Antiviral activity of the dihydropyrone PNU-140690, a new nonpeptidic human immunodeficiency virus protease inhibitor.

PNU-140690 is a member of a new class of nonpeptidic human immunodeficiency virus (HIV) protease inhibitors (sulfonamide-containing 5,6-dihydro-4-hydroxy-2-pyrones) discovered by structure-based design. PNU-140690 has excellent potency against a variety of HIV type 1 (HIV-1) laboratory strains and clinical isolates, including those resistant to the reverse transcriptase inhibitors zidovudine or delavirdine. When combined with either zidovudine or delavirdine, PNU-140690 contributes to synergistic antiviral activity. PNU-140690 is also highly active against HIV-1 variants resistant to peptidomimetic protease inhibitors, underscoring the structural distinctions between PNU-140690 and substrate analog protease inhibitors. PNU-140690 retains good antiviral activity in vitro in the presence of human plasma proteins, and preclinical pharmacokinetic studies revealed good oral bioavailability. Accordingly, PNU-140690 is a candidate for clinical evaluation.

Tröger's base. An alternate synthesis and a structural analog with thromboxane A2 synthetase inhibitory activity.

The synthesis of 2,8-dimethyl-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine (Tröger's base) from p-toluidine and of two Tröger's base analogs from other anilines by reaction with hexamethylenetetramine in trifluoroacetic acid is described. 2,3,6,7-Tetrahydro-9-methyl-2,6-di-p-tolyl-1H,5H-pyrimido[5,6,1-ij] quinazoline is formed as a secondary product in the reaction of p-toluidine and hexamethylenetetramine. One of the Tröger's base analogs, 2,8-bis(3'-pyridylmethyl)-6H,12H-5,11-methanodibenzo[b,f][1,5]d iazocine (5), is an effective inhibitor of the enzyme, thromboxane A2 (TxA2) synthase, with an ED50 of 30 ng/mL in a specified in vitro assay. Three analogs having substituents on the bridging methylene group of the bicyclic nucleus of the Tröger's base structure were prepared, but all were considerably less active than the aforementioned compound in the inhibition assay. The structures of these inhibitors of TxA2 synthase fall outside the classical structure-activity relationship that has been established for this class of enzyme inhibitors.

2-Aminochromones block human platelet aggregation by inhibiting cyclic AMP-dependent phosphodiesterase leading to reduced platelet phospholipase C activity.

We examined a series of 2-aminochromone analogs typified by U-84569 [8-methyl-2-(4-morpholinyl)-7-(1-naphthylenylmethoxy)-4H-1- benzopyran-4-one] as potential antithrombotic agents. U-84569 proved to be a potent inhibitor of human platelet aggregation regardless of the agonist used. Subsequent experiments showed that U-84569 increased platelet cyclic AMP (cAMP) levels in intact cells, but U-84569 did not directly stimulate adenylate cyclase. Our experiments showed that U-84569 was a potent inhibitor of the low Km cAMP-dependent phosphodiesterase with an IC50 of 300 nM in platelet cytosol. Isobutylmethylxanthine had an IC50 of 10 microM in the same system. Although U-84569 elevated cAMP by inhibiting cAMP metabolism, we were interested in the mechanism by which cAMP blocked aggregation. Our first experiments showed that U-84569 concentration-dependently blocked agonist-stimulated, but not phorbol myristate acetate-dependent, phosphorylation of the 47 kDa protein kinase C substrate in platelets. These data suggested that U-84569 could interrupt receptor-mediated signal transduction. In support of this hypothesis, U-84569 proved to be a potent inhibitor of thrombin-stimulated inositol phosphate synthesis, diacylglycerol formation and Ca++ mobilization in intact cells. These data indicate that agonist-stimulated phospholipase C activity was reduced in U-84569-treated cells. There was no direct influence of U-84569 on either basal or thrombin-stimulated phospholipase C activity in broken cells, suggesting that U-84569 (by inhibiting phosphodiesterase and elevating cAMP), indirectly blocked receptor-mediated phospholipase C activation and aggregation in platelets. The 2-aminochromones represent a new class of potent antithrombotic agents.

Activation of human eosinophils by platelet-derived growth factor.

Activated eosinophils are believed to be major contributors to the chronic inflammatory sequelae of asthma, but the details of the mechanism of eosinophil activation in vivo are unknown. In our search for physiologically important modes of eosinophil activation, we studied the effects of recombinant human platelet-derived growth factor (PDGF) on human peripheral blood eosinophils. We compared two activation end-points: secretion of granule contents, exemplified by the release of eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and the generation of active oxygen metabolites (O2- production). PDGFc-sis dose dependently stimulated the secretion of large amounts of EPO and EDN from eosinophils. Higher concentrations of PDGF induced a dose-dependent O2- production, especially if the cells were first primed with low concentrations of phorbol ester. These activities were not seen with the AA homodimer of PDGF, suggesting that the activation was receptor dependent. However, several attempts to directly demonstrate the existence of such receptors were unsuccessful. The magnitude of the secretory response to PDGF, and the realization that eosinophils could be easily exposed to this substance as they travel towards the lung, suggests the possibility that this growth factor may be a physiologically important activator of eosinophils in the pulmonary inflammation which is associated with asthma.

Elevated prostaglandin H synthase gene expression in ras-transformed cells.

1. rPDGF stimulates PGE2 release in wild type, but not ras transformed NIH-3T3 cells. 2. Ras transformation blocks PGE2 release by inhibiting phospholipase C activation, IP3 synthesis, and Ca2+ mobilization. 3. rPDGF stimulation of wild type NIH-3T3 cells increases both prostaglandin H synthase (PGHS) mRNA levels and PGHS enzyme levels as measured by immunoblot. However, PGHS gene transcription is not required for PDGF-stimulated PGE2 release. 4. Ras transformed NIH-3T3 cells display elevated basal PGE2 synthesis, and very high levels of both PGHS mRNA and enzyme. rPDGF does not further stimulate PGHS gene transcription. 5. Exogenous PGE2 attenuates rPDGF-stimulated cell proliferation in both wild type and ras transformed cells. 6. These data suggest that increased PGHS gene expression and enhanced basal PGE2 synthesis may be in response to the unregulated growth of ras transformed cells.

Regulation of prostaglandin H synthase mRNA levels and prostaglandin biosynthesis by platelet-derived growth factor.

Stimulation of serum-starved NIH-3T3 cells with 20 ng/ml recombinant platelet-derived growth factor BB (rPDGF) results in the synthesis of prostaglandin E2 (PGE2) that is detectable within 10 min and which peaks after 2 h. Inhibition of translation with 36 microM cycloheximide inhibits rPDGF-stimulated PGE2 synthesis (greater than 90%), suggesting that de novo synthesis of prostaglandin H synthase (PGHS) is required for growth factor stimulation of PGE2 synthesis. Paradoxically, the addition of 10 microM exogenous arachidonate to the serum-starved cells resulted in 2-fold more PGE2 synthesis, and maximal synthesis occurred at 30 min compared to 2 h following rPDGF treatment. These data suggest that the serum-starved cells have ample biosynthetic capacity to support maximal rPDGF-stimulated PGE2 synthesis without de novo enzyme synthesis. Kinetic analysis of PGHS mRNA levels showed that although rPDGF did elevate the steady-state level of PGHS mRNA, the increase occurred after maximal PGE2 synthesis was achieved. Quantification of PGHS levels by immunoblot showed that there was no significant increase in enzyme levels following rPDGF stimulation even under conditions where PGHS mRNA levels were elevated. Irreversible inactivation of PGHS by treatment with aspirin and subsequent stimulation with rPDGF synchronized the maximal elevation of PGHS mRNA levels and PGE2 synthesis; both peaked at 3 h. Interestingly, aspirin pretreatment increased both the rate and amplitude of rPDGF-stimulated PGHS mRNA induction. The enhanced induction was not simply the result of a loss in PGE2 production since treatment with exogenous PGE2 also induced PGHS mRNA levels. Since rPDGF-stimulated PGE2 synthesis is blocked by both transcription and translation inhibitors but arachidonate-stimulated PGE2 synthesis is unaffected, the requirement for protein synthesis does not appear to involve de novo PGHS synthesis. The synthesis of a protein required for coupling of the rPDGF receptor to phospholipase activation and arachidonic acid mobilization is a more likely interpretation. In summary, our data suggest that rPDGF can induce PGHS mRNA levels, but that rPDGF-stimulated PGE2 synthesis is not dependent upon de novo PGHS synthesis.

Cyclic AMP can partially restore platelet-derived growth factor-stimulated prostaglandin E2 biosynthesis, and calcium mobilization in EJ-ras-transformed NIH-3T3 cells.

NIH-3T3 cells transformed by the EJ-ras oncogene display reduced platelet-derived growth factor (PDGF)-stimulated phospholipase C activity as measured by inositol 1,4,5-triphosphate (IP3) synthesis and Ca2+ mobilization. The lack of PDGF-stimulated Ca2+ mobilization in EJ-ras transformed cells is not due to a loss of IP3 sensitivity, because microinjected IP3 elevates intracellular Ca2+. Treatment of EJ-ras transformed cells with cholera toxin or 8-bromo-cyclic AMP, but not pertussis toxin or the beta-subunit of cholera toxin, results in a slight recovery of PDGF-stimulated IP3 synthesis, a marked increase in intracellular Ca2+ mobilization, and an almost complete recovery of prostaglandin E2 biosynthesis. These data suggest that EJ p21-mediated inhibition of PDGF-stimulated intracellular events can be partially and transiently reversed by cyclic AMP.

Regulation of Na+-H+ exchange in normal NIH-3T3 cells and in NIH-3T3 cells expressing the ras oncogene.

Our laboratory and others have demonstrated that Na+-H+ exchange can be regulated by two different pathways; one that is mediated by an inositol trisphosphate-stimulated increase in intracellular calcium activity, and one that is mediated by an increase in protein kinase C activity. To determine whether one of these pathways is more important than the other, or whether one pathway is physiologically relevant, we employed normal NIH-3T3 cells (3T3 cells) and NIH-3T3 cells expressing the EJ human bladder ras oncogene (EJ cells). The EJ cells were chosen because they provide a genetic model that does not exhibit serum- or platelet-derived growth factor (PDGF)-stimulated inositol trisphosphate release or Ca2+ mobilization. It was found that serum- or PDGF-stimulated Na+-H+ exchange was more pronounced in EJ cells than in control 3T3 cells. As expected, serum- or PDGF-stimulated Na+-H+ exchange in 3T3 cells was inhibited by chelating intracellular Ca2+ with the intracellular Ca2+ chelator quin2, by the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and by the calmodulin antagonist trifluoperazine. In contrast, these agents did not inhibit serum- or PDGF-stimulated Na+-H+ exchange in EJ cells. Activators of protein kinase C (e.g., 1-oleoyl-2-acetylglycerol or biologically active phorbol esters) were found to stimulate Na+-H+ exchange in EJ cells to the same extent as serum. However, these agents were considerably less effective than serum in control 3T3 cells. Despite these findings, PDGF did not stimulate diacylglycerol levels in EJ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Thromboxane synthase activity and platelet function after furegrelate administration in man.

Furegrelate sodium (U-63,557A), a pyridine-derivative thromboxane synthase inhibitor, was administered orally in single doses of 200 to 1600 mg to normal male subjects. Furegrelate produced a dose-related inhibition of thromboxane synthesis for 8-12 hours when measured either ex vivo from platelet-rich plasma (PRP) or in vivo from urine. In general, the extent of thromboxane synthesis inhibition was greater in PRP than in urine. Furegrelate significantly inhibited platelet aggregation, but the effect was variable and measurements of thromboxane synthase did not predict the impact on platelet aggregation. Bleeding times and coagulation parameters were not altered significantly. Furegrelate was well absorbed orally with Tmax = 1 hr and t1/2 = 3.5 to 5 hrs. There was no marked metabolism; elimination was primarily by renal excretion of parent compound. Thus, furegrelate is an effective inhibitor of thromboxane synthase in man with a relatively long biologic and circulating half-life.

Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene.

Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.

NIH-3T3 cells transformed by the EJ-ras oncogene exhibit reduced platelet-derived growth factor-mediated Ca2+ mobilization.

NIH-3T3 cells transformed by the EJ-ras oncogene synthesize only 10-15% as much inositol 1,4,5-trisphosphate (InsP3) as control cells after stimulation with platelet-derived growth factor (PDGF). This is despite the fact that the basal (unstimulated) levels of InsP3 synthesized in control and EJ-ras-transformed cells are not significantly different. Using the fluorescent indicator fura-2 and digital-imaging techniques, we have visualized and quantified changes in intracellular Ca2+ concentrations in control and EJ-ras-transformed NIH-3T3 cells in response to PDGF. Within 3 min after exposure of control cells to PDGF, intracellular Ca2+ levels are increased 3- to 9-fold, paralleling the increase in InsP3. In contrast, the majority (greater than 90%) of the EJ-ras-transformed cells show no increase in Ca2+ levels after PDGF exposure and the few that did respond exhibited only a small transient increase. Pronounced differences in the intracellular localization of Ca2+ increases in control and the responding EJ-ras-transformed cells were also observed. Despite the inhibition of InsP3 synthesis and subsequent Ca2+ mobilization, the EJ-ras-transformed cells respond mitogenically to PDGF. These data do not support the hypothesis that the EJ-ras gene product (p21) stimulates a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C in NIH-3T3 cells; instead they suggest that the EJ-ras p21 may uncouple the PDGF receptor from phospholipase C resulting in inhibition of PDGF-stimulated activity of phospholipase C, InsP3 synthesis, and Ca2+ mobilization.

NIH-3T3 cells expressing high levels of the c-ras proto-oncogene display reduced platelet derived growth factor-stimulated phospholipase activity.

NIH-3T3 cells expressing elevated levels of the normal human c-Ha-ras proto-oncogene (c-ras) exhibit reduced platelet derived growth factor-stimulated phospholipase activity. Three clonal cell lines of NIH-3T3 cells expressing different levels of c-ras have been isolated and characterized. The level of c-ras expression correlates inversely with PDGF-stimulated phospholipase activity as monitored by prostaglandin E2 (PGE2) production. In addition, high levels of c-ras expression produce cells with morphological and biochemical characteristics indistinguishable from NIH-3T3 cells transformed by EJ-ras. These data suggest that abnormal c-ras expression can attenuate growth factor-stimulated phospholipase activity in NIH-3T3 cells, in a manner analogous to that observed in cells transformed by EJ-ras.

Novel molecules that antagonize leukotriene B4 binding to neutrophils.

A series of LTB4 analogues have been synthesized that replace carbons 7-9 of the cis-trans-trans triene unit of LTB4 with a stable ring structure. Meta-substituted pyridine analogues are more potent inhibitors than benzene or furan analogues. C-1 alcohols are often more potent inhibitors than free carboxylic acids, and 5,6-cis double bond compounds are more potent than 5,6-trans compounds. Compounds such as these may prove to be useful in the treatment of inflammatory diseases.

Modular transport ventilation system for twins.

A single modular transport ventilation system has been modified for ventilation of twins by duplicating the gas supply lines, gas blender, and ventilator. This twin ventilator system can be assembled easily in minutes, has essential built-in safety features, and provides simultaneous yet individualized ventilation support to each infant during transport without the need for additional personnel.

The lack of PDGE-stimulated PGE2 release from ras-transformed NIH-3T3 cells results from reduced phospholipase C but not phospholipase A2 activity.

Our previous work demonstrated that NIH-3T3 cells expressing high levels of the mutated cellular ras oncogene (EJ-ras gene) exhibited reduced hormone-sensitive adenylate cyclase and platelet-derived growth factor-stimulated (PDGF) phospholipase A2/C activities. We now report that although the ras-transformed cells display markedly reduced phospholipase C activity, as measured by the levels of inositol 1,4,5-trisphosphate synthesized after PDGF-stimulation, normal levels of phospholipase A2 activity can be uncovered; thus, similar levels of prostaglandin E2 were synthesized in EJ-ras transformed and control cells after stimulation with phorbol myristate acetate (PMA) and/or the calcium ionophore A-23187, agents which stimulate protein kinase C and intracellular Ca2+ levels, respectively. These data suggest that the EJ-ras gene product uncouples the PDGF receptor from the phospholipase C, resulting in reduced PDGF-stimulated Ca2+ mobilization, protein kinase C stimulation and an apparent decrease in Ca2+-dependent phospholipase A2.

Loss of platelet-derived growth factor-stimulated phospholipase activity in NIH-3T3 cells expressing the EJ-ras oncogene.

Data indicating that the 21-kDa protein (p21) Harvey-ras gene product shares sequence homology with guanine nucleotide-binding proteins (G proteins) has stimulated research on the influence(s) of p21 on G-protein-regulated systems in vertebrate cells. Our previous work demonstrated that NIH-3T3 mouse cells expressing high levels of the cellular ras oncogene isolated from the EJ human bladder carcinoma (EJ-ras) exhibited reduced hormone-stimulated adenylate cyclase activity. We now report that in these cells another enzyme system thought to be regulated by G proteins is inhibited, namely phospholipases A2 and C. NIH-3T3 cells incubated in plasma-derived serum release significant levels of prostaglandin E2 (PGE2) as determined by radioimmunoassay when exposed to platelet-derived growth factor (PDGF) at 2 units/ml; the levels of PGE2 released from EJ-ras-transfected cells are only 3% those of controls despite a similar basal (unstimulated) release from control and EJ-ras-transfected cells. The lack of PDGF-stimulated PGE2 release from EJ-ras-transfected cells is not due to a defect in the prostaglandin cyclooxygenase enzyme, since incubation of control cells and EJ-ras-transfected cells in 0.33, 3.3, or 33 microM arachidonate resulted in identical levels of PGE2 release. The lack of PDGF-stimulated PGE2 release from EJ-ras-transfected cells also does not result from the loss of functional PDGF receptors. EJ-ras-transformed cells bind 70% as much 125I-labeled PDGF as control cells and are stimulated to incorporate [3H]thymidine and to proliferate after exposure to PDGF. Moreover, this inhibition is not likely the result of a secondary cellular effect related to the transformed phenotype, since NIH-3T3 cells transformed by v-src released PGE2 at wild-type levels after exposure to PDGF. Determination of total water-soluble inositolphospholipids and changes in the specific activities of phosphatidylcholine in control and EJ-ras-transfected cells demonstrated that PDGF-stimulated phospholipase C and A2 activities are inhibited in the EJ-ras-transfected cells.