Inhibition of canine distemper virus replication by blocking pyrimidine nucleotide synthesis with A77 1726, the active metabolite of the anti-inflammatory drug leflunomide.

Canine distemper virus (CDV) is the aetiological agent that causes canine distemper (CD). Currently, no antiviral drugs have been approved for CD treatment. A77 1726 is the active metabolite of the anti-rheumatoid arthritis (RA) drug leflunomide. It inhibits the activity of Janus kinases (JAKs) and dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. A77 1726 also inhibits the activity of p70 S6 kinase (S6K1), a serine/threonine kinase that phosphorylates and activates carbamoyl-phosphate synthetase (CAD), a second rate-limiting enzyme in the de novo pathway of pyrimidine nucleotide synthesis. Our present study focuses on the ability of A77 1726 to inhibit CDV replication and its underlying mechanisms. Here we report that A77 1726 decreased the levels of the N and M proteins of CDV and lowered the virus titres in the conditioned media of CDV-infected Vero cells. CDV replication was not inhibited by Ruxolitinib (Rux), a JAK-specific inhibitor, but by brequinar sodium (BQR), a DHO-DHase-specific inhibitor, and PF-4708671, an S6K1-specific inhibitor. Addition of exogenous uridine, which restores intracellular pyrimidine nucleotide levels, blocked the antiviral activity of A77 1726, BQR and PF-4708671. A77 1726 and PF-4708671 inhibited the activity of S6K1 in CDV-infected Vero cells, as evidenced by the decreased levels of CAD and S6 phosphorylation. S6K1 knockdown suppressed CDV replication and enhanced the antiviral activity of A77 1726. These observations collectively suggest that the antiviral activity of A77 1726 against CDV is mediated by targeting pyrimidine nucleotide synthesis via inhibiting DHO-DHase activity and S6K1-mediated CAD activation.

4-methylpyrazole decreases 1,4-butanediol toxicity by blocking its in vivo biotransformation to gamma-hydroxybutyric acid.

1,4-Butanediol (1,4-BD), a prodrug converted in vivo to gamma-hydroxybutyric acid by alcohol dehydrogenase, has resulted in life-threatening overdoses and deaths. We investigated whether 4-methylpyrazole (4-MP), an alcohol dehydrogenase antagonist, can be used as an antidote in a murine model of 1,4-BD overdose. CD-1 mice were overdosed with 1,4-BD, 600 mg/kg i.p. Mice then received 4-MP, 25 mg/kg i.p., or control injections after 1 min, 5 min, and symptom appearance. Mice were then evaluated for toxicity by the righting reflex and rotarod test every 10 min after intervention. When 4-MP was administered 1 and 5 min after 1,4-BD overdose, mice completely maintained their righting reflex. Conversely, control mice lost their righting reflex for 110 and 130 min, respectively (P < 0.05). When 4-MP was administered after symptomatic 1,4-BD overdose, mice lost their righting reflex but recovered it by 60 min. Conversely, control mice lost their righting reflex and recovered it by 140 min (P < 0.05). When 4-MP was administered at 1 min after 1,4-BD overdose, mice never failed the rotarod test. Conversely, control mice failed the rotarod test for 210 min (P < 0.05). When 4-MP was administered 5 min after 1,4-BD and after symptomatic 1,4-BD overdose, mice failed the rotarod test for 100 and 110 min, respectively. Conversely, control mice failed the rotarod test for 210 and 180 min, respectively (P < 0.05). In addition, treatment of mice with 4-MP significantly attenuated increases in blood gamma-hydroxybutyric acid concentrations and prevented loss of the righting reflex and failure of the rotarod test. In this murine model of 1,4-BD overdose, 4-MP conferred antidotal effects by inhibiting alcohol dehydrogenase-mediated biotransformation of 1,4-BD to gamma-hydroxybutyric acid.

The "PHB depolymerase inhibitor" of Paucimonas lemoignei is a PHB depolymerase.

A approximately 35 kDa protein that has been described to be secreted by Paucimonas lemoignei during growth on succinate and to inhibit hydrolysis of denatured (crystalline) poly(3-hydroxybutyrate) (dPHB) by extracellular PHB depolymerases of P. lemoignei (PHB depolymerase inhibitor (PDI)) was purified and characterized. Purified PDI (M(r), 36 199 +/- 45 Da) inhibited hydrolysis of dPHB by two selected purified PHB depolymerases (PhaZ2 and PhaZ5) but did not inhibit the hydrolysis of water-soluble substrates such as p-nitrophenylbutyrate by PhaZ5 and PhaZ2. PDI revealed a high binding affinity to dPHB although it was not able to hydrolyze the crystalline polymer. However, purified PDI had a high hydrolytic activity if native (amorphous) PHB (nPHB) was used as a substrate. N-terminal sequencing of PDI revealed that it was identical to recently described extracellular PHB depolymerase PhaZ7 which is specific for nPHB and which cannot hydrolyze dPHB. To confirm that the inhibition of hydrolysis of dPHB by PhaZ7 is an indirect surface competition effect at high depolymerase concentration, the activity of PHB depolymerases PhaZ2 and PhaZ5 in the presence of different amounts of protein mixtures was determined. The components of NB or LB medium inhibited hydrolysis of the polymer in a concentration-dependent manner but had no effect on the hydrolysis of p-nitrophenylbutyrate by PHB depolymerases. In combination with PHB depolymerases PhaZ2 and PhaZ5 the protein PhaZ7 ("PDI") enables the bacteria to hydrolyze dPHB and nPHB simultaneously.

Design and structure-activity relationship analysis of ligands of gamma-hydroxybutyric acid receptors.

With the use of [3H]gamma-hydroxybutyric acid, binding experiments allowed the screening of new compounds as ligands of gamma-hydroxybutyric acid receptors. Starting from the acid-alcohol gamma-hydroxybutyric acid structure, structure-activity relation analysis and lead optimization highlighted gamma-hydroxybutyric acid derivatives with significantly increased affinities, when compared with the affinity of gamma-hydroxybutyric acid. Further pharmacological studies with the use of gamma-hydroxybutyric acid derivatives allowed the characterization of the first competitive antagonist acting at gamma-hydroxybutyric acid receptors (NCS 382).

Immunosuppressive leflunomide metabolite (A77 1726) blocks TNF-dependent nuclear factor-kappa B activation and gene expression.

Leflunomide is a novel immunosuppressive and antiinflammatory agent currently being tested for treatment of autoimmune diseases and transplant rejection. NF-kappa B is a transcription factor activated in response to a wide variety of inflammatory stimuli, including TNF, but whether leflunomide blocks NF-kappa B activation is not known. In the present report we demonstrate that treatment of a human T cell line (Jurkat) with leflunomide blocks TNF-mediated NF-kappa B activation in a dose- and time-dependent manner, with maximum inhibition at 5-10 microM. Inhibition was not restricted to TNF-induced activation, because leflunomide also inhibited NF-kappa B activation induced by other inflammatory agents, including phorbol ester, LPS, H2O2, okadaic acid, and ceramide. Leflunomide blocked the degradation of I kappa B alpha and subsequent nuclear translocation of the p65 subunit, steps essential for NF-kappa B activation. This correlated with inhibition of dual specificity-mitogen-activated protein kinase kinase as well as an Src protein tyrosine kinase, p56lck, by leflunomide. Reducing agents did not reverse the effect of leflunomide. Leflunomide also suppressed the TNF-activated NF-kappa B-dependent reporter gene expression. Our results thus indicate that leflunomide is a potent inhibitor of NF-kappa B activation induced by a wide variety of inflammatory stimuli, and this provides the molecular basis for its anti-inflammatory and immunosuppressive effects.

Inhibition of smooth muscle cell proliferation in vitro by leflunomide, a new immunosuppressant, is antagonized by uridine.

Chronic rejection in the form of graft vascular disease (GVD) continues to plague clinical transplantation of vascularized organs. The histopathology of this lesion is characterized by neointimal hyperplasia, smooth muscle cell proliferation, and obliterative arteriopathy. Due to the lack of effective medical therapy for preventing or reversing these chronic vascular changes, retransplantation remains the final resort in treatment. Some of the newer immunosuppressive agents, including the new isoxazole derivative leflunomide (LFM), have shown efficacy in preventing chronic rejection in animal models of transplantation. Although its mechanism of action remains incompletely elucidated, previous work using lymphocytes in vitro suggests that the drug might act as a tyrosine kinase inhibitor, an inhibitor of de novo pyrimidine biosynthesis, or both. In order to elucidate whether the efficacy of LFM in vivo is attributable not only to anti-proliferative effects on the recipient immune system but also to direct effects on mesenchymal cells in the donor organ, we examined the effects of LFM on a transformed 9E11G murine smooth muscle cell (M-SMC) line in vitro. We demonstrate here that the active metabolite of LFM, A77 1726, dose-dependently inhibits the constitutive and growth-factor stimulated proliferation of M-SMC in vitro. Furthermore, the anti-proliferative effect of the drug can be reversed by the addition of uridine to the culture medium. These results suggest that inhibition of uridine biosynthesis appears to be a mechanism by which LFM exerts anti-proliferative effects on both lymphocytes and smooth muscle cells, and this dual action may be responsible for its efficacy in preventing GVD in vivo.

Mechanism of the antiproliferative action of leflunomide. A77 1726, the active metabolite of leflunomide, does not block T-cell receptor-mediated signal transduction but its antiproliferative effects are antagonized by pyrimidine nucleosides.

BACKGROUND: Leflunomide, a novel immunosuppressive drug, prolongs experimental graft survival effectively and has been well tolerated in patients with rheumatoid arthritis. A77 1726, the active metabolite of leflunomide, inhibits lymphocyte proliferation in vitro. This study was conducted in Jurkat T cells to investigate the effects of A77 1726 on signal transduction pathways initiated by ligands of the T-cell receptor CD3 complex and to evaluate the effects of A77 1726 on nucleotide biosynthesis. METHODS: Tritiated thymidine incorporation and cell counts quantitated cell proliferation. Spectrofluorescence of Indo/AM dye measured intracellular Ca2+ mobilization. A luciferase assay quantitated interleukin-2 gene promoter activity in stimulated cells transfected with an interleukin-2 promoter-luciferase gene construct. Pyrimidine and purine nucleosides were used to assess antagonism of the antiproliferative activity of A77 1726. RESULTS: (1) A77 1726 dose-dependently inhibited the proliferation of Jurkat T cells (inhibitory concentration of 50% = 6 mumol/L); (2) A77 1726 did not decrease mobilization of intracellular Ca2+ stimulated by phytohemagglutinin or anti-CD3 monoclonal antibody; (3) A77 1726 did not inhibit interleukin-2 gene promoter activity in cells stimulated with ionomycin plus phorbol myristate acetate; (4) inhibition of cell proliferation by A77 1726 was antagonized by addition of uridine, cytidine, or 2(+)-deoxycytidine; (5) addition of uridine 24 hours after treatment with A77 1726 antagonized inhibition of proliferation; (6) A77 1726 was not antagonized by 2'-deoxyuridine, thymidine, adenosine, or guanosine. CONCLUSIONS: (1) A77 1726 inhibited Jurkat T-cell proliferation without inhibiting T-cell receptor-mediated signal transduction events, including tyrosine kinase-dependent intracellular Ca2+ mobilization and activation of the interleukin-2 gene promoter; (2) the antiproliferative effects of A77 1726 on Jurkat T cells are primarily due to interruption of de novo pyrimidine nucleotide biosynthesis. These data provide evidence for a novel in vitro mechanism of the antiproliferative action of this immunosuppressant.

Naloxone pretreatment alters the local cerebral metabolic effect of gamma-hydroxybutyrate in rats.

The effect of naloxone on gamma-hydroxybutyrate (GHB)-induced cerebral metabolic depression was studied in rats with the 2-[14C]deoxyglucose method. Naloxone pretreatment statistically significantly antagonized the cerebral metabolic effect of GHB in 10 of 38 structures examined. These results are consistent with previous data showing reversal of the dopaminergic, electroencephalographic, and behavioral effects of GHB by naloxone and suggest that some of the neuropharmacological effects of GHB are mediated by the endogenous opiate system.

Evidence for the importance of cysteine and arginine residues in Pseudomonas fluorescens UK-1 pantoate dehydrogenase.

Homogeneous pantoate dehydrogenase (D-pantoate:NAD+ 4-oxidoreductase, EC 1.1.1.106) was shown to be sensitive to inactivation by p-chloromercuribenzoate (100 microM), 5,5'-dithiobis(2-nitrobenzoic acid) (1 mM), iodoacetic acid (1 mM) and phenylglyoxal (5.3 mM). Potassium D-pantoate and NAD protected against inactivation by p-chloromercuribenzoate, 5,5'-dithiobis (2-nitrobenzoic acid) and iodoacetic acid. NAD and D-pantoate also provided substantial protection against inactivatrion by phenylglyoxal. Titration of the sulphydryl groups by 5,5'-dithiobis(2-nitrobenzoic acid) and incorporation of [14C]carboxymethyl revealed that there are two cysteine residues which are modified and one of those is essential for activity. In the presence of NAD and D-pantoate, incorporation of [14C]phenylglyoxal was decreased by 0.42 mol/mol of subunit.

Antagonism of gamma-hydroxybutyric acid-induced frequency shifts in the cortical EEG of rats by dipropylacetate.

An automated technique for the continuous analysis of selected EEG frequency bands as a function of drug treatment is described. Experiments utilizing this analytical method have shown that the convulsive effects of GHB are antagonized by dipropylacetate, but that the effects of GHB exhibit a latent irreversibility.

Reversal of the anaesthetic action of sodium gamma-hydroxybutyrate.

Physostigmine was administered intravenously to 25 patients, anaesthetised with sodium gamma-hydroxybutyrate (GHB), and their emergence from anesthesia was studied. Physostigmine (2 mg) brought about rapid, safe, reliable and sustained awakening after a "latent period" varying from 2-10 minutes (mean 6-2 minutes+/-S.D. 2-2) in 24 patients. In the one patient not awake at 10 minutes, a second dose of physostigmine produced awakening in an additional 8 minutes. No serious side effects were attributable to the physostigmine. This finding may warrant a reconsideration of the place of GHB in anaesthetic practice.