Exception That Proves the Rule: Investigation of Privileged Stereochemistry in Designing Dopamine D3R Bitopic Agonists

Article abstract of DOI:10.1021/acsmedchemlett.9b00660

In this study, starting from our selective D3R agonist FOB02-04A (5), we investigated the chemical space around the linker portion of the molecule via insertion of a hydroxyl substituent and ring-expansion of the trans-cyclopropyl moiety into a trans-cyclohexyl scaffold. Moreover, to further elucidate the importance of the primary pharmacophore stereochemistry in the design of bitopic ligands, we investigated the chiral requirements of (+)-PD128907 ((+)-(4aR,10bR)-2)) by synthesizing and resolving bitopic analogues in all the cis and trans combinations of its 9-methoxy-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4] oxazine scaffold. Despite the lack of success in obtaining new analogues with improved biological profiles, in comparison to our current leads, a "negative"result due to a poor or simply not improved biological profile is fundamental toward better understanding chemical space and optimal stereochemistry for target recognition. Herein, we identified essential structural information to understand the differences between orthosteric and bitopic ligand-receptor binding interactions, discriminate D3R active and inactive states, and assist multitarget receptor recognition. Exploring stereochemical complexity and developing extended D3R SAR from this new library complements previously described SAR and inspires future structural and computational biology investigation. Moreover, the expansion of chemical space characterization for D3R agonism may be utilized in machine learning and artificial intelligence (AI)-based drug design, in the future.

Full text of DOI:10.1021/acsmedchemlett.9b00660

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Letter
Exception that proves the rule: Investigation of privileged
stereochemistry in designing dopamine D3R bitopic agonists
Francisco O. Battiti, Amy Hauck Newman, and Alessandro Bonifazi
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00660 • Publication Date (Web): 28 Feb 2020
Downloaded from pubs.acs.org on March 3, 2020
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Exception that proves the rule: Investigation of privileged stereochemistry in designing
dopamine D3R bitopic agonists
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Francisco O. Battiti,† Amy Hauck Newman,† and Alessandro Bonifazi†*
†
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
*Corresponding author:
Alessandro Bonifazi:
Phone: (443)-740-2897. Fax: (443)-740-2111
Email: alessandro.bonifazi2@nih.gov
ORCiD: 0000-0002-7306-0114
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ABSTRACT
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In this study, starting from our selective D3R agonist FOB02-04A (5), we investigated the
chemical space around the linker portion of the molecule via insertion of a hydroxyl substituent
and ring-expansion of the trans-cyclopropyl moiety into a trans-cyclohexyl scaffold. Moreover,
to further elucidate the importance of the primary pharmacophore stereochemistry in the design of
bitopic ligands, we investigated the chiral requirements of (+)-PD128907 ((+)-(4aR,10bR)-2)) by
synthesizing and resolving bitopic analogues in all the cis and trans combinations of its 9-
methoxy-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazine scaffold. Despite the lack of
success in obtaining new analogues with improved biological profiles, in comparison to our current
leads, a “negative” result due to a poor or simply not improved biological profile is fundamental
toward better understanding chemical space and optimal stereochemistry for target recognition.
Herein, we identified essential structural information to understand the differences between
orthosteric and bitopic ligand-receptor binding interactions, discriminate D3R active and inactive
states and assist multitarget receptor recognition. Exploring stereochemical complexity and
developing extended D3R SAR from this new library complements previously described SAR and
inspires future structural and computational biology investigation. Moreover, the expansion of
chemical space characterization for D3R agonism may be utilized in machine learning and artificial
intelligence (A.I.)-based drug design, in the future.
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D3R PDB ID: 3PBL
D3R PDB ID: 3PBL
5: D3R Ki = 0.00185 μM
10: D3R Ki = 0.423 μM
11: D3R Ki = 0.0216 μM
(+)-(4aR,10bR)-2: D3R Ki = 0.00167 μM
rel-trans-12: D3R Ki = 5.54 μM
Graphical Abstract
KEYWORDS: Bitopic Ligands, D3R Agonists, GPCRs, Stereochemistry.
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INTRODUCTION
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The dopamine D3 receptor subtype (D3R) is a target of great interest due to its role in
locomotor behavior, motivation, and substance use disorders (SUDs).1-3 D3R belongs to the
dopamine D2-like family which includes D2R, D3R and D4R.4 When activated by endogenous
dopamine (DA), or synthetic agonists binding to the orthosteric binding site (OBS), a signaling
cascade is triggered, mediated via Gi/o inhibitory protein (inhibition of adenylyl cyclase), or
through the -arrestins, a spatially and temporally independent pathway.4
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D3R are highly expressed in neural circuits responsible for emotional and cognitive
functions1 and their involvement in SUDs has been reported.5 In addition, we recently
demonstrated how highly selective D3R antagonists or partial agonists show promise in attenuating
opioid self-administration and reinstatement to drug seeking.6-9
Despite D3R selective agonists being long sought after due to their potential in treatment
of Parkinson’s disease and other locomotor associated disorders, the discovery of highly selective
D3R agonists remains a significant challenge.10-12 Due to high co-localization with D2R in brain
regions mediating similar physiological processes (i.e. reward, motivation and locomotion), the
importance of achieving significant D3R subtype selectivity over D2R,13 to disentangle their
relative functions, remains a challenge in drug development.
In the early stages of drug design, the main approach has been to target the OBS with
compounds identified via screening of large libraries of compounds and comparing SAR.
Advances in X-ray crystallography and cryo-microscopy have enabled the resolution of several
GPCR structures in their active and inactive states, empowering a target-driven drug design
approach.14 Crystal structures of D2R,15 D3R16 and D4R,17 have highlighted the existence of
extended and structurally less conserved secondary binding pockets (SBP).18-20 Targeting this SBP,
which often can function as allosteric binding sites (ABS), whose engagement can positively
(positive allosteric modulator, PAM) or negatively (negative allosteric modulator, NAM)
modulate the OBS functional response, has emerged as an attractive approach to selectively target
GPCRs with small molecules to control specific pharmacological behaviors. Several academic
research laboratories have pioneered the development of allosteric modulators selectively targeting
GPCR subtypes.21-27
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In light of the discovery of a distinct SBP within the dopamine D2-like receptors, we
adopted a bitopic molecular drug design to generate highly selective ligands capable of
discriminating among dopamine receptor subtypes.8, 28, 29 Our approach combines a primary
pharmacophore (PP) – targeting the OBS – with a secondary pharmacophore (SP) – targeting the
SBP – connected via meticulously designed linkers.30 We recently reviewed and detailed how this
approach bridges orthosteric and allosteric pharmacology.30 We posited that the PP primarily
controls target recognition and functional signaling cascades, while the SP mediates potency,
selectivity, bias signaling or allosterism. The linker induces optimal binding pose(s) and is
involved in its own ligand-receptor interactions resulting in unique pharmacological responses,
otherwise impossible to achieve with combinations of two separate molecules.
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We demonstrated how cannonical D3R preferential agonists, such as pramipexole,
PF592379 (1) and (+)-PD128907 ((+)-(4aR,10bR)-2) exhibit low selectivity in optimized binding
assays using the agonist radioligand [3H]-(R)-(+)-7-OH-DPAT.29 Alternatively, through our
bitopic drug design strategy, we have been able to generate the most selective D3R full agonist
reported to date, FOB02-04A (5) (Figure 1).29 A similar drug design has resulted in some of the
most selective and potent D2R G-protein biased agonists reported.28 This synthon-based bitopic
drug design provides a novel approach to selective D2R and D3R agonists, partial agonists, inverse
agonists and antagonists.30,31
Starting from 5, we further investigated the chemical space around the linker, via insertion
of a hydroxyl substituent and ring-expansion into a trans-cyclohexyl scaffold (Figure 1). Inspired
by previous observations,29 we explored the chiral requirements of (+)-(4aR,10bR)-2 (non-
selective D3R agonist) by synthesizing bitopic analogues in all the cis and trans combinations of
its 9-methoxy-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazine scaffold (Figure 1).
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PRIMARY PHARMACOPHORE AND LINKER STEREOCHEMISTRY OPTIMIZATION FOR D3R BINDING AND SELECTIVITY
4
5
6
O
(S)
N
(R)
N
ALTERNATIVE LINKER FOR D3R BINDING
H2N
1: PF592379
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O
(S)
N
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NC
O
7: SB269952
D3R Ki = 0.0039 M
D2R/D3R = 124
N
(r)
H
N
(r)
N
H2N
H
1: AB03-02 (PF592379) diastereomeric mixture
D3R Ki = 0.185 M
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D2R/D3R = 9.4
NC
O
O
(S)
N
H
O
N
(r)
N
(R)
N
N
(r)
H
N
8: SB277011A
D3R Ki = 0.011 M
D2R/D3R = 469
3: AB04-87
D3R Ki = 6.47 M
D2R/D3R = 0.8
N
H
H2N
Cl
O
O
(S)
O
N
H
N
N
(S)
N
N
H
OH
O
4: AB04-88
D3R Ki = 0.00596 M
D2R/D3R = 22.5
H
N
H2N
9: VK04-116
D3R Ki = 0.00684 M
D2R/D3R = 1700
N
N
H
O
O
(S)
(S)
H
N
H
(S)
(R)
N
N
N
(S)
N
N
(S)
N
N
(R)
H
(S)
H
O
O
H2N
H2N
5: FOB02-04A
6: FOB02-04B
D3R Ki = 0.282 M
D2R/D3R = 2.95
D3R Ki = 0.00185 M
D2R/D3R = 47.5
O
(R)
Bitopic O-alkylation
detrimental for D3R affinity
Bitopic N-alkylation
detrimental for D3R affinity
HO
N
(R)
X
X
O
OH
O
(S)
O
H
N
N
(S)
N
N
(+)-(4aR,10bR)-2: (+)-PD128907
D3R Ki = 0.00169 M
D2R/D3R = 12.1
H
H2N
10: FOB02-67
O
(S)
N
(S)
N
O
STEREOCHEMICAL INVESTIGATION
(r)
H
(S)
N
H2N
N
H
11: rel-trans-AB07-08
O
O
O
(S)
HO
N
HO
N
(S)
O
(-)-(4aS,10bS)-2: (-)-PD128907
(±)-cis-2: rel-cis-mixture
O
O
O
O
O
H
H
HO
N
N
HO
N
N
N
N
H
H
O
rel-cis-12: rel-cis-FOB03-56B
rel-trans-12: rel-trans-FOB03-56A
Figure 1. Bitopic molecular approach combining functionalized PPs, linkers and SPs.
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RESULTS
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Drug Design and Chemistry
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We previously demonstrated 1 is a suitable PP to be used in bitopic molecule design.29 We
showed that to generate high affinity and selective bitopic ligands an inversion of configuration at
its (2R) stereocenter was necessary. Indeed, when in a bitopic configuration with a N-butyl-1H-
indole-2-carboxamide SP (3 and 4), the (2S,5S) conformation of the PP in 4 resulted in a privileged
architecture with >1,000-fold increased affinity, and consequent selectivity for the D3R with
respect to its (2R,5S) eutomer.29 Full resolution of the chiral centers in the linker enabled further
analysis of stereochemistry on affinity and selectivity of the bitopic molecules 5 and 6, identifying
5 as the current lead (Figure 1).29
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Inspired by our previous work on D3R antagonists, we expanded the linker chemical space,
utilizing molecular fragments known to induce D3R recognition selectively. Both compounds
SB269952 (7) and SB277011A (8) (Figure 1), present a trans-cyclohexyl moiety in the linker
and both demonstrate selective D3R binding with a unique non-competitive antagonist profile at
D2R for 7.32-34 Similarly, a hydroxyl substituent in the butyl linking chain provided the most D3R
selective antagonists hitherto reported, 98, currently under investigation for treatment of opioid
use disorders. We posited that the presence of a hydroxyl substituent, and the additional chiral
center it introduced, would allow supplementary study of the linker chirality. We have previously
observed that a switch from (R)-OH to (S)-OH in similar structures changes the functional
efficacies from antagonists to partial agonists.18, 35
Following this fragment-inspired approach, we pursued the modification of 4 and 5 to their
corresponding hydroxylated analogue 10, and ring-expansion to the cyclohexyl linker, 11 (Figure
1). Compounds 10 and 11 were obtained as a mixture of diastereomers (d.r. 75:25, Figure S1),
and relative trans enantiomer (ee% >99%, Figure S2), respectively, shown in Schemes 1,2.
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Scheme 1. a) Di-tert-butyl dicarbonate, DMAP, DCM, RT; b) mCPBA, DCM, from 0 oC to RT;
c) K2CO3, toluene, DMF, , microwave 75 W, 120 oC, 275 psi setpoint d) HCl (2N-4N in Et2O),
DCM, from 0 oC to RT; e) EDC.HCl, HOBt, DIPEA, indole-2-carboxylic acid, DCM, RT.
.
Scheme 2. a) BH3 (CH3)2S, THF, from 0 oC to RT; b) HCl (2N-4N in Et2O), DCM, from 0 oC to
RT; c) EDC.HCl, HOBt, DIPEA, indole-2-carboxylic acid, DCM, RT; d) Dess-martin periodinane,
DCM, from 0 oC to RT; e) cat. AcOH, Na(AcO)₃BH, DCE, RT.
Previous observations show that the (4aR,10bR) absolute configuration of (+)-(4aR,10bR)-2 was
privileged for OBS binding but not suitable to bitopic modifications.29 Inspired by our success
optimizing the stereochemistry of 1 to improve its suitability as a bitopic PP, we investigated all
the possible cis and trans bitopic combinations of 2.
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Nor-29 was prepared optimizing precedent literature.36, 37 Previously, cis and trans
mixtures of diastereomers were separated at intermediate 27, however we decided to perform an
overall resolution of diastereomers at the final step (Scheme 3). The ratio of diastereomers
remained constant ~3:1 (trans:cis) throughout the additional steps yielding 29. The O-
demethylation step to obtain nor-29 required optimization, using 48% HBr (water solution) under
reflux and quenching it after 1.5 hours. Longer reaction times led to epimerization at the benzylic
position and overall decrease in the 3:1 ratio of diastereomers. Both (+)-(4aR,10bR)-2 and (-)-
(4aS,10bS)-2 were isolated via preparative chiral HPLC, their absolute configurations assigned by
comparing optical rotations and the analytical chiral HPLC chromatograms of the synthesized (+)-
(4aR,10bR)-2 with the commercially available (+)-PD128907 used as analytical standard
(Figures S3 and S4).
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The (±)-cis-2 diastereoisomer, and its mixture of two enantiomers, were not separated as
previous studies reported the loss of affinity for D2R and D3R for the cis-9-methoxy-3,4,4a,10b-
tetrahydro-2H,5H-chromeno[4,3-b][1,4]oxazine scaffold of 2.37 The lack of affinity for the (±)-
cis-2 does not necessarily predict low affinity when in a bitopic configuration. The intermediate
29, was thus converted into the bitopic analogue 12 via reductive amination in presence of 30. The
mixture of four enantiomers was analyzed by chiral HPLC and the trans and cis diastereomers
were separated by preparative HPLC, into the corresponding mixture of enantiomers rel-trans-12
(er 65:35) and rel-cis-12 which we then carried into biological screening (Figure S5). The relative
enantiomers abundance was not determined for rel-cis-12 because resolution couldn’t be achieved.
It is unknown whether the racemic mixture tested has an equal distribution of the two cis
enantiomers or it is unbalanced toward one, however the lack of biological activity didn’t support
additional efforts in resolving the tested racemate. Cis and trans diastereosiomers’ conformations
for (+)-trans-2, rel-trans-12, rel-cis-12 were readily identifiable by comparison with precedent
1
literature and by H NMR spectroscopy analyzing the coupling constant (J) for the protons in
positions 4a and 10b (Figure 2).
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o
Scheme 3. a) AcONa, NH2OH.HCl, EtOH, H2O; b) p-TsCl, pyridine, from 0 C to RT; c) Na,
EtOH, from 0 oC to RT, 2N HCl in H2O, RT; d) 2-chloroacetyl chloride, DIPEA, EtOAc, RT; e)
NaBH4, EtOH:THF (1:3), RT; f) tBuOK, DCM, 2-PrOH, RT; g) LiAlH4, THF, from 0 oC to RT;
h) 48% HBr in H2O, ; i) Propionaldehyde, cat. AcOH, Na(AcO)₃BH, DCE, RT; preparative chiral
HPLC (Chiralpak AD-H); j) N-(4-oxobutyl)-1H-indole-2-carboxamide,28 cat. AcOH,
Na(AcO)₃BH, DCE, RT; k) preparative chiral HPLC (Chiralpak AD-H)
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Figure 2. Determination of relative stereochemistry for rel-trans-12 (central panel), rel-cis-12
(right panel), and direct comparison to (+)-PD128907 ((+)-trans-2; left panel) by 1H NMR.
Radioligand Binding Studies
All new compounds were tested for their affinities at hD2R and hD3R, in competition with
the agonist [3H]-(R)-(+)-7-OH-DPAT (Table 1). As previously discussed,20, 28, 29 the ability to
evaluate agonist affinities for the receptors of interest in their active states allows for a better
estimation of subtype selectivity and better resembles ligand-receptor interaction under
physiological conditions where only a fraction of the total available receptors are in the active state
and the agonists have to compete with endogenous dopamine (DA) for the OBS. Moreover,
radiolabeled agonist simplifies the binding protocol for testing new agonists’ affinity in cell
membrane preparations, and generates monophasic curves, with optimal fitting, representing the
high-affinity agonist bound state (Figure S6).
Affinities for the orthosteric agonists 1, (+)-(4aR,10bR)-2, (-)-(4aS,10bS)-2, (±)-cis-237
and bitopic agonists 3, 4, 5, and 629 are reported in Table 1 for useful comparison with the
synthesized analogues. Both (+)-(4aR,10bR)-2 and (-)-(4aS,10bS)-2 were tested alongside the
new compounds for assay validation. We confirmed that (+)-(4aR,10bR)-2 has the favored
stereochemical configuration for the 9-methoxy-3,4,4a,10b-tetrahydro-2H,5H-chromeno[4,3-
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b][1,4]oxazine scaffold.29 Inversion of configuration of the PP obtaining (-)-(4aS,10bS)-2 is
detrimental for D3R binding, with a loss of affinity >3,000-fold.37 However, it’s important to study
the cis and trans PP stereochemistry for a complete investigation of the stereochemical space and
suitability of 2 as PP in a bitopic configuration. Both rel-trans-12 and rel-cis-12 complement
extensive SAR observations, suggesting that (+)-(4aR,10bR)-2 is not suitable for either O- or N-
bitopic alkylation;29 we further observed this is not only due to regiochemistry but also
stereochemistry dependent, since (4aR,10bR) appears to be the sole privileged absolute
configuration for the bitopic analogues of 2. Compounds rel-trans-12 and rel-cis-12 showed low
micromolar affinity for D3R, and an overall loss of subtype selectivity in comparison with the
parent lead.
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Compound 529 is reported in Table 1 as reference due to its improved D3R affinity and
selectivity in comparison to its stereoisomer 6 and parent compound 1. Herein, novel bitopic
analogues were designed and synthesized using the 5-((2S,5S)-5-methylmorpholin-2-yl)pyridin-2-
amine PP, and their SAR were directed toward the study of the linker’s chemical space. Both butyl-
hydroxy and trans-cyclohexyl linkers are known to enhance D3R affinity and selectivity in D3R
antagonists and partial agonists (Figure 1).8,18,34 Despite a moderate sub-micromolar D3R affinity,
10, presented decreased binding when compared to both 4 and lead 5. Despite the loss of affinity
and selectivity, the value of this observation resides in the fact that the linker hydroxyl substitution,
though optimal for D3R antagonism is not directly transferable to agonism, probably due to
different receptor active and inactive conformations and pose co-dependence between PP and
linker. Introduction of the trans-cyclohexyl moiety,32 affords an intermediate flexibility between
4 and the more strained 5 and was better tolerated. Analogue 11 presented the highest D3R affinity
of this series, similar to antagonists 7 and 8, in the same order of magnitude with agonist 5, and
~10-15-fold improved with respect to 1. Despite a retained high D3R affinity, 11 yielded just a
moderate selectivity over D2R (6.5-fold). Structural and stereochemical requirements for both PP
and linker are unique in the design of antagonists or agonists; furthermore, for the optimal D3R
agonist affinity/subtype selectivity, (2S,5S)-2-(6-aminopyridin-3-yl)-5-methylmorpholino PP,
(1R,2S)-methyl-cyclopropyl-ethyl linker and 2-indoleamide SP seem to be the privileged synthons.
Nevertheless, compounds 5 and 11 will be further evaluated in functional assays to assess whether
their receptor-induced conformations will induce bias signaling or allosteric modulation, as well
as in computational and molecular simulation studies to correlate the experimentally measured
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structure-functions to molecular interactions at the receptor. Similarly, compounds (-)-(4aS,10bS)-
2, (±)-cis-2, rel-trans-12 and rel-cis-12, despite their limited biological profiles, are valuable tools
for computational biology. For the first time we are observing at a molecular level how changes in
each fragment of bitopic drugs affect the overall poses of the entire molecule, and how this
translates into pharmacological profiles, with the potential to finally identify the structural
fingerprints discriminating D3R agonism and antagonism, as well as D3R and D2R subtype
selectivity.
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Given the established poor D4R affinity associated with these scaffolds,29 a subset of
compounds was selected for off-target binding screening investigating affinities at different
GPCRs highly co-localized and/or functionally related to D3R. We performed competition
experiments on dopamine D1 receptors (D1R), known to form functional heteromers in vivo with
D3R,38 and  opioid receptors (OR), which are located in brain regions associated with reward
and motivation and where D3R modulate opioid self-administration.39 Experiments were
conducted in presence of [3H]-SCH23390 and [3H]-DAMGO, using HEK293 cell membranes
stably expressing hD1R and hOR, respectively. Among all the compounds, 5 presented the
highest affinity for D1R, with a low micromolar Ki. This doesn’t affect the overall pharmacological
applicability of the compound, since it still maintains a very high selectivity for D3R over D1R and
OR. This observation highlights how structurally different GPCR OBS can potentially
accommodate identical PPs, however the linker is responsible for the binding affinity, as small
changes in the linker’s substituents or rigidity result in complete inactivity for D1R.
All the studied compounds were inactive or poor binders for OR, with the exception of
10, whose hydroxyl substituted linker was detrimental for D3R binding, but appears to be an
important structural feature to achieve OR binding. Indeed, 10 is equipotent at D3R and OR,
suggesting that both receptors can be targeted by identical structural scaffolds, with the linker
being the main discriminant. Compound 10 might very well exemplify the first synthetic analogue
probing the possibility of future SAR studies directed toward multitarget ligands for simultaneous
co-modulation of dopaminergic and opioid pharmacology.
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Table 1. Radioligand competition binding assays performed on HEK293 cells stably expressing hD2R, hD3R, hD1R, and hOR
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6
7
8
9
Compounds
Structure
hD2LR
Ki ± SEM (M)
1.74 ± 0.348
(n=5)a
hD3R
hD1R
Ki ± SEM (M)
hOR
Ki ± SEM (M)
Ki ± SEM (M)
0.185 ± 0.0205***
(n=8)a
O
N
1
(d.r. 3:1)
>100b
>100b
N
10
11
12
13
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48
H2N
(+)-(4aR,10bR)-2
(-)-(4aS,10bS)-2
(±)-cis-2
0.0280 ± 0.00559
(n=4)
0.00167 ± 0.000257***
(n=3)
36.6 ± 19.8
(n=4)
>100b
>100b
NT
7.58 ± 2.62
(n=4)
5.62 ± 2.30ns
(n=3)
77.9 ± 41.4
(n=5)
>10a
>10a
NT
NT
5.22 ± 0.735
(n=3)a
6.47 ± 0.675ns
(n=3)a
3
4
NT
0.134 ± 0.0216
(n=4)a
0.00596 ± 0.000477****
(n=4)a
NT
NT
0.0878 ± 0.00981
(n=3)a
0.00185 ± 0.000137****
(n=3)a
5
6
5.63 ± 2.05
(n=3)
16.9 ± 3.10
(n=3)
0.831 ± 0.0995
(n=3)a
0.282 ± 0.024***
(n=3)a
NT
NT
1.01 ± 0.162
(n=5)
0.423 ± 0.164*
(n=5)
10
11
>100b
>100b
0.739 ± 0.0701
(n=4)
0.104 ± 0.0123
(n=4)
0.0216 ± 0.00178****
(n=5)
21.9 ± 7.92
(n=4)
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rel-trans-12
rel-cis-12
5.88 ± 2.03
(n=4)
5.54 ± 1.43ns
12.4 ± 6.36
(n=4)
(n=5)
>100a
>100a
9.42 ± 3.44
(n=4)
13.8 ± 3.13ns
(n=5)
12.3 ± 2.88
(n=4)
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Equilibrium dissociation constants (Ki) were derived from IC50 values using the Cheng−Prusoff equation.40 Each Ki value represents the
arithmetic mean ± S.E.M; n = number of independent experiments, each performed in triplicate. aData previously reported.29, 37 Unpaired
Welch’s t-test performed to compare selectivity for D3R over D2R Ki. ns = P>0.05; * = P<0.05; ** = P<0.01; *** = P<0.001; **** =
P<0.0001. b <50% inhibition at 100 M, from 2 to 5 independent triplicate experiments. NT = Not Tested.
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DISCUSSION
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The current study led us to a series of important conclusions which can guide future drug
design investigations focused on D3R agonist-receptor interactions.
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Our data suggest that when switching from an orthosteric to bitopic binding mode, 1 and
(+)-(4aR,10bR)-2 diverge toward different binding modes, possibly due to different receptor
conformations needed to accommodate them within the OBS and SBP simultaneously. We
highlighted that inverting the stereochemistry of 1, from (2R,5S) to (2S,5S), when designing bitopic
ligands allows the enantiospecific recognition of D3R.29 However, independently from
stereochemical combinations, (+)-(4aR,10bR)-2 has a unique structural motif which binds the D3R
OBS and is unsuitable for modifications into bitopic analogues. This underscores the possible
existence of multiple active states for the D3R when engaged with agonists.
These “negative” observations can be essential to build computational models predicting
how PPs can be modified in their core and stereochemistry to improve affinity and selectivities. A
direct comparison between our compounds, covering a wide range of differences in binding
affinities and selectivity, can feed extensive structural biology studies and molecular dynamic
simulations. Further investigations are elucidating whether these different ligand-induced receptor
conformations potentially affect recruitment of second messengers ultimately inducing bias
signaling.
Our data also suggest that bitopic agonists and antagonists bind differently within the D3R
active and inactive states, respectively. Despite the presence of a hydroxy substituent or a trans-
cyclohexyl group in the linker promoting high affinity and selectivity for various D3R antagonists
(i.e. 7, 8, and 9), the same substitutions applied to our bitopic agonists reduce D3R affinity and
subtype selectivity. These exemplify how SAR directed to explore different linkers’ chemical
space and chirality might represent an important future direction in drug design of highly selective
D3R agonists.
Considering the limited information regarding D2-like receptor active states, these
extensive data sets, along with our previous studies, provide a large library of D3R agonists to
elucidate a greater understanding of ligand-D3R engagement, and clarify the differences ultimately
underlying multiple active and inactive receptor conformations.
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Moreover, the growing interest in drug design assisted by artificial intelligence (A.I.)-
machine learning (M.L.), and the extensive efforts directed towards multi-parameter
physiochemical analyses, would greatly benefit from our D3R agonists library.42, 43 M.L. requires
accurate learning datasets and model validation establishing recognizable structural pattern for
model’s predictive validity. Active compounds used for training sets are often taken from known
experimental observations, however chemogenetic SAR libraries usually lack inactive or poorly
binding compounds with stereochemistry-dependent structural features.44-46 Our molecules, with
multiple characterized chiral centers, and detailed in vitro pharmacology can help M.L. training,
provide structural information on fragment-based drug design, and expand drug prediction
libraries based on chirality and PP suitability for bitopic modifications.
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Linker chirality and chemical space investigation allowed us to identify pharmacological
profiles depending predominantly on its substitutions and rigidity: i) cyclopropyl-containing linker
(5) is privileged for high D3R affinity and selectivity, and is also an important synthon for D1R
recognition directed toward D1R-D3R multitarget drug design; ii) cyclohexyl-containing linker
(11) is well-tolerated at D3R with moderate subtype selectivity, despite a slight loss in overall
affinity; iii) hydroxyl-substituted linker (10) causes a loss in D3R affinity, maintains a moderate
subtype selectivity over D2R, and results in an increased OR affinity. This might inspire new
SAR efforts to generate D3R-OR dual-target chemical tools for studying dopaminergic and opioid
pharmacology in pain, reward and motivation. These data also suggest that identification of new
leads and structural pharmacophores for multi-target GPCR studies can be identified via off-target
binding screening based on receptor co-localization and functional similarities, more than just
simple structural analogies between receptor proteins.
Supplementary Material
Supporting Information containing the following materials is available free of charge.
Experimental methods, compound purity and characterization, cell culture and radioligand
binding protocols, HPLC chromatograms and HRMS-MS/MS analyses.
Authors Contributions
FOB, AHN and AB designed the project, supervised and performed experiments, and wrote the
manuscript.
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AKNOWLEDGMENTS
This project was supported by the National Institute on Drug Abuse – Intramural Research
Program Z1A DA000424 and Z1A DA000609. The authors thank Dr. Ludovic Muller from the
Structural Biology Core at NIDA-IRP for high-resolution MS/MS analyses, Dr. Daryl Guthrie and
Dr. Anerbasha Shaik from NIDA-IRP.
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ABBREVIATIONS
CNS, Central Nervous System; OBS, Orthosteric Binding Site; SBP, Secondary Binding Pocket;
ABS, Allosteric Binding Site; SUD, Substance Use Disorder; M.L., Machine Learning, AI,
Artificial Intelligence.
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