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S1
Selective Homodimerization of Unprotected Peptides Using Hybrid Hydroxydimethylsilane DerivativesCécile Echalier, Aleksandra Kalistratova, Jérémie Ciccione, Aurélien Lebrun, Baptiste Legrand, Emilia Naydenova, Didier Gagne, Jean-Alain Fehrentz, Jacky Marie, Muriel Amblard, Ahmad Mehdi, Jean Martinez, Gilles Subra
Abbreviations ............................................................................................................................4
Material and Method .................................................................................................................4
1,3-bis(3-aminopropyl)tetramethyl disiloxane hydrochloride 1a ...............................................5
1,3-bis(3-aminopropyl)tetramethyl disiloxane trifluoroacetate 1b .............................................8
(3-aminopropyl)dimethylsilanol hydrochloride 2a ...................................................................10
(3-aminopropyl)dimethylsilanol trifluoroacetate 2b .................................................................14
Counter-ion effect on NMR signals.........................................................................................17
DEPT 29Si sequence optimization...........................................................................................18
Stability studies of the siloxane bond in different solutions by 1H-NMR .................................19
Silanol condensation upon lyophilization ................................................................................25
N-ter hybrid monomer 4..........................................................................................................26
N-ter hybrid dimer 5 (JMV 6187) ............................................................................................27
C-ter hybrid monomer 6..........................................................................................................29
C-ter hybrid dimer 7 (JMV 6186) ............................................................................................31
Lys3 hybrid monomer 8 ...........................................................................................................33
Lys3 hybrid dimer 9 (JMV 6185) .............................................................................................35
Lys3(NH(CH2)3Si(CH3)3) monomer 10 (JMV 6246) .................................................................37
Lys3 monomer 11 (JMV 6244) ................................................................................................40
Lys3(Ac) monomer 12 (JMV 6245) .........................................................................................43
Monomeric ligand binding assay ............................................................................................46
Bioactivity of monomeric ligands ............................................................................................47
Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2016
Supporting information
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Figure S1. 1H NMR spectrum of 1a in DMSO-d6 (600 MHz) ....................................................5Figure S2. 13C NMR spectrum of 1a in DMSO-d6 (151 MHz) ..................................................6Figure S3. 29Si NMR spectrum of 1a in DMSO-d6 (119 MHz) ..................................................6Figure S4. 1H NMR spectrum of 1a in D2O (600 MHz).............................................................7Figure S5. 13C NMR spectrum of 1a in D2O (151 MHz) ...........................................................7Figure S6. 29Si NMR spectrum of 1a in D2O (119 MHz)...........................................................8Figure S7. 1H NMR spectrum of 1b in DMSO-d6 (600 MHz)....................................................9Figure S8. 13C NMR spectrum of 1b in DMSO-d6 (151 MHz)...................................................9Figure S9. 29Si NMR spectrum of 1b in DMSO-d6 (119 MHz) ................................................10Figure S10. 1H NMR spectrum of 1a/2a in DMSO-d6 (600 MHz) ...........................................11Figure S11. 13C NMR spectrum of 1a/2a in DMSO-d6 (151 MHz)..........................................11Figure S12. 29Si NMR spectrum of 1a/2a in DMSO-d6 (119 MHz) .........................................12Figure S13. 1H NMR spectrum of 2a in D2O (600 MHz).........................................................13Figure S14. 13C NMR spectrum of 2a in D2O (151 MHz) .......................................................13Figure S15. 29Si NMR spectrum of 2a in D2O (119 MHz).......................................................14Figure S16. 1H NMR spectrum of 1b/2b in DMSO-d6 (600 MHz)...........................................14Figure S17. 13C NMR spectrum of 1b/2b in DMSO-d6 (151 MHz) .........................................15Figure S18.29Si NMR spectrum of 1b/2b in DMSO-d6 (119 MHz) .........................................15Figure S19. 1H NMR spectrum of 1b/2b in D2O (600 MHz)...................................................16Figure S20. 13C NMR spectrum of 1b/2b in D2O (151 MHz) ..................................................16Figure S21. 29Si NMR spectrum of 1b/2b in D2O (119 MHz) .................................................16Figure S22. From hydrochloride to trifluoroacetate salts. Top: 1H NMR spectrum of 1a/2a mixture in D2O. Bottom: 1H NMR spectrum of 1b/2b mixture in D2O......................................17Figure S23. 1H flip angle optimization. ...................................................................................18Figure S24. 1H NMR spectra of 1a in DMSO-d6 ....................................................................19Figure S25.1H NMR spectra of 1a at pH 4.............................................................................20Figure S26. 1a hydrolysis at pH 4 based on 1H NMR kinetic studies ....................................20Figure S27.1H NMR spectra of 1a at pH 7.............................................................................21Figure S28. 1a hydrolysis at pH 7 based on 1H NMR kinetic studies ....................................21Figure S29.1H NMR spectra of 1a at pH 7.4..........................................................................22Figure S30. 1a hydrolysis at pH 7.4 based on 1H NMR kinetic studies .................................22Figure S31. 1H NMR spectra of 1a at pH 10. Signals from the CH2 group in gamma position to silicon..................................................................................................................................23Figure S32. 1a hydrolysis at pH 10 based on 1H NMR kinetic studies ..................................24Figure S33. 1H NMR spectrum of 1a in D2O 0.1% TFA at t = 0.. ...........................................24Figure S34. From monomer to dimer upon lyophilization. .....................................................25Figure S35. LC/MS spectrum of 4..........................................................................................26Figure S36. 1H NMR spectrum of 5 in DMSO-d6 (400 MHz)..................................................27Figure S37. 13C NMR spectrum of 5 in DMSO-d6 (101 MHz).................................................28Figure S38. HR-MS analysis of 5...........................................................................................29Figure S39. LC/MS spectrum of 6..........................................................................................30Figure S40. 1H NMR spectrum of 7 in DMSO-d6 (400 MHz) ..................................................31Figure S41. 13C NMR spectrum of 7 in DMSO-d6 (101 MHz) .................................................32Figure S42. HR-MS analysis of 7...........................................................................................33Figure S43. LC/MS spectrum of 8..........................................................................................34Figure S44. 1H NMR spectrum of 9 in DMSO-d6 (400 MHz)..................................................35Figure S45. 13C NMR spectrum of 9 in DMSO-d6 (101 MHz).................................................36Figure S46. HR-MS analysis of 9...........................................................................................37
Supporting information
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Figure S47. LC/MS spectrum of 10........................................................................................38Figure S48. HR-MS analysis of 10.........................................................................................39Figure S49. LC-MS spectrum of 11 .......................................................................................41Figure S50. HR-MS analysis of 11.........................................................................................42Figure S51. LC-MS spectrum of 12 .......................................................................................44Figure S52. HR-MS spectrum of 12.......................................................................................45Figure S53. Binding of compounds 10, 11 and 12.................................................................46Figure S54. Signaling property of compounds 10, 11 and 12................................................47
Supporting information
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Abbreviations
ACN, acetonitrile; Alloc, allyloxycarbonyl; Boc, t-butyloxycarbonyl; CM, ChemMatrix; DCM, dichloromethane; DEPT, distortionless enhancement by polarization transfer; DIEA, diisopropylethylamine; DMF, N-N’-dimethylformamide; DMSO, dimethylsylfoxide; DPBS, Dulbelcco’s phosphate buffered saline; ESI-MS, electrospray ionization mass spectrometry; Fmoc, fluorenylmethoxycarbonyl; GHRP-6, growth hormone releasing hexapeptide; HBTU, N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; HFIP, hexafluoroisopropanol; HTRF, homogenous time resolved fluorescence; ICPDMCS, 3-isocyanatopropyldimethylchlorosilane; IP1, inositol monophosphate; LC/MS, tandem liquid chromatography/ mass spectrometry; MW, micro-wave; NMP, N-methyl-2-pyrrolidone; NMR, nuclear magnetic resonance; pip, piperidine; PS, polystyrene; RP-HPLC, reversed phase high performance liquid chromatography; RT, room temperature; SPPS, solid phase peptide synthesis; TFA, trifluoroacetic acid; THF, tetrahydrofurane; TIS, triisopropylsilane; Trt, trityl; UV, ultra-violet. Other abbreviations used were those recommended by the IUPAC-IUB Commission (Eur. J. Biochem. 1984, 138, 9-37).
Material and Method
All solvents and reagents were used as supplied. Solvents used for LC/MS were of HPLC grade. Dichloromethane (DCM); N,N-dimethylformamide (DMF) were obtained from Carlo Erba. Fmoc amino acid derivatives, [benzotriazol-1-yloxy(dimethylamino)methylidene]- dimethylazanium hexafluorophosphate (HBTU), Fmoc-Rink amide CM resin, 2-chloro chlorotrityl PS resin, were purchased from Iris Biotech (Marktredwitz, Germany). Diisopropylcarbodiimide (DIC), diisopropylethylamine (DIEA), trifluoroacetic acid (TFA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Hexafluoroisopropanol (HFIP) and triisopropylsilane (TIS) were obtained from Alfa Aeser and Acros respectively. NMR solvents were obtained from Euriso-top.
Samples for LC/MS analyses were prepared in acetonitrile/water (50:50, v/v) mixture, containing 0.1% TFA. The LC/MS system consisted of a Waters Alliance 2695 HPLC, coupled to a Water Micromass ZQ spectrometer (electrospray ionization mode, ESI+). All the analyses were carried out using a Phenomenex Onyx, 25 x 4.6 mm reversed-phase column. A flow rate of 3 mL/min and a gradient of (0-100)% B over 2.5 min were used. Eluent A: water/0.1% HCO2H; eluent B: acetonitrile/0.1% HCO2H. UV detection was performed at 214 nm. Electrospray mass spectra were acquired at a solvent flow rate of 200 µL/min. Nitrogen was used for both the nebulizing and drying gas. The data were obtained in a scan mode ranging from 100 to 1000 m/z or 250 to 1500 m/z to in 0.7 sec intervals.
High Resolution Mass Spectrometric analyses were performed with a Synapt G2-S (Waters) mass spectrometer fitted with an Electrospray Ionisation source. All measurements were performed in the positive ion mode. Capillary voltage: 1000 V; cone voltage: 30 V; source temperature: 120°C; desolvation temperature: 250°C. The data were obtained in a scan mode ranging from 100 to 1500 m/z.
Supporting information
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1,3-bis(3-aminopropyl)tetramethyl disiloxane hydrochloride 1a
SiO
NH3
Si
NH3Cl Cl
Commercially available 1,3-bis(3-aminopropyl)tetramethyl disiloxane (500 µL, 1.8 mmol) was poured into 1 M hydrogen chloride ether solution (7.2 mL, 7.2 mmol, 4 eq) cooled in an ice bath. 1,3-bis(3-aminopropyl)tetramethyl disiloxane hydrochloride precipitated immediately. Diethyl ether was removed under reduced pressure to yield compound 1a as a white powder (100%).
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Figure S1. 1H NMR spectrum of 1a in DMSO-d6 (600 MHz)
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Supporting information
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Figure S2. 13C NMR spectrum of 1a in DMSO-d6 (151 MHz)
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Figure S3. 29Si NMR spectrum of 1a in DMSO-d6 (119 MHz)
Supporting information
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Figure S4. 1H NMR spectrum of 1a in D2O (600 MHz)
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Figure S5. 13C NMR spectrum of 1a in D2O (151 MHz)
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Figure S6. 29Si NMR spectrum of 1a in D2O (119 MHz)
1,3-bis(3-aminopropyl)tetramethyl disiloxane trifluoroacetate 1b
SiO
NH3
Si
NH3
CF3COOCF3COO
Trifluoroacetic acid (220 µL, 2.89 mmol, 4 eq) was added on 1,3-bis(3-aminopropyl)tetramethyl disiloxane (200 µL, 0.722 mmol) in diethyl ether (2 mL) on an ice bath. Diethyl ether and TFA excess were removed under reduced pressure to yield compound 1b as a colorless oil (100%).
Supporting information
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Figure S7. 1H NMR spectrum of 1b in DMSO-d6 (600 MHz)
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Figure S8. 13C NMR spectrum of 1b in DMSO-d6 (151 MHz)
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Figure S9. 29Si NMR spectrum of 1b in DMSO-d6 (119 MHz)
(3-aminopropyl)dimethylsilanol hydrochloride 2a
OHSi
NH3Cl
On the one hand, DCl 35% in D2O (6.00 µL) were added to 1a (20 mg) in DMSO-d6 (600 µL) to trigger 1a hydrolysis into 2a. The 1a/2a mixture in DMSO-d6 was analyzed by NMR.
Supporting information
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Figure S10. 1H NMR spectrum of 1a/2a in DMSO-d6 (600 MHz)
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Figure S11. 13C NMR spectrum of 1a/2a in DMSO-d6 (151 MHz)
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Supporting information
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Figure S12. 29Si NMR spectrum of 1a/2a in DMSO-d6 (119 MHz)
On the other hand, 1,3-bis(3-aminopropyl)tetramethyl disiloxane hydrochloride 1a (20 mg) was solubilized in D2O (600 µL) and the solution was left aside for 8 weeks to allow 1a hydrolysis into 2a. 2a solution in D2O was analyzed by NMR. Traces of 1a are still visible on NMR spectra.
Supporting information
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Figure S13. 1H NMR spectrum of 2a in D2O (600 MHz)
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Figure S14. 13C NMR spectrum of 2a in D2O (151 MHz)
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Supporting information
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Figure S15. 29Si NMR spectrum of 2a in D2O (119 MHz)
(3-aminopropyl)dimethylsilanol trifluoroacetate 2b
OHSi
NH3CF3COO
On the one hand, 3-bis(3-aminopropyl)tetramethyl disiloxane trifluoroacetate 1b (20 mg) was dissolved in DMSO-d6 (600 µL) and the solution was left aside for 5 weeks to allow 1b hydrolysis into 2b. Then the mixture 1b/2b in DMSO-d6 was analyzed by NMR.
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Figure S16. 1H NMR spectrum of 1b/2b in DMSO-d6 (600 MHz)
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Supporting information
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Figure S17. 13C NMR spectrum of 1b/2b in DMSO-d6 (151 MHz)
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Figure S18.29Si NMR spectrum of 1b/2b in DMSO-d6 (119 MHz)
On the other hand, 3-bis(3-aminopropyl)tetramethyl disiloxane trifluoroacetate 1b (20 mg) was dissolved in D2O (600 µL). 1b hydrolysis into 2b occurred. The mixture 1b/2b in D2O was analyzed by NMR.
Supporting information
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Figure S20. 13C NMR spectrum of 1b/2b in D2O (151 MHz)
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Figure S21. 29Si NMR spectrum of 1b/2b in D2O (119 MHz)
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Supporting information
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Counter-ion effect on NMR signals
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Figure S22. From hydrochloride to trifluoroacetate salts. Top: 1H NMR spectrum of 1a/2a mixture in D2O. Bottom: 1H NMR spectrum of 1b/2b mixture in D2O.
Supporting information
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DEPT 29Si sequence optimization
In order to decrease the experiment time, we used a DEPT 29Si sequence, with a relaxation delay of 2 seconds, to take advantage of the polarization transfer from hydrogens of methylene in the alpha position to the silicon atom. An optimization was made on this sequence, where the last 1H flip angle was moved from 17° to 45° (Figure S23). The results showed a factor of 2 gain in favor of the angle of 24° compared to the standard DEPT of 45°.
[ppm]
Figure S23. 1H flip angle optimization. 1H flip angle was moved from 17° to 45°: blue 45°, green 35°, purple 24°, red 21°, orange 19.5°, black 17°
Supporting information
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Stability studies of the siloxane bond in different solutions by 1H-NMR
t = 0 min corresponds to the dissolution of 1,3-bis(3-aminopropyl)tetramethyl disiloxane hydrochloride 1a (20 mg) in DMSO-d6 or in D2O-containing buffer that yields a solution at the desired pH. pH were adjusted in advance with Dulbecco’s phosphate buffered saline, HCl or NaOH aqueous solutions on blank samples. Dimer percent was reported as a function of time based on the integration of 1H signals from the -CH2Si group of monomer and dimer.
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Figure S24. 1H NMR spectra of 1a in DMSO-d6
Supporting information
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Figure S25.1H NMR spectra of 1a at pH 4
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Supporting information
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Figure S27.1H NMR spectra of 1a at pH 7
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Figure S28. 1a hydrolysis at pH 7 based on 1H NMR kinetic studies
Supporting information
S22
[ppm]
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Figure S29.1H NMR spectra of 1a at pH 7.4
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Figure S30. 1a hydrolysis at pH 7.4 based on 1H NMR kinetic studies
Supporting information
S23
We focused our attention on 1H signals from -CH2Si and CH3Si because they are the farthest signals from the peptide sequence in hybrid peptide displaying the dimethylpropylsiloxane dimerization arm. As a consequence, very few differences in chemical shifts and signal shapes are expected when changing the peptide sequence. Thus, NMR studies on the 1,3-bis(3-aminopropyl)tetramethyl disiloxane model can be generalized to hybrid peptides. In addition, these signals appear in a chemical shift area completely different from peptide signals which makes them easily identifiable and integrable. However, the methylene group in gamma position to the silicon is also a proble for the determination of monomer/dimer ratio (figure S31)
0 min
10 min
20 min
30 min
40 min
60 min
80 min
100 min
190 min
420 min
[ppm]
Figure S31. 1H NMR spectra of 1a at pH 10. Signals from the CH2 group in gamma position to silicon
Supporting information
S24
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5 6 7 8
dim
er (%
)
time (h)
Figure S32. 1a hydrolysis at pH 10 based on 1H NMR kinetic studies
0.10.50.91.31.72.12.52.93.3f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000CE-H2O+TFACE-H2O+TFA (7)t 0
6.00
2.03
2.03
1.99
Figure S33. 1H NMR spectrum of 1a in D2O 0.1% TFA at t = 0. Hydrolysis occurred immediately. Signals correspond to monomer 2a.
Supporting information
S25
Silanol condensation upon lyophilization
0.000.100.200.300.400.500.600.700.800.901.001.10f1 (ppm)
1
2
CE-H2O+TFACE-H2O+TFA (7)
CE-15CE-15
-CH2Si dimer
-CH2Si monomer CH3Si
traces of dimer
Figure S34. From monomer to dimer upon lyophilization. Top : 1H NMR spectrum of 2a in D2O/0.1% TFA before lyophilization; bottom : 1H NMR spectrum of the same sample after freeze-drying
Supporting information
S26
N-ter hybrid monomer 4
HN
O
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2SiHO H
NCO
HN
O
Figure S35. LC/MS spectrum of 4
Supporting information
S27
N-ter hybrid dimer 5 (JMV 6187)
HN
O
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2
HN
O
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2
Si
O
HN
Si
HN
OHN
O
O
HN
O
0.01.02.03.04.05.06.07.08.09.010.011.0f1 (ppm)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
4000
4200KAT2-121KAT2-121 / dmso
Figure S36. 1H NMR spectrum of 5 in DMSO-d6 (400 MHz)
-0.4-0.3-0.2-0.10.00.10.20.30.40.50.60.70.80.9f1 (ppm)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500KAT2-121KAT2-121 / dmso
0.19
5.82
2.00
Supporting information
S28
0102030405060708090100110120130140150160170180f1 (ppm)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000KAT2-121KAT2-121 / dmso
Figure S37. 13C NMR spectrum of 5 in DMSO-d6 (101 MHz)
Supporting information
S29
Figure S38. HR-MS analysis of 5
C-ter hybrid monomer 6
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
HN NH Si
OHHN
CO
Supporting information
S30
Figure S39. LC/MS spectrum of 6
Supporting information
S31
C-ter hybrid dimer 7 (JMV 6186)
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
HN
NH
SiO
HNC
O
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
HN
NH
Si
HN
O
0.51.52.53.54.55.56.57.58.59.510.5f1 (ppm)
01002003004005006007008009001000110012001300140015001600170018001900200021002200CE259
CE259 / dmso
Figure S40. 1H NMR spectrum of 7 in DMSO-d6 (400 MHz)
-0.15-0.10-0.050.000.050.100.150.200.250.300.350.400.450.500.550.60f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400CE259CE259 / dmso
0.64
5.36
2.00
Supporting information
S32
0102030405060708090100110120130140150160170f1 (ppm)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000CE259CE259 / dmso
Figure S41. 13C NMR spectrum of 7 in DMSO-d6 (101 MHz)
Supporting information
S33
Figure S42. HR-MS analysis of 7
Lys3 hybrid monomer 8
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2
HN
Si
OH
HNC
O
Supporting information
S34
Figure S43. LC/MS spectrum of 8
Supporting information
S35
Lys3 hybrid dimer 9 (JMV 6185)
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2
HNHN
CO
H2NO
HNN
HN
O
NH
NH O
HN
O
NH
NH O
HN
O
NH2
NH2
HN NH
CO Si
SiO
0.01.02.03.04.05.06.07.08.09.010.011.0f1 (ppm)
0
500
1000
1500
2000
2500
3000
3500
4000
4500CE263CE263 /dmso
Figure S44. 1H NMR spectrum of 9 in DMSO-d6 (400 MHz)
-0.30-0.20-0.100.000.100.200.300.400.500.600.70f1 (ppm)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400CE263CE263 /dmso
0.63
5.35
2.00
Supporting information
S36
0102030405060708090100110120130140150160170180f1 (ppm)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000CE263CE263 /dmso
Figure S45. 13C NMR spectrum of 9 in DMSO-d6 (101 MHz)
Supporting information
S37
Figure S46. HR-MS analysis of 9
Lys3(NH(CH2)3Si(CH3)3) monomer 10 (JMV 6246)
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2
HN
Si
HNC
O
Supporting information
S38
Figure S47. LC/MS spectrum of 10
Supporting information
S39
Figure S48. HR-MS analysis of 10
Supporting information
S40
Lys3 monomer 11 (JMV 6244)
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2
NH2
Supporting information
S41
Figure S49. LC-MS spectrum of 11
Supporting information
S42
Figure S50. HR-MS analysis of 11
Supporting information
S43
Lys3(Ac) monomer 12 (JMV 6245)
H2NO
HNN
NH O
NH
HN
O
NH O
NH
HN
O
NH O
NH2
NH2
HNC
O
Supporting information
S44
Figure S51. LC-MS spectrum of 12
Supporting information
S45
Figure S52. HR-MS spectrum of 12
Supporting information
S46
Monomeric ligand binding assay
Figure S53. Binding of compounds 10, 11 and 12, monomeric analogues of dimer 9, to GHS-R1a. Ligands affinities were determined by HTRF-based competition binding assay performed on intact HEK293T cells as described in Materials and Methods. Results are from one representative experiment of two, each performed in triplicate.
Supporting information
S47
Bioactivity of monomeric ligands
Figure S54. Signaling property of compounds 10, 11 and 12. Efficacy of ligands to stimulate IP1 production was measured on HEK293T cells expressing the GHS-R1a as described in Materials and Methods. Results are one representative experiment of two, each performed in triplicate.
