Effect of flavonoids on androgen and glucocorticoid receptors based on in vitro reporter gene assay

The effect of 32 flavonoids on androgen (AR) and glucocorticoid receptors (GR) was investigated using an MDA-kb2 human breast cancer cell line to predict potential AR and GR activities. Among them, 5-hydroxyflavone (7) had the highest AR antagonistic activity with an IC₅₀ value of 0.3 μM, whereas 6-methoxyflavone (11) had the highest induced luciferase activity with an EC₁₅₀ value of 0.7 μM. Genistein (2) and daizein (1) showed a sufficient increase of luciferase activities as their concentrations increased with EC₁₅₀ values of 4.4 and 10.1 μM, respectively. These findings provide evidence of a fundamental property of their structure–activity relationship with AR and/or GR.     

Anabolic-androgenic steroid interaction with rat androgen receptor in vivo and in vitro: a comparative study

Anabolic steroids are synthetic derivatives of testosterone and are characterized by their ability to cause nitrogen retention and positive protein metabolism, thereby leading to increased protein synthesis and muscle mass. There are disagreements in the literature in regards to the interaction of anabolic steroids with the androgen receptor (AR) as revealed by competitive ligand binding assays in vitro using cytosolic preparations from prostate and skeletal muscle. By use of tissue extracts, it has been shown that some anabolic steroids have binding affinities for the AR that are higher than that of the natural androgen testosterone, while others such as stanozolol and methanedienone have significantly lower affinities as compared with testosterone. In this study we show that stanozolol and methanedienone are low affinity ligands of the rat recombinant AR as revealed by a ligand binding assay in vitro, however, based on a cell-based AR-dependent transactivation assay, they are potent activators of the AR. We also show that a single injection of stanozolol and methanedienone causes a rapid cytosolic depletion of AR in rat skeletal muscle. Based on these results, we conclude that anabolic steroids with low affinity to AR in vitro, can in fact in vivo act on the AR to cause biological responses.     

Reporter gene assay against lipophilic chemicals based on site-specific genomic recombination of a nuclear receptor gene, its response element, and a luciferase reporter gene within a stable HeLa cell line

Genomic recombination was performed in a genetically modified stable HeLa cell line, HeLa55, using a uniquely designed donor vector harboring an exchange cassette comprised of the human glucocorticoid receptor (hGR) gene, its response element, and a luciferase reporter gene, to generate stable hGRLuc clones. After screening for cassette insertion, the selected stable clone, hGRLuc-7, showed high integration stability of the exchange cassette over 20 passages with significantly high luciferase activity and fold inductions of up to 40- to 50-fold. In addition, the cells were evaluated with synthetic glucocorticoid, dexamethasone, and a reasonable EC(50) value of approximately 2.3 x 10(-9) M was obtained. Strong and weak agonists, non-responsive chemicals, and hGR antagonists were also evaluated in which the stable hGRLuc-7 clone showed both high sensitivity and selectivity. The technology presented in this work is simple and reproducible, and shows great potential for the future development of genetically modified stable cell systems which are applicable in both fundamental and application researches of nuclear receptors.     

Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal α7 nicotinic acetylcholine receptor

The pentameric acetylcholine‐binding protein (AChBP) is a soluble surrogate of the ligand binding domain of nicotinic acetylcholine receptors. Agonists bind within a nest of aromatic side chains contributed by loops C and F on opposing faces of each subunit interface. Crystal structures of Aplysia AChBP bound with the agonist anabaseine, two partial agonists selectively activating the α7 receptor, 3‐(2,4‐dimethoxybenzylidene)‐anabaseine and its 4‐hydroxy metabolite, and an indole‐containing partial agonist, tropisetron, were solved at 2.7–1.75 Å resolution. All structures identify the Trp 147 carbonyl oxygen as the hydrogen bond acceptor for the agonist‐protonated nitrogen. In the partial agonist complexes, the benzylidene and indole substituent positions, dictated by tight interactions with loop F, preclude loop C from adopting the closed conformation seen for full agonists. Fluctuation in loop C position and duality in ligand binding orientations suggest molecular bases for partial agonism at full‐length receptors. This study, while pointing to loop F as a major determinant of receptor subtype selectivity, also identifies a new template region for designing α7‐selective partial agonists to treat cognitive deficits in mental and neurodegenerative disorders.     

Structural basis for the interaction between the cytoplasmic domain of the hyaluronate receptor layilin and the talin F3 subdomain

Talin is a large cytoskeletal protein that is involved in coupling the integrin family of cell adhesion molecules to the actin cytoskeleton, colocalising with the integrins in focal adhesions (FAs). However, at the leading edge of motile cells, talin colocalises with the hyaluronan receptor layilin in what are thought to be transient adhesions, some of which subsequently mature into more stable FAs. During this maturation process, layilin is replaced with integrins, which are highly clustered in FAs, where localised production of PI(4,5)P₂ by type 1 phosphatidyl inositol phosphate kinase type 1γ (PIPK1γ) is thought to play a role in FA assembly. The talin FERM F3 subdomain binds both the integrin β-subunit cytoplasmic domain and PIPK1γ, and these interactions are understood in detail at the atomic level. The talin F3 domain also binds to short sequences in the layilin cytoplasmic domain, and here we report the structure of the talin/layilin complex, which shows that talin binds integrins, PIPK1γ and layilin in similar although subtly different ways. Based on structure comparisons, we designed a set of talin F3 mutations that selectively affected the affinity of talin for its targets, as determined by stopped-flow fluorescence measurements. Such mutations will help to assess the importance of the interactions between talin and its various ligands in cell adhesion and migration.     

Synthesis and in vitro evaluation of 18F-labelled S-fluoroalkyl diarylguanidines: Novel high-affinity NMDA receptor antagonists for imaging with PET

Two S-[¹⁸F]fluoroalkylated diarylguanidines were synthesized and evaluated in vitro as potential tracers for imaging of N-methyl-d-aspartate receptors (NMDARs) with positron emission tomography (PET). [¹⁸F]1 and [¹⁸F]10 were synthesized by [¹⁸F]fluoroethylation and [¹⁸F]fluoromethylation of the thiol precursor 6, respectively. [¹⁸F]1 is a promising candidate NMDAR PET tracer, with low nanomolar affinity for the NMDA PCP-site, high selectivity and moderate lipophilicity.     

Synthesis and SAR of derivatives based on 2-biarylethylimidazole as bombesin receptor subtype-3 (BRS-3) agonists for the treatment of obesity

This Letter describes a series of potent and selective BRS-3 agonists containing a biarylethylimidazole pharmacophore. Extensive SAR studies were carried out with different aryl substitutions. This work led to the identification of a compound 2-{2-[4-(pyridin-2-yl)phenyl]ethyl}-5-(2,2-dimethylbutyl)-1H-imidazole 9 with excellent binding affinity (IC₅₀ = 18 nM, hBRS-3) and functional agonist activity (EC₅₀ = 47 nM, 99% activation). After oral administration, compound 9 had sufficient exposure in diet induced obese mice to demonstrate efficacy in lowering food intake and body weight via BRS-3 activation.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

Structural derivatives of pindolol: relationship between in vivo and in vitro potencies for their interaction with central beta-adrenergic receptors

Although (-)-125I-iodopindolol (IPIN) can be used to label beta-adrenergic receptors in the central nervous system (CNS) in vivo, use of this ligand for receptor imaging studies in humans may be limited due to its relatively poor penetration into the CNS. A series of derivatives related to pindolol was therefore studied in an effort to determine the factors that might influence the penetration and interaction of these compounds with central beta-adrenergic receptors in vivo. Evaluation of the ability of these derivatives to displace the binding of IPIN in the brain upon systemic administration provides an assessment of whether the derivatives penetrate and interact with central beta-adrenergic receptors in vivo. Multiple regression analyses showed that the most important factor which influences the ability of the pindolol derivatives to penetrate into the brain and interact with beta-adrenergic receptors in vivo is the affinity of the derivatives for binding to beta-adrenergic receptors in vitro. Both lipophilicity and the molecular weights of the derivatives are important secondary factors which influence their in vivo potency.     

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.     

Inhibition of anion permeability by amphiphilic compounds in human red cell: Evidence for an interaction of niflumic acid with the band 3 protein

Summary In human erythrocyte, permeability to the anion is instantaneously, reversibly, and noncompetitively inhibited by the nonsteroidal anti-inflammatory drug, niflumic acid. The active form of this powerful inhibitor (I ₅₀=6×10^–7 m) is the ionic form. We demonstrated that: (i) The binding of niflumic acid to the membrane of unsealed ghosts shows one saturable and one linear component over the concentration range studied. The saturable component vanishes when chloride transport is fully inhibited by covalently bound 4-acetamido-4-isothiocyano stilbene-2,2-disulfonic acid (SITS). Our estimate of these SITS protectable niflumate binding sites (about 9×10⁵ per cell) agrees with the number of protein molecules per cell in band 3. These sites are halfsaturated with 10^–6 m niflumic acid, a concentration very close toI ₅₀. (ii) Niflumic acid inhibits the binding reaction of SITS with anion controlling transport sites. These results indicate that niflumic acid and SITS are mutually exclusive inhibitors, suggesting that niflumic acid interacts with the protein in band 3.Niflumic acid also decreases glucose and ouabain-insensitive sodium permeabilities. However, these effects are produced at a very high concentration of niflumic acid (in millimolar range), suggesting unspecific action, possibly through lipid phase.     

Relaxin-3: improved synthesis strategy and demonstration of its high-affinity interaction with the relaxin receptor LGR7 both in vitro and in vivo

Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.     

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.     

A single substitution of the insulin receptor kinase inhibits serine autophosphorylation in vitro: evidence for an interaction between the C-terminus and the activation loop

We examined the effects of mutations of tyrosine and serine autophosphorylation sites on the dual specificity of the insulin receptor kinase (IRKD) in vitro using autophosphorylation and substrate phosphorylation and phosphopeptide mapping. For comparable studies, the recombinant kinases were overexpressed in the baculovirus system, purified, and analyzed. The phosphate incorporation into the enzymes was in the range of 3-4.5 mol/mol, and initial velocities of autophosphorylation were reduced up to 2-fold. However, the mutation Y1151F in the activation loop inhibited phosphate incorporation in the C-terminal serine residues 1275 and 1309, due to a 10-fold decrease of the initial velocity of serine autophosphorylation. Although the K(M) and V(MAX) values of this mutant were only slightly altered in substrate phosphorylation reactions using a recombinant C-terminal insulin receptor peptide (K(M): Y1151F, 9.9 +/- 0.4 microM; IRKD, 6.1 +/- 0.2 microM; V(MAX): Y1151F, 72 +/- 4 nmol min(-)(1) mg(-)(1); IRKD, 117 +/- 6 nmol min(-)(1) mg(-)(1)), diminished phosphate incorporation into serine residues of the peptide was observed. In contrast, the phosphorylation of a recombinant IRS-1 fragment, which was shown to be phosphorylated markedly on serine residues by IRKD, was not affected by any kinase mutation. These results underline that IRKD is a kinase with dual specificity. The substrate specificity toward C-terminal serine phosphorylation sites can be modified by a single amino acid substitution in the activation loop, whereas the specificity toward IRS-1 is not affected, suggesting that the C-terminus and the activation loop interact.     

Influence of different transition metals in phthalocyanines on their interaction energies with volatile organic compounds: an experimental and computational study

Experimental partition coefficients were determined for a series of volatile organic compounds (VOCs) (acetonitrile, n-butylamine, n-octane tetrachloroethene, and toluene) for the interaction with 2,3,9,10,16,17,23,24-octakis(octyloxy)-phthalocyaninato complexes, PcM(OR)₈, with varying central metal atoms [M=H₂ (metal-free), Ni, Pd, Cu, Zn]. Large partition coefficients for toluene were observed in the case of the nickel and palladium phthalocyanines, whereas for the corresponding zinc-containing compound, interaction with n-butylamine resulted in a high value for the partition coefficient. Interaction energies for model coordination complexes were obtained at the ab initio LMP2/ LACVP* level of theory. The interaction of various small volatiles with the various PcM(OR)₈ compounds was studied using the PM3 semiempirical Hamiltonian. Large values for interaction energies correspond to particularly strong partition coefficients, suggesting that coordination of the volatiles to the central metal dominates over the often discussed π-system stacking at the PcM(OR)₈’s. Figure: Chemical structure of the phthalocyanines investigated.     

3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-delta8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-delta8-THC (5), 1-deoxy-delta8-THC (6), 1-deoxy-3-butyl-delta8-THC (7), 1-deoxy-3-hexyl-delta8-THC (8) and a series of 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 0-4, 6, 7, where n = the number of carbon atoms in the side chain-2). Three derivatives (17-19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1',1'-dimethylalkyl)-1-deoxy-delta8-THC analogues (2, n = 1-5) have high affinity (Ki = < 20 nM) for the CB2 receptor. Four of them (2, n = 1-4) also have little affinity for the CB1 receptor (Ki = > 295 nM). 3-(1',1'-Dimethylbutyl)-1-deoxy-delta8-THC (2, n = 2) has very high affinity for the CB2 receptor (Ki = 3.4 +/- 1.0 nM) and little affinity for the CB1 receptor (Ki = 677 +/- 132 nM).     

Structural and functional analysis of the gpsA gene product of Archaeoglobus fulgidus: a glycerol-3-phosphate dehydrogenase with an unusual NADP+ preference

NAD(+)-dependent glycerol-3-phosphate dehydrogenase (G3PDH) is generally absent in archaea, because archaea, unlike eukaryotes and eubacteria, utilize glycerol-1-phosphate instead of glycerol-3-phosphate for the biosynthesis of membrane lipids. Surprisingly, the genome of the hyperthermophilic archaeon Archaeoglobus fulgidus comprises a G3PDH ortholog, gpsA, most likely due to horizontal gene transfer from a eubacterial organism. Biochemical characterization proved G3PDH-like activity of the recombinant gpsA gene product. However, unlike other G3PDHs, the up to 85 degrees C thermostable A. fulgidus G3PDH exerted a 15-fold preference for NADPH over NADH. The A. fulgidus G3PDH bears the hallmarks of adaptation to halotolerance and thermophilicity, because its 1.7-A crystal structure showed a high surface density for negative charges and 10 additional intramolecular salt bridges compared to a mesophilic G3PDH structure. Whereas all amino acid residues required for dihydroxyacetone phosphate binding and reductive catalysis are highly conserved, the binding site for the adenine moiety of the NAD(P) cosubstrate shows a structural variation that reflects the observed NADPH preference, for example, by a putative salt bridge between R49 and the 2'-phosphate.