Discovery of small molecule agonists for the bombesin receptor subtype 3 (BRS-3) based on an omeprazole lead

Article abstract of DOI:10.1016/j.bmcl.2008.09.033

Starting from a weak omeprazole screening hit, replacement of the pyridine with a 1,3-benzodioxole moiety, modification of the thioether linkage, and substitution of the benzimidazole pharmacophore led to the discovery of nanomolar BRS-3 agonists.

Full text of DOI:10.1016/j.bmcl.2008.09.033

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5451–5455
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of small molecule agonists for the bombesin receptor
subtype 3 (BRS-3) based on an omeprazole lead
b
David L. Carlton a, , Lissa J. Collin-Smith b, Alejandro J. Daniels c,à, David N. Deaton d, , Aaron S. Goetz ,
*
Christopher P. Laudeman d,§, Thomas R. Littleton b,–, David L. Musso d,|, Ronda J. Ott Morgan e,§§
Jerzy R. Szewczyk d,––, Cunyu Zhang d
,
a Department of Pharmaceutical Development, Physical Properties and Developability, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
b Molecular Discovery Research, Screening & Compound Profiling, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
c Department of Metabolic Diseases, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
d Department of Medicinal Chemistry, GlaxoSmithKline, Research Triangle Park, NC 27709, USA
e Department of Drug Metabolism and Pharmacokinetics, GlaxoSmithKline, Research Triangle Park, NC, 27709, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 15 July 2008
Revised 5 September 2008
Accepted 8 September 2008
Available online 11 September 2008
Starting from a weak omeprazole screening hit, replacement of the pyridine with a 1,3-benzodioxole
moiety, modification of the thioether linkage, and substitution of the benzimidazole pharmacophore
led to the discovery of nanomolar BRS-3 agonists.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Bombesin receptor subtype 3 agonist
G-Protein coupled seven transmembrane
receptor modulation
Omeprazole
Orphan seven transmembrane receptor
BRS-3
BB3
Bombesin
Many drugs exercise their pharmacodynamic effects as ligands
for the G-protein coupled seven transmembrane receptor (7-TM)
protein superfamily.1 In addition to pursuing the discovery of li-
gands for the receptors with known functions, the pharmaceutical
industry has also studied the orphan 7-TM receptors identified
through high homology to known 7-TMs in the human genome,
because of the perceived drugability of this class of proteins. One
such orphan is the 399 amino acid bombesin receptor subtype 3
(BRS-3, BB3) located on the X chromosome (Xq25).2 BRS-3 belongs
to the subfamily of 7-TMs that were originally identified as the
receptors for the bioactive peptide family ligands with high homol-
ogy to bombesin (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-
His-Leu-Met-NH2), a tetradecapeptide originally isolated from the
skin of the frog Bombina bombina.3 The other human members of
this receptor family are the gastrin-releasing peptide (GRP) prefer-
ring receptor (GRP-R, BB2) and the neuromedin B (NMB) preferring
receptor (NMB-R, BB1).4 BRS-3 has 51% identity to GRP-R and 47%
identity to NMB-R, but the natural ligands for these receptors, GRP
and NMB, as well as bombesin, have low affinity for BRS-3. To date,
the natural ligand for BRS-3 is unknown and it remains an
unadopted receptor.
BRS-3 is highly expressed in testes and widely expressed in the
brain, including the hypothalamus, caudate nucleus, pituitary
gland, amygdala, and hippocampus.5 It is also expressed in liver,
* Corresponding author. Tel.: +1 919 483 6270; fax: +1 919 315 0430.
E-mail address: david.n.deaton@gsk.com (D.N. Deaton).
lung, intestine, and pancreas. Through interaction with Gaq, BRS-
3 is coupled to phospholipase C, causing the loss of phosphoinosi-
tides, and leading to calcium mobilization and activation of protein
kinase C.6 BRS-3 activation also stimulates phospholipase D activ-
ity, promoting diacylglycerol formation. BRS-3 (Y/ꢀ) mice are via-
ble and fertile, but become hyperphagic and develop a late onset
mild obesity with increased white adipose tissue mass,7 despite
high leptin levels.8 These knockout mice also have a reduced met-
Present address: 201 Uwharrie Ridge Road, Pittsboro, NC 27312, USA.
Present address: 1345 Grandiflora Drive, Leland, NC 28451, USA.
Present address: 930 Sandlewood Drive, Durham, NC 27712, USA.
Present Address: Esoterix Clinical Trials, 1904 Alexander Drive, Research Triangle
à
§
–
Park, NC 27709, USA.
|
Present address: 1025 Whetstone Court, Raleigh, NC 27615, USA.
Present address: Bayer, 400 Morgan Lane, West Haven, CT 06516, USA.
Present address: 105 Green Willow Court, Chapel Hill, NC 27514, USA.
§§
––
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.033

5452
D. L. Carlton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5451–5455
abolic rate and altered taste preference.9 This obesity is accompa-
nied by hypertension and impaired glucose metabolism10,11 as
well. Male BRS-3 (Y/ꢀ) mice also exhibit altered social interac-
tion.12,13 Furthermore, BRS-3 has been implicated in bronchial epi-
thelial cell proliferation.14 Thus, ligands that modulate BRS-3
signalling may have utility in diabetes, obesity, and cancer.15
Most of these analogs, including the difluoride 1c, the b-naph-
thalene 1e, the pyridine 1f, and the indazole 1g did not activate
BRS-3, as measured by intracellular calcium mobilization, at con-
centrations up to 32,000 nM. In contrast, the 3,5-dimethoxyphenyl
derivative 1d (EC50 = 1,600 nM, %Max = 68%) was a 2-fold more po-
tent BRS-3 partial agonist than omeprazole sulfide 1b. Further-
more, the 1,3-benzodioxole 1h (EC50 = 380 nM, %Max = 67%) was
a 10-fold more potent activator of intracellular calcium mobiliza-
tion than the omeprazole sulfide lead 1b.
With a submicromolar BRS-3 agonist in hand, attention turned
to replacing the thioether linkage with a more metabolically stable
moiety. The synthesis of the amide and methylene amine linked
analogs 1i and 1j are shown in Scheme 2. The purchased aldehyde
4 was oxidized to the carboxylic acid with barium permanganate,
then coupled to the commercially available 2-amino-benzimid-
azole 5 to give the amide 1i (X = O). Whereas, condensation of alde-
hyde 4 with the guanidine 5 to provide an imine, followed by
A non-selective, high affinity peptide ligand for BRS-3, D-Phe-
Gln-Trp-Ala-Val-b-Ala-His-Phe-Nle-NH2 (BB1 Ki = 8.9 nM, BB2
Ki = 0.99 nM, BB3 Ki = 0.36 nM), has been discovered and replace-
ment of the
D
-phenylalanine residue with a 125I-
D
-tyrosine pro-
vided a radioligand for BRS-3 binding studies.16 In addition to
this molecule and other peptide ligands,17–20 a German group has
also disclosed small molecule agonists of BRS-3.21,22
As part of a protein systems-based research strategy, many G-
protein coupled seven transmembrane receptors, including orphan
receptors like BRS-3, were screened by GSK versus a compound set
consisting of the commercially marketed drugs. Surprisingly, the
gastric H+/K+-ATPase proton pump inhibitor omeprazole 1a (Lo-
secÒ), for peptic ulcer treatment, was identified as a weak partial
BRS-3 agonist (BB3 EC50 = 14,000 nM, %Max = 27%). Furthermore,
its sulfide analog 1b was slightly more potent (BB3
EC50 = 3900 nM) with better efficacy (%Max = 64%). Moreover, a lit-
erature search revealed that eleven weeks of treatment with ome-
prazole suppressed body weight gain in rats.23 Was this result
dependent on agonism of BRS-3? With this lead, the intriguing
biology of the knockout mice, and the therapeutic effect of ome-
prazole treatment on body weight in rats, a chemistry program
was started to develop small molecule BRS-3 agonists for the treat-
ment of obesity, based on the omeprazole lead.
O
N
Cl
O
O
O
NH2
O
N
H
a, b or c, d
4
5
Cl
X
N
O
NH
O
N
H
1i-1j
Scheme 2. Reagents and conditions: (a) 4, Ba(MnO4)2, CH2Cl2, rt, 6%; (b) 5, DCC,
HOBt, THF, rt, 23%; (c) 4, 5, piperidine, EtOH, rt to ";, 57%; (d) NaBH4, EtOH, 0 °C to
";, 63%.
H
N
H
N
O
O
O
O
O
S
S
N
N
1a
1b
N
N
Cl
Omeprazole undergoes an acid catalyzed rearrangement at gas-
tric fluid pH, involving the sulfoxide and the pyridine nitrogen, to
irreversibly inhibit the parietal cell H+/K+-ATPase proton pump in
the stomach.24 Replacement of the sulfoxide or removal of the 2-
pyridine nitrogen should prevent the rearrangement and eliminate
proton pump inhibition, improving the off-target selectivity of the
BRS-3 lead. Both strategies were explored. First, a small array of 5-
methoxybenzimidazole thioether analogs was synthesized as
depicted in Scheme 1. Alkylation of the commercially available
thiourea 2 with a group of purchased arylmethyl halides 3a–3f,
followed by removal of the protecting group subsequent to the
reaction of bromide 3e, provided the thioethers 1c–1h.
O
O
O
O
a, b
O
HO
N
6
7
O
O
R
N
c
N
H
1k, 1s-1v
H2N
O
NH2 H2N
NH2 H2N
NH2 H2N
NH2 H2N
NH2
8a
8b
8c
8d
8e
O
Cl
F3C
Cl
Ph
H
O
O
R
N
N
Scheme 3. Reagents and conditions: (a) SO2Cl2, Et2O, rt, 54%; (b) BrCH2CN, K2CO3,
acetone, ";, 89%; (c) 8a–8e, NaOMe, MeOH, rt, 13–76%.
S
S
N
H
N
H
a or a, b
O
2
1c-1h
Cl
O
O
a, b
or
R
Cl
O
O
F
F
HO
a, c, b
O
Br
Br
Br
Br
Cl
4
9a-9c
O
O
Cl
3a
3b
3c
Cl
O
N
O
O
O
O
N
N
O
d
N
H
R
Br
1l-1n
N
O
Scheme
4. Reagents
and
conditions:
(a)
EtO2CCH2P(@O)(OEt)2
or
3d
3e
3f
EtO2CCH(CH3)P(@O)(OEt)2, t-BuO-K+, THF, 0 °C to ";, 29–94%, (b) NaOH, THF, H2O,
rt, 73–99%; (c) H2/10% Pd-C or PtO2, EtOH, EtOAc, rt, 81–87%; (d) HBTU, i-Pr2NEt,
DMF; 8a, rt; AcOH, 100 °C, 40–62%.
Scheme 1. Reagents and conditions: (a) RBr 3a–3e or RCl 3f, Cs2CO3, DMF, 0 °C–rt,
45–80%; (b) TFA, CH2Cl2, 0 °C–rt, 87%.

D. L. Carlton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5451–5455
5453
Table 1
Activation of human BB3
R1
R2
BB3 EC50 (nM)
BB3
27
% (Max %)
a
b
#
O
S
O
O
H
N
O
O
O
R1
1a
14,000
3900
R2
R2
R2
N
N
N
H
N
R1
S
1b
1c
64
—
N
H
N
F
R1
R1
S
S
>32,000
F
N
O
O
H
N
O
1d
1600
68
R2
N
H
N
O
O
O
R1
S
S
1e
1f
>32,000
>32,000
>32,000
—
—
—
R2
R2
R2
N
H
N
R1
N
N
H
N
R1
R1
S
S
1g
N
NH
N
Cl
Cl
Cl
H
N
O
O
O
1h
1i
380
67
—
R2
R2
R2
O
O
O
N
O
H
N
R1 NH
O
>32,000
>32,000
N
O
O
H
N
R1 NH
1j
—
N
Cl
Cl
H
N
R1
O
O
1k
1l
510
210
71
82
R2
O
O
O
N
O
O
H
N
R1
R2
N
Cl
Cl
H
N
R1
O
O
1m
1n
>32,000
350
—
R2
O
O
N
O
O
H
N
R1
83
R2
N
(continued on next page)

5454
D. L. Carlton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5451–5455
Table 1 (continued)
R1
R2
BB3 EC50 (nM)
BB3
—
% (Max %)
a
b
#
Cl
R1
O
O
O
N
R2
1o
1p
>32,000
>32,000
O
O
Cl
R1
S
N
R2
—
O
O
Cl
R1
O
O
N
O
R2
1q
1r
1s
1t
>32,000
>32,000
570
—
O
O
Cl
R1
N
S
R2
—
O
O
Cl
H
R1
Cl
N
59
69
—
R2
O
O
N
O
Cl
H
R1
F3C
N
240
R2
O
O
N
O
Cl
H
R1
Cl
N
1u
>32,000
>32,000
R2
O
O
N
O
Cl
H
N
R1
O
Ph
1v
—
R2
O
O
N
O
a
Activation of BB3 in recombinant BacMam Baculovirus transduced HEK 293 cells grown in EMEM media supplemented with 10% fetal calf serum, 2 nM L-glutamine, and
1% non-essential amino acids at 37 °C, 5% CO2, and 95% humidity for 24 h. Growth media is removed via aspiration and replaced with HBSS media containing 2.5 mM
probenicid and 0.1% bovine serum albumin (w/v) and FLIPR dye. The assay is initiated by the measurement of a background reading, followed by the addition of the test
compound. Activation induced by the test compound is recorded for 2 min. The data points are fitted to a curve using non-linear regression analysis. The EC50 values are the
mean of at least two assays. D-Phe-Gln-Trp-Ala-Val- b-Ala-His-Phe-Nle-NH2 had an EC50 = 3.9 nM in this assay.
b
Maximum percent efficacy of the test compound relative to BB3 activation via
D-Phe-Gln-Trp-Ala-Val- b-Ala-His-Phe-Nle-NH2.
reduction with sodium borohydride, afforded the amine analog 1j
(X = H, H).
tion of these acids 9a–9c with the ortho-dianiline 8a and finally,
acid catalyzed cyclization gave the carbon linked analogs 1l–1n.
As shown in Table 1, the amide linkage 1i was not tolerated by
the receptor, inducing no activation at concentrations up to
32,000 nM. Possibly, the carbonyl group cannot be accommodated
in the receptor binding pocket, but more likely, the trans configu-
ration of the amide bond displays the two appendages in the
The synthesis of the reverse ether linked analog 1k is illustrated
in Scheme 3. First, the purchased phenol 6 was chlorinated with
sulfuryl chloride on the sterically least hindered ortho-phenolic
site. Then, potassium carbonate promoted alkylation of the phenol
with bromoacetonitrile provided the ether 7. Finally, base cata-
lyzed condensation of the commercially available ortho-dianiline
8a with the nitrile 7 yielded the ether linked analog 1k. The differ-
entially substituted benzimidazole analogs 1s–1v were prepared in
a completely analogous manner from the commercially available
ortho-dianilines 8b–8e.
The linker derivatives 1l–1n with all carbon connections were
prepared as depicted in Scheme 4. First, Horner–Wadsworth–Em-
mons stabilized ylide coupling of the aldehyde 4 with the commer-
cially available phosphorus reagents provided the olefin esters
(R = H or R = Me). Hydrolysis of the ester (R = H) with sodium
O
O
X
Y
Cl
3f
Cl
Cl
O
X
Y
O
O
a
O
10a-10d
1o-1r
hydroxide then afforded the a,b-unsaturated acid 9b. Alternatively,
metal catalyzed hydrogenation of the alkenes, followed by hydro-
lysis of the esters yielded the saturated acids 9a and 9c. Condensa-
Scheme 5. Reagents and conditions: (a) 10a–10d, LDA, THF, 0 °C to ꢀ78 °C; 3f,
ꢀ78 °C to rt, 13–39%.

D. L. Carlton et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5451–5455
5455
wrong orientation for receptor activation (vide infra). This implies
that one of the two possible gauche rotamers is the active con-
former. In addition, the amine linker analog 1j is not a BRS-3 ago-
nist. The positive charge at physiological pH is likely not accepted
by the protein. In contrast, the reverse ether analog 1k
(EC50 = 510 nM, %Max = 71%) is an equipotent BRS-3 agonist as
the thioether 1h. Likewise, the carbon linked derivatives 1l
(EC50 = 210 nM, %Max = 82%) and 1n (EC50 = 350 nM, %Max = 83%)
exhibit similar potency to the thioether 1h. Furthermore, analog
1n shows that carbon substitution of the linker is permissible as
well. In contrast, similar to the amide derivative 1i, the trans olefin
1m is inactive, further supporting the hypothesis that an anti rot-
amer population is not the preferred conformation for efficacy.
With two potential linker replacements with perceived greater
metabolic stability identified, focus shifted to exploration of the
benzimidazole pharmacophore. Benzimidazole replacements were
synthesized as shown in Scheme 5. The commercially available
benzoxazoles 10a (X = O, Y = N) and 10c (X = N, Y = O) and benz-
thiazoles 10b (X = S, Y = N) and 10d (X = N, Y = S) were alkylated
with the chloride 3f to give the carbon linked derivatives 1o–1r.
As shown in Table 1, both benzimidazole nitrogens are abso-
lutely required for agonist activity as replacement of either one
of them with oxygen or sulfur, as in analogs 1o-1r, destroys all ago-
nist activity. It is possible that a positive charge on the benzimid-
helped improve metabolic stability, eliminating the omeprazole
rearrangement liability and removing the proton pump inhibition
liability. Finally, modifications to the benzimidazole revealed the
importance of the NH for hydrogen bond donation and showed that
the chloride and trifluoromethane analogs 1s and 1t were also sub-
micromolar BRS-3 agonists. These BRS-3 modulators may prove use-
ful in further defining the physiological roles of BRS-3.
References and notes
1. Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L. Nat. Rev. Drug Discovery 2006, 5,
993.
2. Fathi, Z.; Corjay, M. H.; Shapira, H.; Wada, E.; Benya, R.; Jensen, R.; Viallet, J.;
Sausville, E. A.; Battey, J. F. J. Biol. Chem. 1993, 268, 5979.
3. Jensen, R. T.; Battey, J. F.; Spindel, E. R.; Benya, R. V. Pharmacol. Rev. 2008, 60, 1.
4. Corjay, M. H.; Dobrzanski, D. J.; Way, J. M.; Viallet, J.; Shapira, H.; Worland, P.;
Sausville, E. A.; Battey, J. F. J. Biol. Chem. 1991, 266, 18771.
5. Sano, H.; Feighner, S. D.; Hreniuk, D. L.; Iwaasa, H.; Sailer, A. W.; Pan, J.;
Reitman, M. L.; Kanatani, A.; Howard, A. D.; Tan, C. P. Genomics 2004, 84, 139.
6. Ryan, R. R.; Weber, H. C.; Hou, W.; Sainz, E.; Mantey, S. A.; Battey, J. F.; Coy, D.
H.; Jensen, R. T. J. Biol. Chem. 1998, 273, 13613.
7. Ohki-Hamazaki, H.; Watase, K.; Yamamoto, K.; Ogura, H.; Yamano, M.; Yamada,
K.; Maeno, H.; Imaki, J.; Kikuyama, S.; Wada, E.; Wada, K. Nature 1997, 390, 165.
8. Maekawa, F.; Quah, H.-M. A.; Tanaka, K.; Ohki-Hamazaki, H. Diabetes 2004, 53,
570.
9. Yamada, K.; Wada, E.; Imaki, J.; Ohki-Hamazaki, H.; Wada, K. Physiol. Behav.
1999, 66, 863.
10. Matsumoto, K.; Yamada, K.; Wada, E.; Hasegawa, T.; Usui, Y.; Wada, K. Peptides
2003, 24, 83.
11. Nakamichi, Y.; Wada, E.; Aoki, K.; Ohara-Imaizumi, M.; Kikuta, T.; Nishiwaki,
C.; Matsushima, S.; Watanabe, T.; Wada, K.; Nagamatsu, S. Biochem. Biophys.
Res. Commun. 2004, 318, 698.
12. Yamada, K.; Ohki-Hamazaki, H.; Wada, K. Physiol. Behav. 2000, 68, 555.
13. Yamada, K.; Santo-Yamada, Y.; Wada, E.; Wada, K. Mol. Psychiatry 2002, 7, 113.
14. Liu, H.; Wang, Y.; Qi, M.; Qu, F.; Xiang, Y.; Tan, Y.; Zhang, C.; Qin, X. Zhongnan
Daxue Xuebao, Yixueban 2006, 31, 178.
azole is necessary for receptor activation, but with only
a
moderate basicity (calculated pKa = ꢁ5.9), it is likely that the benz-
imidazole is not charged. Furthermore, analog 1j (calculated
pKa = ꢁ7.4) was not an agonist despite the ability to delocalize its
charge to either ring nitrogen. Therefore, it seems more likely that
a hydrogen bond donor is required for agonist activity and the
benzimidazole NH fulfills this requirement.
15. Gonzalez, N.; Moody, T. W.; Igarashi, H.; Ito, T.; Jensen, R. T. Curr. Opin.
Endocrinol., Diabetes Obes. 2008, 15, 58.
In contrast to benzimidazole replacement, substitution of the
methoxy group with small lipophilic moieties was permissible.
Both the chloro and trifluoromethyl derivatives 1s (EC50 = 570 nM,
%Max = 59%) and 1t (EC50 = 240 nM, %Max = 69%) were equipotent
BRS-3 agonists as the methoxy analog 1k. Larger substituents, like
the phenoxy group in derivative 1v, have no measured activation
and if able to bind likely disturb the receptor conformation, pre-
venting its recruitment of G proteins for signal transduction. Fur-
thermore, substitution of the 6-position of the benzimidazole
ring, as in analog 1u, also abolished agonist activity (compare 1s
with 1u). Representative BRS-3 analogs were profiled versus
NMB-R (BB1)25 and GRP-R (BB2),26 but none of them exhibited
any agonist efficacy as measured by intracellular calcium mobiliza-
tion in a FLIPR-based assay. Although a homology receptor model
of BRS-3, based on the principles of comparative protein modelling,
using the X-ray crystal structure of the seven transmembrane
receptor bovine rhodopsin, was developed, its predictions did not
prove fruitful for further target design. Nor did it provide any dee-
per understanding of the structure/activity relationships of these
analogs.
A representative BRS-3 agonist 1k was dosed i.v. in fasted rats
to ascertain its pharmacokinetic properties. It exhibited a moder-
ate volume of distribution (VSS = 1200 mL/kg) with a short terminal
half-life (t1/2 = 30 min) and a high clearance (Cl = 61 mL/min/kg)
that approached hepatic blood flow. Although these inhibitors con-
form to Lipinski’s Rule of 5 for good bioavailability, analog 1k was
not dosed orally, because of its poor i.v. exposure.
In summary, a directed screeningapproach identified a weak par-
tial BRS-3 agonist, the proton pump inhibitor omeprazole 1a.
Replacements of the pyridine fragment with various groups led to
the identification of a 1,3-benzodioxole submicromolar BRS-3 ago-
nist 1h. Further modifications to the linker region, as in 1k and 1l,
16. Mantey, S. A.; Weber, H. C.; Sainz, E.; Akeson, M.; Ryan, R. R.; Pradhan, T. K.;
Searles, R. P.; Spindel, E. R.; Battey, J. F.; Coy, D. H.; Jensen, R. T. J. Biol. Chem.
1997, 272, 26062.
17. Darker, J. G.; Brough, S. J.; Heath, J.; Smart, D. J. Pept. Sci. 2001, 7, 598.
18. Mantey, S. A.; Coy, D. H.; Pradhan, T. K.; Igarashi, H.; Rizo, I. M.; Shen, L.; Hou,
W.; Hocart, S. J.; Jensen, R. T. J. Biol. Chem. 2001, 276, 9219.
19. Mantey, S. A.; Coy, D. H.; Entsuah, L. K.; Jensen, R. T. J. Pharmacol. Exp. Ther.
2004, 310, 1161.
20. Boyle, R. G.; Humphries, J.; Mitchell, T.; Showell, G. A.; Apaya, R.; Iijima, H.;
Shimada, H.; Arai, T.; Ueno, H.; Usui, Y.; Sakaki, T.; Wada, E.; Wada, K. J. Pept.
Sci. 2005, 11, 136.
21. Weber, D.; Berger, C.; Heinrich, T.; Eickelmann, P.; Antel, J.; Kessler, H. J. Pept.
Sci. 2002, 8, 461.
22. Weber, D.; Berger, C.; Eickelmann, P.; Antel, J.; Kessler, H. J. Med. Chem. 2003,
46, 1918.
23. Cui, G. L.; Syversen, U.; Zhao, C. M.; Chen, D.; Waldum, H. L. Scand. J.
Gastroenterol. 2001, 36, 1011.
24. Lindberg, P.; Nordberg, P.; Alminger, T.; Brandstrom, A.; Wallmark, B. J. Med.
Chem. 1986, 29, 1327.
25. Activation of BB1 in recombinant BacMam Baculovirus transduced HEK 293
cells grown in EMEM media supplemented with 10% fetal calf serum, 2 nM L-
glutamine, and 1% non-essential amino acids at 37 °C, 5% CO2, and 95%
humidity for 24 h. Growth media is removed via aspiration and replaced with
HBSS media containing 2.5 mM probenicid and 0.1% bovine serum albumin (w/
v) and FLIPR dye. The assay is initiated by the measurement of a background
reading, followed by the addition of the test compound. Activation induced by
the test compound is recorded for 2 min. The data points are fitted to a curve
using non-linear regression analysis. The EC50 values are the mean of at least
two assays. D-Phe-Gln-Trp-Ala-Val-b-Ala-His-Phe-Nle-NH2 had an EC50 = 0.55
nM in this assay.
26. Activation of BB2 in recombinant BacMam Baculovirus transduced HEK 293
cells grown in EMEM media supplemented with 10% fetal calf serum, 2 nM L-
glutamine, and 1% non-essential amino acids at 37 °C, 5% CO2, and 95%
humidity for 24 h. Growth media is removed via aspiration and replaced with
HBSS media containing 2.5 mM probenicid and 0.1% bovine serum albumin (w/
v) and FLIPR dye. The assay is initiated by the measurement of a background
reading, followed by the addition of the test compound. Activation induced by
the test compound is recorded for 2 min. The data points are fitted to a curve
using non-linear regression analysis. The EC50 values are the mean of at least
two assays.
D-Phe-Gln-Trp-Ala-Val-b-Ala-His-Phe-Nle-NH2 had an EC50 =
0.060 nM in this assay.