T-cell receptor ligation by peptide/MHC induces activation of a caspase in immature thymocytes: the molecular basis of negative selection.

T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.

Pharmacological profile of NPC 17742 [2R,4R,5S-(2-amino-4,5-(1, 2-cyclohexyl)-7-phosphonoheptanoic acid)], a potent, selective and competitive N-methyl-D-aspartate receptor antagonist.

2R,4R,5S-(2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid) (NPC 17742), the most potent isomer of the mixture 2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid (NPC 12626), was evaluated for activity in tests associated with receptors for excitatory amino acids. In receptor binding assays, NPC 17742 was selective for the N-methyl-D-aspartate (NMDA) receptor with a potency comparable to that of D(-, -3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid. Like (+/-)cis-4-phosphono-methyl-2-piperidine carboxylic acid (CGS 19755) and (+/-)(E)-2-amino-4-methyl-5-phosphono-3-penteneoic acid (CGP 37849), NPC 17742 competitively inhibited NMDA-induced enhancement of 1-[(2-thienyl)cyclohexyl]piperidine binding to the NMDA receptor ionophore and partially inhibited [3H]glycine binding to strychnine-insensitive sites. In contrast, NPC 17742 and CGP 37849 inhibited Mg(++)-stimulated 1-[(2-thienyl)cyclohexyl]piperidine binding in a noncompetitive fashion. In voltage-clamped Xenopus oocytes expressing excitatory amino acid receptors, NPC 17742 (pKB = 6.91) was equipotent with CGP 37849 (pKB = 7.17) in inhibiting NMDA-induced inward currents. Likewise, NPC 17742 (ED50 = 2.68 mg/kg) was equipotent with CGP 37849 and CGS 19755 in blocking NMDA-induced convulsions, but was less potent than these two compounds in the maximal electroshock test. Unlike CGP 37849 or CGS 19755, NPC 17742 potently antagonized seizures induced by pentylenetetrazol. In a model of global ischemia, low doses of NPC 17742 given either before or after ischemic result were effective in blocking damage to hippocampal CA1 neurons. The pharmacologic responses to NPC 17742 occurred at doses 30- to 300-fold lower than the acute lethal dose.(ABSTRACT TRUNCATED AT 250 WORDS)

(Aminoalkoxy)chromones. Selective sigma receptor ligands.

A series of (aminoalkoxy)chromones has been prepared, members of which bind potently (16-100 nM) at the sigma binding site and bind weakly (greater than 1000 nM) at the dopamine D2 receptor and 33 other receptors, second messenger systems, and ion channels. At the sigma receptor, the preferred position of attachment for the aminoalkoxy side chain to the chromone ring followed the rank order: 7-position greater than 5-position greater than 6-position. Chromones that contained a 2-substituent that was not coplanar with the chromone ring system showed improved binding over compounds with coplanar substituents. The most potent compound at the sigma site, 7-[[7-(4-hydroxypiperidyl)heptyl]oxy]-2-phenylchromone (74), had receptor affinities (IC50) of 16 nM at the [3H]DTG site, 19 nM at the [3H]-(+)-3-PPP site, and 4000 nM (Ki) at the dopamine D2 receptor. The most selective compound examined, 6-[[6-(4-hydroxypiperidyl)hexyl]-oxy]-2-cyclopentylchromone (58), exhibited IC50s of 51 nM at the [3H]DTG site, 55 nM at the [3H]-(+)-3-PPP site, and 21,000 nM (Ki) at the dopamine D2 receptor. Compound 44 (6-[[6-(4-hydroxypiperidyl)hexyl]oxy]-3-methylflavone, NPC 16377) was systemically effective (ip and po) in two behavioral models predictive of antipsychotic compounds and systemically active in animal models of ischemia.

Possible cerebroprotective and in vivo NMDA antagonist activities of sigma agents.

The recent finding that ifenprodil binds with high affinity to sigma sites suggests that other sigma agents may have ifenprodil-like cerebroprotectant and functional N-methyl-D-aspartate (NMDA) antagonist effects. The present study, compared the in vivo effects of ifenprodil and the sigma agents, BMY 14802, caramiphen and haloperidol, in three tests sensitive to NMDA antagonists and purported cerebroprotectant drugs. When administered at or below the rotorod TD50 dose, all four compounds significantly increased survival time in an hypoxic environment (4% O2 in nitrogen). Caramiphen and ifenprodil (ED50 = 52 and 61 mg/kg, respectively) also blocked maximal electroshock-induced seizures, whereas BMY 14802 and haloperidol were ineffective. Finally, caramiphen (ED50 = 95 mg/kg) antagonized seizures and lethality induced by administration of NMDA (250 mg/kg, IP). BMY 14802, haloperidol and ifenprodil only partially antagonized NMDA-induced seizures, but did enhance the anticonvulsant potency of the noncompetitive NMDA antagonist, MK-801. Together, these findings suggest that sigma agents may have cerebroprotective effects.

Stereoselective syntheses of the trans-decahydroquinoline-5-carboxylic acid epimers. Diastereomeric zwitterionic probes of gamma-aminobutyric acid related biological properties in vitro and in vivo.

The syntheses of the 5 beta and 5 alpha epimers of trans-(4a alpha,8a beta)-decahydroquinoline-5-carboxylic acids (3 and 4) from vinylogous bicyclic imide 10 are described. The reduction of trans-5-(1,3-dithian-2-ylidene)octahydro-2(1H)-quinolinone (13) to afford the 5 alpha-(1,3-dithian-2-yl) compound 16 was a key step in the synthesis of trans-4 while hydroboration-H2O2 treatment of phenylmethyl trans-octahydro-5-methylene-1(2H)-quinolinecarboxylate (21) to afford the 5 beta-hydroxymethyl compound 22 was a key step in the synthesis of 3. These trans diastereomers 3 and 4 and the previously prepared cis analogues 1 and 2 were investigated for their ability to interact with GABAA and GABAB receptors and picrotoxin binding sites as well as with neuronal GABA transport systems in brain tissue. Like 1 and 2, tonic-clonic seizures were induced when trans-3 or -4 were administered to mice intracerebroventricularly. Only trans-4 weakly inhibited [3H]GABA binding to GABAA and GABAB receptors in vitro. Large doses (10 mg/kg) of diazepam reversed the convulsant activity of both trans-3 and trans-4. Although trans-3 is the more potent convulsant, trans-4 may have GABA antagonist activity in vivo. However, none of the decahydroquinoline diastereomers have a pronounced effect on GABA receptors that can currently be studied in vitro. Results obtained in vivo lead us to propose that these diastereoisomers may serve as unique conformational probes relating certain zwitterionic topographies to stimulatory activity in the central nervous system.

Stereostructure-activity relationships of the GABA-containing cis-decahydroquinoline-5-carboxylic acids; intracerebroventricular studies in mice.

Two cis-decahydroquinoline-5-carboxylic acids, cis-1 and 2 were investigated for their pharmacological activities in vivo. Intracerebroventricular administration of either agent elicited convulsant activity in mice. This convulsant effect can be antagonized by pretreating the mice with n-dipropylacetate (valproate). Our results suggest that cis-1 may act indirectly as a partial gamma-aminobutyric acid (GABA) agonist in vivo. On the other hand, both cis-1 and 2, at higher concentrations, exhibit properties of a GABA-antagonist, but this effect seems not to be due to GABA receptor binding.

Epimeric cis-decahydroquinoline-5-carboxylic acids: effects on gamma-aminobutyric acid uptake and receptor binding in vitro.

The syntheses for two cis-decahydroquinoline-5-carboxylic acid epimers (1 and 2) which contain the =N(C)3CO2H (gamma-aminobutyric acid; GABA) moiety are described. Both intra-and intermolecular [4 + 2] cycloaddition reactions were employed for the construction of key intermediates. 1H NMR studies provided evidence for the preferred solution conformations of the two diastereomers. Pharmacological studies revealed that these isomers have little affinity for GABA receptors in vitro relative to GABA agonists. However, expected but weak stereoselective activity was observed when these analogues were assessed for their ability to inhibit high-affinity [3H]GABA uptake into rat brain synaptosomes. These data are discussed in light of structure-activity studies of other neurotransmitter analogues, and a preliminary hypothesis based upon conformational analysis is presented to explain the results.