Exploring Structural Determinants of Bias among D4 Subtype-Selective Dopamine Receptor Agonists.

The high affinity dopamine D4 receptor ligand APH199 and derivatives thereof exhibit bias toward the Gi signaling pathway over ß-arrestin recruitment compared to quinpirole. Based on APH199, two novel groups of D4 subtype selective ligands were designed and evaluated, in which the original benzyl phenylsemicarbazide substructure was replaced by either a biphenylmethyl urea or a biphenyl urea moiety. Functional assays revealed a range of different bias profiles among the newly synthesized compounds, namely, with regard to efficacy, potency, and GRK2 dependency, in which bias factors range from 1 to over 300 and activation from 15% to over 98% compared to quinpirole. These observations demonstrate that within bias, an even more precise tuning toward a particular profile is possible, which─in a general sense─could become an important aspect in future drug development. Docking studies enabled further insight into the role of the ECL2 and the EPB in the emergence of bias, thereby taking advantage of the diversity of functionally selective D4 agonists now available.

Computational insights into ligand-induced G protein and ß-arrestin signaling of the dopamine D1 receptor.

The dopamine D1 receptor (D1R), is a class A G protein coupled-receptor (GPCR) which has been a promising drug target for psychiatric and neurological disorders such as Parkinson's disease (PD). Previous studies have suggested that therapeutic effects can be realized by targeting the ß-arrestin signaling pathway of dopamine receptors, while overactivation of the G protein-dependent pathways leads to side effects, such as dyskinesias. Therefore, it is highly desirable to develop a D1R ligand that selectively regulates the ß-arrestin pathway. Currently, most D1R agonists are signaling-balanced and stimulate both G protein and ß-arrestin pathways, with a few reports of G protein biased ligands. However, identification and characterization of ß-arrestin biased D1R agonists has been a challenge thus far. In this study, we implemented Gaussian accelerated molecular dynamics (GaMD) simulations to provide valuable computational insights into the possible underlying molecular mechanism of the different signaling properties of two catechol and two non-catechol D1R agonists that are either G protein biased or signaling-balanced. Dynamic network analysis further identified critical residues in the allosteric signaling network of D1R for each ligand at different conformational or binding states. Some of these residues are crucial for G protein or arrestin signals of GPCRs based on previous studies. Finally, we provided a molecular design strategy which can be utilized by medicinal chemists to develop potential ß-arrestin biased D1R ligands. The proposed hypotheses are experimentally testable and can guide the development of safer and more effective medications for a variety of CNS disorders.

Bivalent dopamine agonists with co-operative binding and functional activities at dopamine D2 receptors, modulate aggregation and toxicity of alpha synuclein protein.

To follow up on our previous report on bivalent compounds exhibiting potent co-operative binding at dopamine D2 receptors, we modified the structure of the linker in our earlier bivalent molecules (S)-6-((9-(((R)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)nonyl)-(propyl)amino)-5,6,7,8-tetrahydronaphthalen-1-ol (Ia) and (S)-6-((10-(((R)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)decyl)(propyl)amino)-5,6,7,8-tetrahydronaphthalen-1-ol (Ib) (Fig. 1) connecting the two pharmaophoric moieties to observe any tolerance in maintaining similar affinities and potencies. Specifically, we introduced aromatic and piperazine moieties in the linker to explore their effect. Overall, similar activities at D2 receptors as observed in our earlier study was maintained in the new molecules e.g. (6S,6'S)-6,6'-((1,4-phenylenebis(ethane-2,1-diyl))bis(propylazanediyl))bis(5,6,7,8-tetrahydronaphthalen-1-ol) (D-382) (Ki, D2 = 3.88 nM). The aromatic moiety in D-382 was next functionalized by introducing hydroxyl groups to mimic polyhydroxy natural products which are known to interact with amyloidogenic proteins. Such a transformation resulted in development of compounds like 2,5-bis(2-(((S)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethyl)benzene-1,4-diol (D-666) (Ki, D2 = 7.62 nM) which retained similar affinity and potency at D2 receptors. Such dihydroxyl compounds turned out to be potent inhibitors against aggregation and toxicity of recombinant alpha synuclein protein. The work reported here is in line with our overall goal to develop multifunctional dopamine agonist for symptomatic and disease modifying treatment of Parkinson's disease.

Ligand recognition and biased agonism of the D1 dopamine receptor.

Dopamine receptors are widely distributed in the central nervous system and are important therapeutic targets for treatment of various psychiatric and neurological diseases. Here, we report three cryo-electron microscopy structures of the D1 dopamine receptor (D1R)-Gs complex bound to two agonists, fenoldopam and tavapadon, and a positive allosteric modulator LY3154207. The structure reveals unusual binding of two fenoldopam molecules, one to the orthosteric binding pocket (OBP) and the other to the extended binding pocket (EBP). In contrast, one elongated tavapadon molecule binds to D1R, extending from OBP to EBP. Moreover, LY3154207 stabilizes the second intracellular loop of D1R in an alpha helical conformation to efficiently engage the G protein. Through a combination of biochemical, biophysical and cellular assays, we further show that the broad conformation stabilized by two fenoldopam molecules and interaction between TM5 and the agonist are important for biased signaling of D1R.

2-Phenylcyclopropylmethylamine Derivatives as Dopamine D2 Receptor Partial Agonists: Design, Synthesis, and Biological Evaluation.

Partial agonist activity at the dopamine D2 receptor (D2R) is the primary pharmacological feature of the third-generation antipsychotics─aripiprazole, brexpiprazole, and cariprazine. However, all these drugs share a common phenyl-piperazine moiety as the primary pharmacophore. In this study, we designed and synthesized a series of novel compounds based on the 2-phenylcyclopropylmethylamine (PCPMA) scaffold and studied their pharmacological activity at the D2R. A number of potent D2R partial agonists were identified through binding affinity screening and functional activity profiling in both G protein and ß-arrestin assays. The structure-functional activity relationship results showed that the spacer group is crucial for fine-tuning the intrinsic activity of these compounds. Compounds (+)-14j and (+)-14l showed good pharmacokinetic properties and an unexpected selectivity against the serotonin 2A (5-HT2A) receptor. Preliminary suppressive effects in a mouse hyperlocomotion model proved that these PCPMA-derived D2R partial agonists are effective as potential novel antipsychotics.

Chiral Cyclic Aliphatic Linkers as Building Blocks for Selective Dopamine D2 or D3 Receptor Agonists.

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 Ki = 4.58 nM) and D3R (rel-cis-14a; D3R Ki = 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.

Treatment of subcutaneous nodules after infusion of apomorphine; a biopsy-controlled study comparing 4 frequently used therapies.

This study aimed to provide clinical evidence for existing treatments of subcutaneous nodules after subcutaneous infusion of apomorphine, using a biopsy-controlled prospective crossover design of four treatments. We demonstrated that dilution of apomorphine significantly improved patient satisfaction, while subcutaneous hydrocortisone reduced nodule size, however with no differences in the histopathology.

Evaluation of Substituted N-Phenylpiperazine Analogs as D3 vs. D2 Dopamine Receptor Subtype Selective Ligands.

N-phenylpiperazine analogs can bind selectively to the D3 versus the D2 dopamine receptor subtype despite the fact that these two D2-like dopamine receptor subtypes exhibit substantial amino acid sequence homology. The binding for a number of these receptor subtype selective compounds was found to be consistent with their ability to bind at the D3 dopamine receptor subtype in a bitopic manner. In this study, a series of the 3-thiophenephenyl and 4-thiazolylphenyl fluoride substituted N-phenylpiperazine analogs were evaluated. Compound 6a was found to bind at the human D3 receptor with nanomolar affinity with substantial D3 vs. D2 binding selectivity (approximately 500-fold). Compound 6a was also tested for activity in two in-vivo assays: (1) a hallucinogenic-dependent head twitch response inhibition assay using DBA/2J mice and (2) an L-dopa-dependent abnormal involuntary movement (AIM) inhibition assay using unilateral 6-hydroxydopamine lesioned (hemiparkinsonian) rats. Compound 6a was found to be active in both assays. This compound could lead to a better understanding of how a bitopic D3 dopamine receptor selective ligand might lead to the development of pharmacotherapeutics for the treatment of levodopa-induced dyskinesia (LID) in patients with Parkinson's disease.

Computational study on new natural compound agonists of dopamine receptor.

Dopamine receptor, a polypeptide chain composed of 7 hydrophobic transmembrane regions, is a new and vital drug target, especially Dopamine receptor 2(D2). Targeting dopamine receptors, Dopamine receptor agonists are a class of drugs similar in function and structure to dopamine and can directly act on dopamine receptors and activate it. Clinically, Dopamine receptor agonist drugs have achieved significant therapeutic effects on prolactinoma and Parkinson's Disease. In the study, we virtually screened a series of potential effective agonists of Dopamine receptor by computer techniques. Firstly, we used the Molecular Docking (LibDock) step to screen out some molecules that can dock well with the protein. Then, analysis of toxicity prediction and ADME (adsorption, distribution, metabolism and excretion) were carried out. More precise molecular docking (CDOCKER) and 3-Dimensional Quantitative Structure-Activity Relationship Modeling Study(3D-QSAR) pharmacophore generation were implemented to research and explore these compounds' binding mechanism with Dopamine receptor. Last but not least, to assess compound's binding stabilities, we carried out a molecular dynamic analysis. As the results show, two compounds (ZINC000008860530 and ZINC000004096987) from the small molecule database (ZINC database) were potential effective agonists of Dopamine receptor. These two compounds can combine with Dopamine receptor with higher affinity and proved to be no toxic. The cell experiment showed that two compounds could inhibit the proliferation and PRL secretion of MMQ cells (pituitary tumor cells). Thus, this study provided valuable information about Dopamine receptor agonist-based drug discovery. So, this study will benefit patients with prolactinoma and Parkinson's disease a lot.

Dopamine D2 Receptor Agonist Binding Kinetics-Role of a Conserved Serine Residue.

The forward (kon) and reverse (koff) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Kds derived from the koff and kon estimates correlated well with EC50 values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing D2R agonist candidate drugs.

Effect of pH on Iontophoretic Transport of Pramipexole Dihydrochloride across Human Epidermal Membrane.

PURPOSE: Drugs with higher molecular charges generally show higher flux enhancement when electromigration is the main mechanism in transdermal iontophoresis. This study evaluated the effect of decreasing the formulation pH to increase the positive charges of pramipexole dihydrochloride (PXCl) on its iontophoretic transport across skin. METHODS: In vitro transdermal iontophoresis of PXCl in buffer solution isotonized with either sodium chloride or mannitol were performed in a pH range of 3.0-7.0. Experiments of iontophoresis under symmetric condition with respect to donor and receiver pH and passive transport of the drugs after pretreatment with iontophoresis were conducted to investigate the transport mechanism involved. RESULTS: Iontophoretic permeation of PXCl was pH-dependent in drug solution isotonized with mannitol. The iontophoretic flux of PXCl with valence z = +2 at pH 3.0 was half of that of PXCl with z = +1 at pH 7.0. The results suggest that the decrease in PXCl delivery at higher valence at pH 3 was mainly due to pH-dependent selectivity of PX ion permeation across the skin and not electroosmosis. CONCLUSIONS: Skin permselectivity is a significant factor for iontophoretic transport of PXCl, and reducing formulation pH to increase the positive charges on PX ions did not enhance PXCl delivery.

Tritium labelling of two potent dopamine D2 receptor agonists at high specific activity.

An efficient method is described to radiolabel several dopamine D2 receptor agonists with tritium at high specific activity.

Biosynthesis, total synthesis, and biological profiles of Ergot alkaloids.

While the use of ergot alkaloids in folk medicine has been practiced for millennia, systematic investigations on their therapeutic potential began about 100 years ago. Subsequently, Albert Hofmann's discovery of lysergic acid diethylamide (LSD) and its intense psychedelic properties garnered worldwide attention and prompted further studies of this compound class. As a result, several natural ergot alkaloids were discovered and unnatural analogs were synthesized, and some were used to treat an array of maladies, including Alzheimer's and Parkinson's disease. While LSD was never commercially approved, recent clinical studies have found it can be an innovative and effective treatment option for several psychiatric disorders. Ongoing biosynthetic and total synthetic investigations aim to understand the natural origins of ergot alkaloids, help develop facile means to produce these natural products and enable their continued use as medicinal chemistry lead structures. This review recounts major developments over the past 20 years in biosynthetic, total synthetic, and pharmaceutical studies. Many ergot alkaloid biosynthetic pathways have been elucidated, with some of them subsequently applied toward "green" syntheses. New chemical methodologies have fostered a fast and efficient access to the ergoline scaffold, prompting some groups to investigate biological properties of natural product-like ergot alkaloids. Limited pharmaceutical applications have yet to completely bypass the undesirable side effects of ergotism, suggesting further studies of this drug class are likely needed and will potentially harness major therapeutic significance.

Pharmacological characterization of a new series of carbamoylguanidines reveals potent agonism at the H2R and D3R.

Even today, the role of the histamine H2 receptor (H2R) in the central nervous system (CNS) is widely unknown. In previous research, many dimeric, high-affinity and subtype-selective carbamoylguanidine-type ligands such as UR-NK22 (5, pKi = 8.07) were reported as H2R agonists. However, their applicability to the study of the H2R in the CNS is compromised by their molecular and pharmacokinetic properties, such as high molecular weight and, consequently, a limited bioavailability. To address the need for more drug-like H2R agonists with high affinity, we synthesized a series of monomeric (thio)carbamoylguanidine-type ligands containing various spacers and side-chain moieties. This structural simplification resulted in potent (partial) agonists (guinea pig right atrium, [35S]GTPγS and ß-arrestin2 recruitment assays) with human (h) H2R affinities in the one-digit nanomolar range (pKi (139, UR-KAT523): 8.35; pKi (157, UR-MB-69): 8.69). Most of the compounds presented here exhibited an excellent selectivity profile towards the hH2R, e.g. 157 being at least 3800-fold selective within the histamine receptor family. The structural similarities of our monomeric ligands to pramipexole (6), a dopamine receptor agonist, suggested an investigation of the binding behavior at those receptors. The target compounds were (partial) agonists with moderate affinity at the hD2longR and agonists with high affinity at the hD3R (e.g. pKi (139, UR-KAT523): 7.80; pKi (157, UR-MB-69): 8.06). In summary, we developed a series of novel, more drug-like H2R and D3R agonists for the application in recombinant systems in which either the H2R or the D3R is solely expressed. Furthermore, our ligands are promising lead compounds in the development of selective H2R agonists for future in vivo studies or experiments utilizing primary tissue to unravel the role and function of the H2R in the CNS.

Discovery of a true bivalent dopamine D2 receptor agonist.

Employing two different alkyne-modified dopamine agonists to construct bivalent compounds via click chemistry resulted in the identification of a bivalent ligand (11c) for dopamine D2 receptor homodimer, which, compared to its parent monomeric alkyne, showed a 16-fold higher binding affinity for the dopamine D2 receptor and a 5-fold higher potency in a cAMP assay in HEK 293T cells stably expressing D2R. Molecular modeling revealed that 11c can indeed bridge the orthosteric binding sites of a D2R homodimer in a relaxed conformation via the TM5-TM6 interface and allows to largely rationalize the results of the receptor assays.

Development of pyrimidone D1 dopamine receptor positive allosteric modulators.

MLS1082 is a structurally novel pyrimidone-based D1-like dopamine receptor positive allosteric modulator. Potentiation of D1 dopamine receptor (D1R) signaling is a therapeutic strategy for treating neurocognitive disorders. Here, we investigate the relationship between D1R potentiation and two prominent structural features of MLS1082, namely the pendant N-aryl and C-alkyl groups on the pyrimidone ring. To this end, we synthesized 24 new analogues and characterized their ability to potentiate dopamine signaling at the D1R and the closely related D5R. We identified structure-activity relationship trends for both aryl and alkyl modifications and our efforts afforded several analogues with improvements in activity. The most effective analogues demonstrated an approximately 8-fold amplification of dopamine-mediated D1R signaling. These findings advance the understanding of structural moieties underlying the activity of pyrimidone-based D1R positive allosteric modulators.

The multifunctional dopamine D2/D3 receptor agonists also possess inhibitory activity against the full-length tau441 protein aggregation.

Neurodegeneration leads to variety of diseases which are linked to aberrant protein or peptide aggregation, as a one possible mechanism. Hence, small drug molecules targeting aggregation are of interest. Tau protein aggregation is one of the biomarkers of neurodegenerative diseases and is a viable drug target. Toward multifunctional inhibitors, we aim to incorporate structural elements in a potential drug in order to preserve dopamine agonist activity, which elevates disease symptoms associated with motor skills, and promote inhibitory activity against aggregation of the full-length tau (2N4R, tau441) protein. In our design, we introduced various moieties (catechol, non-catechol, biphenyl, piperazine, and thiazole) to determine which functional group leads to the greatest aggregation inhibition of tau. In vitro, tau aggregation was induced by heparin and monitored by using fluorescence aggregation assay, transmission electron microscopy and 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (Bis-ANS) fluorescence spectroscopy. The catechol containing compounds, D-519 and D-520, prevented aggregation of tau. By contrast, non-catechol and thiazole containing compounds (D-264 and D-636) were poor inhibitors. The Bis-ANS studies revealed that the potent inhibitors bound solvent-exposed hydrophobic sites. Based on the density functional theory calculations on inhibitors tested, the compounds characterized with the high polarity and polarizability were more effective aggregation inhibitors. These findings could lead to the development of small multifunctional drug inhibitors for the treatment of tau-associated neurodegeneration.

Synthesis, pharmacological evaluations, and molecular docking studies on a new 1,3,4,11b-tetrahydro-1H-fluoreno[9,1-cd]azepine framework: Rigidification of D1 receptor selective 1-phenylbenzazepines and discovery of a new 5-HT6 receptor scaffold.

The novel 1,3,4,11b-tetrahydro-1H-fluoreno[9,1-cd]azepine framework, a structurally rigidified variant of the 1-phenylbenzazepine template, was synthesized via direct arylation as a key reaction. Evaluation of the binding affinities of the rigidified compounds across a battery of serotonin, dopamine, and adrenergic receptors indicates that this scaffold unexpectedly has minimal affinity for D1 and other dopamine receptors and is selective for the 5-HT6 receptor. The affinity of these systems at the 5-HT6 receptor is significantly influenced by electronic and hydrophobic interactions as well as the enhanced rigidity of the ligands. Molecular docking studies indicate that the reduced D1 receptor affinity of the rigidified compounds may be due in part to weaker H-bonding interactions between the oxygenated moieties on the compounds and specific receptor residues. Key receptor-ligand H-bonding interactions, salt bridges, and π-π interactions appear to be responsible for the 5-HT6 receptor affinity of the compounds. Compounds 10 (6,7-dimethoxy-2,3,4,11b-tetrahydro-1H-fluoreno[9,1-cd]azepine) and 12 (6,7-dimethoxy-2-methyl-2,3,4,11b-tetrahydro-1H-fluoreno[9,1-cd]azepine) have been identified as structurally novel, high affinity (Ki  = 5 nM), selective 5-HT6 receptor ligands.

Discovery, Optimization, and Characterization of ML417: A Novel and Highly Selective D3 Dopamine Receptor Agonist.

To identify novel D3 dopamine receptor (D3R) agonists, we conducted a high-throughput screen using a ß-arrestin recruitment assay. Counterscreening of the hit compounds provided an assessment of their selectivity, efficacy, and potency. The most promising scaffold was optimized through medicinal chemistry resulting in enhanced potency and selectivity. The optimized compound, ML417 (20), potently promotes D3R-mediated ß-arrestin translocation, G protein activation, and ERK1/2 phosphorylation (pERK) while lacking activity at other dopamine receptors. Screening of ML417 against multiple G protein-coupled receptors revealed exceptional global selectivity. Molecular modeling suggests that ML417 interacts with the D3R in a unique manner, possibly explaining its remarkable selectivity. ML417 was also found to protect against neurodegeneration of dopaminergic neurons derived from iPSCs. Together with promising pharmacokinetics and toxicology profiles, these results suggest that ML417 is a novel and uniquely selective D3R agonist that may serve as both a research tool and a therapeutic lead for the treatment of neuropsychiatric disorders.

Identification of C10 nitrogen-containing aporphines with dopamine D1 versus D5 receptor selectivity.

New aporphines containing C10 nitrogen substituents (viz. nitro, aniline or amide moieties), were synthesized and evaluated for affinity at human serotonin 5-HT1A and 5-HT2A receptors and at human dopamine D1, D2 and D5 receptors. Two series of analogs were investigated: series A which contain a sole C10 nitrogen substituent on the tetracyclic aporphine core and series B which are 1,2,10-trisubstituted aporphines. Remarkably, compounds from both series lacked affinity for the D5 receptor, thus attaining D1 versus D5 selectivity. Compound 20c was the most potent D1 ligand identified. Docking studies at D1 and D5 receptors indicate that the binding mode of 20c at the D1 receptor allows for stronger hydrophobic contacts, (primarily with Phe residues) as compared to the D5 receptor, accounting for its D1 versus D5 selectivity. Considering the lack of affinity for the D5 receptor (and low affinity at other receptors tested), compound 20c represents an interesting starting point for further structural diversification of aporphines as sub-type selective D1 receptor tools.