Human D2 Research Articles

Modeling and protein engineering studies of active and inactive states of human dopamine D2 receptor (D2R) and investigation of drug/receptor interactions.

Homology model structures of the dopamine D2 receptor (D2R) were generated starting from the active and inactive states of β2-adrenergic crystal structure templates. To the best of our knowledge, the active conformation of D2R was modeled for the first time in this study. The homology models are built and refined using MODELLER and ROSETTA programs. Top-ranked models have been validated with ligand docking simulations and in silico Alanine-scanning mutagenesis studies. The derived extra-cellular loop region of the protein models is directed toward the binding site cavity which is often involved in ligand binding. The binding sites of protein models were refined using induced fit docking to enable the side-chain refinement during ligand docking simulations. The derived models were then tested using molecular modeling techniques on several marketed drugs for schizophrenia. Alanine-scanning mutagenesis and molecular docking studies gave similar results for marketed drugs tested. We believe that these new D2 receptor models will be very useful for a better understanding of the mechanisms of action of drugs to be targeted to the binding sites of D2Rs and they will contribute significantly to drug design studies involving G-protein-coupled receptors in the future.

Homology modeling, molecular dynamic simulation, and docking based binding site analysis of human dopamine (D4) receptor.

Human dopamine D4 receptor is a GPCR target in the treatment of neurological and psychiatric conditions such as schizophrenia and Parkinson's disease. The X-ray structure of this receptor has not been resolved so far. Therefore, a proper 3D structure of D4 could provide a good tool in order to design novel ligands against this target. In this study, homology modeling studies were performed to obtain a reasonable structure of the receptor using known templates. The obtained model was subjected to molecular dynamic simulation within a DPPC membrane system. Some structural features of the receptor such as a conserved disulfide bridge and ionic lock were considered in the modeling experiments. The resulted trajectories of simulation were clustered based on the root mean square deviation of the backbone. Some known ligands and decoys were accordingly docked into the representative frames of each cluster. The best final model was finally selected based on its ability to discriminate between active ligands and inactive decoys (ROC = 0.839). The presented model of human D4 receptor could be a promising starting point in future studies of drug design for the described target.

Identification of new inhibitors for human hematopoietic prostaglandin D2 synthase among FDA-approved drugs and other compounds

ObjectiveHematopoietic prostaglandin D2 synthase (HPGDS) is a member of the Sigma class glutathione transferases (GSTs) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2, a mediator of allergy and inflammation responses. Selective inhibitors of human HPGDS are expected to be of therapeutic importance in relieving symptoms related to allergy and asthma. Hence, a collection of diverse FDA-approved compounds was screened for potential novel applications as inhibitors of HPGDS. MethodsThe catalytic activity of purified HPGDS was used for inhibition studies in vitro. ResultsOur inhibition studies revealed 23 compounds as effective inhibitors of HPGDS with IC50 values in the low micromolar range. Erythrosine sodium, suramin, tannic acid and sanguinarine sulfate were characterized with IC50 values of 0.2, 0.3, 0.4, and 0.6μM, respectively. Kinetic inhibition analysis showed that erythrosine sodium is a nonlinear competitive inhibitor of HPGDS, while suramin, tannic acid and sanguinarine sulfate are linear competitive inhibitors. ConclusionThe results show that certain FDA-approved compounds may have pharmacological effects not previously realized that warrant further consideration in their clinical use.

Rotigotine is a potent agonist at dopamine D1 receptors as well as at dopamine D2 and D3 receptors.

Rotigotine acts as a dopamine receptor agonist with high affinity for the dopamine D2, D3, D4 and D5 receptors but with a low affinity for the dopamine D1 receptor. We have investigated this further in radioligand binding and functional studies and compared the profile of rotigotine with that of other drugs used in the treatment of Parkinson's disease (PD). The binding of rotigotine to human dopamine D1, D2, D3, D4 and D5 receptors was determined in radioligand binding studies using [(3)H]rotigotine and compared with that of standard antagonist radioligands. Functional interactions of rotigotine with human dopamine receptors was also determined. [(3)H]rotigotine can be used as an agonist radioligand to label all dopamine receptor subtypes and this can be important to derive agonist affinity estimates. Rotigotine maintains this high affinity in functional studies at all dopamine receptors especially D1, D2 and D3 receptors and, to a lesser extent, D4 and D5 receptors. Rotigotine, like apomorphine but unlike ropinirole and pramipexole, was a potent agonist at all dopamine receptors. Rotigotine is a high-potency agonist at human dopamine D1, D2 and D3 receptors with a lower potency at D4 and D5 receptors. These studies differentiate rotigotine from conventional dopamine D2 agonists, used in the treatment of PD, such as ropinirole and pramipexole which lack activity at the D1 and D5 receptors, but resembles that of apomorphine which has greater efficacy in PD than other dopamine agonists but has suboptimal pharmacokinetic properties.

Human Dopamine Receptor D2 (DRD2) Polymorphisms among Four Tribal Populations of Chhattisgarh, India

Dopamine receptor gene plays an important role in central nervous system and helps in the proper functioning of neural circuits in the human brain. Polymorphisms of this gene affect the functioning of brain activities. Association of Dopamine receptor D2 gene (DRD2) polymorphisms with cognitive disorders and alcoholism has been reported in various studies now-a-days. The interest on DRD2 polymorphism is not only limited to psychiatric disorders or alcoholic behaviors or pharmacology but it is also a promising gene for evolutionary genetic studies and studies on genetic variations among populations. The present study is an attempt to understand the extent of genetic variations in four tribal populations by using the allele frequencies, haplotype frequencies and the distribution patterns of the three Taq I polymorphic sites of DRD2 gene. 197 unrelated blood samples were collected with individual informed consent and analysed by using standard protocols. All the three sites were found to be polymorphic among the four studied populations. The allele frequency distribution patterns and sharing of haplotypes in this study suggest genetic proximity among the populations.

Synthesis, pharmacological evaluation and molecular modeling studies of triazole containing dopamine D3 receptor ligands

A series of 2-methoxyphenyl piperazine analogues containing a triazole ring were synthesized and their in vitro binding affinities at human dopamine D2 and D3 receptors were evaluated. Compounds 5b, 5c, 5d, and 4g, demonstrate high affinity for dopamine D3 receptors and moderate selectivity for the dopamine D3 versus D2 receptor subtypes. To further examine their potential as therapeutic agents, their intrinsic efficacy at both D2 and D3 receptors was determined using a forskolin-dependent adenylyl cyclase inhibition assay. Affinity at dopamine D4 and serotonin 5-HT1A receptors was also determined. In addition, information from previous molecular modeling studies of the binding of a panel of 163 structurally-related benzamide analogues at dopamine D2 and D3 receptors was applied to this series of compounds. The results of the modeling studies were consistent with our previous experimental data. More importantly, the modeling study results explained why the replacement of the amide linkage with the hetero-aromatic ring leads to a reduction in the affinity of these compounds at D3 receptors.

Abstract 257: miR-217 Mediates the Protective Effects of the Dopamine D2 Receptor on Renal Inflammation and Fibrosis.

Some common single nucleotide polymorphisms (SNPs; rs 6276, 6277, and 1800497) in the human dopamine D2 receptor (DRD2) gene are associated with decreased receptor expression and function, as well as high blood pressure. We have reported that human renal proximal tubule cells (hRPTCs) from subjects carrying D2R SNPs had decreased D2R expression and function and increased expression of the pro-fibrotic factor TGFβ1 and its signaling targets Smad3 and Snail1. These cells also showed induction of epithelial mesenchymal transition and production of extracellular matrix proteins fibronectin-1 (FN-1), Collagen 1a (Col 1a), and vimentin. To determine the mechanisms underlying the effects of the D2R SNPs, we studied the expression of several microRNAs (miRs) that are associated with the regulation of chemokines, anti-fibrosis, extracellular matrix remodeling, and cell adhesion through post-transcriptional repression of their target mRNAs. The expression of 4 miRs (miR-217, miR-224, miR-335, miR-1265) was downregulated (-10.3 to -6.0 fold) and that of one miRNA (miR-1290; 5.3 fold) was upregulated in hRPTCs with D2R SNPs. The expression of miR-217 and miR-224 is regulated by the D2R; the expression is decreased by D2R downregulation in cells not carrying D2R SNPs (WT) and normalized by treating cells carrying D2R SNPs with a Drd2 plasmid. Treatment of WT cells with a miR-217 mimic significantly decreased the mRNA expression of TGFβ1 (43±2%), MMP3 (65±4%), and FN-1 (30±4%) while treatment with a miR-217 inhibitor increased TGFβ1 (94± 9%), MMP3 (96±5%), and FN-1 (212±30%). miR-217 mimic treatment of D2R SNPs cells also significantly decreased the expression of TGFβ1, MMP3, and FN-1. However, treatment of these cells with a miR-217 inhibitor had non-significant effects probably because miR-217 level was already reduced. In contrast treatment of either WT or D2R SNPs cells with miR-224 mimic or inhibitor had no significant effects on the expression of TGFβ1, MMP3, or FN-1. These results indicate that the protective effects of D2R on inflammation and fibrosis are at least, in part, mediated by regulation of miR-217 expression. Dysregulation of miR-217 expression may be responsible for the profibrotic phenotype of hRPTCs in individuals with these D2R SNPs.

Prediction blastocyst formation by oxygen consumption in human d3 thawed embryos supernumerary after frozen embryo transfer

Prediction blastocyst formation by oxygen consumption in human d3 thawed embryos supernumerary after frozen embryo transfer

Characterization of [(3) H]LS-3-134, a novel arylamide phenylpiperazine D3 dopamine receptor selective radioligand.

LS-3-134 is a substituted N-phenylpiperazine derivative that has been reported to exhibit: (i) high-affinity binding (Ki value 0.2nM) at human D3 dopamine receptors, (ii) >100-fold D3 versus D2 dopamine receptor subtype binding selectivity, and (iii) low-affinity binding (Ki >5000nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin-dependent activation of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [(3) H]-labeled LS-3-134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10-15min at 37°C) and binds with high affinity to both human (Kd =0.06±0.01nM) and rat (Kd =0.2±0.02nM) D3 receptors expressed in HEK293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [(3) H]LS-3-134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies, we propose that [(3) H]LS-3-134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype.

Multiple fragment docking and linking in primary and secondary pockets of dopamine receptors.

A sequential docking methodology was applied to computationally predict starting points for fragment linking using the human dopamine D3 receptor crystal structure and a human dopamine D2 receptor homology model. Two focused fragment libraries were docked in the primary and secondary binding sites, and best fragment combinations were enumerated. Similar top scoring fragments were found for the primary site, while secondary site fragments were predicted to convey selectivity. Three linked compounds were synthesized that had 9-, 39-, and 55-fold selectivity in favor of D3 and the subtype selectivity of the compounds was assessed on a structural basis.

Differential dopamine receptor subtype regulation of adenylyl cyclases in lipid rafts in human embryonic kidney and renal proximal tubule cells

Dopamine D1-like receptors (D1R and D5R) stimulate adenylyl cyclase (AC) activity, whereas the D2-like receptors (D2, D3 and D4) inhibit AC activity. D1R, but not the D5R, has been reported to regulate AC activity in lipid rafts (LRs). We tested the hypothesis that D1R and D5R differentially regulate AC activity in LRs using human embryonic kidney (HEK) 293 cells heterologously expressing human D1 or D5 receptor (HEK-hD1R or HEK-hD5R) and human renal proximal tubule (hRPT) cells that endogenously express D1R and D5R. Of the AC isoforms expressed in HEK and hRPT cells (AC3, AC5, AC6, AC7, and AC9), AC5/6 was distributed to a greater extent in LRs than non-LRs in HEK-hD1R (84.5±2.3% of total), HEK-hD5R (68.9±3.1% of total), and hRPT cells (66.6±2.2% of total) (P<0.05, n=4/group). In HEK-hD1R cells, the D1-like receptor agonist fenoldopam (1μM/15min) increased AC5/6 protein (+17.2±3.9% of control) in LRs but decreased it in non-LRs (−47.3±5.3% of control) (P<0.05, vs. control, n=4/group). By contrast, in HEK-hD5R cells, fenoldopam increased AC5/6 protein in non-LRs (+67.1±5.3% of control, P<0.006, vs. control, n=4) but had no effect in LRs. In hRPT cells, fenoldopam increased AC5/6 in LRs but had little effect in non-LRs. Disruption of LRs with methyl-β-cyclodextrin decreased basal AC activity in HEK-D1R (−94.5±2.0% of control) and HEK-D5R cells (−87.1±4.6% of control) but increased it in hRPT cells (6.8±0.5-fold). AC6 activity was stimulated to a greater extent by D1R than D5R, in agreement with the greater colocalization of AC5/6 with D1R than D5R in LRs. We conclude that LRs are essential not only for the proper membrane distribution and maintenance of AC5/6 activity but also for the regulation of D1R- and D5R-mediated AC signaling.

Brexpiprazole I: In Vitro and In Vivo Characterization of a Novel Serotonin-Dopamine Activity Modulator

Brexpiprazole (OPC-34712, 7-{4-[4-(1-benzothiophen-4-yl)piperazin-1-yl]butoxy}quinolin-2(1H)-one) is a novel drug candidate in clinical development for psychiatric disorders with high affinity for serotonin, dopamine, and noradrenaline receptors. In particular, it bound with high affinity (Ki < 1 nM) to human serotonin 1A (h5-HT1A)-, h5-HT2A-, long form of human D2 (hD2L)-, hα1B-, and hα2C-adrenergic receptors. It displayed partial agonism at h5-HT1A and hD2 receptors in cloned receptor systems and potent antagonism of h5-HT2A receptors and hα1B/2C-adrenoceptors. Brexpiprazole also had affinity (Ki < 5 nM) for hD3-, h5-HT2B-, h5-HT7-, hα1A-, and hα1D-adrenergic receptors, moderate affinity for hH1 (Ki = 19 nM), and low affinity for hM1 receptors (Ki > 1000 nM). Brexpiprazole potently bound to rat 5-HT2A and D2 receptors in vivo, and ex vivo binding studies further confirmed high 5-HT1A receptor binding potency. Brexpiprazole inhibited DOI (2,5-dimethoxy-4-iodoamphetamine)-induced head twitches in rats, suggestive of 5-HT2A antagonism. Furthermore, in vivo D2 partial agonist activity of brexpiprazole was confirmed by its inhibitory effect on reserpine-induced DOPA accumulation in rats. In rat microdialysis studies, brexpiprazole slightly reduced extracellular dopamine in nucleus accumbens but not in prefrontal cortex, whereas moderate increases of the dopamine metabolites, homovanillic acid and DOPAC (3,4-dihydroxy-phenyl-acetic acid), in these areas also suggested in vivo D2 partial agonist activity. In particular, based on a lower intrinsic activity at D2 receptors and higher binding affinities for 5-HT1A/2A receptors than aripiprazole, brexpiprazole would have a favorable antipsychotic potential without D2 receptor agonist- and antagonist-related adverse effects. In conclusion, brexpiprazole is a serotonin-dopamine activity modulator with a unique pharmacology, which may offer novel treatment options across a broad spectrum of central nervous system disorders.

SKF-83959 is not a highly-biased functionally selective D1 dopamine receptor ligand with activity at phospholipase C

SKF-83959 [6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine] is reported to be a functionally selective dopamine D1 receptor ligand with high bias for D1-mediated phospholipase C (PLC) versus D1-coupled adenylate cyclase signaling. This signaling bias is proposed to explain behavioral activity in both rat and primate Parkinson's disease models, and a D1-D2 heterodimer has been proposed as the underlying mechanism. We have conducted an in-depth pharmacological characterization of this compound in dopamine D1 and D2 receptors in both rat brain and heterologous systems expressing human D1 or D2 receptors. Contrary to common assumptions, SKF-83959 is similar to the classical, well-characterized partial agonist SKF38393 in all systems. It is a partial agonist (not an antagonist) at adenylate cyclase in vitro and ex vivo, and is a partial agonist in D1-mediated β-arrestin recruitment. Contrary to earlier reports, it does not have D1-mediated effects on PLC signaling in heterologous systems. Because drug metabolites can also contribute, its 3-N-demethylated analog also was synthesized and tested. As expected from the known structure-activity relationships of the benzazepines, this compound also had high affinity for the D1 receptor and somewhat higher intrinsic activity than the parent ligand, and also might contribute to in vivo effects of SKF-83959. Together, these data demonstrate that SKF-83959 is not a highly-biased functionally selective D1 ligand, and that its reported behavioral data can be explained solely by its partial D1 agonism in canonical signaling pathway(s). Mechanisms that have been proposed based on the purported signaling novelty of SKF-83959 at PLC should be reconsidered.

Tranylcypromine substituted cis-hydroxycyclobutylnaphthamides as potent and selective dopamine D₃ receptor antagonists.

We report a class of potent and selective dopamine D3 receptor antagonists based upon tranylcypromine. Although tranylcypromine has a low affinity for the rat D3 receptor (Ki = 12.8 μM), our efforts have yielded (1R,2S)-11 (CJ-1882), which has Ki values of 2.7 and 2.8 nM at the rat and human dopamine D3 receptors, respectively, and displays respective selectivities of >10000-fold and 223-fold over the rat and human D2 receptors. Evaluation in a β-arrestin functional assay showed that (1R,2S)-11 is a potent and competitive antagonist at the human D3 receptor.

MicroRNA-9 and MicroRNA-326 Regulate Human Dopamine D2 Receptor Expression, and the MicroRNA-mediated Expression Regulation Is Altered by a Genetic Variant

The human dopamine receptor D2 (DRD2) has been implicated in the pathophysiology of schizophrenia and other neuropsychiatric disorders. Most antipsychotic drugs influence dopaminergic transmission through blocking dopamine receptors, primarily DRD2. We report here the post-transcriptional regulation of DRD2 expression by two brain-expressed microRNAs (miRs), miR-326 and miR-9, in an ex vivo mode, and show the relevance of miR-mediated DRD2 expression regulation in human dopaminergic neurons and in developing human brains. Both miRs targeted the 3'-UTR (untranslated region) of DRD2 in NT2 (neuron-committed teratocarcinoma, which endogenously expresses DRD2) and CHO (Chinese hamster ovary) cell lines, decreasing luciferase activity measured by a luciferase reporter gene assay. miR-326 overexpression reduced DRD2 mRNA and DRD2 receptor synthesis. Both antisense miR-326 and antisense miR-9 increased DRD2 protein abundance, suggesting an endogenous repression of DRD2 expression by both miRs. Furthermore, a genetic variant (rs1130354) within the DRD2 3'-UTR miR-targeting site interferes with miR-326-mediated repression of DRD2 expression. Finally, co-expression analysis identified an inverse correlation of DRD2 expression with both miR-326 and miR-9 in differentiating dopaminergic neurons derived from human induced pluripotent stem cells (iPSCs) and in developing human brain regions implicated in schizophrenia. Our study provides empirical evidence suggesting that miR-326 and miR-9 may regulate dopaminergic signaling, and miR-326 and miR-9 may be considered as potential drug targets for the treatment of disorders involving abnormal DRD2 function, such as schizophrenia.

Dopamine D1‐like receptors differently regulate small G proteins in LRs in human embryonic kidney cells heterologously expressing human D1 or D5 receptors (1097.7)

Small G proteins,e.g.,Rabs (Rab4, Rab5, and Rab11), are involved in G protein‐coupled receptor trafficking, including recycling. We tested the hypothesis that D1‐like receptors (D1R and D5R) differentially regulate the small G proteins Rab4, Rab5, and Rab11 in lipid rafts (LRs) in HEK‐293 cells heterologously expressing either the human D1R or D5R (hD1R or hD5R cells, respectively). In the basal state the Rabs, which co‐fractionated with flotillin‐1, an LR marker protein, were distributed in LR fractions (fractions 2‐6) and nonLR fractions (fractions 7‐12). The D1‐like receptor agonist fenoldopam (1‐5μM/15min), increased Rab4 in LRs in hD1R cells (+38.9±14.8%, vs. control 0.0±4.8 %) and in hD5R cells (+26.9±8.7% vs. control +0.1±3.8 %) (P&lt;0.05, n=4, ANOVA). The effect of fenoldopam on Rab5 was opposite that of Rab4: fenoldopam decreased Rab5 protein in LRs in both cell lines (‐27.4±4.9% hD1R) and (‐19.7±3.0% hD5R) (P&lt;0.05, n=5, ANOVA). Fenoldopam also increased Rab11 protein in LRs in hD1R (+41.6±16.3%) compared to its control (+0.0±0.5%) (P&lt;0.01, n=4, ANOVA) but not in hD5R cells. Moreover, fenoldopam increased Rab11 protein co‐immunoprecipitation with D1R in a time dependent manner which peaked at 1h (22.9±2.5 DU), relative to vehicle‐treatment (9.8±1.9 DU) (P&lt;0.01, n=4, ANOVA). Rab11 protein also co‐immunoprecipitated with D5R but was not affected by fenoldopam. Confocal images corroborated the cell fractionation and co‐immunoprecipitation data: fenoldopam decreased Rab5 in LRs in hD1R and hD5R cells, and increased Rab4 in LRs in hD1R and hD5R cells. Fenoldopam also increased Rab11 protein co‐localization with LR marker protein in LRs in hD1R cells. However, in D5R cells, the D5R and Rab11 co‐localization in LRs was not affected by fenoldopam. We conclude that dopamine D1R and D5R co‐fractionates with Rab4, Rab5, and Rab11 in LRs. These small G proteins may play an important role in D1R and D5R trafficking. Specifically, Rab11 protein may play an important role in D1R recycling.Grant Funding Source: HL023081, HL074940, DK039308

Human dopamine receptor nanovesicles for gate-potential modulators in high-performance field-effect transistor biosensors

The development of molecular detection that allows rapid responses with high sensitivity and selectivity remains challenging. Herein, we demonstrate the strategy of novel bio-nanotechnology to successfully fabricate high-performance dopamine (DA) biosensor using DA Receptor-containing uniform-particle-shaped Nanovesicles-immobilized Carboxylated poly(3,4-ethylenedioxythiophene) (CPEDOT) NTs (DRNCNs). DA molecules are commonly associated with serious diseases, such as Parkinson's and Alzheimer's diseases. For the first time, nanovesicles containing a human DA receptor D1 (hDRD1) were successfully constructed from HEK-293 cells, stably expressing hDRD1. The nanovesicles containing hDRD1 as gate-potential modulator on the conducting polymer (CP) nanomaterial transistors provided high-performance responses to DA molecule owing to their uniform, monodispersive morphologies and outstanding discrimination ability. Specifically, the DRNCNs were integrated into a liquid-ion gated field-effect transistor (FET) system via immobilization and attachment processes, leading to high sensitivity and excellent selectivity toward DA in liquid state. Unprecedentedly, the minimum detectable level (MDL) from the field-induced DA responses was as low as 10 pM in real- time, which is 10 times more sensitive than that of previously reported CP based-DA biosensors. Moreover, the FET-type DRNCN biosensor had a rapid response time (<1 s) and showed excellent selectivity in human serum.

Targeting the sheddase activity of ADAM17 by an anti-ADAM17 antibody D1(A12) inhibits head and neck squamous cell carcinoma cell proliferation and motility via blockage of bradykinin induced HERs transactivation.

A disintegrin and metalloproteinase 17 (ADAM17) regulates key cellular processes including proliferation and migration through the shedding of a diverse array of substrates such as epidermal growth factor receptor (EGFR) ligands. ADAM17 is implicated in the pathogenesis of many diseases including rheumatoid arthritis and cancers such as head and neck squamous cell carcinoma (HNSCC). As a central mediator of cellular events, overexpressed EGFR is a validated molecular target in HNSCC. However, EGFR inhibition constantly leads to tumour resistance. One possible mechanism of resistance is the activation of alternative EGFR family receptors and downstream pathways via the release of their ligands. Here, we report that treating human HNSCC cells in vitro with a human anti-ADAM17 inhibitory antibody, D1(A12), suppresses proliferation and motility in the absence or presence of the EGFR tyrosine kinase inhibitor (TKI) gefitinib. Treatment with D1(A12) decreases both the endogenous and the bradykinin (BK)-stimulated shedding of HER ligands, accompanied by a reduction in the phosphorylation of HER receptors and downstream signalling pathways including STAT3, AKT and ERK. Knockdown of ADAM17, but not ADAM10, also suppresses HNSCC cell proliferation and migration. Furthermore, we show that heregulin (HRG) and heparin-binding epidermal growth factor like growth factor (HB-EGF) predominantly participate in proliferation and migration, respectively. Taken together, these results demonstrate that D1(A12)-mediated inhibition of cell proliferation, motility, phosphorylation of HER receptors and downstream signalling is achieved via reduced shedding of ADAM17 ligands. These findings underscore the importance of ADAM17 and suggest that D1(A12) might be an effective targeted agent for treating EGFR TKI-resistant HNSCC.

Modeling of ligand binding to dopamine D2 receptor

The dopaminic receptors have been for long time the major targets for developing new small molecules with high affinity and selectivity to treat psychiatric disorders, neurodegeneration, drug abuse, and other therapeutic areas. In the absence of a 3D structure for the human D2 dopamine (HDD2) receptor, the efforts for discovery and design of new potential drugs rely on comparative models generation, docking and pharmacophore development studies. To get a better understanding of the HDD2 receptor binding site and the ligand-receptor interactions a homology model of HDD2 receptor based on the X-ray structure of ?2-adrenergic receptor has been built and used to dock a set of partial agonists of HDD2 receptor. The main characteristics of the binding mode for the HDD2 partial agonists set are given by the ligand particular folding and a complex network of contacts represented by stacking interactions, salt bridge and hydrogen bond formation. The characterization of the partial agonist binding mode at HDD2 receptor provide the needed information to generate pharmacophore models which represent essential information in the future virtual screening studies in order to identify new potential HDD2 partial agonists.

Brain human monoclonal autoantibody from sydenham chorea targets dopaminergic neurons in transgenic mice and signals dopamine D2 receptor: implications in human disease.

How autoantibodies target the brain and lead to disease in disorders such as Sydenham chorea (SC) is not known. SC is characterized by autoantibodies against the brain and is the main neurologic manifestation of streptococcal-induced rheumatic fever. Previously, our novel SC-derived mAb 24.3.1 was found to recognize streptococcal and brain Ags. To investigate in vivo targets of human mAb 24.3.1, VH/VL genes were expressed in B cells of transgenic (Tg) mice as functional chimeric human VH 24.3.1-mouse C-region IgG1(a) autoantibody. Chimeric human-mouse IgG1(a) autoantibody colocalized with tyrosine hydroxylase in the basal ganglia within dopaminergic neurons in vivo in VH 24.3.1 Tg mice. Both human mAb 24.3.1 and IgG1(a) in Tg sera were found to react with human dopamine D2 receptor (D2R). Reactivity of chorea-derived mAb 24.3.1 or SC IgG with D2R was confirmed by dose-dependent inhibitory signaling of D2R as a potential consequence of targeting dopaminergic neurons, reaction with surface-exposed FLAG epitope-tagged D2R, and blocking of Ab reactivity by an extracellular D2R peptide. IgG from SC and a related subset of streptococcal-associated behavioral disorders called "pediatric autoimmune neuropsychiatric disorder associated with streptococci" (PANDAS) with small choreiform movements reacted in ELISA with D2R. Reaction with FLAG-tagged D2R distinguished SC from PANDAS, whereas sera from both SC and PANDAS induced inhibitory signaling of D2R on transfected cells comparably to dopamine. In this study, we define a mechanism by which the brain may be altered by Ab in movement and behavioral disorders.