PEGylation of Neuromedin U yields a promising candidate for the treatment of obesity and diabetes

Article abstract of DOI:10.1016/j.bmc.2012.06.003

Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glucose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a metabolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight poly(ethylene) glycol (PEG) ('PEGylation'). PEG size, site of attachment, and conjugation chemistry were optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU displays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1. In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment of obesity and diabetes.

Full text of DOI:10.1016/j.bmc.2012.06.003

Bioorganic & Medicinal Chemistry 20 (2012) 4751–4759
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
PEGylation of Neuromedin U yields a promising candidate for the treatment
of obesity and diabetes
Paolo Ingallinella a, , Andrea M. Peier b, Alessandro Pocai b, Annalise Di Marco a,à, Kunal Desai b,
Karolina Zytko a, Ying Qian b, Xiaobing Du b, Antonella Cellucci a,à, Edith Monteagudo a,à, Ralph Laufer a,à
,
Elisabetta Bianchi a,à, Donald J. Marsh b, ,§, Antonello Pessi a, ,–
⇑
⇑
a IRBM P. Angeletti, 00040 Pomezia (RM), Italy
b Department of Metabolic Disorders, Merck Research Laboratories, Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Neuromedin U (NMU) is an endogenous peptide, whose role in the regulation of feeding and energy
homeostasis is well documented. Two NMU receptors have been identified: NMUR1, expressed primarily
in the periphery, and NMUR2, expressed predominantly in the brain. We recently demonstrated that
acute peripheral administration of NMU exerts potent but acute anorectic activity and can improve glu-
cose homeostasis, with both actions mediated by NMUR1. Here, we describe the development of a met-
abolically stable analog of NMU, based on derivatization of the native peptide with high molecular weight
poly(ethylene) glycol (PEG) (‘PEGylation’). PEG size, site of attachment, and conjugation chemistry were
optimized, to yield an analog which displays robust and long-lasting anorectic activity and significant
glucose-lowering activity in vivo. Studies in NMU receptor-deficient mice showed that PEG-NMU dis-
plays an expanded pharmacological profile, with the ability to engage NMUR2 in addition to NMUR1.
In light of these data, PEGylated derivatives of NMU represent promising candidates for the treatment
of obesity and diabetes.
Received 2 April 2012
Revised 31 May 2012
Accepted 1 June 2012
Available online 9 June 2012
Keywords:
Neuromedin U
Diabetes
Obesity
PEG
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
peripherally and centrally,9,12–14 where it binds to two high affinity
G-protein coupled receptors (GPCRs), NMUR1 and NMUR2, with
With the increasing global prevalence of obesity, the interest is
growing in pharmacological therapies to reduce appetite and in-
crease energy expenditure, and gut peptides which regulate energy
homeostasis represent attractive leads.1–3 Neuromedin U (NMU) is
a highly conserved peptide that is implicated in a number of phys-
iological processes including nociception, stress, inflammation,
blood pressure, feeding, energy homeostasis, and glycemic con-
trol.4,5 Originally isolated from porcine spinal cord based upon its
ability to contract rat uterine smooth muscle6, NMU has since been
found in several species.7–11 It is distributed in the body both
complementary tissue distribution:4,15–21 NMUR1 is predomi-
nantly expressed in the peripheral tissues, particularly the gastro-
intestinal tract, pancreas, uterus and testes,18,19 whereas NMUR2 is
mainly expressed in the central nervous system (CNS) with the
highest levels in the hypothalamus, hippocampus, spinal cord
and paraventricular nucleus.14,15,17
Although the pharmacology of NMU is not yet fully elucidated, a
growing body of evidence highlights its role in the regulation of
energy homeostasis. NMU-deficient mice develop obesity
characterized by hyperphagia, reduced energy expenditure and
hyperglycemia,22 while NMU-overexpression in mice results in a
lean phenotype with improved glucose homeostasis.23 Moreover,
an association has been recently found between NMU gene vari-
ants and excess body weight in humans.24 Central administration
of the peptide reduces food intake and body weight in rodents
and birds,17,25–28 and increases locomotor activity and core body
temperature in rodents.25
⇑
Corresponding authors. Addresses: Department of Safety Assessment and
Laboratory Animal Resources, Merck Research Laboratories, West Point, PA
19486, USA. Tel.: +1 (215) 652 6822; fax: (295) 652 4944 (D.J.M.); PeptiPharma
SRL, Via Dei Castelli Romani 22, 00040 Pomezia (RM), Italy. Tel.: +39 6 91329735;
fax: +39 6 97849266 (A.P.).
E-mail addresses: donald_marsh@merck.com (D.J. Marsh), a.pessi@peptiphar-
ma.it (A. Pessi).
The effects of centrally administered NMU are primarily
mediated by NMUR2.29 However, chronic intracerebroventricular
infusion of NMU in NMUR2-deficient mice still showed a significant
(albeit small) reduction in body weight, an effect which we hypoth-
esized to depend on agonism at peripheral NMUR1 receptors.
DiaSorin Research Center, 21040 Gerenzano (VA), Italy.
IRBM Science Park, 00040 Pomezia (RM), Italy.
Department of Safety Assessment and Laboratory Animal Resources, Merck
à
§
Research Laboratories, West Point, PA 19486, USA.
–
PeptiPharma, 00040 Pomezia (RM), Italy.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.06.003

4752
P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
Accordingly, we showed that both acute and chronic peripheral
administration of NMU resulted in a significant reduction in
cumulative food intake and body weight, and that this effect is
NMUR1-mediated.30 Importantly, the anorectic effect of NMU did
not exhibit tachyphylaxis, as observed for other peptides such as
CCK or Amylin;31,32 rather, the sustained activity observed upon
chronic infusion was similar to other peptides which are being
considered for therapeutic development, like GLP-1 and oxynto-
modulin.33 Finally, independent of its weight loss effects, peripher-
ally administered NMU markedly improved glucose tolerance in
diet-induced obese (DIO) mice.30 The underlying mechanism of
how NMU contributes to the regulation of body weight, feeding
and glucose homeostasis is presently unclear.
Collectively, these findings suggest the possibility of developing
NMU for the treatment of obesity and diabetes. To this end, engi-
neering of the native peptide is required to overcome the poor
pharmacokinetic properties typical of peptide hormones: the
half-life of NMU after subcutaneous (sc) injection is less than
5 min.30
peripheral administration, obtained through conjugation of
poly(ethylene) glycol (PEG) to the native peptide.
2. Materials and methods
A list of all the NMU Analogs discussed in this manuscript is gi-
ven in Table 1, and their analytical data are reported in Table 2.
2.1. Peptide synthesis and purification
Solid-phase peptide synthesis via Fmoc chemistry was per-
formed on ABI433 (Applied Biosystems) or Symphony (Protein
Technologies Inc.) synthesizers. The resin was aminomethylated
polystirene resin (100–200 mesh, 0.41 mmol/g) (Novabiochem)
derivatized with the modified Rink linker p-[(R,S)-a-[9H-Fluoren-
9-yl-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic
acid.34,35 Amino acids were used as 0.5 M solutions in DMF, con-
taining equimolar HOBt (Hydroxybenzotriazole). Activation was
with equimolar HBTU (2-(1H-benzotriazole-1-yl)-1,1,3,3-tetram-
ethyluronium hexafluorophosphate) and a 2-fold molar excess of
DIEA (N,N-diisopropylethylamine) in DMF or NMP. The acylation
Here, we describe the development of a metabolically stable
analog of human NMU with potent and long-lasting efficacy upon
Table 1
In vitro activity at the human NMUR1/R2 receptors of NMU and its PEGylated analogues
b
Peptide
Sequencea
EC50 (nM)
hNMUR1
hNMUR2
1
2
3
4
5
6
7
8
hNMU
Ac-NMU(19-25)
PEG40-NMU
C(PEG40)-NMU
Ac-NMU
C(NEM)-NMU
NMU-Cys4(PEG40)
NMU-Cys8(PEG40)
NMU-Cys12(PEG40)
NMU-Cys16(PEG40)
C(PEG40)-NMU(7-25)
C(PEG40)-NMU(12-25)
C(PEG40)-NMU(19-25)
C(PEG20)-NMU
C(PEG5)-NMU
FRVDEEFQSPFASQSRGYFLFRPRN
Ac-FLFRPRN
2.3 1.1
1.5 0.8
125 16
36 16
1.4 0.7
2.6 1.2
147 21
109 35
PEG40-FRVDEEFQSPFASQSRGYFLFRPRN
Ac-C(PEG40)-FRVDEEFQSPFASQSRGYFLFRPRN
Ac-FRVDEEFQSPFASQSRGYFLFRPRN
Ac-C(NEM)-FRVDEEFQSPFASQSRGYFLFRPRN
Ac-FRVC1EEFQSPFASQSRGYFLFRPRN
Ac-FRVDEEFC1SPFASQSRGYFLFRPRN
Ac-FRVDEEFQSPFC1SQSRGYFLFRPRN
Ac-FRVDEEFQSPFASQSC1GYFLFRPRN
Ac-C1FQSPFASQSRGYFLFRPRN
Ac-C1ASQSRGYFLFRPRN
Ac-C1-Ttds-FLFRPRN
Ac-C(PEG20)-FRVDEEFQSPFASQSRGYFLFRPRN
Ac-C(PEG5)-FRVDEEFQSPFASQSRGYFLFRPRN
4.8 1.4
2
1.2
8.9
2
5.2 0.8
228 75
722 322
659 203
>1000
224 62
>1000
>1000
97 45
212 137
308 162
980 163
227 48
434 126
>1000
9
10
11
12
13
14
15
20
8
37 18
12 2.3
5.8 1.1
a
Ac, acetyl; NEM, N-ethyl-maleimide; PEG40, branched PEG, size 40 kDa; C1 = Cys(PEG40); Ttds = 1-amino-4,7,10-trioxa-13-tridecamine succinimic acid; C(PEG20) =
Cys(PEG20), C(PEG5) = Cys(PEG5), where PEG20 and PEG5 = linear PEG, size 20 and 5 kDa, respectively; all peptides are amidated at the C-terminus.
b
FLIPR assay.
Table 2
Analytical data for the NMU analogs of Table 1
Peptide
Gradienta
Columnb
RTc (min)
Purityd
MW found (Da)
MW calc (D)
1
3
4
5
6
7
8
9
10
11
12
13
14
15
20–40(200)-80(30), 1 mL/min, RT
20–20(10)-60(50)-80(10), 0.4 mL/min, 50 °C
25–50(200)-80(30), 1 mL/min, RT
20–40(200)-80(30), 1 mL/min, RT
15–35(200)-80(30), 1 mL/min, RT
30–60(60)-80(10), 0.6 mL/min, 50 °C
30–60(60)-80(10), 0.6 mL/min, 50 °C
20–40(50)-80(10), 0.6 mL/min, 50 °C
30–60(60)-80(10), 0.6 mL/min, 50 °C
20–60 (50)-80(10), 0.6 mL/min, 50 °C
20–60(50)-80(10), 0.6 mL/min, 50 °C
20–60(50)-80(10), 0.6 mL/min, 50 °C
25–50(200)-80(30), 1 mL/min, RT
30–60(200)-80(30), 1 mL/min, RT
A
B
C
C
C
D
D
D
D
B
B
B
C
C
15.32
4.68
15.83
15.85
14.29
4.12
3.94
3.76
3.81
4.68
>95%
>97%
>97%
>95%
>95%
>97%
>97%
>97%
>95%
>97%
>97%
>95%
>97%
>95%
3081.00
3080.44
46282.73e
46095.57e
3121.00
45893.61e
46226.63e
3120.50
3350.00
3350.75
45769.32e
46318.52e
45726.86e
45776.31e
45747.21e
45167.71e
44910.43e
24606.95e
8951.30e
45710.62e
45307.60e
45798.56e
45796.34e
45674.38e
45069.28e
44619.12e
24525.60e
8925.98e
4.67
4.71
15.97
10.02
a
% of eluent B (time) flow rate, temperature.
A = Phenomenex Jupiter C18 150 ꢀ 4.6 mm 5
b
l
m 300 Å, B = UPLC waters acquity BEH C18 100 ꢀ 2.1 mm 1.7
lm 300 Å, C = Reprosil-Pur C4 150 ꢀ 4.6 mm 5 lm 300 Å,
D = UPLC waters acquity BEH C4 100 ꢀ 2.1 mm 1.7
l
m 300 Å.
c
RT, retention time.
By HPLC.
Average MW.
d
e

P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
4753
reactions were performed for 60 min with 4-fold excess of acti-
vated amino acid over the resin free amino groups. N-terminal
acetylation was performed with 10-fold excess of acetic anhydride
in DMF. The following side chain protecting groups were used:
tert-butyl (OtBu) for Asp, Glu, Ser and Tyr; trityl (Trt) for Asn, Cys
and Gln; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl
(Pbf) for Arg. Fmoc-Ttds (13-amino-4,7,10-trioxa-tridecayl-succi-
namic acid) was purchased from Polypeptide Laboratories (Stras-
bourg, FR). Cleavage was with 88% trifluoroacetic acid (TFA), 5%
phenol, 2% triisopropylsilane and 5% water36 for 1.5 h at room tem-
perature. After filtration, the peptide was precipitated with cold
methyl-t-butyl ether, centrifuged, washed twice with fresh cold
methyl-t-butyl ether, dried, resuspended in 20% acetonitrile/water,
and lyophilized.
carried out on TSK-HW50 (Tosoh) column (21 ꢀ 700 mm) in 0.1%
(w/v) acetic acid, 30% acetonitrile, flow rate 1 mL/min. RP-HPLC
was performed on a semi-preparative Waters RCM Delta-Pak™
C4 cartridge (40 ꢀ 100 mm, 15
lm), using as eluents (A) 0.2% acetic
acid in water and (B) 0.2% acetic acid in acetonitrile and the follow-
ing gradient: 5%–5% B (5 min)–50% B (10 min) ꢁ80% B (2 min), flow
rate 80 mL/min.
The purified PEGylated analogs were characterized by amino
acid analysis (AAA), RP-HPLC and MALDI-Tof Mass Spectrometry.
Purity by HPLC was P98%. The concentration of the PEGylated
peptide solutions used for in vitro and in vivo studies was deter-
mined by quantitative AAA.
2.4. In vitro functional assay (FLIPR)
The crude peptides were purified by reversed-phase HPLC on
Waters RCM Delta-Pak™ C4 cartridges (40 ꢀ 200 mm, 15
l
m)
The NMU receptors signal primarily through Gaq/11 proteins;
using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetoni-
trile. The following gradient of eluent B was used: 20%–20% (5 min)
and 20%–35% (20 min), flow rate 80 mL/min. Analytical HPLC was
performed on an Alliance Waters Chromatograph, using Phenome-
therefore FLIPR, a calcium mobilization assay, was used to measure
functional activity using cell lines stably expressing the human or
mouse NMU receptors. Stable cell lines expressing NMUR1 or
NMUR2 were plated at a density of 12,000 cells/well overnight
on poly-lysine coated 384-well black-walled plates. The following
day, the media was removed from the plates and the cells loaded
with Fluo-3 (Molecular Probes), a calcium sensitive dye, diluted
in FLIPR buffer (1X Hank’s buffered saline containing 20 mM
HEPES, 0.1% BSA, 2.5 mM probenecid (Sigma) and 1.6 mM TR40).
All reagents were from Invitrogen unless otherwise noted. Peptide
solutions were resuspended in saline at a stock concentration of
2 mM, and diluted on the day of the experiment in FLIPR buffer
nex Jupiter C4 (150 ꢀ 4.6 mm,
5 lm, rt) or ACE C4 300 Å
m, t = 45 °C) columns with the following gradi-
(150 ꢀ 4.6 mm, 3
l
ent: 20%–40% B (20 min)-80% (3 min), flow rate 1 mL/min. Alterna-
tively, analytical characterization was performed on a Waters
0
Acquity UPLC, using a BEH 130 ÅA C18 column (2.1 ꢀ 100 mm,
1.7
l
m), with the following gradient: 20%–40% B (4 min) ꢁ80%
(1 min), flow rate 0.4 mL/min, 45 °C. The structure of the purified
(P97%) peptides was confirmed by electrospray mass spectrome-
try on a Micromass LCZ platform.
to a 4 lM working stock solution. After 90 min incubation at rt, cell
plates were loaded onto a FLIPR (Molecular Devices) to monitor
cellular fluorescence (excitation = 488 nM; emission = 540 nM) be-
fore and after peptide addition. Eight to twelve point dose re-
sponses were tested on NMUR-expressing cell lines using FLIPR
2.2. Alkylation with N-ethylmaleimide (NEM)
The control peptide 6 (Table 1) with a blocked thiol group was
prepared by alkylation with 1.5 molar excess of NEM in 0.2 M so-
dium phosphate, 8 M urea, 4 mM EDTA, pH 6.5, for 1 h. The peptide
was purified and characterized as described above.
with 1 lM peptide as the highest dose. The response after peptide
addition was taken as the maximum fluorescence units minus the
fluorescence immediately prior to stimulation for each well. EC50
values were calculated using GraphPad Prism (San Diego, CA) soft-
ware. All the experiments were run at least in duplicate. Note that
the reported endogenous start codon for mouse NMUR1 is a CTG
codon17 but due to low expression/activity in engineered cell lines
we used a mouse NMUR1 cDNA clone that utilized the traditional
ATG start codon to ensure for better receptor expression and activ-
ity in our functional assays.
2.3. PEGylation
For PEGylation at the N-terminal amino group of peptide 3, the
peptide precursor 1 (10 mg, 3.2 lmoles) was dissolved at 6.7 mg/
mL concentration in 0.2 M HEPES, pH 7.3., and reacted with 3-fold
molar excess (413 mg) of 40 kDa methoxy-PEG-N-hydroxy-
succinimide (SUNBRIGHT GL2-400GS2, NOF Corp.), dissolved in
water at 100 mg/mL. The conjugation reaction was monitored by
HPLC, and was complete after 14 h. The solution was acidified with
1% formic acid, and the PEGylated peptide was purified by sequen-
tial cation-exchange chromatography (IXC) and size-exclusion
chromatography (SEC).
For PEGylation through formation of a thioether bond, the Cys-
containing peptide precursors were reacted with branched 40 kDa
methoxy-PEG-maleimide (SUNBRIGHT GL2-400MA, NOF Corp.)
(peptides 4 and 7–13), linear 20 kDa methoxy-PEG-maleimide
(SUNBRIGHT ME-200MA, NOF Corp.) (peptide 14), or 5 kDa meth-
oxy-PEG-maleimide (SUNBRIGHT ME-050MA, NOF Corp.) (peptide
15). In a typical preparation, the peptide precursor was dissolved at
10 mg/mL concentration in 0.15 M sodium phosphate, 8 M urea,
4 mM EDTA, pH 6.5, and reacted with a 1.1-fold molar excess of
PEG reagent dissolved in water at 100 mg/mL. The reaction was
monitored by HPLC or UPLC, and was typically complete after
30 min. The solution was acidified with 1% formic acid, and the
PEGylated peptide was purified by sequential IXC and RP-HPLC.
IXC was carried out on MacroCap SP (GE Healthcare) column
(26 ꢀ 120 mm) with a linear 0–0.6 M gradient of NaCl in 3.5 col-
umn volumes in 0.05% formic acid; the flow rate was 1 mL/min
for column loading and 6 mL/min for gradient elution. SEC was
2.5. Pharmacokinetics
PEGylated NMU was administered intravenously or subcutane-
ously to C57BL/6 mice and plasma samples were obtained at differ-
ent times. Plasma concentrations of biologically active peptide
were determined by bioassay, measuring NMU-induced formation
of inositol-1-phosphate (IP1) in HEK293 cells expressing the hu-
man NMUR1 receptor. IP1 was quantified using a homogeneous
time-resolved fluorescence (HTRF) assay (IPOne, CISbio Interna-
tional) according to the manufacturer’s instructions. Briefly, cells
were plated 24 h before the experiment into 96-well poly-L-Lysine
coated-plates (Becton Dickinson) at a density of 90,000 cells/well
and kept in DMEM medium containing 10% fetal bovine serum,
50 I.U penicillin, 50
lg/ml streptomycin and 2 mM glutamine. On
the day of the assay, the medium was removed and 70
ll of stim-
ulation buffer containing diluted plasma samples or peptide stan-
dards was added. Following incubation at 37 °C for 1 h, the
incubation medium was removed and detection molecules (IP1-
d2 conjugate and anti-IP1 cryptate) were added. An aliquot of this
mixture was transferred to 96-well half-area white plates (Greiner
Bio-one) which were then incubated for 1 h at room temperature.
The HTRF signal was read using a Safire2™ reader (Tecan). Results

4754
P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
were calculated from the HTRF ratio given as [emission (665 nm)/
emission (620 nm)]. A three-point titration was done with the test
plasma samples and a 12-point titration of the peptide was run in
naïve plasma to serve as standard. Peptide concentrations in plas-
ma were extrapolated from the standard curve using non-linear
regression analysis using XLfit 4.2 (IDBS). Pharmacokinetic param-
eters were calculated by non-compartmental analysis using
WinNonlin (Pharsight).
efficacy of drugs and numerous examples exist of its application
to peptides.37–42 The highly hydrated shell of PEG increases the
hydrodynamic radius of the conjugate thus decreasing the rate of
renal clearance, and simultaneously restricts accessibility to prote-
olytic enzymes: this combined effect prolongs the circulation time
of the conjugate.43 By the same token however, PEG conjugation is
typically associated with a reduction of biological potency at the
receptor, caused by steric hindrance of the polymer.44
The key to successful application of PEGylation therefore re-
sides in striking the right balance between the improved pharma-
cokinetics and the reduced receptor potency of the conjugated
peptide.44 We set out to do so for NMU through exploration of a
number of variables like PEG size, PEG structure, PEG site of attach-
ment, and PEGylation chemistry.
2.6. Food intake studies
Animal studies were performed as previously described.30
Briefly, male mice were individually housed under a 12-h light/
12-h dark cycle. For knockout and wild type studies, NMU recep-
tor-deficient animals were weight-matched to littermate controls.
All protocols for use of these animals were approved by the Merck
Research Laboratories Institutional Animal Care and Use Commit-
tee (Rahway, NJ). Animals were ad libitum fed D12492, a high fat
diet composed of 60% Kcal from fat (Research Diets). Male diet-
induced obese (DIO) mice were weighed and dosed subcutane-
ously (sc) 30 min prior to the onset of the dark phase of the light
cycle, and provided with a pre-weighed aliquot of food. Mice and
food were weighed 18 h (day 1), 42 h (day 2), 66 h (day 3), and
90 h (day 4) after the onset of the initial dark phase. In the chronic
studies, food intake and body weight were measured daily. Whole
body composition analysis of conscious live mice was conducted
3.1. PEGylation of NMU. Optimal site of attachment
Human NMU consists of 25 amino acids (Table 1). While the N-
terminal region of the peptide differs in length and/or amino acid
composition between species, the C-terminal region displays
remarkable sequence conservation: the C-terminal pentapeptide
is identical across all species, and the C-terminal heptapeptide
NMU19–25 is conserved in mammals.4 This conserved segment
has been identified as the minimal region able to recapitulate the
in vitro potency of the full length peptide on both NMUR1 and
NMUR24–6,17,45,46 (Table 1). Based on this and in the absence of
any structural information about the peptide active conformation,
we first explored the N-terminus as the site for PEG attachment.
We selected a branched PEG of 40 kDa (PEG40), since this size is
the most efficacious to reduce kidney filtration of PEGylated pep-
tides.38,47,48 Two conjugation chemistries were explored: (i) reac-
tion of the peptide with branched PEG40 N-hydroxysuccinimide,
taking advantage of the absence of any amine functionality in the
peptide other than the N-terminal amine (PEG40-NMU, 3), and
(ii) introduction of an extra-Cys residue at the N-terminus and
using
a
Quantitative Magnetic Resonance (QMR) method
(EchoMR™, Echo Medical Systems). The Minispec (Bruker-Optics)
was used as NMR hardware having an applied static magnetic field
corresponding to radio frequency of 7.5 MHz.
2.7. Glucose tolerance test studies
PEGylated NMU at 0.01, 0.1 or 1 mg/kg was dosed sc in 5% man-
nitol buffer 18 h prior to an oral glucose tolerance test (OGTT, 20%
dextrose challenge, 10 mL/Kg, 2 g/kg) performed in fasted mice at
10:00 A.M. Mice (n = 5/group) were distributed by body weight
into treatment groups. Basal blood glucose concentrations at
T = ꢁ0.5 h and T = 0 h were determined by glucometer from tail
nick blood. Mice were then immediately challenged with dextrose.
Blood glucose levels were determined from tail bleeds taken at 20,
40, 60 and 120 min after dextrose challenge. The blood glucose
excursion profile from T = 0 to T = 120 min was used to integrate
an area under the curve (AUC) for wild-type mice treated with
PEG-NMU.
chemoselective
reaction
with
maleimido-activated
PEG
(C(PEG40)-NMU, 4). Both PEGylated derivatives were full agonists
of the human NMUR1/NMUR2 receptors, with comparable func-
tional EC50 potencies (Table 1). However, depending on the recep-
tor subtype, these values were 15–100 fold higher than those of
native NMU (1) and of the non-PEGylated peptide controls, namely
the N-terminal acetylated NMU (Ac-NMU, 5) and the acetylated
peptide with the cysteine residue alkylated with N-ethyl malei-
mide (C(NEM)-NMU, 6) (Table 1). This decrease in receptor
potency was expected, and is in line with what is typically
observed for other PEGylated peptides and proteins.
2.8. NMU receptor-deficient mice
We then explored PEGylation at internal positions of the
sequence. Four PEGylated peptides were prepared, in which the
PEGylation site was scanned every four residues along the se-
quence up to the C-terminal region17–25, which was unaltered. In
these analogues, PEGylation was achieved by mutating the amino
acid at positions 4, 8, 12 and 16 into cysteine, to enable reaction
with maleimide-activated PEG. The activity of internally PEGylated
analogues 7–10 at the human NMUR1/R2 receptors is shown in
Table 1. The peptides show progressively diminished potency as
the site of PEGylation shifts from the N-terminus towards the C-
terminus, with EC50 values at the human receptors in the following
order: PEG40-NMU (3) < NMU-Cys4(PEG40) (7) < NMU-Cy-
s8(PEG40) (8) < NMU-Cys12(PEG40) (9) < NMU-Cys16(PEG40) (10).
Furthermore, we prepared three PEGylated analogues with
progressive shortening of the NMU sequence starting from the N-
terminus, that is NMU(7–25), NMU(12–25), and NMU (19–25)
(11–13, Table 1). PEGylation of these shortened analogues was
achieved by introduction of an extra-Cys residue at the N-terminus
and reaction with maleimido-activated PEG, as was done with
analogue 4. In the shortest analogue, corresponding to the minimal
NMUR1-deficient (Nmur1ꢁ/ꢁ) and NMUR2-deficient (Nmur2ꢁ/ꢁ
)
&
mice were obtained as previously described.29,30 NMUR1-
NMUR2-deficient mice (Nmur1&2ꢁ/ꢁ) mice were generated by
crossing N6 Nmur1ꢁ/ꢁ mice to N7 Nmur2ꢁ/ꢁ mice. Nmur1&2ꢁ/ꢁ
mice were fertile, and exhibited no gross histological or patholog-
ical abnormalities.
2.9. Statistical analysis
All values are mean SEM. The data were analyzed using a two-
tailed unpaired Student’s t test; p values 60.05 were regarded as
significant and are denoted with an asterisk.
3. Results and discussion
To produce a metabolically stable, long-lasting analog of NMU
we explored conjugation to poly(ethylene) glycol (PEGylation).
PEGylation is a well-established strategy to increase the clinical

P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
4755
bioactive region of NMU, a spacer (Ttds) was introduced between
the N-terminal Cys and the peptide to minimize the shielding ef-
fect of the PEG.
The shortened PEGylated analogs showed a similar trend to the
internally PEGylated series, with the largest loss of potency ob-
served for the shortest analog C(PEG40)-NMU(19–25) (13).
marginal efficacy on day 1, in line with the dramatic decrease of
receptor potency (Table 3).
Because of the large difference in molecular weight due to the
different PEG sizes, the two analogs C(PEG20)-NMU and C(PEG5)-
NMU were tested at doses equimolar with 10 mg/kg of PEG40-
NMU, that is at 5.2 and 2 mg/kg, respectively. C(PEG20)-NMU14
,
was considerably more efficacious than PEG40-NMU on day 1
(99% versus 51% FI reduction), equipotent on day 2, but the effect
was lost on day 3. This likely results from the balance of higher
receptor agonism [C(PEG20)-NMU is >10-fold more potent on
mNMUR1 and twofold more potent on mNMUR2] and poorer
pharmacokinetic profile, both due to the smaller PEG size.
C(PEG5)-NMU (15) exhibited good acute efficacy (79% food intake
reduction) but no significant effect was detected beyond day 1.
C(PEG5)-NMU has EC50 values close to native NMU, but conjuga-
tion with 5 kDa PEG does not seem to significantly improve the
pharmacokinetic profile of the peptide.
Taken together, these data indicated that derivatization of NMU
at the N-terminus with a 40 kDa PEG represents the most appropri-
ate solution to achieve metabolic stability and prolonged duration
of action. For further characterization, the PEG40-NMU derivative
(3) was selected over the similarly active C(PEG40)-NMU (4) and
C(PEG40)-NMU(7-25) (11), because of the recently reported poten-
tial of cys-maleimide derivatives for slow release of the drug upon
exposure to plasma proteins.49
3.2. PEGylation of NMU. Optimal PEG size
Having established the N-terminus as the optimal placement
for PEG, we then explored N-terminal conjugation with different
size PEGs. Although 40 kDa PEG is generally needed to endow on
a peptide a hydrodynamic radius beyond the threshold for glomer-
ular filtration, examples are reported in which the optimal PEG size
for in vivo efficacy is smaller.37,42 Therefore, we prepared NMU
derivatives with 20 kDa and 5 kDa PEG (PEG20 and PEG5), via addi-
tion of an N-terminal Cys and reaction with maleimide-PEG (14
and 15, Table 1). As expected, the smaller the PEG size, the lower
the steric hindrance, and the lower the EC50: C(PEG5)-NMU (15)
was only 2.5-8-fold less potent than the unconjugated peptide at
hNMUR1/R2, while C(PEG20)-NMU (14), showed intermediate
potency between C(PEG5)-NMU and C(PEG40)-NMU.
3.3. In vivo efficacy of PEGylated NMU analogs
While comparison of the in vitro potency of Peptides 7–13 with
Peptides 3 and 4 is sufficient to select the latter because they all
feature PEG of the same size and are expected to have similar
pharmacokinetic properties, the situation is different for Peptides
14–15, which must be compared with Peptides 3–4 in vivo to
determine which peptide has the best overall properties.
Table 3 shows the results of a comparative acute food intake (FI)
study in diet-induced obese (DIO) mice. In the study, we also in-
cluded native NMU (Peptide 1) and the sequentially shortened ana-
logs 11–13. In addition to the FI data, the table shows the EC50 of
the peptides at the mouse NMUR1/R2 receptors, which is generally
similar to the potency observed at the human receptors.
As previously reported (30), treatment of DIO mice subcutane-
ously (sc) with a single 10 mg/kg dose of the native peptide was
highly efficacious, but the effect on feeding peaked at 2 h and
was short-lived; a 45% reduction in FI was measured at day 1, with
no effect at day 2 and 3. By contrast, PEG40-NMU (3) and
C(PEG40)-NMU (4) induced a prolonged reduction of FI, which
was still apparent on the third day. The 7-amino acid deleted ana-
log C(PEG40)-NMU(7–25) (11) showed comparable results, while
further deletion of 5 amino acids in C(PEG40)-NMU (12–25), (12)
reduced the duration of efficacy in vivo to two days. The shortest
PEGylated analog, C(PEG40)-NMU (19–25), (13) showed only
3.4. Pharmacokinetics of PEG40-NMU
The pharmacokinetics of PEG40-NMU (3) was evaluated in lean
C57BL/6 mice upon subcutaneous and intravenous administration,
at 10 and 1 mg/kg, respectively (Fig. 1). Plasma peptide levels were
assessed with a functional bioassay that measures the functional
response of the active peptide on human NMUR1-expressing cell
lines.
Unlike wild type NMU (1) that has a half-life of < 5 minutes in
mice (30), PEG40-NMU (3) showed an excellent pharmacokinetic
profile, with 47% s.c. bioavailability, a half-life of 25 h, and a clear-
ance of 0.03 ml/min/kg.
3.5. Efficacy of PEG40-NMU upon chronic dosing
The pharmacokinetics (Fig. 1) and the acute efficacy studies
(Table 3) indicated that PEG40-NMU (3) is suitable for administra-
tion every other day (Q2D). We then evaluated the effect of PEG40-
NMU upon repeated administration. Ad libitum fed male DIO
C57BL/6 mice were dosed sc with 1, 3 and 10 mg/kg of Peptide 3
Q2D for 10 days (Fig. 2). Daily FI in the treatment groups was
profoundly reduced in a dose-dependent manner for the first
Table 3
In vitro potency and in vivo activity of NMU analogues in diet-induced obese (DIO) mice
Sequencea
EC50 (nM)
mR1
% Food intake reductionc
b
Peptide
mR2
18h
42h
66h
1
3
4
11
12
13
14
15
hNMU
PEG40-NMU
FRVDEEFQSPFASQSRGYFLFRPRN
PEG40-FRVDEEFQSPFASQSRGYFLFRPRN
Ac-C(PEG40)-FRVDEEFQSPFASQSRGYFLFRPRN
Ac-C1FQSPFASQSRGYFLFRPRN
Ac-C1ASQSRGYFLFRPRN
Ac-C1-Ttds-FLFRPRN
0.8 0.3
65 13
110 11
134 13
99 18
>1000
0.5 0.1
35 15
45
51
57
67
71
11
99
79
—
—
57
62
65
40
—
22
23
23
—
—
—
C(PEG40)-NMU
C(PEG40)-NMU(7-25)
C(PEG40)-NMU(12-25)
C(PEG40)-NMU(19-25)
C(PEG20)-NMU
C(PEG5)-NMU
16
9
185 23
363 134
>1000
65 22
nd
Ac-C(PEG20)-FRVDEEFQSPFASQSRGYFLFRPR
Ac-C(PEG5)-FRVDEEFQSPFASQSRGYFLFRPRN
8.6
nd
4
65
—
—
a
b
c
Abbreviations as in Table 1.
FLIPR assay; nd, not done.
Food intake reductions in diet-induced obese (DIO) mice administered a single subcutaneous administration at 10 mg/kg of peptide. Peptides 14 and 15 were tested at 5.2
and 2 mpk, respectively, which is a dose equimolar to 10 mg/kg for the 40 kDa-PEGylated peptides. NMU was dosed at 3250 nmol/kg, whilst the PEGylated peptides were
dosed at ꢂ220 nmol/kg.

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P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
10000
1000
100
10
5 days of the study; by day 6 FI returned to levels comparable to
vehicle treated animals (Fig. 2A). We observed the same initial
reduction and subsequent restoration of food intake upon continu-
ous dosing of native NMU in DIO mice (30), and similar effects have
been reported for other anorexigenic peptides.37,50,51 However, un-
like the effect on FI, BW loss was sustained throughout the course
of the study (Fig. 2B). The cumulative reduction in FI after 10 days
of treatment was 5.8% and 20.3%, respectively, for the 3 and 10 mg/
kg doses (Fig. 2C), while the cumulative reduction in BW was 2.4%
and 7.6%, respectively (Fig. 2D). Weight loss was primarily due to a
decrease in fat mass (vehicle: 0.6 g; 1 mg/kg: 0.8 g; 3 mg/kg:
ꢁ1.4 g; 10 mpk: ꢁ2.1 g). No significant effect on either parameter
was seen for the 1 mg/kg dose. No changes in locomotor activity
were observed with the PEGylated peptide (data not shown). The
sustained reduction in body weight could be partly due to differ-
ences in energy expenditiure, since native NMU has been shown
to have acute effects on metabolic rate.30 Further studies are
warranted to better understand these findings. Encouragingly, we
did not observe any adverse event during the treatment.
1
0
10
20
30
40
50
60
70
80
Time (h)
Figure 1. Pharmacokinetics of PEGylated NMU in mouse. Peptide 3 (PEG40-NMU)
was administered intravenously (h) or subcutaneously (j) to C57BL/6 mice,
respectively at 1 mg/kg and 10 mg/kg in water, and the plasma concentrations
monitored with time. The plasma concentration of peptide were determined by
bioassay as described in the experimental procedures. The curves represent the
mean of three animals.
A
B
3
2
1
0
Vehicle
PEG40-NMU 3 mg/kg
PEG40-NMU 10 mg/kg
30
20
10
0
PEG40-NMU 1 mg/kg
-1
-2
-3
-4
-5
-6
0
1
2
3
4
5
6
7
8
9
10
Vehicle
PEG40-NMU 3 mg/kg
PEG40-NMU 10 mg/kg
-1
0
1
2
3
4
5
6
7
8
9
10
PEG40-NMU 1 mg/kg
C
D
200
175
150
125
100
75
3
2
1
0
*
Vehicle
PEG40-NMU, 1 mg/kg
PEG40-NMU, 3 mg/kg
PEG40-NMU, 10 mg/kg
-1
-2
-3
-4
-5
-6
50
25
0
*
Figure 2. Ad libitum fed male DIO C57BL/6 mice were treated with either vehicle or peptide 3 (PEG40-NMU) at the indicated doses every other day for 10 days. (A) Daily food
intake; (B) cumulative daily change in body weight; (C) cumulative food intake change at the end of the experiment; (D) cumulative body weight change at the end of the
experiment. The asterisk indicates statistical significance (P <0.05) relative to vehicle-treated animals.
A
B
24
20
16
12
8
3
2
1
*
0
WT, Vehicle
WT, PEG-NMU
NMUR1&2-/-, Vehicle
*
-1
-2
*
NMUR1&2-/-, PEG-NMU
*
*
4
-3
-4
0
Day 1 Day 2 Day 3 Day 4
Day 1 Day 2 Day 3 Day 4
Figure 3. Nmur1&2ꢁ/ꢁ, and wild type (WT) mice received a single sc dose of either vehicle or 10 mg/kg PEG40-NMU (3). (A) Daily food intake and (B) daily body weight
change. The asterisk indicates statistical significance (P <0.05) relative to vehicle-treated animals.

P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
4757
A
B
24
20
16
12
8
Vehicle
3
3 (PEG40-NMU), 10 mg/kg
2
1
*
*
0
*
-1
-2
4
*
0
*
WT
NMUR1-/- NMUR2-/-
-3
*
WT
NMUR1-/- NMUR2-/-
Day 1
Day 1
C
D
3
24
20
16
12
8
2
1
0
*
-1
-2
-3
*
4
0
*
*
NMUR1-/- NMUR2-/-
Day 2
WT
NMUR1-/- NMUR2-/-
WT
Day 2
E
F
24
20
16
12
8
3
2
1
*
0
-1
-2
-3
4
0
NMUR1-/- NMUR2-/-
Day 3
WT
NMUR1-/- NMUR2-/-
Day 3
WT
G
H
3
2
24
20
16
12
8
*
1
*
0
-1
-2
-3
4
0
NMUR1-/-
Day 4
NMU2-/-
WT
NMUR1-/- NMUR2-/-
Day 4
WT
Figure 4. Nmur1ꢁ/ꢁ, Nmur2ꢁ/ꢁ, and wild type (WT) mice received a single sc dose of either vehicle or 10 mg/kg PEG40-NMU (3). The panels show the daily food intake and
body weight change measured after dosing at day 1 (A and B), day 2 (C and D), day 3 (E and F) and day 4 (G and H). The asterisk indicates a significant reduction (P <0.05)
relative to vehicle-treated animals.
3.6. Efficacy of PEG40-NMU in NMU receptor-deficient mice
post dose for FI and two days post dose for BW), no significant ef-
fect was observed in the NMUR1/2-deficent animals (Fig. 3). This
demonstrated that, as for the native peptide hormone, the anorec-
tic effect of PEGylated NMU is mediated entirely by interaction
with the NMU receptors.
We then performed studies in single receptor-deficient mice
(Fig. 4). At 10 mg/kg, PEG40-NMU (3) was active in both Nmur1ꢁ/ꢁ
and Nmur2ꢁ/ꢁ animals, but exhibited different efficacy: In
Nmur1ꢁ/ꢁ mice, the effect on FI and BW lasted for 2 days post dose
We had previously studied the anorectic action of native NMU
using NMUR1-deficient (Nmur1ꢁ/ꢁ
)
and NMUR2-deficient
(Nmur2ꢁ/ꢁ) mice.29 We have now produced mice lacking both
receptors, NMUR1/2-deficent mice (Nmur1&2ꢁ/ꢁ). A comparative
study of PEG40-NMU (3) at 10 mg/kg in wild-type (WT) and
NMUR1/2-deficent DIO mice showed that, while in WT mice the
PEGylated peptide had the usual duration of action (three days

4758
P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
600
500
400
300
200
100
0
A
Vehicle
B 18000
PEG-40 NMU, 0.01 mg/kg
PEG-40 NMU, 0.1 mg/kg
PEG-40 NMU, 1 mg/kg
Vehicle
15000
PEG40-NMU, 0.01mpk
12000
PEG40-NMU, 0.1mpk
PEG40-NMU, 1mpk
9000
6000
3000
0
*
*
*
0
15
30
60
120
Time (min)
C
NMUR2-/-, Vehicle
D
500
NMUR1 -/-, Vehicle
500
NMUR2-/-, PEG40-NMU
NMUR1-/-, PEG40-NMU
400
300
200
100
0
400
300
200
100
0
*
*
*
0
15
30
60
120
0
15
30
60
120
Time (min)
Time (Min)
Figure 5. (A). Glucose lowering efficacy of PEG40-NMU (3) in an oral glucose tolerance test (OGTT) in overnight fasted WT DIO mice. (B) Effect of PEG40-NMU on inhibition of
glucose AUC in the OGTT. (C) PEG40-NMU (10 mg/kg sc) suppresses glucose excursion in Nmur1ꢁ/ꢁmice. (D) PEG40-NMU (10 mg/kg sc) did not alter glucose excursion in
Nmur2ꢁ/ꢁ mice. ⁄P <0.05 versus vehicle.
(ꢁ41.8% FI and -2.2% BW at day 1, ꢁ42.6% FI and ꢁ2.1% BW at day
2); in Nmur2ꢁ/ꢁ mice, the peptide was efficacious only on day 1
(ꢁ17.3% in FI and ꢁ1.2% in BW). In the control WT mice, the PEGy-
lated peptide exerted anorectic effects as usual.
Significant reductions were observed at 15, 30 and 60 min post
glucose challenge. Similar results were observed with an intraperi-
toneal GTT (IPGTT) (data not shown). To assess which NMU recep-
tors were contributing to the reductions in glucose, we performed
OGTTs in Nmur1ꢁ/ꢁ and Nmur2ꢁ/ꢁ mice (Fig. 5C–D). Administration
of 10 mg/kg PEG40-NMU significantly reduced glucose excursion
in Nmur1ꢁ/ꢁ mice. In contrast, we found that 10 mg/kg PEG40-
NMU did not significantly alter glucose excursion in Nmur2ꢁ/ꢁ
mice, indicating that the improvement in glucose homeostasis
was mediated by NMUR2 and not by NMUR1. These findings are
in contrast to what we observed with native NMU (GTT efficacy
was mediated by NMUR1) and as we observed for the BW/FI stud-
ies, peripheral administration of the PEGylated peptide exhibits an
altered pharmacological profile. Additionally, the dose titration
study further shows that the MinED for glucose is significantly
lower than what is required for body weight.
This finding highlights that by peripheral administration, native
NMU and PEGylated NMU exert anorectic effects by (at least par-
tially) distinct mechanisms of action. While the anorectic activity
of peripherally administered native NMU is exclusively mediated
by NMUR130, PEG40-NMU exerts its effect by both NMUR1 and
NMUR2 receptors. Indeed, comparison of the efficacy of PEG40-
NMU in Nmur1ꢁ/ꢁ, Nmur2ꢁ/ꢁ, and WT mice seems to indicate that
the three-day efficacy in the latter results from additive agonism
on the two receptor subtypes. We hypothesize that the different
ability of peripherally administered PEG40-NMU and native NMU
to access regions of the brain, where NMUR2 is predominantly ex-
pressed, is due to the fast metabolism of the latter peptide. The
anorectic activity of PEGylated peptides on receptors expressed
in the brain is not unprecedented37,42, and in the case of NMU
PEGylation uncovers the potential for central action of the periph-
erally administered peptide.
4. Conclusions
In conclusion, we developed a metabolically stable analog of
human Neuromedin U, based on conjugation of the native peptide
to poly(ethylene) glycol (PEG). PEG-NMU displays robust and
long-lasting body-weight and glucose lowering activity in vivo,
and an altered pharmacological profile engaging both central
and peripheral receptors. PEG-NMU analogs represent promising
candidates for the treatment of obesity and diabetes. Future work
towards the development of an NMU-based therapeutic will be di-
rected at better understanding the mechanism of how NMU con-
tributes to energy homeostasis. Moreover, given the other
described roles for NMU including the elicitation of the stress re-
ponse (3), it will be important to understand whether any un-
wanted adverse effects become apparent upon chronic treatment
with the peptide.
3.7. PEG40-NMU improves glucose tolerance
We had previously demonstrated that peripherally adminis-
tered NMU improved glucose tolerance during an OGTT29 and
wished to determine if the PEGylated peptide retained this activity.
Initial experiments using 10 mg/kg PEG-40 NMU showed a signif-
icant improvement in glucose excursion during an oral (OGTT) and
intra-peritoneal glucose tolerance test (IPGTT) (data not shown).
To determine the minimal efficacious dose, we performed a dose
titration of PEG40-NMU in an OGTT. Both 0.1 and 1 mg/kg signifi-
cantly reduced the blood glucose excursions observed during OGTT
(Fig. 5A) and the corresponding area under the curve (Fig. 5B).

P. Ingallinella et al. / Bioorg. Med. Chem. 20 (2012) 4751–4759
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