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Introduction
Neuromedin U (NMU) is a structurally, highly-conserved
neuropeptide originally purified from the porcine spinal cord
named for its uterine contractile
property[1]. Two molecular forms with identical C-terminal octapeptide amides, NMU-8
and NMU-25, were characterized simultaneously and showed
similar activities[2]. Several NMU analogs identified from
different species display a high degree of conservation in
amino acid sequences[3]. NMU has been implicated in a
variety of physiological processes, including smooth muscle
contraction[2], peripheral blood flow regulation and ion
transport in the gut[4], food intake, energy
homeostasis[5_7], stress
responses[8], and
pronociception[9]. Recently, its
involvement in the pathogenesis of certain cancer and inflammation
was demonstrated[10_13]. The action of NMU is mediated
through 2 G-protein-coupled receptors (GPCR), NMU-R1 and
NMU-R2. The former is expressed predominantly in
peripheral tissues, and the latter is mainly located in the central
nervous system[14_19]. Since specific roles of these 2
receptor subtypes are not fully understood, non-peptidic ligands
could serve not only as potential drug leads, but also as
powerful molecular probes to functionally characterize
NMU receptors.
In the present study, we describe a robust scintillation
proximity assay (SPA) to measure specific NMU-R1-binding
properties of various ligands and its application in
high-throughput screening (HTS) of 36 000 synthetic compounds
or natural products. Three initial hits were discovered, and
one of them (101586) was chosen as the scaffold for
structural modifications and the subsequent structure_activity
relationship (SAR) analyses. Of the 203 analogs synthesized,
16 retained variable human NMU receptor (hNMU-R)
1-binding abilities, but only 2 elicited calcium influx in both
hNMU-R1 and hNMU-R2-expressing cells without observable
antagonist activity.
Materials and methods
Reagents Sodium chloride, sodium phosphate dibasic
anhydrous, potassium dihydrogen phosphate, potassium
chloride, and EDTA were purchased from Shanghai
Chemical Reagents (Shanghai, China). Bovine serum albumin
(BSA) and fetal bovine serum (FBS) were procured from
Sino-American Biotechnology (Shanghai, China) and Hyclone
(Logan, UT, USA), respectively. Aprotinin, leupeptin, and
G418 were bought from Merck KGaA (Darmstadt, Germany).
The human NMU-25 peptide was obtained from Bachem AG
(Bubendorf, Switzerland). [125I]hNMU-25 was made by
ANAWA Trading SA (Wangen, Switzerland).
FlashBlueTM GPCR beads and
IsoplateTM mircotiter plates were the
products of PerkinElmer (Boston, MA, USA). The FLIPR calcium
3 assay kit was supplied by Molecular Devices (Sunnyvale,
CA, USA), probenecid by Sigma_Aldrich (St Louis, MO,
USA), and cell culture medium F12 was from Invitrogen
(Carlsbad, CA, USA).
Cell culture and membrane preparation Chinese
hamster ovary cells (CHO-K1) stably expressing NMU-R1 or
NMU-R2 were provided by Actelion Pharmaceuticals (Allschwil, Switzerland). They were maintained in F12
medium containing 10% FBS in the presence of G418 (400 mg/L)
at 37 °C in a humidified atmosphere of 5%
CO2. For the membrane preparation, the cells were treated with 0.25%
trypsin (Sigma_Aldrich, USA) for 1 min and centrifuged at
1000×g for 10 min. The pellets were resuspended in the
binding assay buffer (phosphate buffered saline with 1
mmol/L EDTA and 0.5% BSA, pH 7.4) and homogenized with
a BioNeb® cell disruption system (Glas-Col, Terre Haute, IN,
USA) followed by centrifugation at 1200×g at 4 °C for 20 min
to precipitate debris. The supernatant was centrifuged
at 17 000×g for 30 min to pellet the membrane that was
resuspended in the binding assay buffer thereafter. The
protein content was determined using a spectrophotometer
(Thermo Electron, Waltham, MA, USA).
SPA-binding assay Various amounts of the above
membrane receptor preparations, 0.04 nmol/L
[125I]hNMU-25, FlashBlueTM GPCR beads (100 µg/well), different
concentrations of non-labeled hNMU-25, aprotinin (5 µg/mL), and
leupeptin (5 µg/mL) were added to the binding assay buffer
to give a final volume of 0.1 mL. The plates were incubated
at room temperature for 3_4 h before counting on the
Microbeta scintillation counter (PerkinElmer, USA).
Calcium mobilization assay The above cells were
detached and plated onto 96-well clear culture plates (Corning,
Acton, MA, USA) at a density of 30 000_40 000 cells (100
µL/well) and incubated overnight. The cells were loaded
with 100 µL of the calcium 3 assay dye supplemented with
2.5 mmol/L probenecid, and incubated at 37°C for 60 min.
A baseline fluorescence signal was measured for the first
17 s, after which the test compounds prepared as stock with
different concentrations (1 mmol/L_3 µmol/L) in Hanks'
balanced salt solution buffer (supplied with the assay kit)
were added to the plate through an automated pipetter (20
µL/well) equipped within FlexStation
II384 (Molecular Devices, USA). The intracellular calcium influx was
analyzed by the same instrument with an excitation wavelength
of 485 nm and emission wavelength of 525 nm; the
relative fluorescence signal was measured at 1.6 s intervals for
150_300 s. hNMU-25 was used as a positive control in
this homogeneous fluorescence emission assay.
HTS campaign The compound library used for the
screening consisted of 20 000 pure synthetic compounds
and 16 000 natural products. A 10-compound pool per well
mix was applied to the primary screening, with an average
concentration of 7 µmol/L for each compound dissolved in
100% DMSO solution. This matrix system maximizes the
advantage of HTS and allows duplicate screening of each
compound[20]. In each 96-well
IsoplateTM, 14 wells were used as positive controls (hNMU-25) and 2 wells as
negative controls (2.5% DMSO alone). Samples showing
greater than 30% inhibition were considered "hits".
Chemistry Reagents were obtained from Lancaster
(Morecambe, England), Aldrich (St Louis, MO, USA), Acros
(Geel, Belgium), and Shanghai Chemical Reagents (China).
All of the reagents were of analytical pure grade or above
and were used without further purification. The analytical
thin-layer chromatography was performed on HSGF 254
silica gel (150_200 µm thickness; Yantai Huiyou, Yantai,
Shandong, China). Column chromatography was performed
using 200_300 mesh silica gels (Qingdao Haiyang Chemical, Qingdao, Shandong, China). Yields were not
optimized. Melting points were recorded on a capillary
tube on a SGW X-4 melting point apparatus without
correction (Shanghai Precision and Scientific Instrument,
Shanghai, China). 1HNMR was recorded in d-chloroform
on a Varian Mercury 300 or 400 (300 or 400 MHz) NMR
spectrometer (Varian, Fort Collins, CO, USA). Chemical
shifts were reported in parts per million (ppm, δ). Proton
coupling patterns were described as singlet (s), doublet (d),
triplet (t), quartet (q), multiplet (m), and broad
(br). Low-resolution mass spectra (LRMS) were measured by the
electric ionization (EI) method with a Finnigan MAT-95
(Finnigan, Santa Clara, CA, USA). The Liquid
chromatography-electrospray ionization mass (ESI) was carried out
on a Thermo Finnigan LCQDECAXP (Thermo Finnigan, San
Jose, CA, USA).
The general preparation procedure for compounds
1a_1d is illustrated in Figure 1.
1,8-Diazabicyclo(5.4.0)undec-7-ene (2.8 g, 18.2 mmol) and
tert-butyldiphenylchlorosilane (2.2 g, 36.3 mmol) were added to 2-aminoethanol (5.0 g, 18.2
mmol) in 50 mL of acetonitrile, and reacted overnight. The
solvent was evaporated in vacuo, and the residues were
extracted with ethyl acetate, washed with water/brine, and dried
over anhydrous Na2SO4. Evaporation of the solvent gave
5.2 g of crude product 6a (yield: 95.5%). It was
subsequently dissolved (100 mg, 0.33 mmol) in 2 mL
acetonitrile followed by the addition of
2-(methylthio)-4,5-dihydro-1H-imidazole hydroiodide (81.5 mg, 0.33 mmol).
The resulting mixture was heated to reflux for 8 h, and
evaporation of the solvent in vacuo delivered a crude product that
was purified by silica gel chromatography to afford 78 mg of
compound 1a (yield: 64%) as a white amorphous solid. The
structure was determined with 1HNMR (400
MHz, CDCl3) δ 8.43 (br, 1H), 7.96 (br, 1H),
7.40_7.64 (m, 10H), 6.7 (br, 1H), 3.88 (t,
J=4.5 Hz, 2H), 3.74 (m, 2H), 3.44 (m, 4H), 1.09 (s, 9H);
and with LRMS (EI, 70 eV): m/z 367
[M+], 310 (100%). Compounds 1b_1d
were prepared accordingly. The structure of compound 1b (88.2 mg; yield: 69.2%) was
determined with 1HNMR (400 MHz,
CDCl3) δ 7.99 (br, 1H), 7.41_7.51 (m, 10H), 4.12 (t,
J=4.1 Hz, 2H), 3.36 (m, 2H), 3.23 (m, 4H), 1.88 (m,
2H), 1.09 (s, 9H); and with LRMS (EI, 70 eV):
m/z 381 [M+], 310 (100%). The structure of compound
1c (79 mg; yield: 65%) was determined with
1HNMR (400 MHz, CDCl3) δ 7.92 (br,
1H), 7.58_7.62 (m, 10H), 7.0 (br, 1H), 3.81 (t,
J=5.5 Hz, 2H), 3.73 (t, J=8.3 Hz, 2H), 3.46 (m, 4H), 1.74 (m, 2H), 1.09 (s, 9H); and
with LRMS (EI, 70 eV): m/z 381
[M+], 310 (100%). The structure of compound
1d (83.2 mg; yield: 65.9%) was determined with
1HNMR (400 MHz, CDCl3) δ 7.78 (br,
1H), 7.42_7.60 (m, 10H), 3.84 (t, J=5.4 Hz, 2H), 3.39 (m, 2H),
3.19 (m, 4H), 1.90 (m, 2H), 1.1 (s, 9H); and with LRMS (EI, 70 eV):
m/z 395 [M+], 310 (100%).
The general preparation procedure for compounds 2a_2d
is illustrated in Figure 2. TsOH (1.0 g, 5.43 mmol) was added to
diphenylmethanol (10.0 g, 54.3 mmol) and 2-bromoethanol
(13.6 g, 108.6 mmol) in 50 mL anhydrous toluene solution,
and the mixture heated to 60oC for 8 h. Following the
addition of 10 mL saturated NaHCO3 aqueous solution, the
toluene layer was separated and washed with water and
saturated NaCl aqueous solution. It was then dried over
Na2SO4 and evaporation of the solvent in vacuo gave 14.9 g of
compound 7a as a colorless oil (yield: 94.3%). Compound
7a (10.0 g, 34.3 mmol) was dissolved in 40 mL
N,N-dimethylformamide with the addition of 17.9 g (0.27 mol)
NaN3 in an ambient temperature for reaction at
70oC for 3 h. The mixture was then quenched into 100 mL water and the aqueous phase
was extracted with 50 mL ether 3 times. Combining the
organic layer, washing with water and saturated NaCl aqueous
solution, drying over Na2SO4, and removing the solvent in
vacuo afforded compound 9a as pale yellow oil. It was
subsequently dissolved (100 mg, 0.44 mmol) in 2 mL acetonitrile
and mixed with
2-(methylthio)-4,5-dihydro-1H-imidazole hydroiodide (107 mg, 0.44 mmol). Heating to reflux for 8 h
and evaporation of the solvent in vacuo delivered a crude
product, which was purified by silica gel chromatography to
give compound 2a (93 mg; yield: 72%) as a white amorphous
solid. The structure was determined with
1HNMR (300 MHz, CDCl3) δ 8.36 (br, 1H), 7.79 (br, 1H), 7.27_7.38 (m, 10H), 6.81
(br, 1H), 5.44 (s, 1H), 3.69 (m, 2H), 3.65 (br, 2H), 3.49 (m, 2H),
3.26 (br, 2H); and with ESI: m/z (relative intensity) 296 (M+1,
100%), 167. Compounds 2b_2d were made the same. The
structure of compound 2b (92 mg; yield: 67%) was
determined with 1HNMR (300 MHz,
CDCl3) δ 7.82 (t, J=6.0 Hz, 1H),
7.63 (br, 1H), 7.27_7.38 (m, 10H), 5.44 (s, 1H), 3.64 (m, 2H),
3.41 (m, 2H), 3.08 (br, 4H), 1.77 (m, 2H); and with ESI:
m/z (relative intensity) 310 (M+1, 100%), 167. The structure of compound
2c (82 mg; yield: 64%) was determined with
1HNMR (300 MHz, CDCl3) δ 7.27_7.35 (m, 10H), 5.40 (s, 1H),
3.60 (m, 2H), 3.56 (br, 2H), 3.42 (m, 2H), 3.20 (br, 2H),
1.92 (m, 2H); and with ESI: m/z (relative intensity) 310 (M+1,
100%), 167. The structure of compound 2d (102 mg; yield:
75%) was determined with 1HNMR (300 MHz,
CDCl3) δ 7.59 (br, 1H), 7.28_7.36 (m, 10H), 5.38 (s, 1H), 3.60 (t,
J=5.6 Hz, 2H), 3.36 (dd, J=6.7, 12.6 Hz,
2H), 2.95 (br, 4H), 1.38 (m, 2H), 1.75 (m, 2H); and with ESI:
m/z (relative intensity) 324 (M+1, 100%), 167.
The general preparation procedure for compounds
3a_3d is illustrated in Figure 3. Tert-butyl
2-aminoethylcarbamate (1.4 g, 8.63 mmol in 10 mL dichloromethane) was added to
2.0 g (8.63 mmol) diphenylcarbamic chloride in 10 mL
CH2Cl2 (cooled to
0oC) in a drop-wise manner.
N,N'-diisopropylethylamine (1.1 g, 8.63 mmol) and 4-dimethylaminopyridine (105
mg, 0.86 mmol) were introduced thereafter. The stirred
reaction was carried out at room temperature overnight followed
by dilution with an extra 30 mL dichloromethane. The
solution was then sequentially washed with water, 1 mol/L HCl
aqueous solution and saturated NaCl aqueous solution, dried
over Na2SO4, and the solvent removed in vacuo. The
resultant product 10a was recrystallized from petroleum
ether/EtOAc as a white solid (2.6 g; yield: 85%). Compound 10a
was dissolved in a mixture of 10 mL trifluoroacetic acid and
10 mL dichloromethane and stirred at room temperature for
0.5 h. It was poured into a 50 mL saturated
Na2CO3 aqueous solution and extracted with 100 mL dichloromethane. The
organic layer was washed with water and the saturated NaCl
aqueous solution, dried over
Na2SO4, and the solvent evaporated in vacuo to give crude product 11a for use without
further purification. Compound 3a (a white amorphous solid)
was similarly prepared as compound 1a (79 mg; yield: 62%).
The structure was determined with 1HNMR (400 MHz,
CDCl3) δ 8.38 (br, 1H), 8.28 (t,
J=6.3Hz, 1H), 7.26_7.45 (m, 10H), 3.69 (s, 4H), 3.42 (m, 2H), 3.31 (m, 2H); and with ESI:
m/z (relative intensity) 324.2 (M+1, 100%), 239, 155. Compounds 3b_3d
were synthesized accordingly. The structure of compound
3b (a white solid, 88 mg; yield: 67%; melting point:
120_124oC) was determined with
1HNMR (300 MHz, CDCl3) δ 7.66
(br, 1H), 7.59 (m, 1H), 7.23_7.42 (m, 10H), 3.56 (m, 2H), 3.30 (m,
2H), 3.21 (m, 4H), 1.84 (m, 2H); and with ESI:
m/z (relative intensity) 338 (M+1, 100%), 239, 169. The structure of
compound 3c (a white amorphous solid, 72 mg; yield: 57%) was
determined with 1HNMR (300 MHz,
CDCl3) δ 8.23 (br, 1H), 7.78 (br, 1H), 7.20_7.40 (m, 10H), 6.99 (m, 1H), 3.46 (m, 4H),
3.37 (m, 2H), 3.27 (m, 2H), 1.78 (m, 2H); and with ESI:
m/z (relative intensity) 338 (M+1, 100%), 253, 169. The structure
of compound 3d (a white solid, 76 mg; yield: 60%; melting
point: 195_198oC) was determined wuth
1HNMR (300 MHz, CDCl3) δ 8.08 (t,
J=6.0 Hz, 1H), 7.56 (dd, J=1.2, 7.8 Hz, 2H),
7.43 (dd, J=1.4, 4.3 Hz, 2H), 7.39 (td,
J=7.7 Hz, 1.4 Hz, 2H), 7.26 (td, J=7.6, 1.3 Hz, 2H), 7.16 (br, 1H), 5.85 (t,
J=5.5 Hz, 1H), 3.64 (s, 4H), 3.41_3.47 (m, 2H), 3.31_3.37 (m, 2H); and with ESI:
m/z (relative intensity) 354.1 (M+1, 100%).
The general preparation procedure for compounds
4a_4c and 5a_5b is illustrated in Figure 4. Trityl chloride (3.6
g, 0.015 mol) was added to a solution of
N-hydroxylphthalimide in 40 mL dry
CH2Cl2 and N,N'-diisopropylethylamine (2.8 mL,
0.015 mol). The completion of the reaction was confirmed by
thin layer chromatography (TLC) after 20 h. Hydrazine
hydrate (85%, 16 mL) was then introduced to the mixture,
followed by the addition of methanol until it became a
homogenous solution. Upon confirmation by TLC after 1 h, the
completed reaction mixture was diluted with
CH2Cl2, washed with
NaHCO3 aqueous solution, and dried over
Na2SO4. The residue was applied to a silica gel column, eluted with
CH2Cl2:MeOH=50:1, and resulted in 2.49 g of compound 12a. It was
dissolved (100 mg, 0.33 mmol) in 2 mL acetonitrile before
adding 2-(methylthio)-4,5-dihydro-1H-imidazole hydroiodide
(85 mg, 0.33 mmol). The mixture was then heated to reflux for
8 h and the solvent evaporated in vacuo to deliver a crude
product; the subsequent purification by silica gel
chromatography afforded 87.3 mg of compound 4a (yield: 71%) as a
pale yellow solid, melting point:
108~110oC. Its structure was confirmed
with 1HNMR (300 MHz, CDCl3)
δ 8.33 (br, 1H), 7.93 (br, 1H), 7.24_7.38 (m, 15H), 6.97 (br, 1H), 3.70 (m,
2H), 3.45 (m, 4H), 3.37 (m, 2H); and with ESI:
m/z (relative intensity) 372.2 (M+1, 100%), 243.1. Compounds
4b_4c were prepared accordingly. The structure of compound
4b (a pale yellow solid, 93 mg; yield: 76%; melting point:
160_163oC); was determined with
1HNMR (300 MHz, CDCl3) δ 8.08 (br, 1H), 7.61
(br, 1H), 7.23_7.37 (m, 15H), 6.59 (br, 1H), 3.56 (m, 2H), 3.45
(m, 2H), 3.25 (m, 4H), 1.86 (m, 2H); and with ESI:
m/z (relative intensity) 386.1 (M+1, 100%), 243.1. The structure of
compound 4c (amorphous yellow solid, 92 mg, 76%) was
determined with 1HNMR (300 MHz,
CDCl3) δ 7.25_7.39 (m, 15H), 3.58 (m, 4H), 3.09 (m, 2H), 2.98 (m, 2H), 1.56 (m, 4H); and with
ESI: m/z (relative intensity) 400.2 (M+1, 100%), 243.1.
Compound 5a was made from 12a (200 mg, 0.66 mmol), which was
dissolved in 1 mL N,N-dimethylformamide prior to the
addition of 1H-pyrazole-1-carboxamidine hydrochloride (0.11 g,
0.73 mmol) and N,N'-diisopropylethylamine (0.12 mL,
0.73 mmol). Ether (5 mL) was added to the reaction while stirring at room
temperature, and the crude product was precipitated as the
oil. The ether layer was then poured out, and the residue
purified by silica gel chromatography afforded 136 mg of
compound 5a (yield: 60%) as a white solid, melting point:
189_193oC. Its structure was determined with
1HNMR (300 MHz, CDCl3) δ 7.23_7.41 (m, 15H), 3.32 (m, 2H), 2.98 (m, 2H);
and with ESI: m/z (relative intensity) 346.1(M+1, 100%),
243.1. Compound 5b was prepared similarly as a white solid (156
mg; yield: 69%), melting point:
148_151oC. The structure was determined with
1HNMR (300 MHz, CDCl3) δ 7.19_7.39
(m, 15H), 3.11 (m, 2H), 2.99 (m, 2H), 1.77 (m, 2H); and with ESI:
m/z (relative intensity) 360.2 (M+1, 100%), 243.1.
Data analysis Data were analyzed using GraphPad Prism
software (GraphPad, San Diego, CA, USA). Non-linear
regression analyses were performed to generate dose-response
curves to calculate IC50 (the molar concentration of an
antagonist, which produces 50% of maximum possible
inhibition of radiolabeled ligand binding for that antagonist in
the receptor-binding assay) or EC50 (the molar concentration
of an agonist, which produces 50% of the maximum possible
response for that agonist) values.
Ki values were calculated from
IC50 using the equation of Cheng and
Prusoff[21] (see below). The time-course study results from the calcium
mobilization assay were expressed as the relative fluorescence
unit (RFU), and all other calcium influx measurements were
represented as the peak RFU per well. Values presented are
mean±SEM of at least 3 independent experiments. Z' factors
were calculated as described by Zhang et
al[22].
Results
Assay validation We first used various concentrations
of the hNMU-R1 membrane extract to assess the optimal
protein concentration for the SPA assay. Both maximum
binding (MB; using DMSO) and non-specific binding (NSB;
using 1 µmol/L hNMU-25) were investigated, and the optimal
hNMU-R1 concentration was determined to be 2
µg/well (1:230 of the stock solution) that resulted in a
signal to background (S/B) ratio of 5 (Figure 5). To confer HTS
requirements, other assay parameters, such as the quantity
of [125I]hNMU-25, amount of beads, and reaction volume were
also optimized (Figure 6).
Assay performance As shown in Figure 7, the average Z'
value for the SPA assay was 0.81 with a S/B ratio of 6,
suggesting that the system was adequately optimized for HTS.
Assay stability was evaluated by incubating the plates
overnight at room temperature and both the Z' factor and S/B
ratio remained unchanged (data not shown). The coefficient
of variation (CV) values were 3.6% for MB and 10.1% for
NSB, respectively. The known hNMU-R1 ligand hNMU-25
was used to verify the methodology, and the binding
affinity (Ki) observed was consistent with that reported in the
literature (Figure 8)[5].
HTS campaign Of the 36 000 samples screened, 100 hits
(0.28%) showing greater than 30% competitive inhibition on
[125I]hNMU-25 binding to hNMU-R1 were discovered (Figure
9). Secondary (single compound per well) screening
confirmed that 3 of the above hits displayed consistent
inhibitory effects with Ki values around 30 µmol/L. All were
synthetic chemicals with similar structures and newly identified
ligands for hNMU-R1. Compound 101586 was chosen as
the scaffold for the structural modifications (Figures 10, 11).
Structural modifications Five groups of analogs to
101586 were designed and synthesized. Compounds 1a_d
were designed based on the SAR information relative to
porcine NMU-8[23_29]. The diphenyl group was introduced
to resemble the hydrophobic residues in this peptide, as
we believe this group may bind to a hydrophobic site on the
hNMU-R, and that an interaction with an anionic site of the
receptor might be realized by linking to a cyclized guanidine
group with changes in the carbon chain length and ring size.
Such an attempt (substitution with a diphenyl group) yielded
compounds 1a_d and one of them (1a) exhibited an improved
binding affinity to hNMU-R1 (Ki=7.30 µmol/L; Table 1,
Figure 11). When the protective group tert-butyldiphenylsilyl
was substituted by diphenylmethyloxy (2a_d) or diphenylurea group (3a_d) to remove potential toxicity, only
2b and 3a showed weak hNMU-R1-binding ability,
suggesting that the extra tributyl in the tert-butyldiphenylsilyl group
is required for receptor binding. Thus, compounds 4a_b
and 5a_b were prepared by adding a phenyl group onto the
diphenylmethyl site in order to enhance their binding
capability. As a result, these 4 analogs displayed significant
improvements in hNMU-R1-binding affinities, with
Ki values ranging between 2.9 and 8.4 µmol/L (Table 1; Figure 11).
We also replaced the guanidine group with
2-(thiophen-2-yl)ethanyl, 2-(pyridin-4-yl)ethanyl,
3-(1H-imidazol-1-yl)propan-1-yl, 2-(2-chlorophenyl)ethanyl, 4-phenylbutan-2-yl,
2-phenylethanyl, or octan-1-yl, 2-cyclohexenylethanyl
groups, but none of the substituted compounds showed
observable bioactivities (data not shown). The above
limited SAR study led us to postulate that the oxygen atom at
the triphenylmethoxyl group seems important to NMU-R1
binding, while the tritylthio or tritylamino groups do not have
this property.
Functional analysis The pharmacological property
(agonist or antagonist) was evaluated with a calcium
mobilization assay using hNMU-R1 and hNMU-R2-expressing
cells, respectively. Figure 12 depicts the kinetics and
concentration-dependent agonist effects of hNMU-25 on
calcium influx in these 2 types of engineered cells. The
EC50 values estimated from the regression curves were 1.45 nmol/L
(hNMU-R1 cells) and 1.63 nmol/L (hNMU-R2 cells), respectively, consistent with those reported in the
literature[5]. Of the 16 analogs to 101586, 10 possessing
Ki values below 50 µmol/L were functionally tested. Among them, only 5a
and 5b demonstrated moderate and non-selective agonist
activities in both hNMU-R1 (Figure 13A) and hNMU-R2 cells
(Figure 13B). None of the analogs showed any antagonistic
effects (data not shown).
Discussion
Due to their large variety of biological activities, studies
on NMU and its cognate receptors have become an
emerging area that attracts scientists from different disciplines.
Apart from the potential value of using NMU-R1 and/or
NMU-R2 as drug targets, exploration of small molecule ligands for
these receptors could provide powerful tools in further
understanding the physiological roles of this peptide family.
Here we report a rational approach for discovering novel
and non-peptidic modulators for NMU receptors,
combining both molecular and cell-based functional assays.
Taking advantage of our prior experience in the
development of SPA-based, receptor-binding HTS
methods[30,31], efforts were made to expand the knowledge to NMU-R. A
number of assay parameters were sequentially evaluated and
optimized to enhance the S/B ratio. It is well recognized from
other membrane receptor screening settings that assay
sensitivity can be improved by choosing a radiolabeled ligand
concentration at or below its Kd to permit effective
competition by an unlabeled ligand[32]. In the present study, therefore,
we selected 0.04 nmol/L as the concentration for
[125I] hNMU-25, which is well below the
Kd value (0.3 nmol/L) of unlabeled
ligands[5]. A stable signal is highly dependent on protein
stability and its coating ability on the beads. In our assay
system, no signal shift was observed for more than 48 h
(data not shown), implying a very stable interaction among
various reagents.
The Z' factor is a useful indicator for assessing the
quality of HTS assays[22]. In general, a Z' value above 0.5
suggests that an assay is robust enough for use in HTS settings.
The SPA system described in this paper consistently
displayed a Z' value of 0.81. This, and in conjunction with other
parameters, such as the S/B ratio and CV values, suggests
that the assay is of high-quality nature. When reduced to
practice, it led to the discovery of 3 confirmed hits that bind
to NMU-R1 in a high micromolar range (~30 µmol/L).
According to the common molecular skeleton
represented by compound 101586 (as a molecular recognition
chiral building block[33]), 203 analogs were synthesized and
tested. Among the 16 analogs that retained variable
hNMU-R1-binding abilities, 2 (5a and 5b) elicited calcium influx in
both hNMU-R1 and hNMU-R2-expressing cells but none
displayed antagonist activity. Our structural modifications
were carried out based on the SAR analysis of peptidic NMU
analogs, especially for NMU-8. It is known that these
structurally highly conserved neuropeptides share a motif of
FLFRPRX-amide, suggesting the importance of this
sequence in the interaction with its cognate receptors.
Specifically, NMU-8 peptides precede with a large flexibility
of the 4 amino acids sequence rich in hydrophobic residues,
namely Tyr1(or
Phe1)_Phe2_Leu3(or
Val3)_Phe4. This sequence is succeeded by a hydrophilic and base C-terminal
Arg5_Pro6_Arg7_Asn
8-NH2 in all species, implying that an
amphiphilic structure could be
designed[23_29]. Diphenyl groups are widely present in many drugs on the market
targeting GPCR, including adrenergic receptor antagonists,
muscarinic receptor antagonists, analgesic agents (opioid
receptor agonists), anti-allergic medications (histamine
receptor-1 antagonists), and serotonin receptor
modulators[34]. The scaffold of these GPCR modulators can be summarized as
the template in Figure 14.
Indeed, when the diphenyl group was introduced to
resemble the hydrophobic residues in NMU-8, compound 1a
showed an improved binding affinity to hNMU-R1; the
addition of a phenyl group onto the TBDPS' diphenylmethyl
site also increased the receptor-binding capability
(compounds 4a_b and 5a_b); the substitution of TBDPS with
diphenylmethyloxy (2a_d) or the diphenylurea group
(3a_d), and replacement of the guanidine group with other
functional groups did not yield satisfactory results. It is thus
clear that the oxygen atom at the triphenylmethoxyl group is
required for NMU-R1 binding that does not involve the
tritylthio or tritylamino groups. This preliminary SAR
investigation further revealed some common structural features
of this class of 101586 analogs necessary for the observed in
vitro activities: the distance between the hydrophobic part
and cyclized guanidine should not exceed 3 carbons in
length, a 5-membered ring is better than a 6-membered ring in
the cyclic guanidines, and the isolated guanidine was
indispensable for hNMU-R activation.
In summary, a simple and homogeneous SPA-based,
HTS-binding assay was developed and validated for the
identification of novel non-peptidic ligands with specificity and
functionality for NMU receptors. The newly-discovered
NMU-R modulators have distinct structural
features compared to that reported
elsewhere[35]. Knowledge obtained from the present study will certainly facilitate the pursuit of
developing small molecule agonists and/or antagonists
directed at hNMU-R with better potency and efficacy.
Acknowledgements
We are indebted to Mr Jie GAO and Ms Xi-yuan CHENG
for their technical assistance, and to Dr Dale MAIS for
critical review of this manuscript.
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