Analogues of Dehydroacetic Acid as Selective and Potent Agonists of an Ectopic Odorant Receptor through a Combination of Hydrophilic and Hydrophobic Interactions

Article abstract of DOI:10.1002/cmdc.201600612

Identification of potent agonists of odorant receptors (ORs), a major class of G protein-coupled receptors, remains challenging due to complex receptor–ligand interactions. ORs are present in both olfactory and non-chemosensory tissues, indicating roles beyond odor detection that may include modulating physiological functions in non-olfactory tissues. Selective and potent agonists specific for particular ORs can be used to investigate physiological functions of ORs in non-chemosensory tissues. In this study, we designed and synthesized novel synthetic dehydroacetic acid analogues as agonists of odorant receptor 895 (Olfr895) expressed in bladder. Among the synthesized analogues, (E)-3-((E)-1-hydroxy-3-(piperidin-1-yl)allylidene)-6-methyl-2H-pyran-2,4(3H)-dione (10) exhibited extremely high agonistic activity for Olfr895 in Dual-Glo luciferase reporter (EC₅₀=9 nm), Ca²⁺ imaging, and chemotactic migration assays. Molecular docking and site-directed mutagenesis studies suggested that a combination of hydrophilic and hydrophobic interactions is central to the selective and specific binding of 10 to Olfr895. The design of agonists armed with both hydrophilic and hydrophobic portions could therefore lead to highly potent and selective ligands for ectopic ORs.

Full text of DOI:10.1002/cmdc.201600612

DOI: 10.1002/cmdc.201600612
Communications
Analogues of Dehydroacetic Acid as Selective and Potent
Agonists of an Ectopic Odorant Receptor through
a Combination of Hydrophilic and Hydrophobic
Interactions
Bernie Byunghoon Park+,[a] NaHye Lee+,[b, c] YunHye Kim,[a] YoonGyu Jae,[b, c]
Seunghyun Choi,[a] NaNa Kang,[c] Yu Ri Hong,[c] Kiwon Ok,[a] Jeonghee Cho,[d]
Young Ho Jeon,[a] Eun Hee Lee,[a] Youngjoo Byun,*[a] and JaeHyung Koo*[b, c]
Identification of potent agonists of odorant receptors (ORs),
a major class of G protein-coupled receptors, remains challeng-
ing due to complex receptor–ligand interactions. ORs are pres-
ent in both olfactory and non-chemosensory tissues, indicating
roles beyond odor detection that may include modulating
physiological functions in non-olfactory tissues. Selective and
potent agonists specific for particular ORs can be used to in-
vestigate physiological functions of ORs in non-chemosensory
tissues. In this study, we designed and synthesized novel syn-
thetic dehydroacetic acid analogues as agonists of odorant re-
ceptor 895 (Olfr895) expressed in bladder. Among the synthe-
sized analogues, (E)-3-((E)-1-hydroxy-3-(piperidin-1-yl)allyli-
dene)-6-methyl-2H-pyran-2,4(3H)-dione (10) exhibited extreme-
ly high agonistic activity for Olfr895 in Dual-Glo luciferase re-
porter (EC50 =9 nm), Ca2+ imaging, and chemotactic migration
assays. Molecular docking and site-directed mutagenesis stud-
ies suggested that a combination of hydrophilic and hydro-
phobic interactions is central to the selective and specific bind-
ing of 10 to Olfr895. The design of agonists armed with both
hydrophilic and hydrophobic portions could therefore lead to
highly potent and selective ligands for ectopic ORs.
ants with a wide range of binding affinities.[2] Binding of odor-
ants to cognate ORs causes conformational changes in olfacto-
ry-type
G protein (Golf), leading to the activation of
downstream signaling pathways.[3] Although ORs are the larg-
est family of GPCRs (ꢀ50% in human and ꢀ70% in mouse),[4]
no crystal structure of an OR has been elucidated to date.[5] Al-
though most ORs are extensively expressed in nasal olfactory
sensory neurons, where they perform appropriate physiological
functions, ectopic expression of ORs in non-chemosensory tis-
sues has been recently reported.[6] Comprehensive RNA-se-
quence analyses of ectopically expressed ORs have been per-
formed on a broad panel of human tissues.[7] More recently, we
and other researchers demonstrated ectopic expression of ORs
in a variety of tissues including pancreas, bladder, thymus,
heart, brain, skin, kidney, muscle, testis, liver, gut, eye, and thy-
roid.[8–23] ORs might, therefore, play physiological roles beyond
odor perception in non-olfactory tissues, but their exact func-
tions remain unknown.
Given the lack of crystal structures of membrane-bound ORs,
activation mechanisms have been proposed and assessed
based on molecular modelling and site-directed mutagene-
sis.[5,24–30] In general, hydrophobic interactions between ORs
and cognate odorants are the major source of binding energy.
Nevertheless, some ORs such as MOR244-3 (Olfr1509) and
Olfr288 have a very narrow range of ligand specificity.[31] It is
crucial to identify potent ligands that are specific for particular
ORs in order to understand how they recognize and distin-
guish different ligands at the molecular level. Some ectopic
ORs are overexpressed in the cancerous state[32–34] and have
been linked to pathophysiological functions such as the
growth of prostate cancer cells[35] and metastasis of gastric
cancer cells.[36] Sensitive and selective potent ligands for specif-
ic ORs in non-olfactory tissues can be used as biological tool
compounds to elucidate the physiological functions of ectopic
ORs, or to manipulate the activity of ORs and thereby regulate
pathophysiological conditions. Although a number of odorants
for ORs have been reported, all are relatively weak or moderate
agonists for particular ORs, with EC50 values in the mm range.
Furthermore, odorants generally possess one or more function-
al groups (e.g., carboxylic acids, alcohols, amines, aldehydes,
ketones, thiols, and esters) that can activate multiple ORs.[37–39]
Although some odorants display specificity for particular
ORs,[38–40] our knowledge of the molecular pharmacology of ec-
Odorant receptors (ORs), the largest family of G protein-cou-
pled receptors (GPCRs), are responsible for recognizing molec-
ular features of odorant molecules and translating the informa-
tion to the brain.[1] In general, ORs bind to a number of odor-
[a] B. B. Park,+ Y. Kim, S. Choi, K. Ok, Prof. Y. H. Jeon, Prof. E. H. Lee,
Prof. Y. Byun
College of Pharmacy, Korea University, Sejong, 30019 (South Korea)
E-mail: yjbyun1@korea.ac.kr
[b] N. Lee,+ Y. Jae, Prof. J. Koo
Department of Brain and Cognitive Sciences, DGIST, Daegu, 42988 (South
Korea)
E-mail: jkoo001@dgist.ac.kr
[c] N. Lee,+ Y. Jae, Dr. N. Kang, Y. R. Hong, Prof. J. Koo
Department of New Biology, DGIST
[d] Prof. J. Cho
Department of NanoBio Medical Science, Dankook University, Cheonan,
31116 (South Korea)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600612.
ChemMedChem 2017, 12, 1 – 7
1
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Communications
topic ORs remains limited due to the lack of selective and spe-
cific agonists.
are typical GPCRs with seven transmembrane (TM) helices, and
the N-terminus is exposed to the extracellular side of the
plasma membrane. Olfr895-expressing cells exhibited a strong
and non-uniform punctate expression pattern on the plasma
membrane. Expression of the Olfr895 protein was clearly
higher than that in controls. (Figure 1E).
In this study, odorant receptor 895 (Olfr895) was identified
in bladder using Gene Atlas2 microarray data through bioinfor-
matics evaluation as described in previous studies.[8,9] We
quantitatively determined the expression levels of Olfr895 and
three other ORs by PCR and qPCR in the bladder using olfacto-
ry bulb (OB) tissue as a control (Figure 1A,B). The expression
Selective and sensitive binding of the b2 adrenergic receptor
(b2AR) GPCR to ligands such as carazolol and isoproterenol
proceeds via a combination of hydrophilic and hydrophobic in-
teractions.[42] Some ORs including MOR107-1 and MOR271-1 dis-
play high selectivity in the recognition of structurally similar
molecules,[37,43] implying that single OR-mediated signal trans-
duction could be achieved by selective ligands specific for par-
ticular ORs. For instance, the human OR7D4 is selectively acti-
vated by bulky androstenones or structurally related ste-
roids.[40] Meanwhile, mouse eugenol OR is selectively activated
by bulky polycyclic compounds that bind through a combina-
tion of hydrophobic and hydrophilic interactions.[44] However,
most ORs exhibit a degree of affinity for a broad spectrum for
odorant ligands via weak hydrophobic interactions.[29,37] There
exist very few examples of successful screening for potent and
selective agonists that bind through both hydrophobic and hy-
drophilic interactions for targeting ectopically expressed ORs.
In this study, we designed bulky synthetic ligands specific
for Olfr895 by modifying known acetyl-substituted aromatic
odorants such as 2’-hydroxyacetophenone, 3’-hydroxyaceto-
phenone, and dehydroacetic acid (Scheme 1). Commercial
acetyl-substituted compounds activated Olfr895 at >50 mm
(Table 1). To identify selective agonists of Olfr895, acetyl-substi-
tuted odorants were substituted with cyclic amines. 2’-Hydrox-
yacetophenone and 3’-acetophenone analogues were synthe-
sized as described previously.[45] Preparation of compounds 9–
13 derived from dehydroacetic acid was achieved in two steps
as shown in Scheme 1. Briefly, reaction of dehydroacetic acid
with N,N-dimethylformamide dimethylacetal (DMF-DMA) in
xylene followed by amine exchange reaction under acidic con-
ditions afforded enol-functionalized compounds in high yield.
Enolic acid protons in 9–13 were observed at ꢀ14 ppm in
1H NMR spectra.
Figure 1. A) Verification of four ORs from the top-25 ORs selected by refined
microarray by RT-PCR and sequencing. B) Quantification of mRNA for the
four ORs by qPCR in the bladder (BL) and the olfactory bulb (OB) control ol-
factory tissue; values are meansÆSEM. OR mRNA levels in OB and BL tissues
were quantified against the Olfr544 plasmid standard and normalized
against eEF-2 RNA. C) Analysis of Olfr895 expression in the OB and olfactory
epithelium (OE) control tissue and in various other tissues (HT, heart; DD, du-
odenum; TR, thyroid; LG, lung; TT, testis; SP, spleen; TM, thymus; LV, liver;
KD, kidney; PC, pancreas; ST, stomach; SM, smooth muscle; AD, adipose
tissue; M, DNA ladder). D) Plasma membrane expression of Olfr895 verified
using anti-rho antibody in transiently transfected HEK293 cells expressing
rho-Olfr895. E) Confirmation of Olfr895 expression by western blotting with
anti-rho antibody.
level of Olfr895 was higher than the other ORs in bladder
tissue, and was similar to that in the OB. Next, Olfr895 expres-
sion levels in 16 tissues were determined, and the results
showed that bladder is the only non-olfactory tissue express-
ing Olfr895, which was also expressed in the olfactory epitheli-
um and bulb (Figure 1C). Additionally, OR expression was ob-
served in testis, consistent with a previous study.[6] N-terminal
rho-tagged Olfr895 was cloned into a mammalian expression
vector and transiently transfected with accessory proteins in
HEK293 cells.[41] Non-permeable immunofluorescence staining
(Figure 1D) and western blotting (Figure 1E) with anti-rho con-
firmed that the protein was localized at the plasma membrane.
Anti-rho antibody was able to bind to the rho-tagged N-termi-
nal domain of Olfr895 without membrane penetration, as ORs
Scheme 1. Synthesis of compounds 1–13. Reagents and conditions: a) DMF-
DMA, xylene, reflux, 2 h; b) appropriate cyclic amines, acetic acid, EtOH,
reflux, 2 h.
&
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
2
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Communications
Acetophenone, a broad-acting odorant activating ORs with
EC50 values in the range of 0.1–1 mm, was used as a control.[37]
Acetophenone activated Olfr895 with an EC50 of 164 mm, which
is in the reported range.[37] Synthetic analogues 3–7 derived
from 2’-hydroxyacetophenone or 3’-hydroxyacetophenone ex-
hibited moderate agonistic activity for Olfr895 with EC50 values
>1 mm. However, compounds 9–12 derived from dehydroace-
tic acid exhibited increased potency as agonists of Olfr895
with EC50 values <1 mm. In particular, the cyclic tertiary amine
analogues 9 (pyrrolidine) and 10 (piperidine) exhibited ex-
tremely strong activation of Olfr895 with EC50 values of 91 nm
and 9 nm, respectively. However, the morpholine-functional-
ized analogue 11 was less potent with an EC50 value of
679 nm. Introduction of aromatic amines such as aniline (12)
and 2-aminopyridine (13) or acyclic tertiary dimethylamine (8)
also decreased the agonistic activity toward Olfr895 (Table 1).
We confirmed the potency of compound 10 by measuring
the activation of Olfr895 using Ca2+ imaging assays. We deter-
mined intracellular Ca2+ ([Ca2+]i) mobilization using Fura 2-AM
ratiometric calcium imaging in Olfr895-transfected cells treated
with compound 10 at various concentrations (0 nm, 1 nm,
10 nm, 1 mm, 200 mm, and 1 mm). As shown in Figure 2A, treat-
ment of compound 10 at 1 nm and 10 nm concentrations in-
duced the release of [Ca2+]i while the control acetophenone
required a much higher concentration (200 mm and 1 mm),
confirming compound 10 as a strong activator of Olfr895.
Next, we investigated whether compound 10 was a selective
activator for Olfr895 or a broad agonist for many other ORs.
Table 1. Summary of EC50 values of compounds 1–13 and reference
odorants.
Compound
EC50 [mm][a]
1
2
3
4
5
6
7
8
9
10
11
12
27.6Æ0.05
3460Æ0.21
54.4Æ0.32
10.3Æ0.1
307Æ0.17
38.4Æ0.32
447Æ0.12
12.4Æ0.1
0.091Æ0.0007
0.009Æ0.001
0.679Æ0.045
0.386Æ0.054
6.48Æ0.05
258Æ0.14
13
dehydroacetic acid
acetophenone
2’-hydroxyacetophenone
3’-hydroxyacetophenone
164Æ0.02
388Æ0.17
91Æ0.2
[a] Data from luciferase activity assays were statistically analyzed using
GraphPad Prism to generate EC50 values. Values are the meanÆSD from
3–5 independent experiments.
Activation of Olfr895 by synthesized analogues 1–13 was
evaluated by measuring luciferase activity using Dual-Glo luci-
ferase assays.[41] EC50 values of prepared compounds 1–13 and
starting materials including 2’-hydroxyacetophenone, 3’-aceto-
phenone and dehydroacetic acid are summarized in Table 1.
Figure 2. A) Increase in intracellular calcium in mammalian cells transfected with Olfr895 following treatment with compound 10 (bottom panel) or acetophe-
none (control, top panel). Images were assessed using Fura 2-AM ratiometric calcium imaging, revealing an increase in intracellular calcium mobilization after
exposure to agonist for 20 s. B) Luciferase assay responses (n=5) of Olfr895 and 28 other ORs following incubation with compound 10. Controls with pcDNA
mock vector were included for 1 nm and 10 nm treatments. C) Stimulation of chemotactic migration by compound 10 (or acetophenone) at various concen-
trations (NT, 1, 10, and 100 nm) in transiently transfected mammalian cells expressing Olfr895. The migration index was determined by normalization against
the total number of migrating cells in untreated controls. Data are the meanÆSEM of triplicate experiments; ***p<0.001 relative to acetophenone controls.
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
3
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Communications
Agonistic activities for compound 10 against 29 other ORs
were measured using luciferase assays, and only Olfr895 was
strongly activated by compound 10, while all other ORs were
only weakly or negligibly affected (Figure 2B). Furthermore, we
evaluated the effect of compound 10 on chemotactic migra-
tion of Olfr895-expressing mammalian cells. Stimulation of
these cells with compound 10 resulted in high chemotactic mi-
gration (Figure 2C). At a concentration of 100 nm, compound
10 elicited a ꢀ4-fold increase in cell migration, and induced
migration was concentration dependent (Figure 2C).
3.35 ꢁ) as a template (sequence identity=17%, sequence simi-
larity=28%).[46] The ligand binding site was generated with
a radius of 15 ꢁ by selecting two key amino acids (Phe110 in
TM3 and Tyr258 in TM6), which are reported to be the gate
amino acids in ORs.[5] The ligands were docked in the Olfr895
homology model using the Surflex-Dock GeomX module
(SYBYL-X 2.1.1, Tripos Inc.). The highest scoring poses of each
ligand were selected and their binding modes were analyzed.
Interestingly, ligands derived from dehydroacetic acid formed
strong hydrogen bonding interactions with Arg171 in the
active site. The stable enolic OH group of dehydroacetic acid
analogues 8–13 also engaged in strong hydrogen bonding in-
teractions with the guanidinium group of Arg171. In particular,
compound 10 exhibited an extremely high affinity for Olfr895
due to two hydrogen bonding interactions with Arg171, with
intermolecular bond distances of 1.84 and 1.93 ꢁ, and an addi-
tional hydrogen bond with Thr198 (Figure 3B). Hydrophobic
interactions between methylene groups in the cyclic ring and
lipophilic amino acids (Phe110, Leu111, Val114 and Ile213) were
also observed, implying that a combination of hydrophilic and
hydrophobic interactions might contribute to the strong affini-
ty of compound 10 for Olfr895. Indeed, alkylation of com-
pounds 8 and 10 dramatically abolished the agonist activity
for Olfr895 (see Supporting Information). Furthermore, replace-
ment of the piperidine ring in 10 with a hydrophilic morpho-
line group resulted in a significant loss of agonistic activity.
This might be due to the decreased hydrophobicity of 11. A
decrease in agonistic activity for Olfr895 was also observed for
compounds 12–13, even though their binding modes were
similar to that of compound 10 (see Supporting Information),
implying that intermolecular interactions contributed by ani-
line and aminopyridine moieties were less effective than those
by piperidine in 10. It has been reported that most ORs inter-
To investigate the selectivity and sensitivity of compound 10
against Olfr895, its molecular structure was determined and
1
analyzed by single crystal X-ray diffraction and H NMR spec-
troscopy. Due to the delocalization of p electrons in the conju-
gated enolic system, compound 10 has a planar structure with
minor distortions (Figure 3A). The conjugation pattern of 10
was strikingly different from that of 2’-hydroxyacetophenone
analogue 4.[45] The carbonyl group of the conjugation system
in compound 10 exists in a stable enol form (Figure 3A) rather
than a keto tautomer as observed in compound 4. This equilib-
rium was further corroborated by temperature-dependent
1H NMR studies in diverse solvents (see Supporting Informa-
tion). Strong intramolecular hydrogen bonding interactions be-
tween the enolic OH and the carbonyl oxygen of the pyrone
ring were observed in the crystal structure, with an estimated
1
bond length of 1.392 ꢁ. The H NMR spectrum of compound
10 also confirmed the presence of an acidic enolic OH group
which appeared at 14 ppm as a broad singlet.
To investigate whether dehydroacetic acid analogues pos-
sessed strong potency against Olfr895, we performed molecu-
lar docking studies for dehydroacetic acid ligands 1–13 using
a homology model of Olfr895 built using a high-resolution
crystal structure of b1-adrenergic receptor (PDB ID: 5F8U,
Figure 3. A) Crystal structure of compound 10. Selected bond lengths [ꢁ]: N(14)ÀC(13) 1.318 ꢁ, N(14)ÀC(19) 1.467 ꢁ, C(12)ÀC(13) 1.381 ꢁ, C(10)ÀC(12) 1.394 ꢁ,
C(10)ÀO(11) 1.317 ꢁ, C(5)ÀC(10) 1.453 ꢁ, O(9)ÀH(11) 1.392 ꢁ (estimated). B) Highest scoring docked pose of compound 10 in the binding cavity site of Olfr895.
The dotted green lines represent hydrogen bonding interactions between 10 and Olfr895. C) EC50 curves for compound 10 with various Olfr895 mutants
(F110A, V114G, R171I, and T198V). D) Expression of Olfr895 mutants and b-actin.
&
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
4
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

Communications
act with their corresponding odorant ligands via hydrophobic
interactions, with a minimal role for hydrophilic interac-
tions.[5,26,29,38,44,47] However, the strong binding affinity of 10 for
Olfr895 could be explained by a combination of polar and
nonpolar interactions, as was also observed for the binding of
b2AR agonists to b2AR.[48]
Keywords: agonists · dehydroacetic acid · odorant receptors ·
olfr895
[1] L. Buck, R. Axel, Cell 1991, 65, 175–187.
[2] B. Malnic, J. Hirono, T. Sato, L. B. Buck, Cell 1999, 96, 713–723.
[3] D. T. Jones, R. R. Reed, Science 1989, 244, 790–795.
Molecular modelling and site-directed mutagenesis experi-
ments have been used to explore structure-function relation-
ships of ORs with their ligands due to the lack of an available
crystal structure.[5,25,26,38] Docking of compound 10 with the ho-
mology model generated in the present work indicates that
the binding site is amphiphilic and consists of hydrophilic
amino acid residues (Arg171 and Thr198) and hydrophobic en-
vironments (Phe110, Leu111, Val114 and Ile213) as shown in
Figure 3B. When the two residues (Arg171 and Thr198) contri-
buting to hydrophilic interactions were mutated to hydropho-
bic amino acids (Ile and Val), and when the hydrophobic resi-
dues (Phe110 and Val114) were replaced with non-bulky amino
acids (Ala and Gly, respectively), receptor expression levels
were unaffected (Figure 3D). As expected, targeted mutations
R171A and T198V resulted in a significant decrease in relative
luciferase activity for Olfr895 activation (by 71.5% and 67.4%,
respectively) compared with wild-type Olfr895 (Figure 3C). The
F110A mutation resulted in a 3-fold decrease in Olfr895 activa-
tion compared with V114G (Figure 3C). Based on molecular
modelling and site-directed mutagenesis, we concluded that
Arg171 and Thr198 in the loop between TM4 and TM5 are im-
portant residues for the activation of Olfr895 by compound 10.
To the best of our knowledge, this is the first report on
a highly potent agonist that binds specifically to ectopic ORs
via a combination of hydrophilic and hydrophobic interactions.
In conclusion, we successfully identified potent agonists of
Olfr895 that is expressed in bladder, among which the piperi-
dine-functionalized dehydroacetic acid analogue 10 strongly
activated Olfr895 with an EC50 value of 9 nm (based on lucifer-
ase reporter, Ca2+ imaging, and chemotactic migration assays).
This potent compound can be used as a tool compound to in-
vestigate the physiological functions of Olfr895. In addition,
compound 10 may also potentially serve as a useful starting
point for the development of novel antagonists of Olfr895-as-
sociated signaling pathways. Agonist-OR interactions with low
affinity or specificity for particular ORs have been a bottleneck
for understanding the mechanism of odor perception at the
molecular level. The design of a potent and selective Olfr895
agonist by targeting both hydrophilic and hydrophobic sub-
pockets of Olfr895 might prove useful for the identification of
selective and specific agonists for other ectopic ORs.
[4] T. K. Bjarnadꢂttir, D. E. Gloriam, S. H. Hellstrand, H. Kristiansson, R. Fre-
driksson, H. B. Schioth, Genomics 2006, 88, 263–273.
[5] C. A. de March, Y. Q. Yu, M. J. J. Ni, K. A. Adipietro, H. Matsunami, M. H.
Ma, J. Golebiowski, J. Am. Chem. Soc. 2015, 137, 8611–8616.
[6] N. Kang, J. Koo, BMB Rep. 2012, 45, 612–622.
[7] C. Flegel, S. Manteniotis, S. Osthold, H. Hatt, G. Gisselmann, PLoS One
2013, 8, e55368.
[8] N. Kang, H. Kim, Y. Jae, N. Lee, C. R. Ku, F. Margolis, E. J. Lee, Y. Y. Bahk,
M. S. Kim, J. Koo, PLoS One 2015, 10, e0116097.
[9] N. Kang, Y. Y. Bahk, N. Lee, Y. Jae, Y. H. Cho, C. R. Ku, Y. Byun, E. J. Lee,
M. S. Kim, J. Koo, Biochem. Biophys. Res. Commun. 2015, 460, 616–621.
[10] S. H. Kim, Y. C. Yoon, A. S. Lee, N. Kang, J. Koo, M. R. Rhyu, J. H. Park, Bio-
chem. Biophys. Res. Commun. 2015, 460, 404–408.
[11] H. Kim, N. Kang, K. W. Chon, S. Kim, N. Lee, J. Koo, M. S. Kim, Nucleic
Acids Res. 2015, 43, e130.
[12] P. Garcia-Esparcia, A. Schluter, M. Carmona, J. Moreno, B. Ansoleaga, B.
Torrejon-Escribano, S. Gustincich, A. Pujol, I. Ferrer, J. Neuropathol. Exp.
Neurol. 2013, 72, 524–539.
[13] D. Busse, P. Kudella, N. M. Gruning, G. Gisselmann, S. Stander, T. Luger, F.
Jacobsen, L. Steinstrasser, R. Paus, P. Gkogkolou, M. Bohm, H. Hatt, H.
Benecke, J. Invest. Dermatol. 2014, 134, 2823–2832.
[14] A. J. Chang, F. E. Ortega, J. Riegler, D. V. Madison, M. A. Krasnow, Nature
2015, 527, 240–244.
[15] J. L. Pluznick, R. J. Protzko, H. Gevorgyan, Z. Peterlin, A. Sipos, J. Han, I.
Brunet, L. X. Wan, F. Rey, T. Wang, S. J. Firestein, M. Yanagisawa, J. I.
Gordon, A. Eichmann, J. Peti-Peterdi, M. J. Caplan, Proc. Natl. Acad. Sci.
USA 2013, 110, 4410–4415.
[16] C. Pichavant, T. J. Burkholder, G. K. Pavlath, Skelet Muscle 2016, 6, 2.
[17] C. A. Griffin, K. A. Kafadar, G. K. Pavlath, Dev. Cell 2009, 17, 649–661.
[18] A. Grison, S. Zucchelli, A. Urzi, I. Zamparo, D. Lazarevic, G. Pascarella, P.
Roncaglia, A. Giorgetti, P. Garcia-Esparcia, C. Vlachouli, R. Simone, F. Per-
sichetti, A. R. Forrest, Y. Hayashizaki, P. Carloni, I. Ferrer, C. Lodovichi, C.
Plessy, P. Carninci, S. Gustincich, BMC Genomics 2014, 15, 729.
[19] M. Spehr, G. Gisselmann, A. Poplawski, J. A. Riffell, C. H. Wetzel, R. K.
Zimmer, H. Hatt, Science 2003, 299, 2054–2058.
[20] A. Pronin, K. Levay, D. Velmeshev, M. Faghihi, V. I. Shestopalov, V. Z.
Slepak, PLoS One 2014, 9, e96435.
[21] C. Wu, Y. Jia, J. H. Lee, Y. Kim, S. Sekharan, V. S. Batista, S. J. Lee, Int. J.
Biochem. Cell Biol. 2015, 64, 75–80.
[22] D. Priori, M. Colombo, P. Clavenzani, A. J. Jansman, J. P. Lalles, P. Trevisi,
P. Bosi, PLoS One 2015, 10, e0129501.
[23] B. D. Shepard, L. Cheval, Z. Peterlin, S. Firestein, H. Koepsell, A. Doucet,
J. L. Pluznick, Sci. Rep. 2016, 6, 35215.
[24] C. A. de March, S. K. Kim, S. Antonczak, W. A. Goddard, 3rd, J. Golebiow-
ski, Protein Sci. 2015, 24, 1543–1548.
[25] S. Sekharan, M. Z. Ertem, H. Zhuang, E. Block, H. Matsunami, R. Zhang,
J. N. Wei, Y. Pan, V. S. Batista, Biophys. J. 2014, 107, L5–L8.
[26] L. Gelis, S. Wolf, H. Hatt, E. M. Neuhaus, K. Gerwert, Angew. Chem. Int.
Ed. 2012, 51, 1274–1278; Angew. Chem. 2012, 124, 1300–1304.
[27] L. Doszczak, P. Kraft, H. P. Weber, R. Bertermann, A. Triller, H. Hatt, R.
Tacke, Angew. Chem. Int. Ed. 2007, 46, 3367–3371; Angew. Chem. 2007,
119, 3431–3436.
[28] Y. Yu, C. A. de March, M. J. Ni, K. A. Adipietro, J. Golebiowski, H. Matsu-
nami, M. Ma, Proc. Natl. Acad. Sci. USA 2015, 112, 14966–14971.
[29] S. Katada, T. Hirokawa, Y. Oka, M. Suwa, K. Touhara, J. Neurosci. 2005, 25,
1806–1815.
[30] A. Kato, S. Katada, K. Touhara, J. Neurochem. 2008, 107, 1261–1270.
[31] X. Duan, E. Block, Z. Li, T. Connelly, J. Zhang, Z. Huang, X. Su, Y. Pan, L.
Wu, Q. Chi, S. Thomas, S. Zhang, M. Ma, H. Matsunami, G. Q. Chen, H.
Zhuang, Proc. Natl. Acad. Sci. USA 2012, 109, 3492–3497.
[32] L. L. Xu, B. G. Stackhouse, K. Florence, W. Zhang, N. Shanmugam, I. A.
Sesterhenn, Z. Q. Zou, V. Srikantan, M. Augustus, V. Roschke, K. Carter,
Acknowledgements
This work was supported by the National Research Foundation of
Korea (grants 2016R1A2B2009063 and 2016M3A9D5A01952414
to J.K., and grant 2014R1A4A1007304 to Y.B.), the DGIST R&D
Program of the Ministry of Science, ICT & Future Planning (17-
BD-06, 17-BT-02), and the Korea University Research Grant
(K1504321).
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
5
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Communications
D. G. McLeod, J. W. Moul, D. Soppett, S. Srivastava, Cancer Res. 2000, 60,
6568–6572.
[43] K. Yoshikawa, H. Nakagawa, N. Mori, H. Watanabe, K. Touhara, Nat.
Chem. Biol. 2013, 9, 160–162.
[33] T. Cui, A. V. Tsolakis, S. C. Li, J. L. Cunningham, T. Lind, K. Oberg, V. Gian-
domenico, Eur. J. Endocrinol. 2013, 168, 253–261.
[44] O. Baud, S. Etter, M. Spreafico, L. Bordoli, T. Schwede, H. Vogel, H. Pick,
Biochemistry 2011, 50, 843–853.
[34] V. Giandomenico, T. Cui, L. Grimelius, K. Oberg, G. Pelosi, A. V. Tsolakis, J.
Mol. Endocrinol. 2013, 51, 277–286.
[35] D. Massberg, A. Simon, D. Haussinger, V. Keitel, G. Gisselmann, H.
Conrad, H. Hatt, Arch. Biochem. Biophys. 2015, 566, 100–109.
[36] X. Guo, Z. Yang, Q. Zhi, D. Wang, L. Guo, G. Li, R. Miao, Y. Shi, Y. Kuang,
Oncotarget 2016, 7, 30276–30294.
[45] S. Choi, Y. Kim, B. B. Park, S. Park, J. Park, K. Ok, J. Koo, Y. W. Jung, Y. H.
Jeon, E. H. Lee, K. S. Lee, Y. Byun, J. Mol. Struct. 2014, 1076, 600–605.
[46] A. G. Leslie, T. Warne, C. G. Tate, Nat. Struct. Mol. Biol. 2015, 22, 941–
942.
[47] K. L. Portman, J. Long, S. Carr, L. Briand, D. J. Winzor, M. S. Searle, D. J.
Scott, Biochemistry 2014, 53, 2371–2379.
[37] H. Saito, Q. Y. Chi, H. Y. Zhuang, H. Matsunami, J. D. Mainland, Sci. Sig-
naling 2009, 2, ra9.
[48] T. Warne, R. Moukhametzianov, J. G. Baker, R. Nehme, P. C. Edwards,
A. G. Leslie, G. F. Schertler, C. G. Tate, Nature 2011, 469, 241–244.
[38] D. M. Ferrero, D. Wacker, M. A. Roque, M. W. Baldwin, R. C. Stevens, S. D.
Liberles, ACS Chem. Biol. 2012, 7, 1184–1189.
[39] S. D. Liberles, L. B. Buck, Nature 2006, 442, 645–650.
[40] A. Keller, H. Y. Zhuang, Q. Y. Chi, L. B. Vosshall, H. Matsunami, Nature
2007, 449, 468–U466.
[41] H. Zhuang, H. Matsunami, Nat. Protoc. 2008, 3, 1402–1413.
[42] D. M. Rosenbaum, V. Cherezov, M. A. Hanson, S. G. Rasmussen, F. S.
Thian, T. S. Kobilka, H. J. Choi, X. J. Yao, W. I. Weis, R. C. Stevens, B. K. Ko-
bilka, Science 2007, 318, 1266–1273.
Manuscript received: December 8, 2016
Revised: March 3, 2017
Accepted Article published: && &&, 0000
Final Article published: && &&, 0000
&
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

COMMUNICATIONS
Beyond sensory: Dehydroacetic acid
analogues were synthesized and evalu-
ated as agonists of the ectopic odorant
receptor 895 (Olfr895), which is ex-
pressed in the bladder. The most prom-
ising compound exhibited strong activa-
tion of Olfr895, with an EC50 value of 9
nm in luciferase reporter assays. In vitro
Ca2+ imaging, chemotactic migration,
and site-directed mutagenesis experi-
ments indicate that this compound is
a highly potent and selective agonist
for Olfr895 and can be used to investi-
gate its physiological functions.
B. B. Park, N. Lee, Y. Kim, Y. Jae, S. Choi,
N. Kang, Y. R. Hong, K. Ok, J. Cho,
Y. H. Jeon, E. H. Lee, Y. Byun,* J. Koo*
&& – &&
Analogues of Dehydroacetic Acid as
Selective and Potent Agonists of an
Ectopic Odorant Receptor through
a Combination of Hydrophilic and
Hydrophobic Interactions
ChemMedChem 2017, 12, 1 – 7
www.chemmedchem.org
7
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ