Gs protein peptidomimetics as allosteric modulators of the β2-adrenergic receptor

A series of Gs protein peptidomimetics were designed and synthesised based on the published X-ray crystal structure of the active state β2-adrenergic receptor (β2AR) in complex with the Gs protein (PDB 3SN6). We hypothesised that such peptidomimetics may function as allosteric modulators that target the intracellular Gs protein binding site of the β2AR. Peptidomimetics were designed to mimic the 15 residue C-terminal α-helix of the Gs protein and were pre-organised in a helical conformation by (i, i + 4)-stapling using copper catalysed azide alkyne cycloaddition. Linear and stapled peptidomimetics were analysed by circular dichroism (CD) and characterised in a membrane-based cAMP accumulation assay and in a bimane fluorescence assay on purified β2AR. Several peptidomimetics inhibited agonist isoproterenol (ISO) induced cAMP formation by lowering the ISO maximal efficacy up to 61%. Moreover, some peptidomimetics were found to significantly decrease the potency of ISO up to 39-fold. In the bimane fluorescence assay none of the tested peptidomimetics could stabilise an active-like conformation of β2AR. Overall, the obtained pharmacological data suggest that some of the peptidomimetics may be able to compete with the native Gs protein for the intracellular binding site to block ISO-induced cAMP formation, but are unable to stabilise an active-like receptor conformation.


Low Resolution Mass Spectrometry (LRMS) Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI TOF MS)
MALDI TOF MS was performed on a Bruker Microflex LT using a solution of ACCA (αcyano-4-hydroxycinnamic acid) matrix prepared by dissolving 10 mg ACCA in 1 ml of CH 3 CN, H 2 O, TFA 500:475:25 (v/v/v). The data was analysed and processed using Bruker flex Analysis software.

High Resolution Mass Spectrometry (HRMS) HRMS method A: Matrix Assisted Laser Desorption Ionisation (MALDI):
Accurate mass analysis was performed in positive ion mode with MALDI ionisation on a Thermo QExactive Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an AP-SMALDI 10 ion source (TransmitMIT, Giessen, Germany) and operated at mass resolving power 140,000@m/z200. DHB was used as matrix and lock-mass for internal mass calibration. HRMS method B: Electro-Spray Ionisation (ESI): Data was recorded on a Micromass Q-TOF 1.5, UB137 or on a time-of-flight (TOF) MS system, coupled to an analytical HPLC and ESI detector. HRMS HPLC was performed on a C18 column (25 cm × 4.6 mm, 5 µm) with a linear gradient (10% to 100% MeOH in H 2 O, containing 0.1% TFA, in 20 min, v/v) at a flow rate of 1 ml/min and UV detection at 215 nm.

Fourier Transformed Infrared Spectroscopy (FT-IR)
IR spectroscopy was recorded on a Perkin-Elmer Spectrum One IR spectrometer using Spectrum One version 3.02 software. Samples were loaded as solids and signals (ν max ) are reported in wavenumbers (cm -1 ).

Determining net peptide content by quantitative NMR (qNMR)
The net peptide content for all peptides was determined on a Bruker Avance IIIHD 600 MHz NMR spectrometer equipped with a 5 mm cryogenically cooled dual 1 H/ 13 C-probe. The software TopSpin 3.2 was used for data acquisition and analysis. Quantitative determinations were performed with the ERETIC method 1,2 by measuring a separate sample of the 15-mer G αs peptide under identical conditions as the sample in D 2 O (300 K, 12 kHz sweep width, acquisition time 2.73 sec., relaxation delay 4.27 sec.) with appropriate adjustments of tuning and matching of the probe, determination of the correct 90°-pulse, number of scans and receiver gain.

Circular Dichroism Spectroscopy (CD)
CD measurements were recorded on a Jasco J-810 Spectropolarimeter with peltier control using 1-mm Quartz SUPRASIL cuvettes (Hellma Analytics). CD spectra of all peptides were recorded at 25 °C from 260 to 190 nm with a scan speed of 20 nm/min, 10 accumulations, and a response time of 2 s. Peptide samples were dissolved in 10 mM NaH 2 PO 4 buffer at pH 6.0. Concentrations of all peptides were 50 μM, as determined by qNMR. Equivalent spectra of buffers were recorded and subtracted from the spectra of the peptides. The α-helical content was estimated based on the difference between the minimum ellipticity at 222 nm, i.e. the average ellipticity of 5 points around 222 nm, and the maximum at 190 nm, again defined as the average of 5 points around 190 nm. The 12 largest frequency modes of the Fourier transform of the spectra was used to smooth the data.

CHEMISTRY Chemicals
Amino acids, resin and coupling reagents for peptide synthesis were purchased from Iris Biotech GmbH, Marktredwitz, Germany or Chem-Impex International Inc., Wood Dale, Illinois, USA. Other reagents were purchased from Sigma-Aldrich.
The diazotransfer reagent imidazole-1-sulfonyl azide hydrochloride was synthesised according to the published procedure. 3 Fmoc-protected amino acids 1, 2 and 4 were synthesised according to the published procedures. For 1 and 2 the crude products were utilised for SPPS. 4,5 Fmoc-protected amino acid 4 was purified by DCVC prior to use.

Manual Fmoc-based solid phase peptide synthesis (SPPS)
Linear peptides 6A-9A, 8B-9B and 6D-9D were synthesised by standard SPPS using a peptide synthesis manifold. 2-Chlorotrityl chloride resin preloaded with L-Leu (0.67 mmol/g) was used as solid support. For small scale synthesis (0.1 mmol) disposable plastic syringes supplied with pre-cut Teflon filters were used as reactor vessels. For large scale synthesis (2.0 mmol) glass reactors (60 ml volume) with TFA resistant Teflon taps and caps were used. Coupling and deprotection was carried out with shaking. Swelling: Prior to the synthesis the resin was swelled without shaking for minimum 20 min using 3 × bed volume of dichloromethane (DCM).
Amino acid coupling: Single coupling with 3 equivalents of amino acid was used for all amino acids, except all arginine residues that were coupled twice with 3 equivalents of amino acid. Coupling mixture: 1:1:1 amino acid, DIPEA and HBTU (0.488 M in DMF). The coupling mixture was pre-activated for 5 minutes prior to addition. If the volume of the coupling mixture was too small to cover the resin completely additional DMF was added to the reactor.

Kaiser test:
The Kaiser test was performed after every coupling. If the test was positive (blue/purple beads/solution) the coupling was repeated. The Kaiser test solution: Solution A: Phenol in EtOH (4:1); 75 ml/80 g phenol + 20 ml EtOH. Solution B: 0.28 M ninhydrin in EtOH. Solution C: 0.2 mM KCN in pyridine. A small amount of drained resin was taken out with the tip of a Pasteur pipette and added to a test tube. 2 drops of solution A, B and C were added, and the mixture was heated to 110 °C for 2 min. The solution was cooled under running water, and the colour was determined by visual inspection.
Deprotection: Fmoc deprotection was carried out using 20% piperidine in 0.1 M solution of HOBt in DMF (v/v). Prior to deprotection the resins were washed with DCM (3×). 3 × bed volume of the deprotection mixture was added followed by shaking for 5 min. The resin was washed with DMF (3×) and DCM (3×). Additional deprotection mixture was added (3× bed volume) followed by shaking for 20 min. The deprotection mixture was stored at 5 °C, overnight, and freshly prepared every second day.
Capping: To suppress the formation of undesired deletion sequences capping was performed after every arginine residues in the peptide sequences prior to Fmoc-deprotection using DMF, DIPEA, acetic anhydride (8:1:1, v/v/v) with shaking for 30 min. The resin was washed with DMF (3×) and DCM (3×). The capping mixture was freshly prepared, and DIPEA was added to the solution immediately before use.

N-terminal acetylation:
After coupling and Fmoc-deprotection of the final amino acid, the target sequence was N-acetylated. N-acetylation was performed by the same method as capping (described above).

Cleavage and deprotection:
The resins were washed with DCM (3×), dried under vacuum and transferred to a round bottom flask or a Falcon tube. Freshly made cleavage mixture (TFA/TIS/H 2 O, 95:2.5:2.5, v/v/v) was cooled to 0 °C on an ice/water bath and transferred to the resin bound peptide (approx. 1 ml per 0.1 mmol peptide resin or a volume covering the resins). The suspended resins were incubated for ~2.5 hr at room temperature, after which they were removed by filtration using a disposable plastic filter. The resins were washed with TFA and the combined filtrates were concentrated in vacuo using a rotary evaporator or by blowing a N 2 stream on to the solvent surface. The peptide was precipitated by addition of ice cold diethylether. The ether was decanted and the precipitate was washed twice with ice cold ether. The crude peptide was dried in a vacuum desiccator, re-dissolved in CH 3 CN/H 2 O and lyophilized.

Peptide handling and storage:
Short time storage (< 1 week). If the peptide was still attached to the resins, the resin was washed (DCM), dried and stored under vacuum at room temperature. If the peptide was cleaved from the resins, it was lyophilized and stored in a desiccator at room temperature. Long-term storage (> 1 week). If the peptide was still attached to the resins it was washed (DCM), dried under vacuum and stored at -20 °C. If the peptide was cleaved from the resin it was lyophilized and stored at -20 °C.

Microwave assisted Fmoc-based solid phase peptide synthesis (SPPS)
The linear peptides 6B, 6C, 7B, 7C, 8C and 9C were synthesised on chlorotrityl resin preloaded with Fmoc-L-Leu (0.67 mmol/g) using a Biotage Initiator+ SP Wave and the 5 ml reaction vessels supplied with the instrument. A vortex rate of 800 RMP was used unless otherwise stated. Amino acid coupling: All amino acids were coupled twice using 5 equivalents of amino acid. The coupling mixture consisted of 1:1:1 amino acid, DIPEA and HBTU (0.488 M in DMF). The coupling mixture was pre-activated for 5 minutes prior to addition.

Capping:
To suppress the formation of undesired deletion sequences capping was performed after every arginine residues in the peptide sequences prior to Fmoc-deprotection using DMF, DIPEA, acetic anhydride (8:1:1, v/v/v) with shaking for 30 min. The resin was washed with DMF (3×) and DCM (3×). The capping mixture was freshly prepared, and DIPEA was added to the solution immediately before use.

N-terminal acetylation:
After coupling and deprotection of the final amino acid the resins were treated with 8:1:1 (v/v/v) DMF, DIPEA, acetic anhydride for 30 min. at room temperature, vortex rate 800 RMP followed by 4 × 1 min DMF wash at RT and vortex rate 600 RMP.
Cleavage and deprotection: The procedure described above for manual SPPS was followed.
Peptide handling and storage: The procedure described above for manual SPPS was followed.

Membrane-based cAMP accumulation assay
Membrane fractions of adherent human embryonic kidney 293 (HEK293) cell stably overexpressing the β 2 AR 8 were prepared and the peptidomimetics tested as previously described in Martin et al. 9 In brief, HEK293 membranes expressing β 2 AR and peptidomimetics diluted in HBSS buffer supplemented with 20 mM HEPES pH 7.5, 0.1 % BSA and 0.0025 % Tween-20 were preincubated in a Proxiplate-384 plus plate (Perkin Elmer) at room temperature (RT) for 15 min. Then isoproterenol solutions for the concentration response curves or peptidomimetic 10C and Nb80 potency estimation were prepared in ligand buffer (HBSS buffer supplemented with 20 mM HEPES pH 7.5, 0.1 % BSA, 250 µM IBMX, 9 mM MgCl 2 , 100 µM ATP, 10 µM GTP and 0.02 % ascorbic acid), added to the wells and incubated for 30 min at room temperature. To detect cAMP formation the HTRF cAMP dynamic 2 assay kit from CisBio (cat no. 2AM4PEC) was used according to the manufactures protocol. The absolute cAMP levels were determined using a cAMP standard curve and normalised to the maximal cAMP response obtained by full isoproterenol stimulation alone. Significant differences between ISO alone and in presence of the peptides were calculated statistically by use of one-way ANOVA in the GraphPad Prism software.

Conformational bimane fluorescence assay
Baculoviruses containing the β 2 AR-Δ4 construct (full-length β 2 AR with four mutated cysteines (C77V, C327S, C378A and C406A 10,11 ) were generated as previously described by Rosenbaum et al. 12 The modified β 2 AR was expressed in Sf9 insect cells using recombinant baculovirus and purified by M1-anti-FLAG immunoaffinity chromatography. The FLAGpurified receptor was labelled with monobromobimane (mBBr) and purified by alprenolol affinity chromatography as described by Yao et al. 13 with one minor change; the modified β 2 AR was labelled with an excess of 10 µM mBBr (instead of equivalent amounts of β 2 AR and mBBr). Fluorescence spectroscopy was performed as described by Yao et al. 13 with minor changes; a SPEX FluoroMax-3 spectrofluorometer with an excitation and emission bandpass of 2 nm (instead of 4 nm) was used, and 1 µM mBBr-β 2 AR in detergent micelles was used for each scan (instead of 50-100 nM). The mBBr-β 2 AR was incubated at room temperature and in the dark in absence and presence of different ligand and/or peptide concentrations and with a final DMSO concentration of 1% for 30 min. Hereafter, emission scans at wavelengths from 430-530 nm were performed for each sample. Background emission spectra of buffer, ligands and peptides were subtracted from the samples. Data was analysed by use of the GraphPad Prism software and all samples were normalised against the spectrum of receptor alone. Figure S1. Helicity vs. efficacy. (%)-Helicity determined by CD (the most helical peptidomimetic 10C was set to 100%) vs. the observed lowering of the ISO induced efficacy (100% -maximum efficacy, adjusted for the basal level).