Light-Activatable, Cell-Type Specific Labeling of the Nascent ProteomeClick to copy article linkArticle link copied!
- H. T. EvansH. T. EvansCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by H. T. Evans
- T. KoT. KoDepartment of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United StatesMore by T. Ko
- M. M. OliveiraM. M. OliveiraCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by M. M. Oliveira
- A. YuA. YuCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by A. Yu
- S. V. KalavaiS. V. KalavaiCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by S. V. Kalavai
- E. N. GolhanE. N. GolhanCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by E. N. Golhan
- A. PolavarapuA. PolavarapuCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by A. Polavarapu
- E. BalamotiE. BalamotiCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by E. Balamoti
- V. WuV. WuCenter for Neural Science, New York University, New York, New York 10003, United StatesMore by V. Wu
- E. Klann*E. Klann*Email: [email protected]Center for Neural Science, New York University, New York, New York 10003, United StatesMore by E. Klann
- D. Trauner*D. Trauner*Email: [email protected]Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United StatesMore by D. Trauner
Abstract
Elucidating the mechanisms by which protein synthesis contributes to complex biological processes has remained a challenging endeavor. This is particularly true in the field of neuroscience, where multiple, tightly regulated periods of new protein synthesis in different cell-types are thought to facilitate intricate neurological functions, such as memory formation. Current methods for labeling the de novo proteome have lacked the spatial and temporal resolution to accurately discriminate these overlapping and often competing windows of mRNA translation. To address this technological limitation, here we describe a novel, light-inducible specific method for labeling newly synthesized proteins within a targeted cell-type.By developing Opto-ANL, a photocaged version of the nonendogenous amino acid azidonorleucine (ANL), we can selectively label newly synthesized proteins in specific cell-types through the targeted expression of a mutant methionyl-tRNA synthetase (L274G-MetRS). We demonstrate that Opto-ANL can be rapidly uncaged by UV light treatment in both cell culture and mouse brain slices, with Opto-ANL labeled proteins being able to be visualized via fluorescent noncanonical amino acid tagging. We also reveal that pretreatment with Opto-ANL not only allows for the period of de novo proteomic labeling to be tightly controlled, but also improves labeling efficiency compared to regular ANL. To demonstrate the potential applications of this novel technique, we use Opto-ANL to detect insulin-induced increases in protein synthesis and to label the excitatory neuronal de novo proteome in mouse brain slices. We believe that this application of photopharmacology will allow researchers to generate novel insights into how the translational landscape is altered across cell-types during complex neurological phenomena such as memory formation.
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License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
*Disclaimer
This summary highlights only some of the key features and terms of the actual license. It is not a license and has no legal value. Carefully review the actual license before using these materials.
License Summary*
You are free to share(copy and redistribute) this article in any medium or format and to adapt(remix, transform, and build upon) the material for any purpose, even commercially within the parameters below:
Creative Commons (CC): This is a Creative Commons license.
Attribution (BY): Credit must be given to the creator.
*Disclaimer
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Introduction
Results
Design and Synthesis of Opto-ANL
Opto-ANL Enables Light-Triggered and Cell-Type Specific Proteomic Labeling
Opto-ANL can Detect Changes in mRNA Translation with High Temporal Resolution
Opto-ANL can Distinguish between Different Cell-Types in Mice Brain Slices
Discussion
Methods
Chemical Synthesis
Absorbance Standard Curve
Uncaging Kinetics
NMR Analysis of Uncaged Products
Cell Culture
De Novo Proteomic Labeling with Opto-ANL in Cell Culture
FUNCAT Western Blot Analysis
Cytotoxicity Assay
De Novo Proteomic Labeling with Opto-ANL in Mouse Brain Slices
FUNCAT and Immuno-Staining
Imaging and Image Analysis
Statistical Analysis
Supporting Information
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.4c00274.
Supporting Information figures, additional experimental details, synthetic procedures, and characterization data for all compounds (PDF)
Terms & Conditions
Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.
Acknowledgments
The authors would like to acknowledge the contributions Maggie Donohue to this work.
References
This article references 46 other publications.
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- 29Walbert, S.; Pfleiderer, W.; Steiner, U. E. Photolabile Protecting Groups for Nucleosides: Mechanistic Studies of the 2-(2-Nitrophenyl)Ethyl Group. Helv. Chim. Acta 2001, 84 (6), 1601– 1611, DOI: 10.1002/1522-2675(20010613)84:6<1601::aid-hlca1601>3.0.co;2-sGoogle Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXls1Whtrs%253D&md5=f56eb91a7643335fcfbec9814a71f43bPhotolabile protecting groups for nucleosides: mechanistic studies of the 2-(2-nitrophenyl)ethyl groupWalbert, Stefan; Pfleiderer, Wolfgang; Steiner, Ulrich E.Helvetica Chimica Acta (2001), 84 (6), 1601-1611CODEN: HCACAV; ISSN:0018-019X. (Verlag Helvetica Chimica Acta)The photochem. of several 2-(2-nitrophenyl)ethyl-caged compds. including caged thymidine nucleosides was studied by nanosecond laser flash photolysis and stationary illumination expts. with quant. HPLC anal. for quantum yields and product distribution. Effects of solvent basicity and acidity were investigated by varying the H2O content and HCl concn., resp., in MeCN/H2O mixts. For all compds. investigated, intramol. H abstraction by the nitro group from the exocyclic α-position with respect to the aryl moiety was found to be the primary process. The protolytic dissocn. equil. of the resulting aci-nitro compd. was kinetically characterized in the 0.1 - 10 μs time region. In general, two reaction channels compete for the aci-nitro compd. and its anion: β-elimination of the caged compd. occurs from the anion, while from the undissociated aci-nitro compd., a nitrosobenzene deriv. is formed with no release of the caged compd. The yield ratio of these two reaction channels can be controlled through shifts in the protolytic dissocn. equil. of the aci-nitro compd. In solns. with either low basicity (H2O-free MeCN) or high acidity (higher concn. of HCl in H2O/MeCN), two as yet unidentified products are formed, each one specifically for one of the mentioned conditions.
- 30Walker, J. W.; McCray, J. A.; Hess, G. P. Photolabile Protecting Groups for an Acetylcholine Receptor Ligand. Synthesis and Photochemistry of a New Class of o-Nitrobenzyl Derivatives and Their Effects on Receptor Function. Biochemistry 1986, 25 (7), 1799– 1805, DOI: 10.1021/bi00355a052Google ScholarThere is no corresponding record for this reference.
- 31Deforest, C. A.; Tirrell, D. A. A Photoreversible Protein-Patterning Approach for Guiding Stem Cell Fate in Three-Dimensional Gels. Nat. Mater. 2015, 14 (5), 523– 531, DOI: 10.1038/nmat4219Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFems7o%253D&md5=85decd4a0ad635de28d205339cb4ec01A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gelsDeForest, Cole A.; Tirrell, David A.Nature Materials (2015), 14 (5), 523-531CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)Although biochem. patterned hydrogels are capable of recapitulating many crit. aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomol. signaling cues in such systems proved difficult. Here, the authors demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, the authors accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. The authors' protein-patterning approach should provide further avenues to probe and direct changes in cell physiol. in response to dynamic biochem. signaling.
- 32Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P.; Hasserodt, J.; Rehberg, M.; Delattre, M.; Zahler, S.; Vollmar, A.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162 (2), 403– 411, DOI: 10.1016/j.cell.2015.06.049Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFCkur7M&md5=0fdc2af4eb68494cbf55e6383a4ecd47Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell DeathBorowiak, Malgorzata; Nahaboo, Wallis; Reynders, Martin; Nekolla, Katharina; Jalinot, Pierre; Hasserodt, Jens; Rehberg, Markus; Delattre, Marie; Zahler, Stefan; Vollmar, Angelika; Trauner, Dirk; Thorn-Seshold, OliverCell (Cambridge, MA, United States) (2015), 162 (2), 403-411CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Small mols. that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biol. research and as clin. anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, the authors introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biol., and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
- 33Proud, C. G. Regulation of Protein Synthesis by Insulin. Biochem. Soc. Trans. 2006, 34 (2), 213– 216, DOI: 10.1042/BST0340213Google ScholarThere is no corresponding record for this reference.
- 34Tsien, J. Z.; Chen, D. F.; Gerber, D.; Tom, C.; Mercer, E. H.; Anderson, D. J.; Mayford, M.; Kandel, E. R.; Tonegawa, S. Subregion- and Cell Type–Restricted Gene Knockout in Mouse Brain. Cell 1996, 87 (7), 1317– 1326, DOI: 10.1016/S0092-8674(00)81826-7Google ScholarThere is no corresponding record for this reference.
- 35Josa-Culleré, L.; Llebaria, A. In the Search for Photocages Cleavable with Visible Light: An Overview of Recent Advances and Chemical Strategies. ChemPhotoChem 2021, 5 (4), 296– 314, DOI: 10.1002/cptc.202000253Google ScholarThere is no corresponding record for this reference.
- 36Frank, J. A.; Antonini, M.-J.; Chiang, P.-H.; Canales, A.; Konrad, D. B.; Garwood, I. C.; Rajic, G.; Koehler, F.; Fink, Y.; Anikeeva, P. In Vivo Photopharmacology Enabled by Multifunctional Fibers. ACS Chem. Neurosci. 2020, 11 (22), 3802– 3813, DOI: 10.1021/acschemneuro.0c00577Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFKis7rM&md5=d37052959e3769e84a20b21dd32b4c0fIn Vivo Photopharmacology Enabled by Multifunctional FibersFrank, James A.; Antonini, Marc-Joseph; Chiang, Po-Han; Canales, Andres; Konrad, David B.; Garwood, Indie C.; Rajic, Gabriela; Koehler, Florian; Fink, Yoel; Anikeeva, PolinaACS Chemical Neuroscience (2020), 11 (22), 3802-3813CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Photoswitchable ligands can add an optical switch to a target receptor or signaling cascade and enable reversible control of neural circuits. The application of this approach, termed photopharmacol., to behavioral expts. has been impeded by a lack of integrated hardware capable of delivering both light and compds. to deep brain regions in moving subjects. Here, the authors devise a hybrid photochem. genetic approach to target neurons using a photoswitchable agonist of the capsaicin receptor TRPV1, red-AzCA-4. Using multifunctional fibers with optical and microfluidic capabilities, the authors delivered a transgene coding for TRPV1 into the ventral tegmental area (VTA). This sensitized excitatory VTA neurons to red-AzCA-4, allowing the authors to optically control conditioned place preference in mice, thus extending applications of photopharmacol. to behavioral expts. Applied to endogenous receptors, the authors' approach may accelerate future studies of mol. mechanisms underlying animal behavior.
- 37Dong, M.; Babalhavaeji, A.; Samanta, S.; Beharry, A. A.; Woolley, G. A. Red-Shifting Azobenzene Photoswitches for in Vivo Use. Acc. Chem. Res. 2015, 48 (10), 2662– 2670, DOI: 10.1021/acs.accounts.5b00270Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFKktrzJ&md5=c016585816dac3f40a35d4836e5d3fdaRed-Shifting Azobenzene Photoswitches for in Vivo UseDong, Mingxin; Babalhavaeji, Amirhossein; Samanta, Subhas; Beharry, Andrew A.; Woolley, G. AndrewAccounts of Chemical Research (2015), 48 (10), 2662-2670CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Recently, there has been a great deal of interest in using the photoisomerization of azobenzene compds. to control specific biol. targets in vivo. These azo compds. can be used as research tools or, in principle, could act as optically controlled drugs. Such "photopharmaceuticals" offer the prospect of targeted drug action and an unprecedented degree of temporal control. A key feature of azo compds. designed to photoswitch in vivo is the wavelength of light required to cause the photoisomerization. To pass through tissue such as the human hand, wavelengths in the red, far-red, or ideally near IR region are required. This Account describes our attempts to produce such azo compds. Introducing electron-donating or push/pull substituents at the para positions delocalizes the azobenzene chromophore and leads to long wavelength absorption but usually also lowers the thermal barrier to interconversion of the isomers. Fast thermal relaxation means it is difficult to produce a large steady state fraction of the cis isomer. Thus, specifically activating or inhibiting a biol. process with the cis isomer would require an impractically bright light source. We have found that introducing substituents at all four ortho positions leads to azo compds. with a no. of unusual properties that are useful for in vivo photoswitching. When the para substituents are amide groups, these tetra-ortho substituted azo compds. show unusually slow thermal relaxation rates and enhanced sepn. of n-π* transitions of cis and trans isomers compared to analogs without ortho substituents. When para positions are substituted with amino groups, ortho methoxy groups greatly stabilize the azonium form of the compds., in which the azo group is protonated. Azonium ions absorb strongly in the red region of the spectrum and can reach into the near-IR. These azonium ions can exhibit robust cis-trans isomerization in aq. solns. at neutral pH. By varying the nature of ortho substituents, together with the no. and nature of meta and para substituents, long wavelength switching, stability to photobleaching, stability to hydrolysis, and stability to redn. by thiols can all be crafted into a photoswitch. Some of these newly developed photoswitches can be used in whole blood and show promise for effective use in vivo. It is hoped they can be combined with appropriate bioactive targets to realize the potential of photopharmacol.
- 38Morstein, J.; Trauner, D. New Players in Phototherapy: Photopharmacology and Bio-Integrated Optoelectronics. Curr. Opin. Chem. Biol. 2019, 50, 145– 151, DOI: 10.1016/j.cbpa.2019.03.013Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpvFOgsb8%253D&md5=23ee3fbfdf33ec9c2cb5e5292f0bbc4aNew players in phototherapy: photopharmacology and bio-integrated optoelectronicsMorstein, Johannes; Trauner, DirkCurrent Opinion in Chemical Biology (2019), 50 (), 145-151CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Photodynamic therapy and phototherapy are used in the clinic to treat dermatol. conditions, cancer, macular degeneration, and a variety of other diseases. Despite their long history and widespread application, the scope of these therapeutic approaches has been limited by a lack of specificity and challenges with light delivery. In recent years, much progress has been made in these regards. Photopharmacol. has provided drug-like mols. that change their efficacy upon irradn. and allow for the optical control of a wide range of defined biol. targets. Many photopharmaceuticals are now used in vivo and some show promising results in preclin. development. At the same time, new bioelectronics for subdermal light delivery have been engineered that could enable phototherapy deep in tissue, for example within the human brain. These developments could increase the impact of photodynamic therapy in human precision medicine.
- 39Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chem. Rev. 2018, 118 (21), 10710– 10747, DOI: 10.1021/acs.chemrev.8b00037Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1OmsrrK&md5=9d82ae8b341d68f2e88ec4f6bf3915e0In Vivo PhotopharmacologyHull, Katharina; Morstein, Johannes; Trauner, DirkChemical Reviews (Washington, DC, United States) (2018), 118 (21), 10710-10747CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Synthetic photoswitches have been known for many years, but their usefulness in biol., pharmacol., and medicine has only recently been systematically explored. Over the past decade photopharmacol. has grown into a vibrant field. As the photophys., pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biol. targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), sol. proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, the authors provide an overview of photopharmacol. using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. The authors discuss the scope and limitations of this approach to study biol. function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.
- 40Broichhagen, J.; Levitz, J. Advances in Tethered Photopharmacology for Precise Optical Control of Signaling Proteins. Curr. Opin. Pharmacol. 2022, 63, 102196, DOI: 10.1016/j.coph.2022.102196Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XkvFWmsbY%253D&md5=122036d2b6a03de14b2246a0c05e60acAdvances in tethered photopharmacology for precise optical control of signaling proteinsBroichhagen, Johannes; Levitz, JoshuaCurrent Opinion in Pharmacology (2022), 63 (), 102196CODEN: COPUBK; ISSN:1471-4892. (Elsevier Ltd.)A review. To overcome the limitations of traditional pharmacol., the field of photopharmacol. has developed around the central concept of using light to endow drug action with spatiotemporal precision. Tethered photopharmacol., where a photoswitchable ligand is covalently attached to a target protein, offers a particularly high degree of spatiotemporal control, as well as the ability to genetically target drug action and limit effects to specific protein subtypes. In this review, we describe the core engineering concepts of tethered pharmacol. and highlight recent advances in harnessing the power of tethered photopharmacol. for an expanded palette of targets and conjugation modes using new, complementary strategies. We also discuss the various applications, including mechanistic studies from the mol. biophys. realm to in vivo studies in behaving animals, that demonstrate the power of tethered pharmacol.
- 41Leippe, P.; Koehler Leman, J.; Trauner, D. Specificity and Speed: Tethered Photopharmacology. Biochemistry 2017, 56 (39), 5214– 5220, DOI: 10.1021/acs.biochem.7b00687Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVelu73F&md5=20f2d6ff8335b1173d849be095e33e6aSpecificity and Speed: Tethered PhotopharmacologyLeippe, Philipp; Koehler Leman, Julia; Trauner, DirkBiochemistry (2017), 56 (39), 5214-5220CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Genetics and pharmacol. are often seen as two distinct approaches to interrogate, elucidate, and manipulate biol. systems. The former is renowned for its precision whereas the latter for its fast kinetics, reversibility, and practicality. Here, we show that both can be joined as "tethered pharmacol.", wherein a genetically programmed bioconjugation site provides selectivity and a tethered pharmacophore provides function. The speed of onset, and esp. offset, of pharmacol. activity can be greatly enhanced by incorporating photoswitches and using light as the trigger ("tethered photopharmacol."). Genetically encoded, tethered photopharmacol. is a variant of Optogenetics and could even play a role in medicine wherever gene therapy is viable. However, gene therapy may not be necessary if sufficiently selective tethering strategies can be developed that operate on wild-type receptors.
- 42Sandoz, G.; Levitz, J.; Kramer, R. H.; Isacoff, E. Y. Optical Control of Endogenous Proteins with a Photoswitchable Conditional Subunit Reveals a Role for TREK1 in GABAB Signaling. Neuron 2012, 74 (6), 1005– 1014, DOI: 10.1016/j.neuron.2012.04.026Google Scholar42https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XptVKhu7s%253D&md5=6aca3e4fe1db6f0cb3df62082896b063Optical Control of Endogenous Proteins with a Photoswitchable Conditional Subunit Reveals a Role for TREK1 in GABAB SignalingSandoz, Guillaume; Levitz, Joshua; Kramer, Richard H.; Isacoff, Ehud Y.Neuron (2012), 74 (6), 1005-1014CODEN: NERNET; ISSN:0896-6273. (Cell Press)Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins contg. a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins using a "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABAB response.
- 43Lemoine, D.; Mondoloni, S.; Tange, J.; Lambolez, B.; Faure, P.; Taly, A.; Tricoire, L.; Mourot, A. Probing the Ionotropic Activity of Glutamate GluD2 Receptor in HEK Cells with Genetically-Engineered Photopharmacology. Elife 2020, 9, 1– 40, DOI: 10.7554/eLife.59026Google ScholarThere is no corresponding record for this reference.
- 44Wessel, D.; Flügge, U. I. A Method for the Quantitative Recovery of Protein in Dilute Solution in the Presence of Detergents and Lipids. Anal. Biochem. 1984, 138 (1), 141– 143, DOI: 10.1016/0003-2697(84)90782-6Google Scholar44https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXhvVygsrc%253D&md5=6738c52467e3e397159c36861a0ee6fdA method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipidsWessel, D.; Fluegge, U. I.Analytical Biochemistry (1984), 138 (1), 141-3CODEN: ANBCA2; ISSN:0003-2697.A rapid method based on a defined MeOH-CHCl3-H2O mixt. for the quant. pptn. of sol. as well as hydrophobic proteins from dil. solns. (e.g., column chromatog. effluents) was developed. The effectiveness of this method is not affected by the presence of detergents, lipids, salt, buffers, and β-mercaptoethanol.
- 45Longo, F.; Mancini, M.; Ibraheem, P. L.; Aryal, S.; Mesini, C.; Patel, J. C.; Penhos, E.; Rahman, N.; Mamcarz, M.; Santini, E.; Rice, M. E.; Klann, E. Cell-Type-Specific Disruption of PERK-EIF2α Signaling in Dopaminergic Neurons Alters Motor and Cognitive Function. Mol. Psychiatry 2021, 26 (11), 6427– 6450, DOI: 10.1038/s41380-021-01099-wGoogle ScholarThere is no corresponding record for this reference.
- 46Longo, F.; Aryal, S.; Anastasiades, P. G.; Maltese, M.; Baimel, C.; Albanese, F.; Tabor, J.; Zhu, J. D.; Oliveira, M. M.; Gastaldo, D.; Bagni, C.; Santini, E.; Tritsch, N. X.; Carter, A. G.; Klann, E. Cell-Type-Specific Disruption of Cortico-Striatal Circuitry Drives Repetitive Patterns of Behavior in Fragile X Syndrome Model Mice. Cell Rep. 2023, 42 (8), 112901, DOI: 10.1016/j.celrep.2023.112901Google ScholarThere is no corresponding record for this reference.
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- 10Nessen, M. A.; Kramer, G.; Back, J. W.; Baskin, J. M.; Smeenk, L. E. J.; de Koning, L. J.; van Maarseveen, J. H.; De Jong, L.; Bertozzi, C. R.; Hiemstra, H.; De Koster, C. G. Selective Enrichment of Azide-Containing Peptides from Complex Mixtures. J. Proteome Res. 2009, 8 (7), 3702– 3711, DOI: 10.1021/pr900257z10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXmtlKitbg%253D&md5=a117803d2b4aa02b9e12aa13a92c8b37Selective Enrichment of Azide-Containing Peptides from Complex MixturesNessen, Merel A.; Kramer, Gertjan; Back, JaapWillem; Baskin, Jeremy M.; Smeenk, Linde E. J.; de Koning, Leo J.; van Maarseveen, Jan H.; de Jong, Luitzen; Bertozzi, Carolyn R.; Hiemstra, Henk; de Koster, Chris G.Journal of Proteome Research (2009), 8 (7), 3702-3711CODEN: JPROBS; ISSN:1535-3893. (American Chemical Society)A general method is described to sequester peptides contg. azides from complex peptide mixts., aimed at facilitating mass spectrometric anal. to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-contg. peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-contg. cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link contg. peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.
- 11Hinz, F.; Dieterich, D.; Schuman, E. Teaching Old NCATs New Tricks: Using Non-Canonical Amino Acid Tagging to Study Neuronal Plasticity. Curr. Opin. Chem. Biol. 2013, 17 (5), 738– 746, DOI: 10.1016/j.cbpa.2013.07.02111https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXht1Gjs7vL&md5=0fd99be89250b08a93cbb20d8ae4a080Teaching old NCATs new tricks: using non-canonical amino acid tagging to study neuronal plasticityHinz, FI; Dieterich, DC; Schuman, EMCurrent Opinion in Chemical Biology (2013), 17 (5), 738-746CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. The non-canonical amino acid labeling techniques BONCAT (bioorthogonal non-canonical amino acid tagging) and FUNCAT (fluorescent non-canonical amino acid tagging) enable the specific identification and visualization of newly synthesized proteins. Recently, these techniques have been applied to neuronal systems to elucidate protein synthesis dynamics during plasticity, identify stimulation-induced proteomes and subproteomes and to investigate local protein synthesis in specific subcellular compartments. The next generation of tools and applications, reviewed here, includes the development of new tags, the quant. identification of newly synthesized proteins, the application of NCAT to whole animals, and the ability to genetically restrict NCAT labeling. These techniques will enable not only improved detection but also allow new scientific questions to be tackled.
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- 16Carlisle, A. K.; Götz, J.; Bodea, L.-G. Three Methods for Examining the de Novo Proteome of Microglia Using BONCAT Bioorthogonal Labeling and FUNCAT Click Chemistry. STAR Protoc. 2023, 4 (3), 102418, DOI: 10.1016/j.xpro.2023.102418There is no corresponding record for this reference.
- 17Schanzenbächer, C.; Sambandan, S.; Langer, J. D.; Schuman, E. M. Nascent Proteome Remodeling Following Homeostatic Scaling at Hippocampal Synapses. Neuron 2016, 92 (2), 358– 371, DOI: 10.1016/j.neuron.2016.09.058There is no corresponding record for this reference.
- 18Evans, H. T.; Bodea, L.; Götz, J. Cell-Specific Non-Canonical Amino Acid Labelling Identifies Changes in the de Novo Proteome during Memory Formation. Elife 2020, 9, 1– 19, DOI: 10.7554/eLife.52990There is no corresponding record for this reference.
- 19Dörrbaum, A. R.; Kochen, L.; Langer, J. D.; Schuman, E. M. Local and Global Influences on Protein Turnover in Neurons and Glia. Elife 2018, 7, 1– 24, DOI: 10.7554/eLife.34202There is no corresponding record for this reference.
- 20Ngo, J. T.; Schuman, E. M.; Tirrell, D. a. Mutant Methionyl-TRNA Synthetase from Bacteria Enables Site-Selective N-Terminal Labeling of Proteins Expressed in Mammalian Cells. Proc. Natl. Acad. Sci. U.S.A. 2013, 110 (13), 4992– 4997, DOI: 10.1073/pnas.1216375110There is no corresponding record for this reference.
- 21Mahdavi, A.; Hamblin, G. D.; Jindal, G. A.; Bagert, J. D.; Dong, C.; Sweredoski, M. J.; Hess, S.; Schuman, E. M.; Tirrell, D. A. Engineered Aminoacyl-TRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis. J. Am. Chem. Soc. 2016, 138 (13), 4278– 4281, DOI: 10.1021/jacs.5b0898021https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28XksFCisbg%253D&md5=03a54561183578fb7cdc5c3ada204e07Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein SynthesisMahdavi, Alborz; Hamblin, Graham D.; Jindal, Granton A.; Bagert, John D.; Dong, Cathy; Sweredoski, Michael J.; Hess, Sonja; Schuman, Erin M.; Tirrell, David A.Journal of the American Chemical Society (2016), 138 (13), 4278-4281CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)Methods for cell-selective anal. of proteome dynamics will facilitate studies of biol. processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNAMet in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective anal. of mammalian protein synthesis.
- 22Alvarez-Castelao, B.; Schanzenbächer, C. T.; Hanus, C.; Glock, C.; tom Dieck, S.; Dörrbaum, A. R.; Bartnik, I.; Nassim-Assir, B.; Ciirdaeva, E.; Mueller, A.; Dieterich, D. C.; Tirrell, D. A.; Langer, J. D.; Schuman, E. M. Cell-Type-Specific Metabolic Labeling of Nascent Proteomes in Vivo. Nat. Biotechnol. 2017, 35 (12), 1196– 1201, DOI: 10.1038/nbt.4016There is no corresponding record for this reference.
- 23Alvarez-Castelao, B.; Schanzenbächer, C. T.; Langer, J. D.; Schuman, E. M. Cell-Type-Specific Metabolic Labeling, Detection and Identification of Nascent Proteomes in Vivo. Nat. Protoc. 2019, 14 (2), 556– 575, DOI: 10.1038/s41596-018-0106-6There is no corresponding record for this reference.
- 24Shrestha, P.; Klann, E. Spatiotemporally Resolved Protein Synthesis as a Molecular Framework for Memory Consolidation. Trends Neurosci. 2022, 45 (4), 297– 311, DOI: 10.1016/j.tins.2022.01.004There is no corresponding record for this reference.
- 25Sun, C.; Nold, A.; Fusco, C. M.; Rangaraju, V.; Tchumatchenko, T.; Heilemann, M.; Schuman, E. M. Prevalence and Specificity of Local Protein Synthesis during Neuronal Synaptic Plasticity. Sci. Adv. 2021, 7, eabj0790 DOI: 10.1126/sciadv.abj0790There is no corresponding record for this reference.
- 26Mangubat-Medina, A. E.; Ball, Z. T. Triggering Biological Processes: Methods and Applications of Photocaged Peptides and Proteins. Chem. Soc. Rev. 2021, 50 (18), 10403– 10421, DOI: 10.1039/D0CS01434F26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhs1Kgs7vM&md5=878df041ef552c7a4c9c3cab7332d74aTriggering biological processes: methods and applications of photocaged peptides and proteinsMangubat-Medina, Alicia E.; Ball, Zachary T.Chemical Society Reviews (2021), 50 (18), 10403-10421CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. There has been a significant push in recent years to deploy fundamental knowledge and methods of photochem. toward biol. ends. Photoreactive groups have enabled chemists to activate biol. function using the concept of photocaging. By granting spatiotemporal control over protein activation, these photocaging methods are fundamental in understanding biol. processes. Peptides and proteins are an important group of photocaging targets that present conceptual and tech. challenges, requiring precise chemoselectivity in complex polyfunctional environments. This review focuses on recent advances in photocaging techniques and methodologies, as well as their use in living systems. Photocaging methods include genetic and chem. approaches that require a deep understanding of structure-function relationships based on subtle changes in primary structure. Successful implementation of these ideas can shed light on important spatiotemporal aspects of living systems.
- 27Adelmund, S. M.; Ruskowitz, E. R.; Farahani, P. E.; Wolfe, J. V.; DeForest, C. A. Light-Activated Proteomic Labeling via Photocaged Bioorthogonal Non-Canonical Amino Acids. ACS Chem. Biol. 2018, 13 (3), 573– 577, DOI: 10.1021/acschembio.7b0102327https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXitlarsro%253D&md5=1d9133335ad550225d03a962d1d3e75fLight-Activated Proteomic Labeling via Photocaged Bioorthogonal Non-Canonical Amino AcidsAdelmund, Steven M.; Ruskowitz, Emily R.; Farahani, Payam E.; Wolfe, Julie V.; De Forest, Cole A.ACS Chemical Biology (2018), 13 (3), 573-577CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)This work introduces light-activated bioorthogonal noncanonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging L-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compd. remains stable for many hours in culture, but can be photochem. liberated rapidly and on demand with spatial control. Upon directed light exposure, the uncaged amino acid is available for local translation, enabling downstream proteomic interrogation via bioorthogonal conjugation. Exploiting the reactive azide moiety present on Aha's amino acid side chain, we demonstrate that newly synthesized proteins can be purified for quant. proteomics or visualized in synthetic tissues with a new level of spatiotemporal control. Shedding light on when and where proteins are translated within living samples, we anticipate that laBONCAT will aid in understanding the progression of complex protein-related disorders.
- 28Wöll, D.; Smirnova, J.; Galetskaya, M.; Prykota, T.; Bühler, J.; Stengele, K.; Pfleiderer, W.; Steiner, U. E. Intramolecular Sensitization of Photocleavage of the Photolabile 2-(2-Nitrophenyl)Propoxycarbonyl (NPPOC) Protecting Group: Photoproducts and Photokinetics of the Release of Nucleosides. Chem.─Eur. J. 2008, 14 (21), 6490– 6497, DOI: 10.1002/chem.200800613There is no corresponding record for this reference.
- 29Walbert, S.; Pfleiderer, W.; Steiner, U. E. Photolabile Protecting Groups for Nucleosides: Mechanistic Studies of the 2-(2-Nitrophenyl)Ethyl Group. Helv. Chim. Acta 2001, 84 (6), 1601– 1611, DOI: 10.1002/1522-2675(20010613)84:6<1601::aid-hlca1601>3.0.co;2-s29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXls1Whtrs%253D&md5=f56eb91a7643335fcfbec9814a71f43bPhotolabile protecting groups for nucleosides: mechanistic studies of the 2-(2-nitrophenyl)ethyl groupWalbert, Stefan; Pfleiderer, Wolfgang; Steiner, Ulrich E.Helvetica Chimica Acta (2001), 84 (6), 1601-1611CODEN: HCACAV; ISSN:0018-019X. (Verlag Helvetica Chimica Acta)The photochem. of several 2-(2-nitrophenyl)ethyl-caged compds. including caged thymidine nucleosides was studied by nanosecond laser flash photolysis and stationary illumination expts. with quant. HPLC anal. for quantum yields and product distribution. Effects of solvent basicity and acidity were investigated by varying the H2O content and HCl concn., resp., in MeCN/H2O mixts. For all compds. investigated, intramol. H abstraction by the nitro group from the exocyclic α-position with respect to the aryl moiety was found to be the primary process. The protolytic dissocn. equil. of the resulting aci-nitro compd. was kinetically characterized in the 0.1 - 10 μs time region. In general, two reaction channels compete for the aci-nitro compd. and its anion: β-elimination of the caged compd. occurs from the anion, while from the undissociated aci-nitro compd., a nitrosobenzene deriv. is formed with no release of the caged compd. The yield ratio of these two reaction channels can be controlled through shifts in the protolytic dissocn. equil. of the aci-nitro compd. In solns. with either low basicity (H2O-free MeCN) or high acidity (higher concn. of HCl in H2O/MeCN), two as yet unidentified products are formed, each one specifically for one of the mentioned conditions.
- 30Walker, J. W.; McCray, J. A.; Hess, G. P. Photolabile Protecting Groups for an Acetylcholine Receptor Ligand. Synthesis and Photochemistry of a New Class of o-Nitrobenzyl Derivatives and Their Effects on Receptor Function. Biochemistry 1986, 25 (7), 1799– 1805, DOI: 10.1021/bi00355a052There is no corresponding record for this reference.
- 31Deforest, C. A.; Tirrell, D. A. A Photoreversible Protein-Patterning Approach for Guiding Stem Cell Fate in Three-Dimensional Gels. Nat. Mater. 2015, 14 (5), 523– 531, DOI: 10.1038/nmat421931https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXjtFems7o%253D&md5=85decd4a0ad635de28d205339cb4ec01A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gelsDeForest, Cole A.; Tirrell, David A.Nature Materials (2015), 14 (5), 523-531CODEN: NMAACR; ISSN:1476-1122. (Nature Publishing Group)Although biochem. patterned hydrogels are capable of recapitulating many crit. aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomol. signaling cues in such systems proved difficult. Here, the authors demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, the authors accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. The authors' protein-patterning approach should provide further avenues to probe and direct changes in cell physiol. in response to dynamic biochem. signaling.
- 32Borowiak, M.; Nahaboo, W.; Reynders, M.; Nekolla, K.; Jalinot, P.; Hasserodt, J.; Rehberg, M.; Delattre, M.; Zahler, S.; Vollmar, A.; Trauner, D.; Thorn-Seshold, O. Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162 (2), 403– 411, DOI: 10.1016/j.cell.2015.06.04932https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFCkur7M&md5=0fdc2af4eb68494cbf55e6383a4ecd47Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell DeathBorowiak, Malgorzata; Nahaboo, Wallis; Reynders, Martin; Nekolla, Katharina; Jalinot, Pierre; Hasserodt, Jens; Rehberg, Markus; Delattre, Marie; Zahler, Stefan; Vollmar, Angelika; Trauner, Dirk; Thorn-Seshold, OliverCell (Cambridge, MA, United States) (2015), 162 (2), 403-411CODEN: CELLB5; ISSN:0092-8674. (Cell Press)Small mols. that interfere with microtubule dynamics, such as Taxol and the Vinca alkaloids, are widely used in cell biol. research and as clin. anticancer drugs. However, their activity cannot be restricted to specific target cells, which also causes severe side effects in chemotherapy. Here, the authors introduce the photostatins, inhibitors that can be switched on and off in vivo by visible light, to optically control microtubule dynamics. Photostatins modulate microtubule dynamics with a subsecond response time and control mitosis in living organisms with single-cell spatial precision. In longer-term applications in cell culture, photostatins are up to 250 times more cytotoxic when switched on with blue light than when kept in the dark. Therefore, photostatins are both valuable tools for cell biol., and are promising as a new class of precision chemotherapeutics whose toxicity may be spatiotemporally constrained using light.
- 33Proud, C. G. Regulation of Protein Synthesis by Insulin. Biochem. Soc. Trans. 2006, 34 (2), 213– 216, DOI: 10.1042/BST0340213There is no corresponding record for this reference.
- 34Tsien, J. Z.; Chen, D. F.; Gerber, D.; Tom, C.; Mercer, E. H.; Anderson, D. J.; Mayford, M.; Kandel, E. R.; Tonegawa, S. Subregion- and Cell Type–Restricted Gene Knockout in Mouse Brain. Cell 1996, 87 (7), 1317– 1326, DOI: 10.1016/S0092-8674(00)81826-7There is no corresponding record for this reference.
- 35Josa-Culleré, L.; Llebaria, A. In the Search for Photocages Cleavable with Visible Light: An Overview of Recent Advances and Chemical Strategies. ChemPhotoChem 2021, 5 (4), 296– 314, DOI: 10.1002/cptc.202000253There is no corresponding record for this reference.
- 36Frank, J. A.; Antonini, M.-J.; Chiang, P.-H.; Canales, A.; Konrad, D. B.; Garwood, I. C.; Rajic, G.; Koehler, F.; Fink, Y.; Anikeeva, P. In Vivo Photopharmacology Enabled by Multifunctional Fibers. ACS Chem. Neurosci. 2020, 11 (22), 3802– 3813, DOI: 10.1021/acschemneuro.0c0057736https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXitFKis7rM&md5=d37052959e3769e84a20b21dd32b4c0fIn Vivo Photopharmacology Enabled by Multifunctional FibersFrank, James A.; Antonini, Marc-Joseph; Chiang, Po-Han; Canales, Andres; Konrad, David B.; Garwood, Indie C.; Rajic, Gabriela; Koehler, Florian; Fink, Yoel; Anikeeva, PolinaACS Chemical Neuroscience (2020), 11 (22), 3802-3813CODEN: ACNCDM; ISSN:1948-7193. (American Chemical Society)Photoswitchable ligands can add an optical switch to a target receptor or signaling cascade and enable reversible control of neural circuits. The application of this approach, termed photopharmacol., to behavioral expts. has been impeded by a lack of integrated hardware capable of delivering both light and compds. to deep brain regions in moving subjects. Here, the authors devise a hybrid photochem. genetic approach to target neurons using a photoswitchable agonist of the capsaicin receptor TRPV1, red-AzCA-4. Using multifunctional fibers with optical and microfluidic capabilities, the authors delivered a transgene coding for TRPV1 into the ventral tegmental area (VTA). This sensitized excitatory VTA neurons to red-AzCA-4, allowing the authors to optically control conditioned place preference in mice, thus extending applications of photopharmacol. to behavioral expts. Applied to endogenous receptors, the authors' approach may accelerate future studies of mol. mechanisms underlying animal behavior.
- 37Dong, M.; Babalhavaeji, A.; Samanta, S.; Beharry, A. A.; Woolley, G. A. Red-Shifting Azobenzene Photoswitches for in Vivo Use. Acc. Chem. Res. 2015, 48 (10), 2662– 2670, DOI: 10.1021/acs.accounts.5b0027037https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhsFKktrzJ&md5=c016585816dac3f40a35d4836e5d3fdaRed-Shifting Azobenzene Photoswitches for in Vivo UseDong, Mingxin; Babalhavaeji, Amirhossein; Samanta, Subhas; Beharry, Andrew A.; Woolley, G. AndrewAccounts of Chemical Research (2015), 48 (10), 2662-2670CODEN: ACHRE4; ISSN:0001-4842. (American Chemical Society)A review. Recently, there has been a great deal of interest in using the photoisomerization of azobenzene compds. to control specific biol. targets in vivo. These azo compds. can be used as research tools or, in principle, could act as optically controlled drugs. Such "photopharmaceuticals" offer the prospect of targeted drug action and an unprecedented degree of temporal control. A key feature of azo compds. designed to photoswitch in vivo is the wavelength of light required to cause the photoisomerization. To pass through tissue such as the human hand, wavelengths in the red, far-red, or ideally near IR region are required. This Account describes our attempts to produce such azo compds. Introducing electron-donating or push/pull substituents at the para positions delocalizes the azobenzene chromophore and leads to long wavelength absorption but usually also lowers the thermal barrier to interconversion of the isomers. Fast thermal relaxation means it is difficult to produce a large steady state fraction of the cis isomer. Thus, specifically activating or inhibiting a biol. process with the cis isomer would require an impractically bright light source. We have found that introducing substituents at all four ortho positions leads to azo compds. with a no. of unusual properties that are useful for in vivo photoswitching. When the para substituents are amide groups, these tetra-ortho substituted azo compds. show unusually slow thermal relaxation rates and enhanced sepn. of n-π* transitions of cis and trans isomers compared to analogs without ortho substituents. When para positions are substituted with amino groups, ortho methoxy groups greatly stabilize the azonium form of the compds., in which the azo group is protonated. Azonium ions absorb strongly in the red region of the spectrum and can reach into the near-IR. These azonium ions can exhibit robust cis-trans isomerization in aq. solns. at neutral pH. By varying the nature of ortho substituents, together with the no. and nature of meta and para substituents, long wavelength switching, stability to photobleaching, stability to hydrolysis, and stability to redn. by thiols can all be crafted into a photoswitch. Some of these newly developed photoswitches can be used in whole blood and show promise for effective use in vivo. It is hoped they can be combined with appropriate bioactive targets to realize the potential of photopharmacol.
- 38Morstein, J.; Trauner, D. New Players in Phototherapy: Photopharmacology and Bio-Integrated Optoelectronics. Curr. Opin. Chem. Biol. 2019, 50, 145– 151, DOI: 10.1016/j.cbpa.2019.03.01338https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpvFOgsb8%253D&md5=23ee3fbfdf33ec9c2cb5e5292f0bbc4aNew players in phototherapy: photopharmacology and bio-integrated optoelectronicsMorstein, Johannes; Trauner, DirkCurrent Opinion in Chemical Biology (2019), 50 (), 145-151CODEN: COCBF4; ISSN:1367-5931. (Elsevier B.V.)A review. Photodynamic therapy and phototherapy are used in the clinic to treat dermatol. conditions, cancer, macular degeneration, and a variety of other diseases. Despite their long history and widespread application, the scope of these therapeutic approaches has been limited by a lack of specificity and challenges with light delivery. In recent years, much progress has been made in these regards. Photopharmacol. has provided drug-like mols. that change their efficacy upon irradn. and allow for the optical control of a wide range of defined biol. targets. Many photopharmaceuticals are now used in vivo and some show promising results in preclin. development. At the same time, new bioelectronics for subdermal light delivery have been engineered that could enable phototherapy deep in tissue, for example within the human brain. These developments could increase the impact of photodynamic therapy in human precision medicine.
- 39Hüll, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chem. Rev. 2018, 118 (21), 10710– 10747, DOI: 10.1021/acs.chemrev.8b0003739https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXht1OmsrrK&md5=9d82ae8b341d68f2e88ec4f6bf3915e0In Vivo PhotopharmacologyHull, Katharina; Morstein, Johannes; Trauner, DirkChemical Reviews (Washington, DC, United States) (2018), 118 (21), 10710-10747CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)A review. Synthetic photoswitches have been known for many years, but their usefulness in biol., pharmacol., and medicine has only recently been systematically explored. Over the past decade photopharmacol. has grown into a vibrant field. As the photophys., pharmacodynamic, and pharmacokinetic properties of photoswitches, such as azobenzenes, have become established, they have been applied to a wide range of biol. targets. These include transmembrane proteins (ion channels, transporters, G protein-coupled receptors, receptor-linked enzymes), sol. proteins (kinases, proteases, factors involved in epigenetic regulation), lipid membranes, and nucleic acids. In this review, the authors provide an overview of photopharmacol. using synthetic switches that have been applied in vivo, i.e., in living cells and organisms. The authors discuss the scope and limitations of this approach to study biol. function and the challenges it faces in translational medicine. The relationships between synthetic photoswitches, natural chromophores used in optogenetics, and caged ligands are addressed.
- 40Broichhagen, J.; Levitz, J. Advances in Tethered Photopharmacology for Precise Optical Control of Signaling Proteins. Curr. Opin. Pharmacol. 2022, 63, 102196, DOI: 10.1016/j.coph.2022.10219640https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XkvFWmsbY%253D&md5=122036d2b6a03de14b2246a0c05e60acAdvances in tethered photopharmacology for precise optical control of signaling proteinsBroichhagen, Johannes; Levitz, JoshuaCurrent Opinion in Pharmacology (2022), 63 (), 102196CODEN: COPUBK; ISSN:1471-4892. (Elsevier Ltd.)A review. To overcome the limitations of traditional pharmacol., the field of photopharmacol. has developed around the central concept of using light to endow drug action with spatiotemporal precision. Tethered photopharmacol., where a photoswitchable ligand is covalently attached to a target protein, offers a particularly high degree of spatiotemporal control, as well as the ability to genetically target drug action and limit effects to specific protein subtypes. In this review, we describe the core engineering concepts of tethered pharmacol. and highlight recent advances in harnessing the power of tethered photopharmacol. for an expanded palette of targets and conjugation modes using new, complementary strategies. We also discuss the various applications, including mechanistic studies from the mol. biophys. realm to in vivo studies in behaving animals, that demonstrate the power of tethered pharmacol.
- 41Leippe, P.; Koehler Leman, J.; Trauner, D. Specificity and Speed: Tethered Photopharmacology. Biochemistry 2017, 56 (39), 5214– 5220, DOI: 10.1021/acs.biochem.7b0068741https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhsVelu73F&md5=20f2d6ff8335b1173d849be095e33e6aSpecificity and Speed: Tethered PhotopharmacologyLeippe, Philipp; Koehler Leman, Julia; Trauner, DirkBiochemistry (2017), 56 (39), 5214-5220CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)A review. Genetics and pharmacol. are often seen as two distinct approaches to interrogate, elucidate, and manipulate biol. systems. The former is renowned for its precision whereas the latter for its fast kinetics, reversibility, and practicality. Here, we show that both can be joined as "tethered pharmacol.", wherein a genetically programmed bioconjugation site provides selectivity and a tethered pharmacophore provides function. The speed of onset, and esp. offset, of pharmacol. activity can be greatly enhanced by incorporating photoswitches and using light as the trigger ("tethered photopharmacol."). Genetically encoded, tethered photopharmacol. is a variant of Optogenetics and could even play a role in medicine wherever gene therapy is viable. However, gene therapy may not be necessary if sufficiently selective tethering strategies can be developed that operate on wild-type receptors.
- 42Sandoz, G.; Levitz, J.; Kramer, R. H.; Isacoff, E. Y. Optical Control of Endogenous Proteins with a Photoswitchable Conditional Subunit Reveals a Role for TREK1 in GABAB Signaling. Neuron 2012, 74 (6), 1005– 1014, DOI: 10.1016/j.neuron.2012.04.02642https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XptVKhu7s%253D&md5=6aca3e4fe1db6f0cb3df62082896b063Optical Control of Endogenous Proteins with a Photoswitchable Conditional Subunit Reveals a Role for TREK1 in GABAB SignalingSandoz, Guillaume; Levitz, Joshua; Kramer, Richard H.; Isacoff, Ehud Y.Neuron (2012), 74 (6), 1005-1014CODEN: NERNET; ISSN:0896-6273. (Cell Press)Selective ligands are lacking for many neuronal signaling proteins. Photoswitched tethered ligands (PTLs) have enabled fast and reversible control of specific proteins contg. a PTL anchoring site and have been used to remote control overexpressed proteins. We report here a scheme for optical remote control of native proteins using a "photoswitchable conditional subunit" (PCS), which contains the PTL anchoring site as well as a mutation that prevents it from reaching the plasma membrane. In cells lacking native subunits for the protein, the PCS remains nonfunctional internally. However, in cells expressing native subunits, the native subunit and PCS coassemble, traffic to the plasma membrane, and place the native protein under optical control provided by the coassembled PCS. We apply this approach to the TREK1 potassium channel, which lacks selective, reversible blockers. We find that TREK1, typically considered to be a leak channel, contributes to the hippocampal GABAB response.
- 43Lemoine, D.; Mondoloni, S.; Tange, J.; Lambolez, B.; Faure, P.; Taly, A.; Tricoire, L.; Mourot, A. Probing the Ionotropic Activity of Glutamate GluD2 Receptor in HEK Cells with Genetically-Engineered Photopharmacology. Elife 2020, 9, 1– 40, DOI: 10.7554/eLife.59026There is no corresponding record for this reference.
- 44Wessel, D.; Flügge, U. I. A Method for the Quantitative Recovery of Protein in Dilute Solution in the Presence of Detergents and Lipids. Anal. Biochem. 1984, 138 (1), 141– 143, DOI: 10.1016/0003-2697(84)90782-644https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXhvVygsrc%253D&md5=6738c52467e3e397159c36861a0ee6fdA method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipidsWessel, D.; Fluegge, U. I.Analytical Biochemistry (1984), 138 (1), 141-3CODEN: ANBCA2; ISSN:0003-2697.A rapid method based on a defined MeOH-CHCl3-H2O mixt. for the quant. pptn. of sol. as well as hydrophobic proteins from dil. solns. (e.g., column chromatog. effluents) was developed. The effectiveness of this method is not affected by the presence of detergents, lipids, salt, buffers, and β-mercaptoethanol.
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Supporting Information
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