charged mGluR2 ("SNAG-mGluR2") evoked robust OFF responses to light, but not in wild-type retinas, revealing selectivity for RGCs that have lost photoreceptor input. SNAG-mGluR2 enabled animals to discriminate parallel from perpendicular lines and parallel lines at varying spacing. Simultaneous viral delivery of the inhibitory SNAG-mGluR2 and excitatory light-activated ionotropic glutamate receptor LiGluR yielded a distribution of expression ratios, restoration of ON, OFF and ON-OFF light responses and improved visual acuity. Thus, SNAG-mGluR2 restores patterned vision and combinatorial light response diversity provides a new logic for enhanced-acuity retinal prosthetics.
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http://dx.doi.org/10.1038/s41467-017-01990-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5709376PMC
November 2017

Potent Prearranged Positive Allosteric Modulators of the Glucagon-like Peptide-1 Receptor.

ChemistryOpen 2017 08 5;6(4):501-505. Epub 2017 Jun 5.

École Polytechnique Fédérale de Lausanne ISIC SB, Laboratory of Protein Engineering Av. Forel 21015 Lausanne Switzerland.

Drugs that allosterically modulate G protein-coupled receptor (GPCR) activity display higher specificity and may improve disease treatment. However, the rational design of compounds that target the allosteric site is difficult, as conformations required for receptor activation are poorly understood. Guided by photopharmacology, a set of prearranged positive allosteric modulators (PAMs) with restricted degrees of freedom was designed and tested against the glucagon-like peptide-1 receptor (GLP-1R), a GPCR involved in glucose homeostasis. Compounds incorporating a -stilbene comprehensively outperformed those with a -stilbene, as well as the benchmark BETP, as GLP-1R PAMs. We also identified major effects of ligand conformation on GLP-1R binding kinetics and signal bias. Thus, we describe a photopharmacology-directed approach for rational drug design, and introduce a new class of stilbene-containing PAM for the specific regulation of GPCR activity.
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http://dx.doi.org/10.1002/open.201700062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542757PMC
August 2017
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