Non-catechol Agonists Research Articles

Crystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist

Dopamine D1 receptor (D1R) is an important drug target implicated in many psychiatric and neurological disorders. Selective agonism of D1R are sought to be the therapeutic strategy for these disorders. Most selective D1R agonists share a dopamine-like catechol moiety in their molecular structure, and their therapeutic potential is therefore limited by poor pharmacological properties in vivo. Recently, a class of non-catechol D1R selective agonists with a distinct scaffold and pharmacological properties were reported. Here, we report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 at 3.8 Å resolution. The structure reveals the ligand bound to D1R in an extended conformation, spanning from the orthosteric site to extracellular loop 2 (ECL2). Structural analysis reveals that the unique features of D1R ligand binding pocket explains the remarkable selectivity of this scaffold for D1R over other aminergic receptors, and sheds light on the mechanism for D1R activation by the non-catechol agonist.

Ligand recognition and allosteric regulation of DRD1-Gs signaling complexes

Dopamine receptors, including D1- and D2-like receptors, are important therapeutic targets in a variety of neurological syndromes, as well as cardiovascular and kidney diseases. Here, we present five cryoelectron microscopy (cryo-EM) structures of the dopamine D1 receptor (DRD1) coupled to Gs heterotrimer in complex with three catechol-based agonists, a non-catechol agonist, and a positive allosteric modulator for endogenous dopamine. These structures revealed that a polar interaction network is essential for catecholamine-like agonist recognition, whereas specific motifs in the extended binding pocket were responsible for discriminating D1- from D2-like receptors. Moreover, allosteric binding at a distinct inner surface pocket improved the activity of DRD1 by stabilizing endogenous dopamine interaction at the orthosteric site. DRD1-Gs interface revealed key features that serve as determinants for G protein coupling. Together, our study provides a structural understanding of the ligand recognition, allosteric regulation, and G protein coupling mechanisms of DRD1.

Characterization of PF-6142, a Novel, Non-Catecholamine Dopamine Receptor D1 Agonist, in Murine and Nonhuman Primate Models of Dopaminergic Activation.

Selective activation of dopamine D1 receptors remains a promising pro-cognitive therapeutic strategy awaiting robust clinical investigation. PF-6142 is a key example from a recently disclosed novel series of non-catechol agonists and partial agonists of the dopamine D1/5 receptors (D1R) that exhibit pharmacokinetic (PK) properties suitable for oral delivery. Given their reported potential for functionally biased signaling compared to known catechol-based selective agonists, and the promising rodent PK profile of PF-6142, we utilized relevant in vivo assays in male rodents and male and female non-human primates (NHP) to evaluate the pharmacology of this new series. Studies in rodents showed that PF-6142 increased locomotor activity and prefrontal cortex acetylcholine release, increased time spent in wakefulness, and desynchronized the EEG, like known D1R agonists. D1R selectivity of PF-6142 was supported by lack of effect in D1R knock-out mice and blocked response in the presence of the D1R antagonist SCH-23390. Further, PF-6142 improved performance in rodent models of NMDA receptor antagonist-induced cognitive dysfunction, such as MK-801-disrupted paired-pulse facilitation, and ketamine-disrupted working memory performance in the radial arm maze. Similarly, PF-6142 reversed ketamine-induced deficits in NHP performing the spatial delayed recognition task. Of importance, PF-6142 did not alter the efficacy of risperidone in assays predictive of antipsychotic-like effect in rodents including pre-pulse inhibition and conditioned avoidance responding. These data support the continued development of non-catechol based D1R agonists for the treatment of cognitive impairment associated with brain disorders including schizophrenia.

Novel Strategies To Activate the Dopamine D1 Receptor: Recent Advances in Orthosteric Agonism and Positive Allosteric Modulation.

The five dopamine receptor subtypes (D1-5) are activated by the endogenous catecholamine dopamine. Sustained research has sought to identify selective ligands for receptor subtypes. In particular, activation of the D1 receptor has attracted attention due to its promising role in neurological diseases. Initial attempts to identify agonists yielded catechol derivatives, mimicking dopamine, with suboptimal DMPK parameters and low selectivity over the D5 subtype. However, more recent efforts to identify ligands capable of activating the D1 receptor have made substantial progress with the identification of non-catechol agonists with suitable properties to progress to clinical studies. In addition, several research groups have identified positive allosteric modulators that offer new potential. Furthermore, structural studies have surprisingly uncovered two potential allosteric binding sites, the most characterized of which appears to be on intracellular loop 2 (ICL2). This review highlights the recent progress in the field, covering both orthosteric and allosteric modes of activation, discusses the elucidation of the allosteric binding sites, and summarizes the clinical development status of various compounds.

Impaired \u03b2-arrestin recruitment and reduced desensitization by non-catechol agonists of the D1 dopamine receptor

Selective activation of dopamine D1 receptors (D1Rs) has been pursued for 40 years as a therapeutic strategy for neurologic and psychiatric diseases due to the fundamental role of D1Rs in motor function, reward processing, and cognition. All known D1R-selective agonists are catechols, which are rapidly metabolized and desensitize the D1R after prolonged exposure, reducing agonist response. As such, drug-like selective D1R agonists have remained elusive. Here we report a novel series of selective, potent non-catechol D1R agonists with promising in vivo pharmacokinetic properties. These ligands stimulate adenylyl cyclase signaling and are efficacious in a rodent model of Parkinson's disease after oral administration. They exhibit distinct binding to the D1R orthosteric site and a novel functional profile including minimal receptor desensitization, reduced recruitment of β-arrestin, and sustained in vivo efficacy. These results reveal a novel class of D1 agonists with favorable drug-like properties, and define the molecular basis for catechol-specific recruitment of β-arrestin to D1Rs.

NOVEL NON-CATECHOL D1 RECEPTOR AGONISTS EXHIBIT SUSTAINED PHARMACOLOGICAL ACTIVITY

Increasing evidence associates dysfunctional dopamine signaling with the motor and cognitive deficits in Alzheimer's disease. While D1 receptor (D1R) specific pathologies are reported, current D1R compounds have limited therapeutic potential due to their poor CNS penetration, negligible oral bioavailability, rapid metabolism and propensity to cause receptor desensitization. We describe here a novel class of D1R agonists that show reduced receptor desensitization in vitro as well as reduced tachyphylaxis in vivo. For in vitro D1 receptor desensitization experiments cAMP activity was measured from rat striatal neurons using the Cisbio HTRF cAMP dynamic range assay kit. For in vivo testing of tachyphylaxis, Sprague-Dawley rats with unilateral 6-OHDA lesions (n=17) were individually monitored for rotational behavior using infrared cameras and dosed using a crossover design (2-week washout period) in their home cages. In each treatment period animals received six doses of either a catechol (A-77636, 0.32 mg/kg S.C.) or non-catechol (PF-2334, 10.78 mg/kg, P.O.) D1R agonist administered every 12 h followed by a single dose of the D2 agonist quinpirole (0.1 mg/kg S.C.). In vitro, pre-treatment with catecholamine agonists (dopamine, A-77636, dihydrexidine, or SKF-38393 all at 10 mM) blunted the cAMP increase induced by subsequent D1R agonist challenge by 40–60% (1 mM SKF-81297). In contrast, pretreatment with the non-catechol agonist PF-2334 did not diminish the subsequent elevation in cAMP by D1R agonist challenge. In vivo, after 72 hours of chronic treatment with a catechol agonist (A-77636), animals displayed near-zero contralateral rotational behavior. In contrast, non-catechol agonist (PF-2334) treated animals were still rotating at the 72-hour time point. The absence of rotational behavior under A-77636 was likely due to D1R tachyphylaxis since treatment with a D2 agonist caused a significant increase in the number of contralateral rotations. The persistent in vitro and in vivo pharmacodynamic response of PF-2334 supports limited receptor desensitization by non-catechol agonists and suggests durable D1R agonism by PF-2334. These data highlight the therapeutic potential of this novel class of compounds and warrants further investigation in relevant Alzheimer's disease animal models.

Computational Mapping of the Conformational Transitions in Agonist Selective Pathways of a G-Protein Coupled Receptor

The active state conformation of a G-protein coupled receptor (GPCR) is influenced by the chemical structure and the efficacy of the bound ligand. Insight into the active state conformation as well as the activation pathway for ligands with different efficacies is critical in designing functionally specific drugs for GPCRs. Starting from the crystal structure of the beta2-adrenergic receptor, we have used coarse grain computational methods to understand the modulation of the potential energy landscape of the receptor by two full agonists, two partial agonists, and an inverse agonist. Our coarse grain method involves a systematic conformational spanning of the receptor transmembrane helices followed by an energy minimization and ligand redocking in each sampled conformation. We have derived the activation pathways for several agonists and partial agonists, using a Monte Carlo algorithm, and these are in agreement with fluorescence spectroscopy measurements. The calculated pathways for the full agonists start with an energy downhill step leading to a stable intermediate followed by a barrier crossing leading to the active state. We find that the barrier crossing involves breaking of an interhelical hydrogen bond between helix5 and helix6, and polarization of the binding site residues by water facilitates the barrier crossing. The uphill step in the partial agonist salbutamol induced activation is distinct from full agonist norepinephrine, and originates from steric hindrance with the aromatic residues on helix6. Virtual ligand screening with the salbutamol-stabilized conformation shows enrichment of noncatechol agonists over the norepinephrine-stabilized conformation. Our computational method provides an unprecedented opportunity to derive hypotheses for experiments and also understand activation mechanisms in GPCRs.

Activation Pathways of Agonists, Partial Agonists and Inverse Agonist in Beta1 and Beta2 Adrenergic Receptors

Modulation of cell signaling by ligands of different efficacies via G-protein coupled receptors (GPCRs), depends intrinsically on the effect of the ligand on the dynamics between the multiple conformational states of these proteins. Ligands with different efficacies can remodel the energy landscape of the receptors, thereby perturbing this conformational equilibrium in many ways depending on the nature of the ligand, and the G-proteins that the receptor couples to, thereby conferring functional specificity. Understanding activation dynamics and pathways is vital in designing functionally specific drugs for GPCRs.Starting from the crystal structure of beta2-adrenergic receptor (B2AR), we have used LITiCon computational method to predict the ligand stabilized receptor state with full (epinephrine and norepinephrine), partial (salbutamol and dopamine), and inverse agonists (carazolol) bound. We have calculated the minimum energy pathway going from the inactive to the ligand stabilized state for each of the ligand/receptor complex. The activation pathways derived for all the agonists studied here are in agreement with fluorescence lifetime measurements1,2. We have also studied the norepinephrine activation pathway for the mutant m23 avian beta1-adrenergic receptor (B1AR) that has been crystallized, and the wild type B1AR. We show that the activation barrier for activation of the mutant m23B1AR by norepinephrine, is larger than for the wildtype B1AR, which is in agreement with the previous experimental finding that m23B1AR requires higher concentration of norepinephrine for activation3.Virtual ligand screening with the salbutamol-stabilized B2AR conformation shows enrichment of non-catechol agonists over norepinephrine-stabilized conformation. Our computational method provides an unprecedented opportunity to understand activation mechanisms in GPCRs.1. Swaminath G, et al. (2004), J. Biol. Chem. 279:686-691.2. Swaminath G, et al. (2005) , J. Biol. Chem. 280:22165-22171.3. Warne T., et. Al., (2008), Nature, 454, 486-491.

Dopamine D 2 synthesis-modulating receptors are present in the striatum of the guinea pig

Dopamine and selective agonists of D 1 [(1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride, SKF 38393] and D 2 [(3-[2-[ N-(3-hydroxyphenylethyl)- N-propylamino]ethyl] phenol, RU 24926] receptors were examined as inhibitors of the activity of tyrosine hydroxylase in the striatum of the guinea pig. In soluble enzyme preparations, the agonists were weak inhibitors of the activity of tyrosine hydroxylase. However, the catechol-containing agonists dopamine (EC 50 = 44.7 μM) and SKF 38393 (EC 50 = 35.5 μM) were more potent than the non-catechol agonist RU 24926 (EC 50 = 447 μM). All of the agonists were much more potent in synaptosome-rich preparations of guinea pig striatum, where stimulation of autoreceptors mediated inhibition of the enzyme (SKF 38393, D 1, EC 50 = 27 nM; RU 24926, D 2, EC 50 = 30nM; dopamine, non-selective, EC 50 = 1.5 μM). The D 1 antagonist, SCH 23390 [(R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-(-−)-3-benzazepine hydrochloride], did not significantly reduce the action of SKF 38393 or dopamine. Furthermore, the D 2 antagonist, (—)-sulpiride, significantly antagonized the inhibitory activity of both RU 24926 and dopamine. Studies in synaptosome-rich preparations from the striatum of the rat showed that both SKF 38393 (EC 50 = 398 nM) and RU 24926 (EC 50 = 58 nM) were also effective autoreceptor-mediated inhibitors of the activity of tyrosine hydroxylase in the rat. However, in the rat, SCH 23390 and (−)-sulpiride were equally effective in attenuating the inhibitory actions of dopamine. These results suggest that: (1) dopamine autoreceptors, similar to the D 2 type, modulate tyrosine hydroxylase in the striatum of the guinea pig. (2) In the striatum of the rat, dopamine synthesis-modulating autoreceptors are either a heterogeneous population of D 1, and D 2 receptors or a single population of receptors, with combined features of classic D 1 and D 2 receptors.

Is inhibition of striatal synaptosomal tyrosine hydroxylation by dopamine agonists a measure of dopamine autoreceptor function?

Rat striatal synaptosomal tyrosine hydroxylation was inhibited dose- and pH dependently by a number of dopamine agonists. The catecholic agonists apomorphine and (-)N-n-propylnorapomorphine inhibited synaptosomal tyrosine hydroxylase completely, with IC50 values of around 0.3 mumol/l at pH 6.6. The noncatechol agonists pergolide and bromocriptine and the putative dopamine autoreceptor agonists 3-PPP(-), 3-PPP(+), HW-165 and B-HT 920 produced only partial inhibition of synaptosomal tyrosine hydroxylation at high concentrations. Comparison of the inhibition of synaptosomal and soluble tyrosine hydroxylase indicated that the inhibition produced by apomorphine could be ascribed to a direct effect on the enzyme, whereas this was not the case for the noncatechol agonists. The inhibition produced by pergolide and 3-PPP(-) was not antagonised by either dopamine receptor or alpha-adrenoceptor antagonists. The present results have been compared with results reported in the literature for inhibition of synaptosomal tyrosine hydroxylation and for two other tests of dopamine autoreceptor agonist activity (inhibition of dopamine release from striatal slices in vitro, and inhibition of the gamma-butyrolactone induced increase in dopamine synthesis in vivo). It is concluded that inhibition of striatal synaptosomal tyrosine hydroxylation by dopamine agonists does not fulfil the criteria required for it to be considered as a useful measure of dopamine autoreceptor function.