Dopamine D4 Receptor-Selective Compounds Reveal Structure-Activity Relationships that Engender Agonist Efficacy

Article abstract of DOI:10.1021/acs.jmedchem.9b00231

The dopamine D₄ receptor (D₄R) plays important roles in cognition, attention, and decision making. Novel D₄R-selective ligands have promise in medication development for neuropsychiatric conditions, including Alzheimer's d

Full text of DOI:10.1021/acs.jmedchem.9b00231

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Article
Dopamine D4 Receptor-Selective Compounds Reveal Structure-
Activity Relationships that Engender Agonist Efficacy
Thomas M Keck, R. Benjamin Free, Marilyn M Day, Sonvia L Brown, Michele S Maddaluna, Griffin Fountain,
Charles Cooper, Brooke Fallon, Matthew Holmes, Christopher Stang, Russell Burkhardt, Alessandro
Bonifazi, Michael P Ellenberger, Amy Hauck Newman, David R. Sibley, Chun Wu, and Comfort A. Boateng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00231 • Publication Date (Web): 18 Mar 2019
Downloaded from http://pubs.acs.org on March 18, 2019
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Dopamine D Receptor-Selective Compounds
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Reveal Structure-Activity Relationships that
Engender Agonist Efficacy
Thomas M. Keck,+ R. Benjamin Free, Marilyn M. Day, Sonvia L. Brown, Michele S.
,‡,#
≠,#
≠
†
†
+
+
+
Maddaluna, Griffin Fountain, Charles Cooper, Brooke Fallon, Matthew
+
≠
$
$
Holmes, Christopher T. Stang, Russell Burkhardt, Alessandro Bonifazi, Michael P.
$
$
≠
+
†,#,*
Ellenberger, Amy H. Newman, David R. Sibley, Chun Wu, and Comfort A. Boateng
+Department of Chemistry & Biochemistry, Department of Molecular & Cellular Biosciences,
College of Science and Mathematics, Rowan University, 201 Mullica Hill Road, Glassboro, New
Jersey, 08028, United States
‡Cooper Medical School of Rowan University, 401 Broadway, Camden, New Jersey 08103,
United States
†Department of Basic Pharmaceutical Sciences, Fred Wilson School of Pharmacy, High Point
University, One University Parkway, High Point, North Carolina, 27268, United States
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$Medicinal Chemistry Section, Molecular Targets and Medications Discovery Branch, National
Institute on Drug Abuse-Intramural Research Program, National Institutes of Health, 333 Cassell
Drive, Baltimore, Maryland, 21224, United States
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≠Molecular Neuropharmacology Section, National Institute of Neurological Disorders and
Stroke-Intramural Research Program, National Institutes of Health, Bethesda, Maryland, 20892,
United States
#
Equally Contributing Authors, * Corresponding Author
KEYWORDS. Dopamine D receptor, partial agonist, antagonist, phenylpiperazine, cognition,
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molecular modeling and simulation
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ABSTRACT
The dopamine D receptor (D R) plays important roles in cognition, attention, and decision
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making. Novel D R-selective ligands have promise in medication development for
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neuropsychiatric conditions, including Alzheimer’s disease and substance use disorders (SUD).
To identify new D R-selective ligands, and to understand the molecular determinants of agonist
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efficacy at D R, we report a series of eighteen novel ligands based on the classical D R agonist A-
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12997 (1, 2-(4-(pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide). Compounds were profiled
using radioligand binding displacement assays, β-arrestin recruitment assays, cAMP inhibition
assays, and molecular dynamic computational modeling. We identified several novel D R-
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selective (K ≤ 4.3 nM and >100-fold vs. other D -like receptors) compounds with diverse partial
i
2
agonist and antagonist profiles, falling into three structural groups. These compounds highlight
receptor-ligand interactions that control efficacy at D -like receptors and may provide insights to
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targeted drug discovery leading to a better understanding of the role of D Rs in neuropsychiatric
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disorders.
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INTRODUCTION
The dopamine D receptor (D R) is a G protein-coupled receptor and a member of the D -like
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subfamily of dopamine receptors (including D R, D R and D R). D -like receptors have high
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sequence homology and share a G -coupled signaling mechanism, but differ substantially in
αi/o
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localization within the brain and at the subcellular level. Compared with D Rs and D Rs, D Rs
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have the lowest level of expression in the brain, and show a unique distribution pattern with most
located in the prefrontal cortex (PFC) and hippocampus. The other D -like receptors are primarily
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in the striatum, basal ganglia and pituitary gland regions; regions associated D R-targeting
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antipsychotic drugs and the motor and endocrine side-effects commonly observed with them.2-3 In
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contrast, D Rs expressed in PFC and hippocampus affect attention, exploratory behavior, and
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performance in novel object recognition4-5 and inhibitory avoidance cognitive tasks. Therefore,
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pharmacological activation of D Rs may be useful to treat cognitive deficits associated with
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schizophrenia7-10 and ADHD.10-11 Additional research has explored D R agonism as a strategy to
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reduce the adverse effects of opioid drugs like morphine.12-13 D R antagonism may be useful to
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treat substance use disorders (SUDs), particularly psychostimulant addiction, and L-DOPA-
induced dyskinesias.10, 14-20 The importance of targeting D Rs in treating these complex
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pathologies, especially in regards to the extent of receptor activation or inhibition, remains
unknown, partially due to a lack of suitable compounds for investigating these pathways.
A-412997 (1, 2-(4-(pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide, Figure 1) was initially
characterized as a “full agonist” (83% intrinsic activity) at D R, with high selectivity over D R
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21-22
and D R and in vivo effects that included induction of penile erection in rats.
Subsequent in
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vivo evaluations showed improved cognitive performance in social recognition tasks, novel object
recognition tasks, and 5-trial repeated acquisition inhibitory avoidance tasks following treatment
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by 1 (or similar D R agonists PD168077 (2) and CP226269 (3)); suggesting an important role for
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D R signaling in mediating short-term memory and cognition.5, 23
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O
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CN
NH
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(PD 168077)
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(A-412997)
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H
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(CP-226269)
Figure 1. Three classic D R-selective partial agonists.
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The goals of this study were to develop new D R agonists with a range of efficacy levels and to
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identify the molecular components that engender ligand efficacy at D R. To that end, we employed
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a rational drug design strategy incorporating classic structure-activity relationship (SAR) analysis
around lead compound 1. These studies were enhanced by detailed in silico molecular dynamics
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simulations exploiting the recently reported crystal structure of D R. Furthermore, comparative
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analyses were done using the D R crystal structure and the recently reported D R structure.
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We synthesized a library of analogues primarily featuring modifications in the phenylpiperidinyl
region of 1, with additional variations in linker chain length and substitutions on the amidylphenyl
region. Following extensive in vitro analyses, including binding and functional studies, we
determined that selected modifications resulted in novel analogues with improved subtype
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selectivity. Furthermore, we identified three classes of modifications that resulted in altered
efficacy profiles at all D -like receptors. In order to determine key receptor-ligand interactions,
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and identify the molecular substrates of a putative “efficacy switch,” the library was docked in
receptor models of D R, D R, and D R using molecular dynamics simulations.
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CHEMISTRY
Ligands were synthesized as outlined in Scheme 1 using routine N-alkylation reactions previously
reported.21, 27 The commercially available m-toluidine 4 was converted to intermediate 2-chloro-
N-(m-tolyl)acetamide 5 by reacting with 2-chloroacetyl chloride in the presence of triethylamine
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and ethyl acetate at room temperature. Using the same procedure, intermediates 14, 19, and 24
were synthesized in a similar manner, as indicated in Scheme 1, with either a one- or two-carbon
linker. The intermediate compounds 5, 14, 19, and 24 were used to alkylate different commercially
available arylpiperazine or arylpiperidine amines in the presence of K CO in CH CN under reflux
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conditions to yield the desired target compounds 6–9, 10–13, 15–17, 20–22, and 25–28,
respectively, with the exception of the synthesis of 1-(naphthalen-1-yl)piperazine which was
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previously reported via nucleophilic substitution reaction with naphthalen-1-amine.
Scheme 1. Synthesis of 2-(4-(pyridin-2-yl)piperidin-1-yl)-N-(m-tolyl)acetamide analoguesa
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(
A)
Ar
Ar
O
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Cl
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NH
NH
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10-13
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NH2
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a
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N
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Cl
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Ar
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(B)
Ar
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O
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O
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N
NH
NH
Cl
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NH2
NH
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19
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21-22
O
Ar
O
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N
N
X
Cl
b
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X
a
N
NH2
NH
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NH
X
2
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25; X = CH
2
2
2
6; X = CH
7; X = N
8; X = N
CH3
Cl
*
*
*
*
*
Ar
=
Cl
N
N
N
*
N
*
a
Reagents and Conditions: (a) Triethylamine, EtOAc, RT; (b) CH CN, K CO , Reflux,
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appropriate arylpiperazine or arylpiperidine.
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PHARMACOLOGICAL RESULTS AND DISCUSSION
Structure-Activity Relationships at Dopamine D -Like Receptors.
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subtype selectivity. The compound 1 and several designed analogs are shown in Figure 2. In order
to obtain D R ligands with high affinity and selectivity, using compound 1 as our lead compound,
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we employed three modification strategies, creating 2-(piperidin-4-yl)pyridinyl analogs, altering
the linker chain length, and creating N-(m-tolyl)acetamide analogs.
Of note, when 1 was evaluated in two different functional assays, its profile was clearly that of
a partial agonist rather than a full agonist as it is often described in the literature. In the agonist
mode for both the cAMP accumulation and β-arrestin recruitment assays, 1 had an Emax of 61.9%
and 22.5%, respectively, when normalized to dopamine.
The 2-pyridine moiety of 1 was replaced with a phenyl in 6, para-tolyl in 7, 4-chlorophenyl in
8
, and 5-methylpyridin-2-yl in 9. The piperidine attached to the linker chain was replaced with a
piperazine to form 10, replaced with a pyrimidine to form 11, replaced with a 5-chloropyridin-2-
yl to form 12, and replaced with a naphthyl substituent to obtain 13. To evaluate the contribution
of the alkyl chain to the binding affinity and selectivity, we synthesized alkyl chain length analogs
of compounds 1, 10 and 11, adding an extra methylene to the linker chain in compounds 15, 16,
and 17, respectively. Finally, we probed the contribution of the N-(3-methylphenyl)acetamide
moiety via replacement of the methyl with ethyl (compounds 20, 21 and 22, compared to
compounds 1, 10, and 12, respectively) or replaced of the entire N-(3-methylphenyl)acetamide
moiety with heteroaromatics (compounds 25-28).
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In order to best evaluate comparative affinities, two different radioligands were used in
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competition binding studies: [ H]N-methylspiperone, a high-affinity D -like antagonist, and [ H]-
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(R)-(+)-7-OH-DPAT, a D -like agonist. Importantly, the binding affinities of D -like agonists and
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high-efficacy partial agonists are considerably higher when competing against an agonist
radioligand because high-affinity agonist binding incorporates an efficacy measure in that the
greater the efficacy for inducing G protein coupling, the greater the “apparent” affinity will be. On
the other hand, antagonist binding, and competition for it, is unlinked from efficacy and therefore
unbiased. Therefore, because these radioligands probe different receptor states, they provide
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complimentary views of ligand binding, which are particularly valuable when examining affinity
of partial agonists.
Several modifications of 1 resulted in modest improvements in D R affinity as measured by
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competition assays with [ H]N-methylspiperone (up to ~3-fold) and [ H]-(R)-(+)-7-OH-DPAT (up
to ~3-fold). However, marked improvements in D R selectivity over D R and D R resulted from
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a variety of modifications, typically driven by a loss of affinity at D R and D R.
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-pyridine substitutions resulted in a potency gain when the piperidinyl moiety was replaced
with piperazinyl (e.g., 10 and 21). Adding an extra methylene to the linker chain, as in compounds
5, 16, and 17, significantly diminished D R affinity and selectivity. These results are consistent
1
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with previous studies that determined the importance of carboxamide linker length for D -like
2
receptor selectivity.31 Replacement of the methyl with an ethyl at the N-(3-
methylphenyl)acetamide moiety (compounds 20, 21 and 22) did not substantially alter affinity or
selectivity for D R compared to methyl analogues 1, 10, and 12, respectively. Replacement of the
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entire N-(3-methylphenyl)acetamide moiety with heteroaromatics (compounds 25-28) uniformly
led to loss of affinity and selectivity.
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Overall, we noted three broader classes of modifications with distinct binding and efficacy
profiles across the D -like receptors; as outlined in Figure 2, these include 1) substitution of the
2
piperidine ring for piperazine, 2) substitution of the pyridine ring with a phenyl or napthyl moiety,
and 3) para-substituted pyridine rings. These classes formed the basis for further structure-activity
relationship profiling and modeling studies using molecular dynamics simulations.
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A. Class 1 modifications
O
N
N
N
N
H
10 (CAB02-140)
O
N
N
N
O
N
N
H
1
N
N
H
21 (CAB02-017)
Similar D R binding/efficacy profile, improved subtype
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selectivity, gain in efficacy at D R & D R
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B. Class 2 modifications
C. Class 3 modifications
Cl
O
N
N
O
N
N
N
H
N
H
12 (CAB02-003)
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(CAB03-015)
CH3
O
N
O
N
N
N
N
H
N
H
13 (CAB02-011)
9 (CAB02-005)
Lower efficacy D R partial agonists, improved affinity
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Highly selective full D R antagonists with no agonist
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& subtype selectivity, no efficacy at D R & D R
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activity at D -like receptors
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Figure 2. Three classes of modifications to the structure of 1 resulting in differing binding and
efficacy profiles at D -like receptors. A. Substitution of piperidine ring for piperazine induced a
2
gain of efficacy at D R and D R with insubstantial changes to D R efficacy. B. Substitution of the
2
3
4
pyridine ring with a phenyl or napthyl moiety produced modest D R subtype selectivity
4
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improvements and lowered partial agonist efficacy at D R with no agonist activity at D R or D R.
4
2
3
C. para-substituted pyridine rings produced highly D R-selective antagonists.
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The parent compound, 1, showed 115-fold and 31-fold higher affinity for D R over D R and D R,
4
2
3
3
3
respectively, as measured by [ H]N-methylspiperone competition. When examined using [ H]-(R)-
(+)-7-OH-DPAT competition, 1 had higher affinity at all subtypes (consistent with an agonist
3
0
radioligand being displaced by a compound that favors the activated receptor ), and showed a
similar selectivity profile of 64-fold and 42-fold higher affinity for D R over D R and D R,
4
2
3
respectively. Full binding results are presented in Table 1. Functional characterization revealed 1
to be a partial agonist at D R as measured in β-arrestin assays (E = 22.5%, EC = 473 nM)
4
max
50
(
Figure 3A and B) and cAMP inhibition assays (Emax = 61.9%, EC = 2.7 nM) (Figure 3B and C).
50
The higher efficacy observed in the cAMP assay is likely due to spare receptors and/or
amplification of cAMP accumulation vs. recruitment of β-arrestin. Consistent with a partial agonist
profile, 1 and related analogs were partial antagonists when run in antagonist mode (Figure 3B and
D), blocking function to a similar degree as their maximal agonist activity. This would be expected
for a compound that is a partial agonist that maintains affinity for the orthosteric part of the receptor
thereby acting as a partial antagonist in antagonist assays. Importantly, 1 showed no measurable
agonist response on D R-mediated β-arrestin recruitment but behaved as a low affinity full
2
antagonist (Figure 3E). Furthermore, 1 has very low potency and efficacy at the D R (Figure 3F).
3
Complete functional results are presented in Tables 2 and 3. These data indicate that 1 is a potent
and highly selective partial agonist at the D R.
4
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
Table 1. Human dopamine D -like receptor binding data in HEK293 membranes for ligands with varying arylpiperazine and arylamide
2
moieties.a
K
i
(nM) ± SEM
i
K (nM) ± SEM
[ H]7-OH-DPAT
3
3
Compound
Structure
[ H]N-methlyspiperone
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
D
2
R
D
3
R
D
4
R
D
2
R/D
4
R D
3
R/D
4
R
D
2
R
D
3
R
D
4
R
D
2
R/D
4
R D
3
4
R/D R
O
N
N
3
1.41
.95 ±
1
; A-41299721-22
6250 ± 375
821 ± 34.9
1680 ± 446
433 ± 137
54.2 ± 7.01
25.8 ± 9.01
110.4 ± 55
115.2 ± 42
41.7 ± 7.0
115
31
251 ± 72.2 167 ± 38.8
64
42
NH
O
1
0.42
.4 ±
6; (CAB03-015)22
32
71
17
33
127 ± 35.8 777 ± 141
91
555
N
NH
O
CH3
7
; (CAB02-007HP)
; (CAB02-009HP)
; (CAB02-005HP)
7824 ± 347 3681 ± 1237
N
NH
Cl
O
26,320 ±
>50,000
8
9
>434
228
12,028
N
NH
CH3
O
N
6.87 ±
0
>50,000
>50,000
>1198
>1198
>10,000
>10,000
>1455
>1455
.73
N
NH
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2
2,
O
O
N
N
N
N
10; (CAB02-140)
3320 ±
450
1.89 ±
0.38
>
10,000
>10,000
>10,000
>50,000
212 ± 62.9
318 ± 95.1
95 ± 26
>47
>47
>31
6480 ± 972
1757
428
3429
1058
>800
755
27
N
N
NH
NH
N
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
2420 ±
5990 ±
2040
5.66 ±
0.42
11; (CAB02-110)
12; (CAB02-
6400 ± 3800
20
219
Cl
O
N
N
12.5 ±
1.85
>
50,000
>526
>526
402
>10,000
>10,000
>800
121
003HP)
N
NH
O
N
13; (CAB02-
1.65 ±
0.21
1489 ± 95 11,459 ± 3085
28.4 ± 8
52
200 ± 38.1 1246 ± 195
011HP)
N
NH
NH
O
O
O
N
N
80.3 ±
76.4 ±
7.09
15; (CAB02-120)
16; (CAB02-142)
17; (RNB01-007)
5940 ± 556 3040 ± 1350
>10,000
297 ± 34.2
526 ± 128
<0.6
40
<0.31
1276
8.6
510 ± 57.3
1.1
6.7
24
28.6
N
NH
NH
11,900 ±
32.2 ±
8.28
3790 ± 462
200 ± 18 781 ± 68.6
6
N
2580
N
N
N
1490 ±
1850 ± 333 4530 ± 2010
3.5
9350 ± 288 33 ± 2.52
45
2833
N
N
275
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8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
O
N
20; (CAB02-
21HP)
N
1159 ± 241
496 ± 36
>50,000
82.3 ± 36
14
6.0
0
NH
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
O
N
N
21; (CAB02-
2.23 ±
0.93
N
>50,000
67.9 ± 24
>736
>736 603 ± 220 1490 ± 275
270
668
017HP)
NH
Cl
O
N
N
22; (CAB02-
19HP)
46,260 ±
3740
N
>50,000
172.2 ± 28
269
>290
0
NH
25; (CAB02-
033HP)
O
N
N
>
50,000
>50,000
>50,000
6407 ± 1678
5000 ± 296
>7.8
>10
>7.8
>10
N
N
N
NH
Cl
26; (CAB02-
O
N
N
>50,000
035HP)
N
NH
27; (CAB02-
29HP)
39,290 ±
10,710
O
N
N
>
50,000
>50,000
>50,000
>1.2
>1.2
0
N
N
NH
N
Cl
28; (CAB02-
31HP)
O
N
N
>50,000
>50,000
0
N
N
N
NH
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a
3
3
K values determined by competitive inhibition of [ H]N-methylspiperone or [ H]-(R)-(+)-7-OH-DPAT binding in membranes
i
harvested from HEK 293 cells stably expressing hD R, hD R, or hD R. All K values are presented as means ± SEM.
2
3
4
i
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
A
B
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
2
3
4
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7
8
9
0
C
D
E
F
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Figure 3. Compounds 10 (red) and 21 (gray) show similar pharmacology to parent
compound 1 (black). D R-expressing stable cells lines were plated and compounds were
4
assayed for agonist (A) and antagonist (B) activity on β-arrestin recruitment. Similarly, D R-
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
mediated inhibition of cAMP accumulation was also examined in both agonist (C), and
antagonist (D) modes, as indicated. Assays were conducted as described in the Experimental
Procedures; briefly, agonist assays were conducted by incubating the cells with the indicated
concentration of test compound and measuring luminescence. Antagonist assays were conducted
by incubating the compound with an EC concentration of dopamine (1 µM for β-arrestin and
8
0
1
0 nM in cAMP) and the indicated concentration of test compound. For cAMP assays cells were
first stimulated with 10 µM forskolin. Agonist mode assays are expressed as a percentage of the
maximum dopamine response, whereas antagonist mode assays are expressed as a percentage
of dopamine's EC response. E and EC values are shown in Table 2 and 3. Data were fit
8
0
max
50
using nonlinear regression of individual experiments performed in triplicate and are shown as
means ± SEM; n = 3. Dopamine and sulpiride were run during each assay as positive controls
for a full agonist and full antagonist respectively (data not shown). Compounds were also tested
for both agonist and antagonist activity on cells stably expressing the closely related D R (E) or
2
D R (F). Assays were conducted as described in the Experimental Procedures. Agonist mode
3
assays (open symbols) are expressed as a percentage of the maximum dopamine response
observed for each receptor, whereas antagonist mode assays (solid symbols) are expressed as a
percentage of dopamine's EC response. E and EC values are shown in Table 2 and 3. Data
8
0
max
50
were fit using nonlinear regression of individual experiments performed in triplicate and are
shown as means ± SEM; n = 3.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Replacing the piperidinyl ring of 1 with a piperazine (Figure 2, Class 1)—typified by 10 and 21—
resulted in similar binding and agonist efficacy profiles at D R, improved subtype selectivity
4
(Tables 1 and 2), and a gain in efficacy at both D R and D R (Figure 3 and Table 2 and 3).
2
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Replacing the pyridinyl ring of 1 with a phenyl or napthyl moiety (Figure 2, Class 2),—typified
by 6 and 13—resulted in improved subtype selectivity, and importantly a diminished-efficacy
partial agonist profile at D R. These compounds showed no measurable agonist efficacy at either
4
D R or D Rs (Figure 4). A para- substitution on the pyridinyl ring of 1 (Figure 2, Class 3)—
2
3
typified by 12 and 9—resulted in compounds that lost all agonist efficacy but retained high-affinity
binding at D R, with very minimal binding at D R or D R. The compounds showed potent
4
2
3
antagonism of the D R response with minimal low potency D R blockade and no measurable
4
2
affinity or efficacy at D R. Therefore, this class of compounds represents highly selective D R
3
4
antagonists with no measurable agonist efficacy on any D -like receptor (Figure 5, Table 1-3).
2
Individual compounds within Classes 1-3 resulted in modest changes to overall efficacy and
potency as overviewed in Tables 1-3. For this reason, we chose to focus on typified examples of a
range of agonist efficacy (higher, medium, and none) at the D R. Using these classes, we
4
performed molecular dynamic simulations to identify interaction sites on the receptor that may
play a pivotal role in engendering agonist selectivity and efficacy.
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Table 2. Efficacy as measured via modulation of cAMP accumulation.a
D
2
R efficacy
D
4
R efficacy
EC50
IC50
Compound
cAMP Emax
cAMP EC50
cAMP Ant.
cAMP Emax
cAMP EC50
cAMP Ant.
cAMP IC50 (nM)
cAMP IC50 (nM)
D
2
R/D
4
R D
2
4
R/D R
b
c
b
c
%
(nM)
%
%
(nM)
%
1
6
Inactive
Inactive
Inactive
Inactive
ND
>50,000
61.9 ± 4.7
2.7 + 0.9
53.8 ± 6.0
46.7 ± 6.0
68.4 ± 32.8
2.0 ± 0.05
ND
ND
>735
8224
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
100 + 0.00 16,447 + 3540 32.9 ± 3.9
15.4 ± 13.2
4
2
4,834 ±
8,125
7
Inactive
Inactive
Inactive
Inactive
100 ± 0
Inactive
Inactive
Inactive
Inactive
95.8 ± 2.2
100 ± 0
3064 ± 1220
ND
ND
15
71,437 ±
70,157 ±
20,766
8;
97.5 ± 2.5
1.0
28,563
9
Inactive
18.96 ± 5.2
54.4 ± 7.5
83.1 ± 4.2
79.7 ± 8.4
Inactive
Inactive
763 ± 386
2092 ± 46
50.1 ± 25
154 ± 31
Inactive
100 ± 0
ND
71065 ± 20585
Inactive
Inactive
3.6 ± 1.3
3.4 + 2.0
349 ± 75
612 ± 563
568 ± 456
100 ± 0
453 ± 15
82.7 ± 37.9
463 ± 157
ND
214
612
0.14
0.25
ND
157
10
>100,000
>100,000
>100,000
>100,000
>100,000
64.2 ± 5.7
64.6 ± 4.2
28.1 ± 1.6
30.0 ± 2.1
13.7 ± 1.2
43.2 ±1.8
45.0 ± 7.7
77.6 ± 5.2
84.2 ± 6.0
87.0 ± 3.4
>1210
>216
>16
11
15
16
17
ND
ND
6343 ± 2524
1629 ± 255
2120 ± 534
ND
>61
ND
>47
6
1
6,077 ±
8,646
1
1
2
2
3
0
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
98.3 ± 1.7
100 ± 0
Inactive
27.8 ± 8.4
25.6 ± 7.2
58.0 ± 1.8
Inactive
93.4 ± 2.6
4701 ± 1466
2521 ± 1067
1908 ± 242
1311 ± 814
ND
ND
ND
56
14
27
9
68,329 ±
108.5 ± 94.3 73.8 ± 13.6
3
1,671
16,278 ±
1,601
0,466 ±
96.6 ± 3.5
88 ± 6.1
539 ± 151
28.7 ± 9.9
70.8 ± 15
58.4 ± 9.7
1
4
21
18.80 ± 8.19 1600 ± 396
31
2
9,968
4
6,795 ±
2
2
2
2
5
6
Inactive
38.6 ± 3
Inactive
Inactive
Inactive
Inactive
1965 ± 44
Inactive
Inactive
Inactive
100 ± 0
100 ± 0
100 ± 0
Inactive
47.3 ± 7.9
Inactive
Inactive
Inactive
Inactive
1075 ± 390
Inactive
100 ± 0
100 ± 0
100 ± 0
100 ± 0
100 ± 0
7059 ± 1136
86,493 ± 3130
40,000 ± 9421
>100,000
ND
2.0
ND
ND
ND
7
>1.1
2
27,644
>100,000
86,617 ±
13,383
27
98.1 ± 1.5 94,255 ± 5745
Inactive
<1
<1
72,516 ±
1
28
76.2 ± 17.6
Inactive
>100,000
8,052
a
values determined by nonlinear regression of individual experiments run in triplicate as detailed in materials and methods under
cAMP accumulation assays. All EC , IC , and E values are presented as means ± SEM; n = 3-4. ND indicates Not Determined due
5
0
50
max
to an incomplete curve. Inactive indicates no measurable activity in indicated assay.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
bA measure of agonism as defined by the maximum inhibition of cyclic AMP observed for each compound.
cA measure of antagonism as defined by the maximum blockade of dopamine mediated cAMP inhibition by each compound.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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0
1
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Table 3. Efficacy as measured via modulation of β-arrestin recruitment.a
D
2
R efficacy
D
3
R efficacy
D
4
R efficacy
EC50
IC50
Compound
β-arr
β-arr
EC50 (nM)
β-arr
Ant. %
β-arr
β-arr β-arr
EC50 (nM) Ant. %
β-arr
β-arr β-arr
EC50 (nM) Ant. %
β-arr IC50
β-arr IC50
>100,000
>50,000
β-arr IC50
191 ± 98
135 ± 65
D
2
R/D
ND
ND
ND
ND
ND
4
R D
3
R/D
4
R
D
2
R/D
4
R
3 4
D R/D R
E
max
E
max
E
max
94.8 ±
5846 ±
1802
7692 ±
2301
22.5 ± 473 ±
81.7 ±
2.7
93.3 ±
1.8
1
6
7
8
9
Inactive Inactive
Inactive Inactive
Inactive Inactive
Inactive Inactive
Inactive Inactive
ND
>100,000
ND
ND
>4
31
>524
>371
ND
2
.8
9.7 ±
.3
3.98
14 ±
0.3
457
242 ±
89
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Inactive Inactive
ND
ND
ND
ND
57
0
89,153 ±
7352 ±
1749
42,357 ±
100 ± 0
100 ± 0
100 ± 0
Inactive Inactive Inactive Inactive Inactive Inactive 100 ± 0
Inactive Inactive Inactive Inactive Inactive inactive 100 ± 0
12
10,847
>100,000
>2
ND
30,018
98.1 ±
>100,000
Inactive Inactive Inactive Inactive Inactive Inactive
394 ± 78
>254
51
ND
2
2
6,175
±
2
3.9 ±
76.5 ±
6.9
16,010 ±
5174
49.4 ±
2.0
6354 ±
2617
30.7 ± 394 ±
78.9 ±
3.1
313 ±
215
10
11
Inactive >100,000
66
>16
>320
5.1
6.4
294
12,448
3
9.6 ±
.3
8.9 ±
.8
44.1 ±
.3
8321 ±
3455
2480 ±
1834
1455 ±
572
78.9 ±
8.6
72.1 ±
7.9
77.7 ±
3.7
25,008 ±
5017
7627 ±
1573
7067 ±
2290
11,847 ±
2000
58.4 ±
6.6
55.4 ±
1.57
41.4 ±
5.83
5581 ±
1614
970 ±
115
1601 ±
867
278 ±
167
80.6 ±
3.0
98.7 ±
1.0
97.9 ±
1.2
97.1 ±
0.8
197 ±
115
3805 ±
2944
1086 ±
597
430 ±
195
Inactive >100,000 24.7± 5
30
12
>20
>5
127
2.0
7.0
28
>509
>26
>46
>116
ND
5
4
1
5
6
ND
ND
ND
>50,000 Inactive Inactive
>50,000 Inactive Inactive
>50,000 Inactive Inactive
6
1
ND
ND
ND
ND
ND
29
>1
6
94.6 ±
17
Inactive Inactive
Inactive Inactive
Inactive Inactive
Inactive Inactive
Inactive Inactive
ND
ND
ND
ND
21
5.4
7777 ±
1
2
3
100 ± 0
100 ± 0
100 ± 0
100 ± 0
100 ± 0
>100,000
>100,000
Inactive Inactive Inactive Inactive Inactive Inactive 100 ± 0
>13
>24
26
2166
4252 ±
1077
2622 ±
678
370 ±
105
16.4 ± 9212 ± 96.7 ±
1
Inactive Inactive Inactive Inactive
ND
3.9
6238
2.7
91.7 ±
6.8
59.7 ±
4.6
80.4 ± 10,449 ±
67,613 ±
20
Inactive Inactive Inactive Inactive Inactive Inactive
ND
17,748
18.7 ±
3887 ±
1878
88,447 ±
11,553
70,703 ±
44.7 ±
5.9
2755 ±
472
88205 ± 26.2 + 133 ±
9631 5.1 60.3
2
1
2
100 ± 0
239
6.8
238
ND
0.4
2
Inactive Inactive
Inactive Inactive Inactive Inactive Inactive Inactive
ND
ND
29,297
6.0
2482
4
8,503
±
21.3 ±
.8
18.6 ±
2.7
2802 ±
2507
19.0 ± 2657 ±
25,780 ±
9773
2
5
6
Inactive
Inactive
Inactive
Inactive Inactive
100 ± 0
18
1.0
ND
ND
ND
ND
ND
6
2.7
121
27,571
93.1 ± 23,450 ±
11,959
2
Inactive Inactive
Inactive
Inactive Inactive Inactive Inactive Inactive Inactive
ND
7
2
7
8
Inactive Inactive
Inactive Inactive
Inactive
Inactive
Inactive
Inactive
Inactive Inactive Inactive Inactive Inactive Inactive Inactive Inactive
Inactive Inactive Inactive Inactive Inactive Inactive 100 ± 0 >100,000
ND
ND
ND
ND
ND
ND
ND
ND
2
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Journal of Medicinal Chemistry
Page 22 of 66
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2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
a
values determined by nonlinear regression of individual experiments run in triplicate as detailed in materials and methods under β-
arrestin assays. All EC , IC , and E values are presented as means ± SEM; n = 3-4. ND indicates Not Determined due to an
max
5
0
50
incomplete curve. Inactive indicates no measurable activity in indicated assay.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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Page 23 of 66
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
A
B
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
D
Figure 4. Compounds 13 (yellow) and 6 (blue) show diminished agonist activity at the D R
4
compared to parent compound 1 (black). D R-expressing stable cells lines were plated and
4
compounds were assayed for agonist (A) and antagonist (B) activity on β-arrestin recruitment.
Similarly, D R-mediated inhibition of cAMP accumulation was also examined in both agonist
4
(C), and antagonist (D) modes, as indicated. Assays were conducted as described in the
Experimental Procedures; briefly, agonist assays were conducted by incubating the cells with
the indicated concentration of test compound and measuring luminescence. Antagonist assays
were conducted by incubating the compound with an EC concentration of dopamine (1 µM for
8
0
β-arrestin and 10 nM in cAMP) and the indicated concentration of test compound. For cAMP
assays cells were first stimulated with 10 µM forskolin. Agonist mode assays are expressed as
a percentage of the maximum dopamine response, whereas antagonist mode assays are
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Journal of Medicinal Chemistry
Page 24 of 66
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2
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
expressed as a percentage of dopamine's EC response. E and EC values are shown in Table
8
0
max
50
2
and 3. Dopamine and sulpiride were run during each assay as positive controls for a full agonist
and full antagonist respectively (data not shown). Data were fit using nonlinear regression of
individual experiments performed in triplicate and are shown as means ± SEM; n = 3.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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Page 25 of 66
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
A
B
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
D
Figure 5. Compounds 12 (green) and 9 (purple) are full antagonists at the D R. D R-
4
4
expressing stable cells lines were plated and compounds were assayed for agonist (A) and
antagonist (B) activity on β-arrestin recruitment. Similarly, D R-mediated inhibition of cAMP
4
accumulation was also examined in both agonist (C), and antagonist (D) modes, as indicated.
Assays were conducted as described in the Experimental Procedures; briefly, agonist assays
were conducted by incubating the cells with the indicated concentration of test compound and
measuring luminescence. Antagonist assays were conducted by incubating the compound with
an EC concentration of dopamine (1 µM for β-arrestin and 10 nM in cAMP) and the indicated
8
0
concentration of test compound. For cAMP assays cells were first stimulated with 10 µM
forskolin. Assays were conducted as described in the Experimental Procedures. Agonist mode
assays are expressed as a percentage of the maximum dopamine response, whereas antagonist
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Journal of Medicinal Chemistry
Page 26 of 66
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
mode assays are expressed as a percentage of dopamine's EC (1 µM in β-arrestin and 10 nM
8
0
in cAMP) response. Emax and EC values are shown in Table 2 and 3. Dopamine and sulpiride
50
were run during each assay as positive controls for a full agonist and full antagonist respectively
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(data not shown). Data were fit using nonlinear regression of individual experiments performed
in triplicate and are shown as means ± SEM; n = 3.
Molecular Dynamics (MD) Studies.
To gain insights on probable ligand interactions at D R, a set of seven ligands from the parent
4
compound and the three class of modifications (i.e., 1, 6, 9, 10, 12, 13, and 21) were docked to the
2
6
25
24
crystal structures of D R, D R, and D R. Each receptor-ligand combination was subjected to
2
3
4
1
00 ns MD simulations, followed by the simulation interaction diagram and clustering analysis as
described in the method section. The results are included in the supporting documents (Tables S2-
S4 and Figures S2-S39). Comparisons of structural and dynamic properties of each ligand-receptor
system, with reference to the parent compound 1 for each receptor system, are listed in Table S1.
Although the same class modifications caused similar changes in the majority of the analyzed
properties, some subtle differences are also identified. A representative ligand-receptor system
from each class modification is presented here.
In order to explore Class 1 modifications that showed a gain of efficacy at D R and D R with
2
3
minimal changes in D R binding or efficacy, 10 was selected to be presented here along with
4
parent compound 1. The comparative ligand binding at of 10 D R (Figure 6) and D R (Figure 7)
2
3
revealed that the modest ligand change—the substitution of a piperazine for a piperidine—induced
a dramatic shift in the binding orientation at D R and D R: compared to the parent compound 1,
2
3
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Page 27 of 66
Journal of Medicinal Chemistry
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7
8
9
1
0 took on a rotated orientation in both receptors, in which the arylamide portion of the 10 occupies
a region of the binding pocket that accommodates the 2-(piperidin-4-yl)pyridinyl portion of 1. This
pose allows 10 to better engage with the conserved transmembrane (TM) 3 aspartate residue (D3.32
)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
located within the orthosteric binding pocket of biogenic amine receptors like dopaminergic
receptors. Additionally, there was new engagement with conserved V2.61, and additional TM5
3
2
and TM6 helix shifts in both receptors. In contrast, the binding orientation of 10 at D R is similar
4
to that of 1 (Figure S7), although a shift in the orientation of the pyridinylpiperidine ring system
deeper into the receptor was observed. 21 docked similarly to 10 at D R and at D R (i.e., rotated
2
4
1
80° in comparison to 1), but differed at D R in which the pose was similar to that of 1, possibly
3
indicating a different activation mechanism for D R by this compound.
3
1
9
3 (Figure 8), representing Class 2 modifications that showed a partial loss of efficacy at D R, and
4
(Figure 9), representing Class 3 modifications that showed a complete loss of efficacy at D R,
4
are shown in models of D R alongside the parent compound 1. 13 uniquely engaged with S2.64,
4
E2.65, T7.39, and induced conformational shifts in several TM domains and intra/extracellular loops.
9
showed grater engagement with ECL2 and uniquely interacted with C3.25 and W6.48. Whereas 13
adopted pose similar to 1, 9 adopted a rotated orientation in which the arylamide portion of the 9
occupies a region of the binding pocket that accommodates the 2-(piperidin-4-yl)pyridinyl portion
of 1. (Table S1). The results seen in these two comparisons are consistent with previous
observations in which regiosubstitutions on an aryl ring of a terminal arylpiperazine can modulate
3
3-34
efficacy at D R.
In particular, the inclusion of a para substitution on the terminal
4
arylpiperazine has reliably produced D R antagonists for a wide variety of molecules with diverse
4
substituents on the secondary pharmacophore.
CONCLUSIONS
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Journal of Medicinal Chemistry
Page 28 of 66
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6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Evidence from human genetic studies and animal models suggest D R signaling may mediate
4
3
5
35-38
fear and anxiety,39-40 and sensitivity
behavioral traits including impulsivity, novelty-seeking,
to drugs of abuse.40-43 While modulation of postsynaptic D R expression in the prefrontal cortex
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
is typically hypothesized to mediate the reported in vivo effects of D R agonists and antagonists,
4
4
4
evidence suggests important roles of D R expression in the nucleus accumbens shell and within
4
4
5
the lateral habenula, in which the receptor may be preferentially activated by norepinephrine
rather than dopamine.46-47 Furthermore, little is known about the physiological relevance of
independent D R-mediated signaling pathways (e.g., cAMP and β-arrestin) in the manifestation of
4
behavioral outputs. A recent report identified a D R-selective compound containing an
4
unsubstituted phenylpiperazine that potently and partially activated Gα but inhibited β-arrestin2
i
recruitment, and identified likely ligand-residue interactions that affect receptor activation states.48
There remains much left to determine about the physiological role of D R signaling in modulating
4
attention and cognitive processes and new selective agonists and antagonists of these receptors
will be valuable tools for deduction of signaling importance by these receptors.
New highly selective D R partial agonists and antagonists will be useful to better characterize the
4
role of D R signaling in vivo. While we have primarily focused on selectivity against the very
4
closely related D R and D R, it will be important to establish global selectivity of these compounds
2
3
for in vivo experimentation through a broader screen of biogenic amine receptors. To this end, we
investigated a subset of these compounds on the related D -like DARs. None of the tested
1
compounds showed any measurable agonist or antagonist effect at either the D R or D R (Figure
1
5
S1). Comprehensive binding and functional studies, in concert with detailed molecular modeling
analyses using newly published crystal structures, provides a platform for developing high-affinity
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Page 29 of 66
Journal of Medicinal Chemistry
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2
3
4
5
6
7
8
9
and highly subtype selective ligands of varying efficacies. This study aimed to identify key
molecular interactions that dictate D R potency, efficacy and subtype selectivity.
4
The parent compound, 1, was confirmed to be a high-affinity (low nM K value) partial agonist
i
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
Emax = 23-62%) at D R. Illustrative of broader trends from our library, the compound K values
4
i
3
for all D -like receptors determined using [ H]-(R)-(+)-7-OH-DPAT competition assays tended to
2
3
be lower (more potent) than those obtained using [ H]N-methylspiperone. As expected, the
divergence between these values increased with agonist efficacy, consistent with previous
experience regarding agonist versus antagonist radioligands.30
Key modifications to the 1 pharmacophore provided modest gains to D R affinity, but dramatic
4
gains in selectivity over D R and D R, likely due to a substantial decrease in D R and D R
2
3
2
3
engagement by the analogs. Interestingly, the manner of these substitutions produced three classes
of lead compound: 1) those with binding and efficacy profiles similar to 1 at D R but gains in
4
efficacy at D R and D R; 2) those with improved D R binding and subtype selectivity with lower
2
3
4
partial agonist efficacy; and 3) those with improved D R binding and subtype selectivity with full
4
antagonist characteristics. Molecular dynamics simulations suggest that the gain in D R and D R
2
3
efficacy seen in compounds like 10 could be partially due to achieving a rotated ligand pose that
more fully engages the conserved TM3 aspartate. Similarly, the complete shift to antagonism at
D R seen in compounds like 9 could be partially due to inducing an alternate binding pose that
4
either no longer allows full engagement of the orthosteric binding site and occupation of an
alternative secondary binding pocket, or a ligand-dependent alteration of the receptor energy
landscape leading to the stabilization of a different receptor conformation.
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