A Biomimetic C-Terminal Extension Strategy for Photocaging Amidated Neuropeptides

Photoactivatable neuropeptides offer a robust stimulus–response relationship that can drive mechanistic studies into the physiological mechanisms of neuropeptidergic transmission. The majority of neuropeptides contain a C-terminal amide, which offers a potentially general site for installation of a C-terminal caging group. Here, we report a biomimetic caging strategy in which the neuropeptide C-terminus is extended via a photocleavable amino acid to mimic the proneuropeptides found in large dense-core vesicles. We explored this approach with four prominent neuropeptides: gastrin-releasing peptide (GRP), oxytocin (OT), substance P (SP), and cholecystokinin (CCK). C-terminus extension greatly reduced the activity of all four peptides at heterologously expressed receptors. In cell type-specific electrophysiological recordings from acute brain slices, subsecond flashes of ultraviolet light produced rapidly activating membrane currents via activation of endogenous G protein-coupled receptors. Subsequent mechanistic studies with caged CCK revealed a role for extracellular proteases in shaping the temporal dynamics of CCK signaling, and a striking switch-like, cell-autonomous anti-opioid effect of transient CCK signaling in hippocampal parvalbumin interneurons. These results suggest that C-terminus extension with a photocleavable linker may be a general strategy for photocaging amidated neuropeptides and demonstrate how photocaged neuropeptides can provide mechanistic insights into neuropeptide signaling that are inaccessible using conventional approaches.


Chemical Synthesis
Large-scale synthesis of racemic Fmoc-DMNBA (4) and the synthesis of all four NPP-caged peptides was conducted by BaChem.NPP-caged peptides were purified by high-pressure liquid chromatography and their identities confirmed by high resolution mass spectrometry (HRMS).Diastereomers were observed in ~equal proportion and were readily resolved for SP-NPP but not for GRP(14-27)-NPP, OT-NPP, and CCK(8S)-NPP.
Commercial reagents were used as received.All solvents were purchased as septum-sealed bottles stored under an inert atmosphere.All reactions were sealed with septa through which a nitrogen atmosphere was introduced unless otherwise noted.Reactions were conducted in round-bottomed flasks or septum-capped amber screw-cap vials containing Teflon-coated magnetic stir bars.Reactions were monitored by liquid chromatography-mass spectrometry (Agilent 1260 Infinity II) using C-18 column (4.6 × 50 mm, 1.8 μm, Agilent) with a linear gradient (water/MeCN 5%/95% → MeCN 100%, 0-8 min with 0.1% formic acid, 1 ml/min flow, electrospray ionization, positive ion mode, UV detection at 220 nm, 280 nm, and 350 nm).Highresolution mass spectrometry data were obtained at the UCSD Chemistry and Biochemistry Mass Spectrometry Facility on an Agilent 6230 time-of flight mass spectrometer (TOFMS).
Proton ( 1 H) and carbon ( 13 C) NMR spectra were recorded at room temperature in DMSO, MeCN, D2O or base-filtered CDCl3 on a Bruker AVA-400, Varian VX-500, or Jeol ECA-500 spectrometer operating at 400 MHz for proton and 100 or 101 MHz for carbon nuclei.For 1 H NMR spectra, signals arising from the residual protioforms of the solvent were used as the internal standards. 1H NMR data are reported as follows: chemical shift (δ) [multiplicity, coupling constant(s) J (Hz), relative integral] where multiplicity is defined as: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet or combinations of the above.All NMR spectra were processed using MestReNova 14.2.1.UV-visible spectra were recorded on a NanoDrop 2000 UV-VIS spectrophotometer (Thermo-Fisher).Room lights were covered with Roscolux Canary Yellow #312 film (Rosco Laboratories, Stamford, CT) to filter out wavelengths of light that could lead to unintentional photolysis during purification and handling.
The reaction mixture was refluxed overnight (18 hours) to find a milky thick white solution.This solution was then filtered and washed with EtOH until the filtrate was colorless.Solvent removal afforded 3-amino-3-(  3-amino-3-(4,5-dimethoxy-2-nitrophenyl)propanoic acid (1.13 g, 5.02 mmol, 1 eq) was placed in a round bottom flask, sealed and purged with nitrogen 3 times before dissolving in trifluoroacetic anhydride (21.0 mL, 150.5 mmol, 30 eq) and mixed vigorously (1 hour) or sonicated until the reaction mixture becomes fully homogenous.This mixture was then cooled down to 0°C followed by the addition of acetyl chloride (3.6 ml, 50.17 mmol, 10 eq) and slow dropwise addition of anhydrous methanol (6.09 ml, 150.5 mmol, 30 eq).The reaction mixture was allowed to stir for 1 hour before quenching with water (500 mL) followed by 3 washes with CH 2 Cl 2 .The organic layers were combined, concentrated in vacuo, and purified by silica gel chromatography (0-30% EtOAc in hexane for 10 column volumes) to give methyl 3-(
In vitro GPCR activation assays.GloSensor assay of G-protein signaling.Human embryonic kidney 293T cells were grown in Complete DMEM (Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) containing 5% fetal bovine serum (Corning), 50 U/mL Penicillin-Streptomycin (Invitrogen), and 1 mM sodium pyruvate (Corning)) and maintained at 37 °C in an atmosphere of 5% CO 2 in 10 cm TC dishes.Media in 10 cm TC dishes with HEK 293T cells (at around 70% confluence) were replaced with Opti-MEM (Invitrogen).Then the GPCR-plasmid, cAMP dependent reporter plasmid (pGloSensor -22F), and Lipofectamine 2000 (Invitrogen) in Opti-MEM were added.The dishes with transfection media were incubated at 37°C in an atmosphere of 5% CO 2 for 6 h before replacing the media with complete DMEM.After incubating at 37°C in an atmosphere of 5% CO 2 for 16 h, transfected cells were plated in poly-Dlysine coated 96-well plates at ~40,000 cells/well and incubated at 37 °C in an atmosphere of 5% CO 2 for 16 h.On the day of assay, media in each well were replaced with 50 µL of assay buffer (20 mM HEPES, 1x HBSS, pH 7.2, 2 g/L d-glucose), followed by addition of 25 µL of 4x drug solutions for 15 min at room temperature.Subsequently, 25 µL of 4 mM GloSensor cAMP Reagent (luciferin) was added, and, following gentle mixing, luminescence counting was performed using a plate reader (iD5, Molecular Devices) after 25 min.For antagonism experiments, the candidate antagonist was added to the assay buffer (50 uL/well) at 2x the final concentration and allowed to incubate for 5 minutes prior to the addition of agonist.
power was set to 5 mW in the sample plane (∼80 mW of an ∼20 mm diameter "beam" at the back aperture).
Data analysis.Electrophysiology data were analyzed in Igor Pro (Wavemetrics).Current response to bath application of peptide were calculated as the average current over a 20 sec window surrounding the peak current measured for each cell.For each parent peptide, peak responses were obtained at different time windows after peptide addition (GRP(14-27): 30-60 sec; OT: 2-3 min; SP: 2-3 min; CCK(8S): 1.5-2.5 min).For the NPP-caged peptides, responses were calculated within the time window in which peak responses to the corresponding parent peptide were obtained.Peak responses to photo-uncaging were calculated by taking the average current over 200 ms window surrounding the peak current measured for each cell.Time constants describing the photo-uncaging-evoked currents were obtained by fitting either a single exponential or double exponential function to the waveform of the average uncaging response for all cells in a given condition.Summary values are reported as mean ± SEM.Data were found to be normally distributed using a Shapiro-Wilk test and subsequently analyzed using an Ordinary one-way ANOVA followed by Sidak's test for multiple comparisons.All statistical tests were performed in GraphPad Prism v.9.5.1.Specific statistical tests and corrections are described for each figure in the text and figure legends.