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H Hong

Publications and source records attributed to H Hong.

At least 73 records · Page 4Linked to original sources
PubMed1997

Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors.

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate beta-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.

Animals↗
PubMed1997

HIV-1 integrase pharmacophore: discovery of inhibitors through three-dimensional database searching.

Starting from a known inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN); caffeic acid phenethyl ester (CAPE), a putative three-point pharmacophore for binding of inhibitors to IN was derived. This pharmacophore was used to search the National Cancer Institute three-dimensional (3D) structural database. Out of the open, nonproprietary part of this database, comprising approximately 200000 compounds, 267 structures were found to match the pharmacophore in at least one conformation, and 60 of those were tested in an in vitro assay against HIV-1 IN. Out of these, 19 were found to inhibit both the 3'-processing and strand transfer of IN at micromolar concentrations. In order to test the validity of this pharmacophore, a small 3D database of 152 published IN inhibitors was built. A search in this database yielded a statistically significant correlation of the presence of this pharmacophore and the potency of the compounds. An automated pharmacophore identification procedure performed on this set of compounds provided additional support for the importance of this pharmacophore for binding of inhibitors to IN and hinted at a possible second pharmacophore. The role of aromatic moieties in the binding of ligands to HIV-1 IN through interactions with divalent metal cations, which are known to be necessary for activity of the enzyme, was explored in ab initio calculations.

Caffeic Acids↗
PubMed1997

Discovery of HIV-1 integrase inhibitors by pharmacophore searching.

Based upon a class of known HIV-1 integrase inhibitors, several pharmacophore models were proposed from molecular modeling studies and validated using a 3D database of 152, compounds for which integrase assay data are known. Using the most probable pharmacophore model as the query, the NCI 3D database of 206,876 compounds was searched, and 340 compounds that contain the pharmacophore query were identified. Twenty-nine of these compounds were selected and tested in the HIV-1 integrase assay. This led to the discovery of 10 novel, structurally diverse HIV-1 integrase inhibitors, four of which have an IC50 value less than 30 microM and are promising lead compounds for further HIV-1 integrase inhibitor development.

4-Hydroxycoumarins↗
PubMed1997

Hydrazide-containing inhibitors of HIV-1 integrase.

Inhibitors of HIV integrase are currently being sought as potential new therapeutics for the treatment of AIDS. A large number of inhibitors discovered to date contain the o-bis-hydroxy catechol structure. In an effort to discover structural leads for the development of new HIV integrase inhibitors which do not rely on this potentially cytotoxic catechol substructure, NSC 310217 was identified using a three-point pharmacophore search based on its assigned structure N-(2-hydroxybenzoyl)-N-(2-hydroxy-3-phenoxypropyl)hydrazine (1). When a sample of NSC 310217 was obtained from the NCI repository, it was shown to exhibit potent inhibition of HIV-1 integrase (3'-processing IC50 = 0.6 microgram/mL). In work reported herein, we demonstrate that NSC 310217, rather than containing 1, which has no inhibitory potency against HIV-1 integrase, is comprised of roughly a 1:1 mixture of N-(2-hydroxybenzoyl)-N'-(2-hydroxy-3-phenoxypropyl)hydrazine (6) and N,N'-bis-salicylhydrazine 7, with all inhibitory potency residing with compound 7(IC50 = 0.7 microM for strand transfer). In subsequent structure-activity studies on 7, it is shown that removing a single amide carbonyl (compound 14, IC50 = 5.2 microM) or replacing one aromatic ring system with a naphthyl ring (compound 19, IC50 = 1.1 microM) can be accomplished with little loss of inhibitory potency. Additionally, replacing a single hydroxyl with a sulfhydryl (compound 23, IC50 = 5.8 microM) results in only moderate loss of potency. All other modifications examined, including the replacement of a single hydroxyl with an amino group (compound 22), resulted in complete loss of potency. Being potent, structurally simple, and non-catechol-containing, compounds such as 7 and 14 may provide useful leads for the development of a new class of HIV integrase inhibitor.

Databases, Factual↗
PubMed1997

Depsides and depsidones as inhibitors of HIV-1 integrase: discovery of novel inhibitors through 3D database searching.

Seventeen lichen acids comprising despides, depsidones, and their synthetic derivatives have been examined for their inhibitory activity against HIV-1 integrase, and two pharmacophores associated with inhibition of this enzyme have been identified. A search of the NCI 3D database of approximately 200,000 structures yielded some 800 compounds which contain one or the other pharmacophore. Forty-two of these compounds were assayed for HIV-1 integrase inhibition, and of these, 27 had inhibitory IC50 values of less than 100 microM; 15 were below 50 microM. Several of these compounds were also examined for their activity against HIV-2 integrase and mammalian topoisomerase I.

Anti-HIV Agents↗
PubMed1997

Coumarin-based inhibitors of HIV integrase.

The structures of a large number of HIV-1 integrase inhibitors have in common two aryl units separated by a central linker. Frequently at least one of these aryl moieties must contain 1,2-dihydroxy substituents in order to exhibit high inhibitory potency. The ability of o-dihydroxy-containing species to undergo in situ oxidation to reactive quinones presents a potential limitation to the utility of such compounds. The recent report of tetrameric 4-hydroxycoumarin-derived inhibitor 5 provided a lead example of an inhibitor which does not contain the catechol moiety. Compound 5 represents a large, highly complex yet symmetrical molecule. It was the purpose of the present study to determine the critical components of 5 and if possible to simplify its structure while maintaining potency. In the present study, dissection of tetrameric 5 (IC50 = 1.5 microM) into its constituent parts showed that the minimum active pharmacophore consisted of a coumarin dimer containing an aryl substituent on the central linker methylene. However, in the simplest case in which the central linker aryl unit consisted of a phenyl ring (compound 8, IC50 = 43 microM), a significant reduction in potency resulted by removing two of the original four coumarin units. Replacement of this central phenyl ring by more extended aromatic systems having higher lipophilicity improved potency, as did the addition of 7-hydroxy substituents to the coumarin rings. Combining these latter two modifications resulted in compounds such as 3,3'-(2-naphthalenomethylene)bis[4,7-dihydroxycoumarin] (34, IC50 = 4.2 microM) which exhibited nearly the full potency of the parent tetramer 5 yet were structurally much simpler.

Anti-HIV Agents↗
PubMed1997

Effect of methadone addiction on glucose metabolism in rats.

Female albino rats were exposed to methadone over a 35-day period by addition of the drug in their drinking water. The final dose of the drug was 1.8 mg/kg body weight per day. After this period, the drug was withdrawn from some animals for 30 days (postexposure). Compared to unexposed controls, serum glucose levels rose during exposure and returned to control levels postexposure. Oral glucose tolerance tests showed impairment in 35-day drug-exposed animals compared to controls and postexposure. The activities of three key enzymes of glycolysis and three key enzymes of gluconeogenesis were measured in liver during and at the end of the exposure period, as well as postexposure. Compared to unexposed controls and postexposure, specific activities of two glycolytic enzymes in livers of exposed animals-hexokinase and phosphofructokinase 1-were significantly reduced, whereas the activity of a third glycolytic enzyme-pyruvate kinase-was unchanged. The specific activities of two gluconeogenic enzymes-glucose-6-phosphatase and fructose-1,6-biphosphatase-were significantly elevated in the drug-exposed animals compared to controls, whereas the activity of a third enzyme-phosphoenolpyruvate carboxykinase-was unchanged. These data indicate that methadone addiction produces a metabolic state similar to insulin-resistant diabetes.

Animals↗
PubMed1997

Potent inhibitors of human immunodeficiency virus type 1 integrase: identification of a novel four-point pharmacophore and tetracyclines as novel inhibitors.

A four-point pharmacophore was constructed from energy-minimized structures of chicoric acid and dicaffeoylquinic acid. The search of 206,876 structures in the National Cancer Institute 3D database yielded 179 compounds that contain this pharmacophore. Thirty-nine of these compounds were tested in an in vitro assay specific for human immunodeficiency virus type 1 integrase (IN). Each retrieved structure was fit to the pharmacophore, and the conformation that afforded the best fit was identified. Twenty of the 39 compounds tested exhibited IC50 values of < 20 microM. Among the most potent inhibitors, tetracyclines emerged as a new class of inhibitors. Although the parent tetracycline exhibited marginal potency against purified IN, all substituted tetracyclines tested showed 5-100-fold increased potency. Disintegration assays with truncated IN mutants indicated that tetracyclines inhibit the IN catalytic core domain. To investigate whether chelation of divalent metals is implicated in differential potency of tetracyclines, enzyme assays were performed in the presence of both Mn2+ or Mg2+; no significance difference in potency was observed. Rolitetracycline inhibited IN/DNA complex formation in the presence of EDTA, which suggests that inhibition was metal independent. Rolitetracycline reversed DNA binding of IN after the complex was allowed to form before the addition of drug. Selectivity of tetracyclines was also examined in an assay specific for topoisomerase I, and none of the tetracyclines tested induced topoisomerase I-mediated cleavable complex or inhibited camptothecin-induced cleavable complex. Remarkable potency against the IN in the absence of divalent metals and the core enzyme coupled with water solubility makes tetracyclines potential candidates for X-ray crystal structure determination with IN.

Anti-HIV Agents↗
PubMed1997

GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors.

After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.

Amino Acid Sequence↗
PubMed1997

Identification of amino acids in the tau 2-region of the mouse glucocorticoid receptor that contribute to hormone binding and transcriptional activation.

The tau 2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding tau 2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the tau 2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the tau 2-region of the mouse glucocorticoid receptor (mGR, amino acids 533-562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum tau 2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum tau 2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum tau 2-fragment and the full-length GR indicated that the tau 2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the tau 2-region (mGR 541-547) was primarily involved in hormone binding, whereas the C-terminal portion of the tau 2-region (mGR 548-561) was primarily involved in transactivation.

Amino Acid Sequence↗
PubMed1997

Magnesium modulates ouabain action on angiotensin II-induced aldosterone synthesis in vitro.

Ouabain can block the action of angiotensin II (AII on aldosterone secretion in both rat and human adrenal tissue although its action is much more potent on human zona glomerulosa cells (ZG). Since magnesium can antagonize digitalis action in vivo, we have examined the interaction of both agents on aldosterone secretion in both rat and human adrenal cells. Ouabain itself at high concentration (10(-5) M) in Mg++ buffer blocks AII action on aldosterone secretion on rat ZG cells and at 1000-fold lower concentrations inhibits AII-action in human cells as previously reported by us. When buffer Mg++ is higher (4mM) than normal (0.7 mM) in cultured human adrenal cells it decreases both basal aldosterone secretion (8.6 +/- 0.4 vs. 6.8 +/- 0.2, p < 0.001) and AII action on aldosterone (13.9 +/- 0.6 vs. 9.7 +/- 0.7 ng/10(6) cells/h, p < 0.01). High Mg++ buffer had this effect at both 10(-8) and 10(-7) M concentrations of ouabain (control 8.6 +/- 0.4, AII [10(-9) M] 13.9 +/- 0.7, AII + Ouabain [5 x 10(-8) M] 10.9 +/- 0.6, AII + ouabain [10(-7) M] 7.8 +/- 0.3 ng/10(6) cells/h, both p < 0.01 vs. AII). In contrast, a low Mg++ buffer stimulated both basal (6.9 +/- 0.2 vs. 8.3 +/- 0.4 ng/10(6) cells/h, p < 0.01) and AII stimulation of aldosterone secretion (8.4 +/- 0.2 vs. 9.8 +/- 0.4 ng/10(6) cells/h, p < 0.01). When ouabain was added to low Mg++ buffer there was further inhibition of AII induced aldosterone secretion than with normal Mg++ buffer. However, the effect of ACTH on aldosterone was not altered by changes in Mg++. We conclude that ouabain actions and the effect of angiotensin II on aldosterone is inhibited by an increased level of Mg++ while low levels of Mg++ are stimulatory to both basal and AII action on aldosterone.

Aldosterone↗
PubMed1996

GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors.

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.

Amino Acid Sequence↗
PubMed1996

Instability of Rts1 (drug-resistant factor) replicon: stabilization by DNA fragments derived from Rts1.

Rts1 is a large naturally occurring plasmid which has a kanamycin resistance gene and exhibits various temperature-sensitive phenotypes. A smaller derivative of plasmid, pOK, contains the Rts1 replicon and the kanamycin resistance gene of Rts1. This plasmid, pOK, is much more unstable than Rts1 at 42.5 degrees C. A DNA fragment, G3, 1590 nucleotides long from Rts1 DNA, stabilized pOK completely at 42.5 degrees C but only in the cis configuration. G3 did not change the copy number of pOK. The pOK derivative containing G3 was destabilized by the presence of a compatible plasmid containing G3. G3 has four inverted repeats, two 14-base direct repeats, and three ORFs. Smaller fragment of G3 also had a stabilization effect and these studies showed that the ORF does not play any role in stabilization.

Base Sequence↗
PubMed1995

Antioxidant enzyme activities and lipid peroxidation levels in exercised and hypertensive rat tissues.

Previous studies have shown that exercise-induced changes in muscle antioxidant status occur shortly after exercise. The present studies were designed to determine if longer-term exercise-related changes in antioxidant enzyme activities in both normotensive (WKY) and hypertensive rats (SHR) occurred, and if these changes were related to the levels of lipid peroxidation. WKY and SHR rats were exercised over a 10-week period using a progressive treadmill regimen. After a 1-week detraining period, the animals were euthanized and measurements of tissue antioxidant enzyme activities and lipid peroxide levels were determined in both exercised and cage-sedentary groups. Decreases in antioxidant activities (particularly glutathione peroxidase and catalase) in liver, kidney, skeletal and cardiac were associated with exercise training in both WKy and SHR rats (e.g. left ventricular glutathione peroxidase specific activity in WKY rats was decreased from 234 +/- 25 [SD, n = 12] to 187 +/- 17 [SD, n = 11] units/mg protein). Elevations in activities of antioxidant enzymes were generally associated with hypertension in these tissues (e.g. left ventricular glutathione peroxidase specific activity in SHR rats was 275 +/- 30 [SD, n = 12] units/mg protein), but changes in activities were more variable than those seen in response to exercise. Exercise-related changes in tissue levels of thiobarbituric acid-reactive substances (an indirect measure of tissue lipid peroxide levels) generally did not correlate with exercise-related antioxidant enzyme activity changes, and hypertension had no effect on these levels except in liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Magnetic backprojection imaging of the vascular lumen.

Current injected into a phantom model generated a magnetic field which was distorted above a simulated atherosclerotic lesion. The output of a Hall effect magnetic sensor was used in a backprojection to reconstruct the centroid of the simulated blood flow and thus localize the modeled atherosclerotic plaque region.

Arteries↗