Carbon chain shape selectivity by the mouse olfactory receptor OR-I7

Article abstract of DOI:10.1039/c8ob00205c

The rodent OR-I7 is an olfactory receptor exemplar activated by aliphatic aldehydes such as octanal. Normal alkanals shorter than heptanal bind OR-I7 without activating it and hence function as antagonists in vitro. We report a series of aldehydes designed to probe the structural requirements for aliphatic ligand chains too short to meet the minimum approximate 6.9 ? length requirement for receptor activation. Experiments using recombinant mouse OR-I7 expressed in heterologous cells show that in the context of short aldehyde antagonists, OR-I7 prefers binding aliphatic chains without branches, though a single methyl on carbon-3 is permitted. The receptor can accommodate a surprisingly large number of carbons (e.g. ten in adamantyl) as long as the carbons are part of a conformationally constrained ring system. A rhodopsin-based homology model of mouse OR-I7 docked with the new antagonists suggests that small alkyl branches on the alkyl chain sterically interfere with the hydrophobic residues lining the binding site, but branch carbons can be accommodated when tied back into a compact ring system like the adamantyl and bicyclo[2.2.2]octyl systems.

Full text of DOI:10.1039/c8ob00205c

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Carbon chain shape selectivity by the mouse
olfactory receptor OR-I7†
Cite this: DOI: 10.1039/c8ob00205c
a
Min Ting Liu,‡a,b Jianghai Ho,‡c Jason Karl Liu,d Radhanath Purakait,
Uriel N. Morzan,d Lucky Ahmed, d Victor S. Batista,d Hiroaki Matsunami*c and
a,b,e
Kevin Ryan
*
The rodent OR-I7 is an olfactory receptor exemplar activated by aliphatic aldehydes such as octanal.
Normal alkanals shorter than heptanal bind OR-I7 without activating it and hence function as antagonists
in vitro. We report a series of aldehydes designed to probe the structural requirements for aliphatic ligand
chains too short to meet the minimum approximate 6.9 Å length requirement for receptor activation.
Experiments using recombinant mouse OR-I7 expressed in heterologous cells show that in the context of
short aldehyde antagonists, OR-I7 prefers binding aliphatic chains without branches, though a single
methyl on carbon-3 is permitted. The receptor can accommodate a surprisingly large number of carbons
(e.g. ten in adamantyl) as long as the carbons are part of a conformationally constrained ring system. A
rhodopsin-based homology model of mouse OR-I7 docked with the new antagonists suggests that small
alkyl branches on the alkyl chain sterically interfere with the hydrophobic residues lining the binding site,
but branch carbons can be accommodated when tied back into a compact ring system like the adamantyl
and bicyclo[2.2.2]octyl systems.
Received 24th January 2018,
Accepted 14th March 2018
DOI: 10.1039/c8ob00205c
rsc.li/obc
TM6 and TM7.6–10 Some receptors, like the rodent I7 receptor
(OR-I7; a.k.a. MOR103-15 and Olfr2),11 which we study here,
Introduction
The mammalian olfactory (a.k.a. odorant) receptors (ORs) appear to be highly specific for odorant ligand traits such as
form the largest family of G-protein coupled receptors (GPCRs) functional group and carbon chain length, but others appear
in the human and rodent genomes.1–3 Mice, for example, are to lack ligand specificity, at least in vitro.12,13 The requirement
predicted to have over 1000 different OR genes, while humans for volatility puts a limit on an odorant’s molecular weight and
have approximately 390 ORs out of a total of about 825 pre- number of polar functional groups, but terrestrial ORs have
dicted GPCR genes. Within the context of the GPCR struc- nevertheless evolved to bind innumerable small, usually hydro-
ture,4,5 each OR is expected to form within its 7-transmem- phobic ligands which offer a receptor limited opportunity for
brane alpha helical (TM) bundle a unique binding site with hydrogen-bonding and other polar interactions. Aliphatic
distinct ligand-binding properties resulting from the conver- odorants such as monoterpenoids (e.g. geraniol), sesquiterpen-
gence of receptor-specific residues, mainly from TM3, TM5, oids (e.g. santalols), and those derived from fatty acid biogenic
precursors (e.g. octanal) typically have only one polar func-
tional group, and many hydrocarbons are found among
aDepartment of Chemistry and Biochemistry, The City College of New York,
fragrant natural products. The specific manner in which the
New York, NY 10031, USA. E-mail: kryan@ccny.cuny.edu
bPh.D. Program in Chemistry, The Graduate Center of the City University of
ORs interact with the hydrocarbon portion of an odorant
remains entirely unknown.
New York, New York, NY 10016, USA
cDepartment of Molecular Genetics and Microbiology, and Neurobiology, Duke
Institute for Brain Sciences, Duke University Medical Center, Durham, NC 27710,
USA. E-mail: matsu004@mc.duke.edu
The terpenoid, fatty acyl-derived and hydrocarbon odorants
therefore present interesting molecular recognition puzzles
because their odor character appears to depend heavily on
attributes of their carbon skeletons, including features such as
length, size and shape. Using calcium imaging of dissociated
olfactory receptor neurons (ORNs, a.k.a. odorant sensory
neurons, OSNs), evidence for a correlation was previously
found between ligand conformational flexibility and the
number of different ORs a ligand activated when the sole polar
functional group (an aldehyde) and the number of carbons
dDepartment of Chemistry, Yale University, New Haven, CT 06520, USA
ePh.D. Program in Biochemistry, The Graduate Center of the City University of
New York, New York, NY 10016, USA
†Electronic supplementary information (ESI) available: Synthetic procedures
and characterization of analogues 2, 5, 6, 7, 8, 9, 10, 11 and 12; time-course dose
response data for mOR-I7 in Hana3A cells; and mOR-I7 homology model com-
parison with rhodopsin and rat OR-I7 structures. This material is available free
of charge via the journal website. See DOI: 10.1039/c8ob00205c
‡Contributed equally to this work.
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(eight) were kept constant.14 This evidence suggested that for about the same chain length as 2, was also found to be an
some odorants, carbon chains with more well-defined shapes antagonist, but was less potent than 2. When two carbons
activate fewer receptors than more flexible analogs, and may were added back to 2, to make (4-ethylcyclohexyl)ethanal, com-
thereby achieve unique olfactory codes15 by activating smaller pound 4, or to pentanal to make heptanal, the aldehydes
subsets of sensory neurons which, when mature, express only regained their ability to function as agonists. These and prior
one type of OR. For example, the cyclic muscone family of findings led us to conclude that the OR-I7 receptor requires a
odorants appears to activate only one major human OR,13,16–19 minimum of two molecular features in a ligand, and a third
while octanal, an odorant with an acyclic, conformationally feature if binding is to trigger activation. For binding, either as
unrestricted chain, activates 33–55 rat ORs.20 By having better- an agonist or antagonist, the aldehyde group is required
defined shapes and also lower entropy loss upon binding, con- (Fig. 1A, CHO recognition).11,14 Next, for binding, but not
formationally restricted ligands may be limited to binding necessarily activation, an aliphatic chain of at least five
fewer ORs than their more flexible relatives. To date, there are carbons total (as in pentanal, e.g.) is required.14 We refer to
no atomic-level structural data on how odorants bind olfactory octanal carbons-2 through -5 or -6, as the ligand “mid-region.”
receptors. In this report we attempt to study the variable of For aldehydes longer than hexanal, for example, the mid-
carbon chain shapes in the context of a known OR-antagonist region connects the aldehyde to the third feature, a small
pair.
hydrophobic group (e.g. carbons 7 and 8 in octanal) that must
While studying conformational restriction of the octanal reach a putative small hydrophobic group binding pocket for
carbon chain as a determinant of OR-I7 activation, a relation- OR-I7 activation. Aliphatic aldehydes shorter than the
ship was previously found between restriction of the chain and threshold of about 6.9 Å bind, but do not activate, OR-I7 and
whether an eight carbon aldehyde activated the receptor or can thus function as antagonists.14 We note that a similar
bound without activation.14 The present work is a follow-up to dependence of activation on alkyl chain length has been found
that study, whose findings we summarize here. Compound 1, for another Class A GPCR, the cannabinoid CB1 receptor. The
octanal, is a natural product activating ligand, or agonist, of CB1 agonist Δ9-tetrahydrocannabinol (THC) contains a simple
OR-I7. Conceptually joining octanal’s third and eighth n-pentyl chain whereas the CB1 antagonist Δ9-tetrahydrocan-
carbons, to make cyclohexylethanal (compound 2, Fig. 1), con- nabivarin (THCV) has an n-propyl chain but is otherwise iden-
formationally restricts the chain and prevents it from unfur- tical.21 Based on our initial short aldehyde data using rat
ling to an extended conformation. We found that this change OR-I7 there appeared to be a possible correlation between the
converted octanal into a non-activating ligand, or antagonist number of carbons in the ligand’s mid-region and the strength
(Fig. 1A).14 Pentanal, compound 3, which when extended has of the estimated IC50 for antagonists that can inhibit the acti-
vation of OR-I7 by co-applied octanal.14
In this study, to extend the previous OR-I7 findings we have
made a series of new OR-I7 antagonists to probe the mid-
region requirements of aldehyde antagonists. Each compound
was designed to have an extended length less than 6.9 Å to
avoid receptor activation. We used methyl and ethyl groups
and a variety of rings to increase the number of carbons within
the mid-region. We find that the OR-I7 binding site in contact
with the ligand’s mid-region has a surprising capacity for ali-
phatic carbons, but prefers dense, compact rings with no
branches, such as the bicyclo[2.2.2]octyl and adamantyl ring
systems.
Experimental
Method to estimate the maximum extended length of
aldehydes
Chem3D Ultra 12.0 software (CambridgeSoft) was used. The
structure of the aldehyde was drawn in its most extended con-
formation. The energy was minimized using the MM2 force
Fig. 1 OR-I7 ligands and structural design of new antagonists. (A) The
rodent OR-I7 olfactory receptor requires an aldehyde group and an ali-
field. The length was then measured from the carbonyl carbon
phatic carbon chain (mid-region) of at least five carbons for antagonist
ligand binding.14 A small alkyl group such as ethyl, extending beyond to the most remote carbon.
≈6.9 from the aldehyde, is required for receptor activation.
Compounds and are agonists; and are antagonists. (B)
Å
1
4
2
3
Chemical synthesis and characterization
Compounds designed in this study for antagonist structure–activity
relationship analysis. Estimated lengths are for the most extended con-
formations after energy minimization.
The synthesis and characterization of the tested compounds is
described in detail in the ESI.†
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Hana3A GloSensor cAMP assay
radiating from carbon-3 in 8 were also conformationally
restricted by incorporating them into the bicyclo[2.2.2]octyl
ring system (compound 9). For the set based on antagonist 2,
carbons were added incrementally at carbon-3 to make 10 and
11. Compound 11 can be viewed as an incompletely restricted
version of compound 9. Lastly, the adamantyl ring in 12,
which is larger than 2 by four carbons but, like compound 9,
is highly restricted and has no protruding small alkyl groups,
was used to probe the size limit of the mid-region.
The GloSensor cAMP Assay System (Promega) was used accord-
ing to the manufacturer’s instructions with slight modifi-
cations. A plasmid encoding Rho-tagged mouse OR-I7 (80 ng
per well) was transfected into the Hana3A cell line in 96-well
plate (Biocoat; Becton Dickinson Biosciences) format along
with plasmids encoding the human receptor trafficking
protein, RTP1S (10 ng per well), type 3 muscarinic acetyl-
choline receptor (M3-R) (10 ng per well), and pGloSensor
TM-22F (10 ng per well). Then, 18 to 24 h following transfec-
tion, cells were loaded with 2% GloSensor reagent for 2 h and
Synthesis of OR-I7 antagonists
The synthesis and characterization of designed antagonists
5–12 is described in detail in the ESI.†
treated with compounds in
a total volume of 74 µL.
Luminescence was measured using a Polarstar Optima plate
reader (BMG) with a time interval of 90 seconds per well. Data
were analyzed and IC50s were estimated using Prism 5.0 and
Microsoft Excel. Responses over t = 3–7.5 minutes were
summed, base-lined, normalized, and plotted versus odorant
concentration.
Antagonist testing
The IC50 values previously estimated for compounds 2 and 3
were obtained in dissociated rat ORNs infected with an adeno-
virus vector carrying the rat OR-I7.14,28 Despite one seminal
report that the mouse ortholog, which has 15 amino acid
differences overall but only 2 predicted to be in the TM2–
TM7 helical regions, prefers heptanal over octanal when
expressed in HEK293 cells,29 we and others have found that
both rat and mouse orthologs are aldehyde-specific and
respond similarly to octanal and heptanal.30,31 Here, we opted
to continue using the mouse ortholog but as expressed in
Hana3A cells. The Hana3A system consists of specially modi-
fied HEK293T cells, including the odorant receptor specific
G-protein, Golf, and avoids the need to use live rodents.32 We
found that in these cells the mouse OR-I7 gave a more consist-
ent octanal-induced cAMP response than rat OR-I7. To mini-
mize the possibility of any differential evaporation among the
ligands, which vary in molecular weight, we also switched from
the original luciferase reporter gene system to the GloSensor™
reporter, which detects the second messenger cAMP as it is pro-
duced in response to agonist-receptor binding.33
The response vs. time plot for different concentrations of
octanal applied alone is shown in Fig. 2A. To be an antagonist,
a compound must not activate the receptor, and using this
reporter system we confirmed that compound 2 is not a mouse
OR-I7 agonist (Fig. 2B). When compound 2 was co-applied at
increasing concentrations with a constant concentration of
octanal (5 μM), the response to octanal was reduced in a dose-
dependent manner (Fig. 2C). This result showed that 2 antago-
nized the activation of mouse OR-I7 by octanal, as it did in the
rat homolog expressed in neurons.14 To obtain an OR-I7
response vs. concentration plot for estimating IC50s, we
summed the reporter response over the period of highest
Homology model construction and ligand docking
A mouse OR-I7 homology model was constructed beginning
with a previously published rat OR-I7 ortholog model.22 The
initial rat model was based on crystallographic data taken
from rhodopsin PDB entry 1U19. The mouse model included
the following rat-to-mouse ortholog substitutions (single-letter
amino acid abbreviations; underscore indicates a predicted
helical position in TM2-TM7, superscripts are Ballesteros–
Weinstein numbering23 for TM residues): V26A1.33, M44I1.51
I48T1.55, K90E, V I5.41 L7.49
, F , D301E and R304K. A rat/
,
̲
̲
̲
̲
̲
̲2̲9̲0̲ ̲
mouse OR-I7 alignment with predicted helical regions can be
found in this reference.24 The ligand binding pocket of the
mOR-I7 was predicted using the SiteMap module of Maestro
software package (Version 10.2, Schrödinger, LLC, New York,
NY, 2015). Protonation states of amino acids were assigned by
using the PROPKA module, followed by structural relaxation
using the preparation wizard tool.25 The docking configur-
ations of the ligands were analyzed by using the Glide module
of Maestro.26 Short (20 ns) molecular dynamics simulations
were performed using the CHARMM force field, as
implemented in NAMD.27 During simulations, both octanal
and the antagonists were observed to sample the different
aldehyde group orientations shown in Fig. 5.
Results and discussion
Antagonist design
To probe the structural requirements of the OR-I7 ligand mid- cAMP production, from 3 to 7.5 minutes following addition of
region, but without extending into the receptor’s small hydro- the odorant(s) to the stimulation medium (Fig. 2C). This
phobic binding pocket beyond 6.9 Å from the aldehyde group, measurement allowed us to estimate the EC50 of octanal
we began with two previously identified antagonists, pentanal (0.7 μM, confidence interval 0.35–1.4 μM) and then to estimate
(IC50 460 μM in rat OR-I7) and cyclohexylethanal (compound 2, the IC50 values for the designed aldehyde antagonists. We note
IC50 37 μM, also in rat OR-I7).14 As shown in Fig. 1B, carbons that day-to-day variation in the Hana3A cells, plasmid transfec-
were added incrementally to pentanal at carbon-3, to produce tion efficiency and the cAMP assay response prevented the cal-
compounds 5–8. (Compound 5 is the only chiral compound in culation of absolute IC50 values, but relative efficacies
the set and was made in racemic form.) The three ethyl groups remained consistent during preliminary testing. For this
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Fig. 2 Relative mOR-I7 response elicited by octanal (an agonist) with or
without antagonist 2. Hana3A cells expressing the mouse OR-I7 recep-
tor were exposed to increasing concentrations of octanal and/or com-
pound 2 (cyclohexylethanal) and the rise in cAMP was monitored for
24 minutes using the GloSensor system. (A) Time course plot for
increasing concentration of octanal. (B) Time course plot for increasing
concentration of cyclohexylethanal, 2. (C) Time course plot for octanal
at 5 µM co-applied with increasing concentrations of 2. The summed
response between 3 min and 7.5 min (indicated by dash lines) was used
to create a point for dose–response curves. Here and in Fig. 3 and 4,
error bars indicate the average SEM of six replicates run in the same
plate.
Fig. 3 Antagonist dose–response plots for aldehyde antagonists 3, 5, 6,
7, 8, 9. Activation dose–response curves for octanal, and octanal co-
applied with each designed antagonist. The summed cAMP level for
each concentration between 3 and 7.5 min provided one data point in
the dose–response plots. (A) Compounds were applied individually to
Hana3A cells expressing mouse OR-I7. The responses were compared to
that of octanal, a known agonist of OR-I7. Octanal EC50 in this experi-
ment was estimated at 0.21 µM (confidence interval, 0.11–0.4 µM). (B)
Inhibition dose–response curves. Each compound shown in panel C was
tested for its ability to antagonize mouse OR-I7 in the presence of 5 μM
co-applied octanal. The “octanal” curve indicates that additional octanal
was added in place of antagonist (filled circles) to show whether agonist
was saturating or close to saturating. (C) IC50 values estimated from the
reason, we evaluated the candidate antagonists side-by-side
with previously studied antagonists 2 and 3 to obtain the most
accurate structure–activity comparison. Compounds grouped dose–response curves.
within Fig. 3 and 4 were tested with six replicates in the same
experiment.
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shorter than 6.9 Å, activated mouse OR-I7. The results, sum-
marized in Fig. 3A and 4A showed that none of these alde-
hydes activated Hana3A cells expressing the mouse OR-I7.
These experiments provided further evidence that the aldehyde
length vs. activation relationship previously found in the rat
OR-I7 also held for the mouse OR-I7 ortholog.
Observing the response to 5 μM octanal in the presence of
increasing concentrations of each inhibitor allowed us to esti-
mate the IC50 of each compound, subject to the limitations of
the assay described above. Inhibition plots are shown for com-
pounds 3, and 5–9 (Fig. 3B), and for compounds 2 and 9–12
(Fig. 4B). The estimated IC50 values are listed below the struc-
tures in the C panels of the same figures. Within each figure,
all compounds were tested side-by-side on the same day on
cells from the same Hana3A culture.
Structure activity relationship of the antagonists
Pentanal, 3, but not butanal, was previously shown to function
as a weak rat OR-I7 antagonist,14 while compound 2, which is
about the same length as pentanal but has eight carbons, was
eight-fold more potent as an antagonist. One simplistic
interpretation of this difference is that increasing the number
of carbons on the aldehyde buries more hydrophobic surface
area upon binding OR-I7 (i.e. a favorable hydrophobic effect
contribution) and also increases the van der Waals contact
with the part of the receptor binding site in contact with the
ligand’s mid-region. As shown in Fig. 3, we tested this possi-
bility by adding carbons to pentanal, beginning at carbon-3
rather than carbon-2 so as not to risk interfering with aldehyde
recognition.11 Adding one carbon to make compound 5
increased potency 5-fold, but additional carbons progressively
decreased potency (compounds 6, 7, 8) until the three alkyl
groups of compound 8 were tied back into the conformation-
ally restricted bicyclo[2.2.2]octyl ring system (compound 9),
which was the most potent antagonist in this series. Thus,
adding one carbon to carbon-3 was favorable but additional
carbons were unfavorable unless they were conformationally
restricted in the bicyclo[2.2.2]octyl ring system.
We used a similar approach beginning with antagonist 2,
and a similar trend was observed: the methyl and ethyl groups
of 10 and 11, respectively, were unfavorable, but tying com-
pound 11’s ethyl group back, as in compound 9, was once
again favorable in comparison (Fig. 4). We expanded the size
of the bicyclo[2.2.2]octyl ring system − without adding methyl
or ethyl groups and without exceeding 6.9 Å in length − by
attaching the adamantyl group to ethanal (compound 12).
This aldehyde, which was noticed to have a distinct camphor-
aceous odor, was 3-fold weaker in potency than 9, suggesting
that while the OR-I7 binding site mid-region can accommodate
this large ring system, it may be approaching the site’s size
limit.
Fig. 4 Antagonist dose–response plots for aldehyde compounds 2, 9,
10, 11, and 12. Activation dose–response curves for octanal and octanal
co-applied with each designed antagonist. The summed cAMP level for
each concentration between 3 and 7.5 min provided one data point in
the dose–response plots. (A) Compounds were applied individually to
Hana3A cells expressing mouse OR-I7. The responses were compared to
that of octanal, a known agonist of OR-I7. Octanal EC50 in this experi-
ment was estimated at 0.7 µM (confidence interval 0.35–1.4 µM). (B)
Inhibition dose–response curves. Each compound shown in panel C was
tested for its ability to antagonize mouse OR-I7 in the presence of 5 μM
co-applied octanal. The “octanal” curve indicates that additional octanal
was added in place of antagonist (filled circles) to show whether agonist
was saturating or close to saturating. (C) IC50 values estimated from the
dose–response curves.
In combination with previous reports on the rodent
OR-I7,11,14,20,22,31,34,35 our results are consistent with the view
Antagonists bind to their receptors without activating them. that the part of the OR-I7 binding site in contact with the
The first step in evaluating the new compounds was therefore ligand’s mid-region has evolved to accommodate carbon
to see whether any of the proposed antagonists, which are all chains with unbranched alkyl chains, i.e. a chain of methylene
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groups, such as carbons-3 through -6 of octanal. This interpret- conformation of the receptor. Without structural information
ation suggests that the small alkyl protrusions of compounds it is impossible to discern between these scenarios. Overall,
6, 7, 8, 10 and 11 may be interpreted by the receptor as alkyl these data support an interpretation where OR-I7 detects
chain branches, which are not found on the typical fatty acyl unbranched aliphatic aldehydes and citronellal-like terpene
chain aldehyde odorants that OR-I7 is known to detect.11 aldehydes, but when the chain is shorter than about 6.9 Å,
Thus, the middle of the OR-I7 site accommodates a chain of binding fails to stabilize the activated form of the receptor and
methylene groups passing through it, but anchored at one end the ligand acts as an antagonist.
by the aldehyde recognition site and, for activation only, at the
other end by the small hydrophobic group binding site. The
Structural model of the mouse OR-I7 binding site
ability to accommodate the bicyclo[2.2.2]octyl system may We previously built a rhodopsin-based homology model of
reflect accommodation of a “jump rope” movement of the the unactivated rat OR-I7 and predicted an orthosteric
octanal’s methylene groups between the anchors. An apparent binding site by looking for voids large enough to accommo-
exception to the unbranched chain preference was compound date octanal.22 To understand the in vitro data presented
5, whose single 3-methyl group improved potency compared to above, we adapted this model to the mouse I7 ortholog. The
pentanal. Compound 5 is identical to the first five carbons in ligand binding sites for aldehydes 2, 8, 9, 10 and octanal were
the carbon chain of the terpene citronellal. Interestingly, citro- predicted using the SiteMap module of the Maestro software
nellal, which has an aldehyde group and a length exceeding package.
6.9 Å, is almost as good a rat OR-I7 agonist as octanal.11 We
The predicted ligand binding site, modeled using com-
suggest that the OR-I7 features that allow citronellal binding pound 10, is shown in relation to the overall predicted recep-
in the mid-region also allow compound 5 to bind − but as an tor structure in Fig. 5A. The site is in the upper half of the
antagonist because its chain does not extend beyond the 6.9 Å receptor (closer to the extracellular side) and formed by TM3-
threshold required for activation.14 The idea that shortened TM6. The site is approximately the same binding site pre-
forms of good OR-I7 agonists make good OR-I7 antagonists dicted for octanal in the rat I7 ortholog and that found experi-
(compare 5 with citronellal, and 2 with 4) raises the possibility mentally for retinal in rhodopsin (see the ESI Fig. S5† for a
that the binding site’s mid-region is the same for agonists and comparison).22 In Fig. 5A we have highlighted four residues
antagonists alike, and the ligand does not change its location of mouse OR-I7 homologous to residues predicted in the
during activation. Alternatively, longer aldehydes may be able mouse MOR256-3 to mark the ligand binding site in that
to move into a different location where they stabilize the active odorant receptor: F1093.32, G1133.36, A2085.43 and Y2576.48
,
Fig. 5 A rhodopsin-based mouse OR-I7 homology model docked with selected antagonists. Representative docking configurations for the mouse
OR-I7 homology model and antagonist 10 (panel A), 2 (panel B), 8 (panel C), 9 (panel D) and octanal (panel E). In panel A, the global location of the
predicted binding site is shown, with ligand presented as a space-filling model. The four numbered OR-I7 residues, 109, 113, 208 and 257, corres-
pond to residues predicted in homology models for other odorant receptors to define the most likely orthosteric ligand-binding site, as noted in the
text.
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which correspond in MOR256-3 to F1043.32
, ,
G1083.36
Conflicts of interest
G2035.43 and Y2526.48, respectively.36 Though not coinciding
exactly, this comparison predicts that the binding cavity is The authors have no conflicts of interest to declare.
close to that of MOR256-3 and the binding sites predicted for
several other odorant receptors for which models have been
made.8,37–39 In the continuing absence of any OR structural
biology data, a consensus among binding site predictions is
building increased confidence in their validity. Closer inspec-
tion of the mOR-I7 site reveals a binding cavity lined with
hydrophobic amino acids, such as F1093.32, L1103.33, and the
aromatic rings of Y2576.48 and Y2646.55 (Fig. 5B–E).
Hydrogen-bonding interactions with the aldehyde were pre-
dicted for these two tyrosines and K1644.60, a protonated
amino acid residue that is also capable of forming a hydrogen
bond with Y2646.55 and a salt-bridge with the negatively
charged D2045.39. Based on this model, we speculate that the
conformationally flexible ethyl groups found in relatively
lower potency ligands like 8 (e.g. Fig. 5C) and 11 sterically
interfere with some of the hydrophobic residues lining the
site, e.g. L1103.33, while the conformationally restricted ring
systems of 2 (Fig. 5B) and 9 (Fig. 5D), being more compact
and unbranched, are better accommodated by mOR-I7. For
comparison, a representative view of octanal in the model’s
binding site is shown in Fig. 5E.
Acknowledgements
This work was supported in part by the U. S. Army Research
Laboratory and the U. S. Army Research Office under grant
number W911NF-13-1-0148 (to K. R.), NIH grants DC012095
and DC014423 (to H. M.) and NSF grant CHE-1465108 (to
V. S. B.). Additional infrastructural support at the City College
of New York was provided through grant 3G12MD007603-30S2
from the National Institute on Minority Health and Health
Disparities. R. P. gratefully acknowledges support from a
National Science Foundation REU grant (DBI-1560384). We
thank Dr Lijia Yang for mass spectroscopy analysis, and
NERSC for high-performance computing time. We thank an
anonymous reviewer for bringing to our attention the THC/
THCV analogy.
References
1 L. Buck and R. Axel, Cell, 1991, 65, 175–187.
2 G. Glusman, I. Yanai, I. Rubin and D. Lancet, Genome Res.,
2001, 11, 685–702.
3 T. Olender, D. Lancet and D. W. Nebert, Hum. Genomics,
2008, 3, 87–97.
Conclusions
The new aldehyde odorants studied here were designed to probe
the carbon chain requirements for antagonizing the mouse
OR-I7 receptor. The results show that the receptor prefers chains
of methylene groups, disfavors branches except for a single
methyl on carbon-3 and can accommodate a surprisingly large
number of carbons (e.g. ten in adamantyl) as long as they are
part of conformationally constrained ring system like cyclohexyl,
bicyclo[2.2.2]octyl or adamantyl. Thus, in the context of antagon-
ist ligands, the part of the receptor in contact with the mid-
region imposes shape selectivity for compact carbon rings. In the
context of an agonist, the ligand mid-region has to also serve to
spatially orient the two end groups − the aldehyde and last two
4 V. Katritch, V. Cherezov and R. C. Stevens, Annu. Rev.
Pharmacol. Toxicol., 2013, 53, 531–556.
5 B. K. Kobilka, Biochim. Biophys. Acta, 2007, 1768, 794–807.
6 T. Abaffy, A. Malhotra and C. W. Luetje, J. Biol. Chem.,
2007, 282, 1216–1224.
7 C. A. de March, Y. Yu, M. J. Ni, K. A. Adipietro,
H. Matsunami, M. Ma and J. Golebiowski, J. Am. Chem.
Soc., 2015, 137, 8611–8616.
8 L. Gelis, S. Wolf, H. Hatt, E. M. Neuhaus and K. Gerwert,
Angew. Chem., Int. Ed., 2012, 51, 1274–1278.
9 O. Man, Y. Gilad and D. Lancet, Protein Sci., 2004, 13, 240–
254.
carbons of octanal, separated optimally by five carbons − as 10 K. Schmiedeberg, E. Shirokova, H. P. Weber, B. Schilling,
required for activation. A homology model predicts the location
of the antagonist binding site, which is close to the ligand site
predicted for several other ORs and rhodopsin.
W. Meyerhof and D. Krautwurst, J. Struct. Biol., 2007, 159,
400–412.
11 R. C. Araneda, A. D. Kini and S. Firestein, Nat. Neurosci.,
2000, 3, 1248–1255.
12 X. Grosmaitre, S. H. Fuss, A. C. Lee, K. A. Adipietro,
H. Matsunami, P. Mombaerts and M. Ma, J. Neurosci.,
2009, 29, 14545–14552.
Abbreviations
GPCR G protein-coupled receptor
13 K. Nara, L. R. Saraiva, X. Ye and L. B. Buck, J. Neurosci.,
2011, 31, 9179–9191.
OR
Olfactory or odorant receptor
ORN Odorant receptor neuron, aka OSN, olfactory sensory 14 Z. Peterlin, Y. Li, G. Sun, R. Shah, S. Firestein and K. Ryan,
neuron
Chem. Biol., 2008, 15, 1317–1327.
TM
Transmembrane
15 B. Malnic, J. Hirono, T. Sato and L. B. Buck, Cell, 1999, 96,
713–723.
cAMP Cyclic adenosine monophosphate
IC50
EC50
Half maximal inhibition constant
Half maximal binding constant
16 E. Block, V. S. Batista, H. Matsunami, H. Zhuang and
L. Ahmed, Nat. Prod. Rep., 2017, 34, 529–557.
This journal is © The Royal Society of Chemistry 2018
Org. Biomol. Chem.

View Article Online
Paper
Organic & Biomolecular Chemistry
17 E. Block, S. Jang, H. Matsunami, S. Sekharan, B. Dethier, 27 J. C. Phillips, R. Braun, W. Wang, J. Gumbart,
M. Z. Ertem, S. Gundala, Y. Pan, S. Li, Z. Li, S. N. Lodge,
M. Ozbil, H. Jiang, S. F. Penalba, V. S. Batista and
E. Tajkhorshid, E. Villa, C. Chipot, R. D. Skeel, L. Kale and
K. Schulten, J. Comput. Chem., 2005, 26, 1781–1802.
H. Zhuang, Proc. Natl. Acad. Sci. U. S. A., 2015, 112, E2766– 28 H. Zhao and S. Firestein, Science, 1998, 279, 237–242.
E2774.
29 D. Krautwurst, K. W. Yau and R. R. Reed, Cell, 1998, 95,
917–926.
18 T. S. McClintock, K. Adipietro, W. B. Titlow, P. Breheny,
A. Walz, P. Mombaerts and H. Matsunami, J. Neurosci., 30 T. Bozza, P. Feinstein, C. Zheng and P. Mombaerts,
2014, 34, 15669–15678. J. Neurosci., 2002, 22, 3033–3043.
19 M. Shirasu, K. Yoshikawa, Y. Takai, A. Nakashima, 31 Y. Li, Z. Peterlin, J. Ho, T. Yarnitzky, M. T. Liu, M. Fichman,
H. Takeuchi, H. Sakano and K. Touhara, Neuron, 2014, 81,
165–178.
M. Y. Niv, H. Matsunami, S. Firestein and K. Ryan, ACS
Chem. Biol., 2014, 9, 2563–2571.
20 R. C. Araneda, Z. Peterlin, X. Zhang, A. Chesler and 32 H. Saito, M. Kubota, R. W. Roberts, Q. Chi and
S. Firestein, J. Physiol., 2004, 555, 743–756. H. Matsunami, Cell, 2004, 119, 679–691.
21 A. Thomas, L. A. Stevenson, K. N. Wease, M. R. Price, 33 F. Fan, B. F. Binkowski, B. L. Butler, P. F. Stecha,
G. Baillie, R. A. Ross and R. G. Pertwee, Br. J. Pharmacol.,
2005, 146, 917–926.
M. K. Lewis and K. V. Wood, ACS Chem. Biol., 2008, 3, 346–
351.
22 M. D. Kurland, M. B. Newcomer, Z. Peterlin, K. Ryan, 34 M. S. Singer, Chem. Senses, 2000, 25, 155–165.
S. Firestein and V. S. Batista, Biochemistry, 2010, 49, 6302–6304. 35 R. S. Smith, Z. Peterlin and R. C. Araneda, Methods Mol.
23 J. A. Ballesteros and H. Weinstein, Methods Neurosci., 1995,
25, 366–428.
24 Y. Li, Z. Peterlin, J. Ho, T. Yarnitzky, M. T. Liu, M. Fichman,
M. Y. Niv, H. Matsunami, S. Firestein and K. Ryan, ACS
Chem. Biol., 2014, 9, 2563–2571.
Biol., 2013, 1003, 203–209.
36 C. A. de March, Y. Yu, M. J. Ni, K. A. Adipietro,
H. Matsunami, M. Ma and J. Golebiowski, J. Am. Chem.
Soc., 2015, 137, 8611–8616.
37 O. Baud, S. Etter, M. Spreafico, L. Bordoli, T. Schwede,
H. Vogel and H. Pick, Biochemistry, 2011, 50, 843–853.
25 G. M. Sastry, M. Adzhigirey, T. Day, R. Annabhimoju and
W. Sherman, J. Comput.-Aided Mol. Des., 2013, 27, 221– 38 S. Katada, T. Hirokawa, Y. Oka, M. Suwa and K. Touhara,
234. J. Neurosci., 2005, 25, 1806–1815.
26 R. A. Friesner, R. B. Murphy, M. P. Repasky, L. L. Frye, 39 S. Sekharan, M. Z. Ertem, H. Zhuang, E. Block,
J. R. Greenwood, T. A. Halgren, P. C. Sanschagrin and
H. Matsunami, R. Zhang, J. N. Wei, Y. Pan and V. S. Batista,
D. T. Mainz, J. Med. Chem., 2006, 49, 6177–6196.
Biophys. J., 2014, 107, L5–L8.
Org. Biomol. Chem.
This journal is © The Royal Society of Chemistry 2018