The genesis of designing
bivalent or bitopic molecules that engender
unique pharmacological properties began with Portoghese’s work
directed toward opioid receptors, in the early 1980s. This strategy
has evolved as an attractive way to engineer highly selective compounds
for targeted G-protein coupled receptors (GPCRs) with optimized efficacies
and/or signaling bias. The emergence of X-ray crystal structures of
many GPCRs and the identification of both orthosteric and allosteric
binding sites have provided further guidance to ligand drug design
that includes a primary pharmacophore (PP), a secondary pharmacophore
(SP), and a linker between them. It is critical to note the synergistic
relationship among all three of these components as they contribute
to the overall interaction of these molecules with their receptor
proteins and that strategically designed combinations have and will
continue to provide the GPCR molecular tools of the future.
Because
of the large degree of homology among dopamine D2-like
receptors, discovering ligands capable of discriminating between
the D2, D3, and D4 receptor subtypes
remains a significant challenge. Previous work has exemplified the
use of bitopic ligands as a powerful strategy in achieving subtype
selectivity for agonists and antagonists alike. Inspired by the potential
for chemical modification of the D3 preferential agonists
(+)-PD128,907 (1) and PF592,379 (2), we
synthesized bitopic structures to further improve their D3R selectivity. We found that the (2S,5S) conformation of scaffold 2 resulted in a privileged
architecture with increased affinity and selectivity for the D3R. In addition, a cyclopropyl moiety incorporated into the
linker and full resolution of the chiral centers resulted in lead
compound 53 and eutomer 53a that demonstrate
significantly higher D3R binding selectivities than the
reference compounds. Moreover, the favorable metabolic stability in
rat liver microsomes supports future studies in in vivo models of
dopamine system dysregulation.
The need for safer pain-management
therapies with decreased abuse
liability inspired a novel drug design that retains μ-opioid
receptor (MOR)-mediated analgesia, while minimizing addictive liability.
We recently demonstrated that targeting the dopamine D3 receptor (D3R) with highly selective antagonists/partial
agonists can reduce opioid self-administration and reinstatement to
drug seeking in rodent models without diminishing antinociceptive
effects. The identification of the D3R as a target for
the treatment of opioid use disorders prompted the idea of generating
a class of ligands presenting bitopic or bivalent structures, allowing
the dual-target binding of the MOR and D3R. Structure–activity
relationship studies using computationally aided drug design and in vitro binding assays led to the identification of potent
dual-target leads (23, 28, and 40), based on different structural templates and scaffolds, with moderate
(sub-micromolar) to high (low nanomolar/sub-nanomolar) binding affinities.
Bioluminescence resonance energy transfer-based functional studies
revealed MOR agonist–D3R antagonist/partial agonist
efficacies that suggest potential for maintaining analgesia with reduced
opioid-abuse liability.
In this study, starting from our selective D 3 R 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 transcyclohexyl 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 D 3 R active and inactive states, and assist multitarget receptor recognition. Exploring stereochemical complexity and developing extended D 3 R 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 D 3 R agonism may be utilized in machine learning and artificial intelligence (AI)based drug design, in the future.
The dopamine D2/D3 receptor (D2R/D3R) agonists are used as therapeutics for Parkinson’s disease (PD) and other motor disorders. Selective targeting of D3R over D2R is attractive because of D3R’s restricted tissue distribution with potentially fewer side-effects and its putative neuroprotective effect. However, the high sequence homology between the D2R and D3R poses a challenge in the development of D3R selective agonists. To address the ligand selectivity, bitopic ligands were designed and synthesized previously based on a potent D3R-preferential agonist PF592,379 as the primary pharmacophore (PP). This PP was attached to various secondary pharmacophores (SPs) using chemically different linkers. Here, we characterize some of these novel bitopic ligands at both D3R and D2R using BRET-based functional assays. The bitopic ligands showed varying differences in potencies and efficacies. In addition, the chirality of the PP was key to conferring improved D3R potency, selectivity, and G protein signaling bias. In particular, compound AB04-88 exhibited significant D3R over D2R selectivity, and G protein bias at D3R. This bias was consistently observed at various time-points ranging from 8 to 46 min. Together, the structure-activity relationships derived from these functional studies reveal unique pharmacology at D3R and support further evaluation of functionally biased D3R agonists for their therapeutic potential.
Linkers
are emerging as a key component in regulating the pharmacology
of bitopic ligands directed toward G-protein coupled receptors (GPCRs).
In this study, the role of regio- and stereochemistry in cyclic aliphatic
linkers tethering well-characterized primary and secondary pharmacophores
targeting dopamine D2 and D3 receptor subtypes
(D2R and D3R, respectively) is described. We
introduce several potent and selective D2R (
rel-trans-16b; D2R K
i = 4.58 nM) and D3R (
rel-cis-14a; D3R K
i = 5.72 nM) agonists while modulating subtype
selectivity in a stereospecific fashion, transferring D2R selectivity toward D3R via inversion of the stereochemistry
around these cyclic aliphatic linkers [e.g., (−)-(1S,2R)-43 and (+)-(1R,2S)-42]. Pharmacological observations
were supported with extensive molecular docking studies. Thus, not
only is it an innovative approach to modulate the pharmacology of
dopaminergic ligands described, but a new class of optically active
cyclic linkers are also introduced, which can be used to expand the
bitopic drug design approach toward other GPCRs.
The
crystal structure of the dopamine D3 receptor (D3R) in complex with eticlopride inspired the design of bitopic
ligands that explored (1) N-alkylation of the eticlopride’s
pyrrolidine ring, (2) shifting of the position of the pyrrolidine
nitrogen, (3) expansion of the pyrrolidine ring system, and (4) incorporation
of O-alkylations at the 4-position. Structure activity
relationships (SAR) revealed that moving the N- or
expanding the pyrrolidine ring was detrimental to D2R/D3R binding affinities. Small pyrrolidine N-alkyl groups were poorly tolerated, but the addition of a linker
and secondary pharmacophore (SP) improved affinities. Moreover, O-alkylated analogues showed higher binding affinities compared
to analogously N-alkylated compounds, e.g., O-alkylated 33 (D3R, 0.436 nM and
D2R, 1.77 nM) vs the N-alkylated 11 (D3R, 6.97 nM and D2R, 25.3 nM).
All lead molecules were functional D2R/D3R antagonists.
Molecular models confirmed that 4-position modifications would be
well-tolerated for future D2R/D3R bioconjugate
tools that require long linkers and or sterically bulky groups.
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