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1.
Mol Pharmacol ; 94(4): 1232-1245, 2018 10.
Article in English | MEDLINE | ID: mdl-30111649

ABSTRACT

The binding site for DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one], a positive allosteric modulator (PAM) of the dopamine D1 receptor, was identified and compared with the binding site for CID 2886111 [N-(6-tert-butyl-3-carbamoyl-4,5,6,7-tetrahydro-1-benzothiophen-2-yl)pyridine-4-carboxamide], a reference D1 PAM. From D1/D5 chimeras, the site responsible for potentiation by DETQ of the increase in cAMP in response to dopamine was narrowed down to the N-terminal intracellular quadrant of the receptor; arginine-130 in intracellular loop 2 (IC2) was then identified as a critical amino acid based on a human/rat species difference. Confirming the importance of IC2, a ß2-adrenergic receptor construct in which the IC2 region was replaced with its D1 counterpart gained the ability to respond to DETQ. A homology model was built from the agonist-state ß2-receptor structure, and DETQ was found to dock to a cleft created by IC2 and adjacent portions of transmembrane helices 3 and 4 (TM3 and TM4). When residues modeled as pointing into the cleft were mutated to alanine, large reductions in the potency of DETQ were found for Val119 and Trp123 (flanking the conserved DRY sequence in TM3), Arg130 (located in IC2), and Leu143 (TM4). The D1/D5 difference was found to reside in Ala139; changing this residue to methionine as in the D5 receptor reduced the potency of DETQ by approximately 1000-fold. None of these mutations affected the activity of CID 2886111, indicating that it binds to a different allosteric site. When combined, DETQ and CID 2886111 elicited a supra-additive response in the absence of dopamine, implying that both PAMs can bind to the D1 receptor simultaneously.


Subject(s)
Allosteric Regulation/physiology , Allosteric Site/physiology , Receptors, Dopamine D1/metabolism , Allosteric Regulation/drug effects , Allosteric Site/drug effects , Amino Acids/metabolism , Animals , Cell Line , Conserved Sequence/drug effects , Conserved Sequence/physiology , Dopamine/metabolism , HEK293 Cells , Humans , Isoquinolines/pharmacology , Rats
2.
Behav Pharmacol ; 22(5-6): 531-9, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21836463

ABSTRACT

The dopamine D(1) receptor agonist dihydrexidine (DHX) [(±)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a] phenanthridine hydrochloride] has shown efficacy in animal models of Parkinson's disease and improved cerebral blood flow and working memory of schizophrenic patients. Although the discriminative stimulus effects of DHX, an in-vivo predictor of human subjective effect profile, have only been characterized with respect to activity at D(1) receptors, DHX also has significant affinity for D(2) receptors. This study was designed to characterize the role of D(1) and D(2)/D(3) receptors in mediating the discriminative stimulus effects of DHX. Rats were trained to discriminate DHX [3 mg/kg, intraperitoneally (i.p.)] from the vehicle. The selective dopamine D(1) receptor partial agonist SKF 38393 was fully substituted for DHX. The D(1) receptor antagonist SCH 23390 (0.1 mg/kg, s.c.) and the D(3)-selective antagonist U99194 (10 mg/kg, i.p.) significantly attenuated the discriminative stimulus effects of the training dose of DHX by 80 and 60%, respectively, suggesting that both D(1) and D(3) receptors mediate the discriminative stimulus effects of DHX. In contrast, raclopride (1 mg/kg, i.p.) did not significantly alter the discriminative stimulus effects of DHX, indicating a lack of D(2)-mediated effects. The D(2)/D(3) receptor preferring agonists, quinpirole and (+)-PD 128907 were fully substituted, whereas (+)-7-OH-DPAT was partially substituted for DHX. The DHX bound to D(2) receptors with a Ki of 4.3+0.7 nmol/l was compared with 33.7+4.6 nmol/l at D(3) receptors. Determinations of activity at second messenger systems revealed that DHX functioned as a full agonist at D(3) receptors and a partial agonist at D(2) receptors in vitro. These activities at D(2)/D(3) receptors have shown effects in some preclinical models and clinical disease states. Therefore, the prominent in-vivo agonist activity of DHX at both D(1) receptors and D(2)/D(3) receptors should be considered while making predictions of effects in humans.


Subject(s)
Discrimination Learning/drug effects , Dopamine Agonists/pharmacology , Phenanthridines/pharmacology , Animals , Dopamine Antagonists/pharmacology , Humans , Male , Rats , Rats, Sprague-Dawley , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/drug effects , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D3/drug effects , Receptors, Dopamine D3/metabolism , Species Specificity
3.
J Pharmacol Exp Ther ; 320(1): 475-85, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17038507

ABSTRACT

The hemagglutinin-tagged human trace amine-associated receptor1 (TAAR1) was stably coexpressed with rat Galpha(s) in the AV12-664 cell line, and receptor activation was measured as the stimulation of cAMP formation. After blockade of endogenously expressed alpha2- and beta-adrenoceptors with 2-[2-(2-methoxy-1,4-benzodioxanyl)]-imidazoline hydrochloride (2-methoxyidazoxan, RX821002) and alprenolol, respectively, the resulting pharmacology was consistent with that of a unique receptor subtype. beta-Phenylethylamine (beta-PEA), the putative endogenous ligand, gave an EC50 of 106 +/- 5 nM in the assay. For a series of beta-PEA analogs used to explore the pharmacophore, small substituents at ring positions 3 and/or 4 generally resulted in compounds having lower potency than beta-PEA, although several were as potent as beta-PEA. However, small substituents at ring position 2 resulted in a number of compounds having potencies as good as or better than beta-PEA. A number of nonselective antagonists known to share affinity for multiple monoaminergic receptors were evaluated for their ability to inhibit beta-PEA stimulation of the human TAAR1. None had an IC50 <10 microM. For comparison, the rat TAAR1 receptor was expressed in the AV12-664 cell line. A number of agonist compounds had significantly different relative potencies between the rat and human TAAR1, demonstrating a significant species difference between the rat and human TAAR1. The TAAR1 receptor exhibits a pharmacologic profile uniquely different from those of classic monoaminergic receptors, consistent with the structural information that places them in a distinct family of receptors. This unique pharmacologic profile suggests the potential for development of TAAR-selective agonists and antagonists to study their physiologic roles.


Subject(s)
Receptors, G-Protein-Coupled/drug effects , Animals , Base Sequence , Cyclic AMP/biosynthesis , Humans , Idazoxan/analogs & derivatives , Idazoxan/pharmacology , Isoproterenol/pharmacology , Molecular Sequence Data , Phenethylamines/pharmacology , Rats , Receptors, Adrenergic, alpha-2/physiology , Receptors, Adrenergic, beta/physiology , Receptors, G-Protein-Coupled/agonists , Species Specificity , Structure-Activity Relationship
4.
J Neurochem ; 91(6): 1249-59, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15584902

ABSTRACT

Beta-amyloid peptides (Abeta) are produced by a sequential cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. The lack of Abeta production in beta-APP cleaving enzyme (BACE1)(-/-) mice suggests that BACE1 is the principal beta-secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild-type and BACE1(-/-) mice supports cleavage of APP at the canonical beta-secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as beta', resulting in Abeta peptides starting at Glu11. The apparent inability of human BACE1 to make this beta'-cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species-specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the beta'-cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the beta- and beta'-cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the beta- and beta'-sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated beta'-cleavage site. It does not appear to be a significant species specificity to this cleavage.


Subject(s)
Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Amino Acid Sequence , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Cell Line , Endopeptidases , Guinea Pigs , Humans , Kinetics , Mice , Molecular Conformation , Molecular Sequence Data , Recombinant Proteins/antagonists & inhibitors , Species Specificity
5.
Clin Chem ; 50(4): 709-16, 2004 Apr.
Article in English | MEDLINE | ID: mdl-14764643

ABSTRACT

BACKGROUND: Human kallikrein 7 (hK7), also known as human stratum corneum chymotryptic enzyme, is a chymotrypsin-like serine protease first identified in human skin extracts and predicted to be a secreted protease. The aim of this study was to develop a sensitive and specific immunoassay for hK7 and to examine the distribution of hK7 in tissue extracts and biological fluids. METHODS: Recombinant hK7 was produced in human embryonic kidney cells (HEK293T) and purified by a three-step column chromatographic procedure. The purified hK7 was injected into mice for antibody generation. A sandwich-type immunoassay was developed with the anti-hK7 monoclonal antibodies. RESULTS: The assay had imprecision (CV) <10% through the dynamic range of 0.2-20 microg/L and had no detectable cross-reactivity from other members in the human kallikrein gene family. Highest concentrations were found in skin, esophagus, and kidney. hK7 was also found in amniotic fluid, ascites from ovarian cancer patients, breast milk, cerebrospinal fluid, saliva, seminal plasma, serum, sweat, synovial fluid, and urine. CONCLUSIONS: This study describes the first ELISA-type immunoassay for hK7 protein quantification. hK7 is found many human tissues and in various biological fluids.


Subject(s)
Body Fluids/chemistry , Kallikreins/analysis , Tissue Extracts/chemistry , Adult , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Cell Line , Electrophoresis, Polyacrylamide Gel , Female , Humans , Immunoassay , Kallikreins/immunology , Kallikreins/isolation & purification , Male , Mass Spectrometry , Mice , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sensitivity and Specificity
6.
Eur J Pharmacol ; 484(2-3): 127-39, 2004 Jan 26.
Article in English | MEDLINE | ID: mdl-14744596

ABSTRACT

The human 5-HT(1E) receptor gene was cloned more than a decade ago. Little is known about its function, and there have been no reports of its existence in the genome of small laboratory animals. In this study, attempts to clone the 5-HT(1E) gene from the rat and mouse were unsuccessful. In fact, a search of the mouse genome database revealed that the 5-HT(1E) receptor gene is missing from the mouse genome. However, the 5-HT(1E) gene was cloned from guinea pig genomic DNA and was characterized. The guinea pig 5-HT(1E) receptor gene encodes a protein of 365 amino acids. It shares 88% (nucleic acid) and 95% (amino acid) homology with the human receptor. The guinea pig 5-HT(1E) receptor showed similar pharmacology to the human 5-HT(1E) receptor in radioligand binding assays. Serotonin (5-hydroxytryptamine, 5-HT) dose-dependently stimulated [35S]GTPgammaS binding to the guinea pig 5-HT(1E) receptor with an EC(50) of 13.6+/-1.92 nM, similar to that of the human 5-HT(1E) receptor (13.7+/-1.78 nM). Activation of the guinea pig 5-HT(1E) receptor was also achieved by ergonovine, alpha-methyl-5-HT, 1-naphthylpiperazine, methysergide, tryptamine, and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI). Methiothepin exhibited antagonist activity. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that 5-HT(1E) mRNA was present in the guinea pig brain with the greatest abundance in the hippocampus, followed by the olfactory bulb. Lower levels were detected in the cortex, thalamus, pons, hypothalamus, midbrain, striatum, and cerebellum. Our current study marks the first identification of the 5-HT(1E) receptor gene in a commonly used laboratory animal species. This finding should allow the elucidation of the receptor's role(s) in the complex coordination of central serotonergic effects.


Subject(s)
Cloning, Molecular/methods , Receptors, Serotonin/genetics , Receptors, Serotonin/metabolism , Serotonin Agents/metabolism , Serotonin Agents/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Brain/drug effects , Brain/metabolism , Chickens , Cricetinae , Dose-Response Relationship, Drug , Gerbillinae , Guinea Pigs , Haplorhini , Humans , Molecular Sequence Data , Protein Binding/physiology , Rabbits , Serotonin Agents/chemistry , Swine
7.
Biochem Biophys Res Commun ; 307(4): 948-55, 2003 Aug 08.
Article in English | MEDLINE | ID: mdl-12878203

ABSTRACT

Human kallikrein 6 (hK6) is a trypsin-like serine protease, member of the human kallikrein gene family. Studies suggested a potential involvement of hK6 in the development and progression of Alzheimer's disease. The serum levels of hK6 might be used as a biomarker for ovarian cancer. To gain insights into the physiological role of this enzyme, we sought to determine its substrate specificity and its interactions with various inhibitors. We produced the proform of hK6 and showed that this enzyme was able to autoactivate, as well as proteolyse itself, leading to inactivation. Kinetic studies indicated that hK6 cleaved with much higher efficiency after Arg than Lys and with a preference for Ser or Pro in the P2 position. The efficient degradation of fibrinogen and collagen types I and IV by hK6 indicated that this kallikrein might play a role in tissue remodeling and/or tumor invasion and metastasis. We also demonstrated proteolysis of amyloid precursor protein by hK6 and determined the cleavage sites at the N-terminal end of the protein. Inhibition of hK6 was achieved via binding to different serpins, among which antithrombin III was the most efficient.


Subject(s)
Kallikreins/metabolism , Amino Acid Sequence , Amyloid beta-Protein Precursor/chemistry , Amyloid beta-Protein Precursor/metabolism , Enzyme Activation , Humans , Kinetics , Molecular Sequence Data , Serine Proteinase Inhibitors/pharmacology , Substrate Specificity
8.
Brain Res ; 955(1-2): 191-9, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12419536

ABSTRACT

We have recently reported a critical role for apolipoprotein E (apoE) in the process of amyloid deposition and neuritic plaque formation in APP(V717F) transgenic (Tg) mice, an animal model of Alzheimer's disease (AD). In the present study, we have investigated whether the presence or absence of apoE alters the processing of the amyloid precursor protein (APP) to various fragments, including the beta-amyloid peptides (Abeta). Here we show that, in contrast to APP(V717F) Tg mice expressing apoE, APP(V717F) Tg mice deficient in apoE develop anti-Abeta immunoreactive multifocal aggregates, which contain the beta-cleaved C-terminal fragments (beta-CTFs) of APP. Tg mice deficient in apoE also display altered levels of mature full-length APP, increased amounts of beta-CTFs, as well as elevated levels of Abeta(1-40) and Abeta(1-42) in an age- and region-dependent manner when compared to Tg mice expressing apoE. Taken together, these data support a role for apoE in APP processing in vivo.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Apolipoproteins E/deficiency , Brain/metabolism , Protein Processing, Post-Translational/genetics , Age Factors , Amyloid beta-Protein Precursor/biosynthesis , Amyloid beta-Protein Precursor/genetics , Animals , Apolipoproteins E/biosynthesis , Apolipoproteins E/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Phenylalanine/genetics , Valine/genetics
9.
Anticancer Res ; 22(5): 2725-32, 2002.
Article in English | MEDLINE | ID: mdl-12529988

ABSTRACT

BACKGROUND: PSA mediates growth factor responses that stimulate proliferation of prostatic and other cellular types. Androgen-sensitive TE85 human osteosarcoma cells were used to study PSA as a potential mediator of prostatic cancer growth and osseous metastasis. MATERIALS AND METHODS: TE85 cells were probed for PSA mRNA and protein levels under testosterone (T)-replete and--depleted conditions. TE85 proliferative responses to PSA were evaluated in the absence and presence of LY312340, a monocyclic beta-lactam inhibitor of PSA enzymatic activity. RESULTS: A 3.1-fold increase in PSA mRNA was observed following T stimulation. Low levels of immunoreactive PSA (iPSA) were detected in media of androgen-stimulated TE85 cells while iPSA was not found in control media. Conversely, iPSA was detected in TE85 cell pellets from control but not in androgen-stimulated cell cultures. Exogenously added enzymatically active PSA stimulated TE85 proliferation in a bi-phasic manner. LY312340 inhibited PSA-induced increases in TE85 cell numbers but had no effect on basal or T- stimulated cellular proliferation. CONCLUSION: While the PSA levels produced by TE-85 cells in response to androgen stimulation are too low to be biologically active, PSA produced by metastatic PCa cells may mediate paracrine stimulation of osteogenic PCa metastasis. Inhibitors of PSA enzymatic activity could be useful therapeutic agents.


Subject(s)
Benzoates/pharmacology , Bone Neoplasms/pathology , Lactams/pharmacology , Neoplasms, Hormone-Dependent/pathology , Osteosarcoma/pathology , Prostate-Specific Antigen/antagonists & inhibitors , Adolescent , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Cell Division/drug effects , Cell Division/physiology , Female , Humans , Neoplasms, Hormone-Dependent/genetics , Neoplasms, Hormone-Dependent/metabolism , Osteosarcoma/genetics , Osteosarcoma/metabolism , Prostate-Specific Antigen/biosynthesis , Prostate-Specific Antigen/genetics , Prostate-Specific Antigen/physiology , Testosterone/deficiency , Testosterone/pharmacology , Testosterone/physiology , Tumor Cells, Cultured
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