Identification of cb1 receptor allosteric sites using force-biased mmc simulated annealing and validation by structure-activity relationship studies

Article abstract of DOI:10.1021/acsmedchemlett.9b00256

Positive allosteric modulation of the cannabinoid 1 receptor (CB1R) has demonstrated distinct therapeutic advantages that address several limitations associated with orthosteric agonism and has opened a promising therapeutic avenue for further drug development. To advance the development of CB1R positive allosteric modulators, it is important to understand the molecular architecture of CB1R allosteric site(s). The goal of this work was to use Force-Biased MMC Simulated Annealing to identify binding sites for GAT228 (R), a partial allosteric agonist, and GAT229 (S), a positive allosteric modulator (PAM) at the CB1R. Our studies suggest that GAT228 binds in an intracellular (IC) TMH1-2-4 exosite that would allow this compound to act as a CB1 allosteric agonist as well as a CB1 PAM. In contrast, GAT229 binds at the extracellular (EC) ends of TMH2/3, just beneath the EC1 loop. At this site, this compound can act as CB1 PAM only. Finally, these results were successfully validated through the synthesis and biochemical evaluation of a focused library of compounds.

Full text of DOI:10.1021/acsmedchemlett.9b00256

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Letter
Identification of CB1 Receptor Allosteric Sites Using Force-
Biased MMC Simulated Annealing and Validation by SAR Studies
Dow P. Hurst, Sumanta Garai, Pushkar M Kulkarni, Peter
Carlton Schaffer, Patricia H. Reggio, and Ganesh A Thakur
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00256 • Publication Date (Web): 10 Jul 2019
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Identification of CB1 Receptor Allosteric Sites Using Force-Biased
MMC Simulated Annealing and Validation by SAR Studies
Dow P. Hurst, Φ Sumanta Garai, † Pushkar M Kulkarni, † Peter C. Schaffer, † Patricia H. Reggio* Φ and
Ganesh A. Thakur*†
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Φ Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro NC USA
† Department of Pharmaceutical Sciences, Bouve
chusetts 02115, USA
́ College of Health Sciences, Northeastern University, Boston, Massa-
KEYWORDS: CB1 cannabinoid receptor, PAM binding site, MMC Simulated Annealing, structure-activity relationship.
ABSTRACT: Positive allosteric modulation of the CB1R has demonstrated distinct therapeutic advantages that addresses sev-
eral limitations associated with orthosteric agonism and has opened a promising therapeutic avenue for further drug devel-
opment. To advance the development of CB1R positive
allosteric modulators, it is important to understand the
molecular architecture of CB1R allosteric site(s). The
goal of this work was to use Force-Biased MMC Simulated
Annealing to identify binding sites for GAT228 (R), a par-
tial allosteric agonist and GAT229 (S), a PAM at the CB1R.
Our studies suggest that GAT228 binds in an intracellular
(IC) TMH1-2-4 exosite that would allow this compound
to act as a CB1 allosteric agonist as well as a CB1 PAM. In
contrast, GAT229 binds at the extracellular (EC) ends of
TMH2/3, just beneath the EC1 loop. At this site, this com-
pound can act as CB1PAM only. Finally, these results
were successfully validated through the synthesis and biochemical evaluation of a focused library of compounds.
INTRODUCTION:
CB1R PAM,5 whereas pregnenolone has been demonstrated
to have CB1R NAM activity.6 A small molecule GAT211 (Fig-
ure 1) was the first well- characterized CB1R ago-PAM.7 It
exhibited agonism through allosteric site to varying degrees
in the absence of orthosteric ligand and also potentiated
CB1R orthosteric activation.7 It displayed both PAM and ag-
onist activity in multiple cell lines expressing human recom-
binant CB1R and in mouse-brain membranes rich in native
CB1R.14 GAT211 was instrumental in establishing the ther-
apeutic role of CB1R PAMs in treating several diseases in
preclinical studies.8-11 Enantiomeric resolution of GAT211
led to identification of R-(+)- enantiomer GAT228 as a par-
tial allosteric agonist with a weak PAM activity. The S-(-) en-
antiomer GAT229, was a pure CB1 PAM and lacked intrinsic
activity in the biological systems examined. Our current
preclinical studies suggest that CB1R allosteric agonists,
pure PAMs and ago-PAMs can provide distinct therapeutic
advantages depending on the disease to be treated. To facil-
itate drug discovery and development of CB1R allosteric
modulators of this structural class, it is essential to under-
stand the molecular architecture of CB1R allosteric site(s).
Although CB1R has been crystallized recently in the active
and inactive form, it does not provide understanding of the
therapeutically relevant allosteric site(s).12-15 The goal of
The cannabinoid 1 receptor (CB1R) is one of the most
widely expressed metabotropic G protein-coupled receptor
(GPCR) in brain. It’s participation in various (patho)physio-
logical processes have made CB1 (in)activation a viable
therapeutic modality for treating prevalent unsolved medi-
cal problems including neurological/neurodegenerative
diseases, chronic and neuropathic pain, substance-use dis-
orders, obesity, and diabetes.1 Naturally occurring CB1R ag-
onists, including the endocannabinoids anandamide (AEA)
and 2-arachidonoylglycerol (2-AG) and the principal psy-
choactive cannabis constituent, Δ9-tetrahydrocannabinol
(9-THC), activate the receptor by engaging its orthosteric
site.2 Orthosterically acting agonists or antagonists have
been met with significant challenges in clinical advance-
ment due to their association with undesirable CNS side ef-
fects. From the alternative approaches pursued to modulate
the CB1R activity, allosteric modulation that targets topo-
graphically distinct site(s) has met with significant success
so far in preclinical settings.3-4 Positive allosteric modula-
tors (PAMs) enhance orthosteric ligand binding or receptor
activity, whereas negative allosteric modulators (NAMs) re-
duce orthosteric ligand binding or receptor activity. Endog-
enous ligands such as LipoxinA4 has been shown to act as a
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the present work was to use Force-Biased MMC Simulated
Annealing to identify binding sites for the allosteric agonist
GAT228 and pure PAM GAT229.
Page 2 of 6
F2.42 and W4.50) in the TMH1-2-4 region 1 (PDB-ID:
5UO9; see Figure 2).18 In the CB1R* state complexed with Gi
protein (PDB-ID: 6N4B; see Figure 2),18 a fourth aromatic
residue moves into the TMH1-2-4 region, F4.46. This resi-
due changes its position to occupy the TMH1-2-4 exosite by
undergoing a rotameric change (γ1g+ ➔ trans). In addition,
TMH2 comes closer to TMH4 on the IC side of the bundle.
This conformation of F4.46 allows it to form an aromatic
stack with W4.50.18
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RESULTS AND DISCUSSION:
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The GAT228 and GAT229 structures were broken into
fragments as shown in Figure 3. All fragments were pre-
pared with partial charges using the Amber 2002, a point-
charge force field for molecular mechanics simulations of
proteins
Figure 1. Representative CB1R allosteric modulators
Transmembrane helices (TMHs) in all Class A GPCRs have
varying lipid face sizes depending on their tilt and also their
relative position in the TMH bundle. Due to this architec-
ture, distinctive crevices exist on the lipid face of all Class A
GPCRs. Some of these crevices are large enough to form
binding exosites for lipid components like cholesterol or for
allosteric modulators. Hanson and co-workers described a
consensus IC binding site for cholesterol in a TMH1-2-4 ex-
osite at the β2-AR (PDB code 2RH1) that is present in 26%
of human Class A GPCRs.16 Another cholesterol IC exosite
has been identified at TMH1-7-Hx8 in the β2-AR (PDB code
2RH1) and the 5HT-2B (PDB code 4IB4) receptors.17 On the
extracellular (EC) side, cholesterol has been found to bind
at TMH2-3 and the EC1 loop in the α2A-AR (PDB code
4EIY).17
Figure 3. The fragments of GAT228 and GAT229 created for
MMC studies.
based on condensed-phase quantum mechanical calcula-
tions.19 Four MMC runs were performed in which our CB1R*
model was immersed in a box filled with copies of one of
these fragments (For details see SI, Figure S1). Analysis of
the MMC runs for GAT229 revealed that while each frag-
ment bound to multiple positions on the CB1R*, there was
only one region in which all fragments clustered in the cor-
rect spatial proximity for the S stereoisomer. This was at the
extracellular end of TMH2/3, just beneath the EC1 loop.
Y2.59, a polar residue that faces lipid was found to attract
the nitro group of the AS stereoisomer fragment, while
D2.63 consistently was found interacting with the N-H of
the indole fragment (Figure S1).
We have shown that the CB1 biased NAM, pregnenolone,
binds in the IC TMH1-7-Helix8 exosite that allows the mod-
ulator to limit the movement of TMH7, a movement im-
portant for β-arrestin biased signaling. 6 Recent CB1R crys-
tal structures reveal that at the CB1R IC TMH1-2-4 lipid fac
ing crevice, there are several aromatic residues (H2.41,
GAT228 AGO-PAM BINDING AT IC TMH1-2-4 EXOSITE
Figure 4 illustrates the binding site identified for GAT228
by MMC. This site is in the IC TMH1-2-4 exosite, that accom-
modates cholesterol in many Class A GPCRs. It is important
to note that CB1 does not have the cholesterol consensus
sequence motif identified for 26% of human Class A GPCRs16
at the TMH1-2-4 exosite. So, it is less likely that the CB1
TMH1-2-4 exosite binds cholesterol, leaving it available for
other ligands. The principal GAT228 interactions at this site
includes a strong dual hydrogen bond between the GAT228
NO2 group and R(148) (R(148) NH1 to Nitro O1, N-O dis-
tance, 2.8Å ; N-H—O, 169° and R(148) NH2 to Nitro O2, N-
O distance, 3.1 Å; N-H—O, 173°), while R(148) also is in-
volved in a cation-π interaction with the indole ring of
GAT228 (distance from R(148) Cz to indole five-membered
ring centroid, 3.8 Å; distance from R(182) Cz to indole six-
membered ring centroid, 4.6 Å).20 GAT228 also has a hy-
drogen bond between the indole N-H and H2.41 (H2.41 ND
to Nitro O2, N-O distance, 2.9Å; N-H—O, 175°). There are
also several aromatic-aromatic interactions: an aromatic
tilted T-stack between the phenyl ring attached to stereo-
center and F4.46 (tilted T, centroid-centroid distance 5.0 Å,
angle, 83°); an off-set parallel aromatic interaction between
Figure 2. The location of four key aromatic residues in the
IC TMH1-2-4 exosite region is shown. While H2.41, F2.42
and W4.50 have similar orientations in R and R*, F4.46
points into the orthosteric site in the inactive state (R), but
points into the TMH1-2-4 exosite in the CB1 activated state
in R*.
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F4.46 and the indole ring phenyl substituent (centroid-cen-
troid distance 4.2 Å, angle, 15°); and, an aromatic tilted T-
stack between W4.50 and the indole ring phenyl substituent
(centroid-centroid distance 5.0Å, angle, 79°). The total In-
teraction Energy for GAT228 at this site is -37.18 kcal/mol
and Glide Score was -6.5 kcal/mol (see Interaction Energy
Table, Table S1).
of CB1, making this ligand a non-orthosteric agonist. Be-
cause GAT228 promotes a receptor active state confor-
mation, it can also act as a PAM. In this case, F4.46 will be in
its R* conformation (γ1 = trans), because an orthosteric ag-
onist like CP55940 has activated CB1. GAT228 binding to
the exosite in this situation will stabilize the activated state
of CB1, thus acting as a PAM.
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Figure 5. This figure provides a view of the inactive (R; Figure
6A) and activated state (R*; Figure 6B) CB1 models. The view
is from the IC side of the TMH bundle looking towards EC. (A)
In the inactive state, F4.46 is in a γ1 =g+, it points into the or-
thosteric TMH2-3-4 pocket. (B) When CB1 activates, F4.46 un-
dergoes a γ1 g+ ➔ trans change, causing F4.46 to leave the or-
thosteric pocket and face into the TMH1-2-4 exosite.
Figure 4. (A) This figure illustrates the binding site identified
for GAT228 by MMC. This site is in the IC TMH1-2-4 exosite.
Interacting receptor residues are shown contoured at their
VDW radii and colored green. Atoms in GAT228 are also con-
toured at their VDW radii and colored magenta. (B) GAT228
docked in the TMH1-2-4 exosite is shown.
To study an allosteric ligand that affects the binding and
activity of an agonist, it is important to study its effect on the
agonist bound CB1 activated (R*) state. Recently, Saleh and
colleagues reported multiple adjunct binding sites in the
Ago-PAM activity of 2-phenylindole modulators such as
GAT228 using metadynamics studies in the ligand binding
pocket of the CB1 inactive (R) state crystal structure (PDB
5U09)22 in the absence or presence of the CB1 agonist,
CP55940. Since the authors did not see the receptor convert
to R* during their simulations, the GAT228 site they identi-
fied is a site that may influence the low-affinity state for
CP55940 that does not signal because it remains the R state.
Therefore, the GAT228 binding site identified may be less
relevant to the pharmacology of GAT228.
How could a ligand bound in an exosite be an agonist?
Like all Class A GPCRs, CB1 has charged residues at the IC
side of TMH3 (R3.50) and TMH6 (D6.30) that form a salt
bridge (called the “Ionic Lock”) that keeps CB1 in its inactive
state, unable to couple to G protein. When the ionic lock is
broken, either by ligand binding or as consequence of a mu-
tation, CB1 assumes its activated state conformation (R*).
We reported several years ago that a missense mutation
(F4.46(238)L) in the rat Cnr1 gene that encodes for CB1
represents a gain of function mutation that results in an ad-
olescent-like phenotype. 21 Cell based assays found that the
F4.46L mutant was highly constitutively active and con-
sistent with these results, molecular dynamics calculations
revealed that the toggle switch for CB1 activation and the
“Ionic Lock” was more frequently broken in the F4.46L mu-
tant compared to WT. When GAT228 binds in the IC TMH1-
2-4 exosite (see Figure 4), it forms an extended aromatic
cluster, interacting directly with F4.46 (holding the F4.46 in
its activated state γ1 = trans conformation). At the same
time, GAT228 can interact with H2.41 and W4.50.
GAT229 PAM BINDING AT TMH2-3-EC1 SITE
Figure 6 illustrates the EC TMH2-3-EC1 binding site iden-
tified for GAT229 by MMC. GAT229 (magenta) can bind in
this site in the presence of CP55940 (lavender). Figure 6
shows that when GAT229 interacts at its EC TMH2-3-EC1
site, its indole ring N-H hydrogen bonds with D2.63 (N-O
distance, 3.0Å; N-H—O, 141°), while D2.63 maintains its in-
teraction with K(373). In addition, the GAT229 NO2 group
hydrogen bonds with Y2.59 (O-O distance, 2.9Å ; O-H—O,
137°); the EC1 loop residue R(182) has a cation-π interac-
tion with the indole ring (distance from R(182) Cz to indole
five-membered ring centroid, 4.1 Å; distance from R(182)
Cz to indole six-membered ring centroid, 3.8 Å) and Y2.59
has an off-set parallel aromatic stack with the GAT229 in-
dole ring phenyl substituent (centroid-centroid distance
4.1 Å, angle, 11°). This last aromatic interaction is made
stronger by the creation of a triple stack as F3.27 has a
tilted-T aromatic stacking interaction with Y2.59 (centroid-
centroid distance 5.3 Å, angle, 80°). The net interaction en-
ergy of GAT229 in this complex is -32.73 kcal/mol and its
Glide Score was -4.5 kcal/mol (see Table S2 for Interaction
Energy Table).
Figure 5 provides a view of the inactive (R; Figure 5A) and
activated state (R*; Figure 5B) CB1R models. The view is
from the IC side of the TMH bundle looking towards EC. In
the inactive state, F4.46 is in a γ1 =g+ and points into the
orthosteric TMH2-3-4 pocket (Figure 5A). When CB1R acti-
vates, F4.46 undergoes a γ1 g+ ➔ trans change and F4.46
leaves the orthosteric pocket and faces into the TMH1-2-4
exosite (Figure 5B). In this latter case, TMH2 and TMH3 (on
IC end) can move toward TMH4 and away from TMH6,
stretching the R3.50/D6.30 Ionic Lock until it breaks and ac-
tivates CB1 (see Figure 5B). Thus, by its binding at the
TMH1-2-4 exosite, GAT228 binding can promote the F4.46
γ1 g+ ➔ trans rotameric change that may cause activation
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As discussed in the Introduction, the α2A-AR (PDB code
Page 4 of 6
yield. 2-Phenyl-1H-pyrrole, 14 was synthesized from aceto-
phenone, 13 using Trofimov Reaction. Finally, 14 was
treated with -nitro styrene using CF3CO2NH4 to yield Mi-
chael adduct, 15 in 91% yield (for details see Supporting In-
formation).
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4EIY) structure illustrates another cholesterol binding site
in Class A GPCRs that is on the extracellular (EC) side, where
cholesterol binds at the EC ends of TMH2-3 and the EC1
loop. As shown in Figure 6, our MMC results place GAT229
in this same site. Positive allosteric interactions typically
stabilize the receptor/orthosteric ligand complex in its acti-
vated state conformation. Although there are clear changes
on the IC side of GPCRs when they are activated (“Ionic
Lock” broken and TMH6 straightening), there are also
changes in EC loop conformations that take place during ac-
tivation. CB1 EC2 loop mutation studies performed in the
Kendall lab indicate that the EC2 loop moves toward the
TMH bundle when CB1 activates. 23 CB1 EC3 loop mutations
in our lab have indicated that the EC3 loop residue K(373)
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interacts directly with D2.63 when CB1 activates. In its
docking position, GAT229 stabilizes the EC3 loop confor-
mation in the activated state and therefore should act as a
CB1 PAM.
Figure 6. (A) This figure illustrates the EC TMH2-3-EC1 bind-
ing site identified for GAT229 by MMC. Receptor residues that
interact with GAT229 are shown contoured at their VDW radii
and colored green. Atoms in GAT229 are also contoured at their
VDW radii and colored magenta. (B) This figure shows key in-
teractions of GAT229 at its EC site.
CHEMISTRY and STRUCTURE-ACTIVITY RELATIONSHIP
STUDIES: To validate MMC results, we designed a focused
library of GAT211 analogs (Table 1) in which the key phar-
macophoric groups were removed and evaluated them for
their CB1R PAM activity using our previous published pro-
tocols.7
Significance of the NO2 group: Replacement of nitro
group of GAT211 (cAMP: EC50=230 nM and Emax=110%; -
arrestin2: EC50=940 nM and Emax=46%) with amine (-NH2,
2) and -CF3 (Compound 37)25 leads to dramatic loss in po-
tency and efficacy in both cAMP and -arrestin2 assays.
These data strongly support the MMC studies prediction
that a strong hydrogen bond interaction exist between the -
NO2 group and R(148) for GAT228 and Y2.59 amino acid for
GAT229 respectively (Fig. 4B and 6B) and it is important for
PAM activity. The CF3 group does not serve as efficient bi-
oisosteric replacement to the nitro group.
The amine analog of GAT211, 2 was synthesized by the
nitro-reduction of 1 (GAT211). Accordingly, nitro com-
pound, 1 was treated with NiCl2.6H2O and NaBH4 in metha-
nol to yield amine 2 in 81% yield. α, β-Unsaturated nitro
compound, 4 was synthesized from 2-phenyl-1H-indole-3-
carbaldehyde, 3 by treating with nitromethane in presence
of ammonium acetate. The double bond of 4 was selectively
reduced with NaBH4 to get compound, 5 in 72% yield. On
the other hand, 3-(2-nitro-1-phenylethyl)-1H-indole, 8 and
1-methyl-3-(2-nitro-1-phenylethyl)-2-phenyl-1H-indole, 9
were synthesized using Et4NBr and -nitro styrene under
reflux condition from 6 and 7 respectively in good yields. N-
Arylation of 8 was carried out using iodobenzene under mi-
crowave irradiation in DMSO to yield N-arylated analog 10.
The C2-cyclohexyl analog, 12 was synthesized from 2-cyclo-
hexyl-1H-indole, 11using literature known protocol in 63%
Significance of C2 phenyl ring: MMC results suggested
the C2 aromatic ring is important for allosteric activity due
to an aromatic tilted T-stack between C-2 phenyl ring of
GAT228 and F4.46 and triple stack as F3.27 has a tilted-T
aromatic stacking interaction with Y2.59 for GAT229 re-
spectively (Fig. 4B and 6B). Both analogs 8 and 10 lacking
the C2 phenyl ring (R2=H) were found to be inactive thus
signifying the importance of C2 aromatic ring. Interestingly,
when C2-phenyl group of GAT211 was replaced by cyclo-
hexyl ring the resulting compound 12 showed slight
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improvement in cAMP potency as well in efficacy (cAMP:
optimizations of a 2-phenylindole class of CB1R PAM for im-
proved potency and efficacy.
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8
EC50=992 nM and Emax=121%) when compared to 8 in
which the C2 phenyl ring was absent. This may be due to the
Van der Waals interaction of cyclohexyl group with key
amino acid, F4.46.
ASSOCIATED CONTENT
Supporting Information: Supporting Information: The
Supporting Information is available free of charge on the ACS
Publications website at DOI:
We also observed considerable loss in efficacy and po-
tency when the phenyl ring (R3) of 1 was replaced with hy-
drogen (R3=H, 5), which leads to the interruption of the pre-
dicted π-π interaction.
Synthetic procedures, analytical data, and MMC method (PDF)
9
Table 1. PAM activity of all GAT211 analogs.
AUTHOR INFORMATION
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Corresponding Authors
cAMP
EC50(nM)
-Arrestin2
EC50(nM)
Compound
*Email: g.thakur@northeastern.edu and phreggio@uncg.edu
Emax (%)
Emax (%)
1,
230
Author Contributions
110 ± 6.8
940 (540-1,800)
>10,000
46 ± 9.5
GAT211
(140-370)
GAT, PHR, DPH and SG designed and conducted experiments,
analyzed data, and contributed to the writing of the manu-
script. SG, PMK and PCS synthesized the analogs.
2
>10,000
NA
12
12
Compound
3725
Moderate PAM ac-
tivity @1uM
NA
NA
ACKNOWLEDGMENT
Funding Sources
5
8
5,528
>10,000
>10,000
>10,000
992
85
0
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
0
0
0
0
0
0
This work was supported by NIH/National Eye Institute, RO1
EY024717 (GAT), RO1 DA003934 (PHR) and KO5 DA021358
(PHR).
9
ABBREVIATIONS
0
CB1, cannabinoid receptor type 1; PAM, positive allosteric
modulator; ago-PAM, allosteric agonist and positive allosteric
modulator; EC, extracellular; TMH, IC, intracellular; TMHs,
Transmembrane helices; MMC, Metropolis Monte Carlo.
10
12
15
0
121
0
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Compounds were evaluated by Eurofins DiscoverX in inde-
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EC20 CP55,940.7
Significance of Indole ring: The pyrrole analog of
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