N-aryl-piperidine-4-carboxamides as a novel class of potent inhibitors of MALT1 proteolytic activity

Starting from a weak screening hit, potent and selective inhibitors of the MALT1 protease function were elaborated. Advanced compounds displayed high potency in biochemical and cellular assays. Compounds showed activity in a mechanistic Jurkat T cell activation assay as well as in the B-cell lymphoma line OCI-Ly3, which suggests potential use of MALT1 inhibitors in the treatment of autoimmune diseases as well as B-cell lymphomas with a dysregulated NF-κB pathway. Initially, rat pharmacokinetic properties of this compound series were dominated by very high clearance which could be linked to amide cleavage. Using a rat hepatocyte assay a good in vitro-in vivo correlation could be established which led to the identification of compounds with improved PK properties.     

Synthetic Analogues of Conantokin-G: NMDA Antagonists Acting Through a Novel Polyamine-Coupled Site

Abstract: Conantokin-G (con-G) is a 17-amino-acid polypeptide that acts as an N-methyl-d -aspartate (NMDA) antagonist. This action has been attributed to a specific but noncompetitive inhibition of the positive modulatory effects of polyamines at NMDA receptors. Con-G possesses several unusual structural features, including five γ-carboxyglutamate (Gla) residues and a high degree of helicity in aqueous media. Previous structure-activity studies indicated that one or more Gla residues are necessary for NMDA antagonist activity. Con-G analogues were synthesized with alanine (Ala), serine (Ser), and phosphoserine substituted for Gla to assess the contribution of individual Gla residues to biological activity and secondary structure. Replacement of Gla in positions 3 and 4 resulted in polypeptides with markedly reduced and no NMDA antagonist actions, respectively. In contrast, Gla residues in positions 7, 10, and 14 are not required for NMDA antagonist actions because the potencies of con-G analogues containing Ser⁷, Ser¹⁰, Ala¹⁴, and Ser¹⁴ to inhibit spermine-stimulated [³H]MK-801 binding are similar to the parent peptide. Moreover, the Ala⁷ derivative of con-G was about fourfold more potent than the parent peptide both as an inhibitor of spermine-stimulated increases in [³H]MK-801 binding (IC₅₀ of ～45 nM) and in reducing NMDA-stimulated increases in cyclic GMP levels (IC₅₀ of ～77 nM) in cerebellar granule cell cultures. Although con-G and its analogues assumed mixtures of 3₁₀ and α-helices, no clear-cut relationship was evinced between the NMDA antagonist properties of these peptides and the degree of helicity they assumed in aqueous solutions. Together with the inability of con-G to affect 5,7-dichloro[³H]kynurenic acid, [³H]CGP-39653, and [³H]ifenprodil binding, these data are consistent with the hypothesis that this polypeptide acts at a unique, polyamine-associated site on NMDA receptors.     

Diminished plasma cGMP during weightlessness

We performed an experiment within the project "RLF" (Russian long-term flight) on a cosmonaut onboard the space station MIR. For creating an analogue to orthostatic stress, we used lower body negative pressure (LBNP) as stimulus. Decrease in central and peripheral baroreceptor load by LBNP can be used as a cardiovascular countermeasure in cosmonauts or for inducing endocrine responses. Altered steady-state plasma concentration values of volume sensitive hormones have been observed inflight as well as postflight. Within this project we measured plasma ANP and cGMP as second messenger. Changes in plasma cGMP concentration are generally considered to be a good indicator of those in ANP activity. However, in our experiments depression of cGMP during space flight was more impressive than ANP decline. We are not aware of previous measurements of plasma cGMP under these conditions, and believe to be the first to report complete suppression of plasma cGMP during long-term stay in space.     

Isolation of Relatively Large Amounts of Endomorphin-1 and Endomorphin-2 From Human Brain Cortex

Hackler, L., J. E. Zadina, L-J. Ge and A. J. Kastin. Isolation of relatively large amounts of endomorphin-1 and endomorphin-2 from human brain cortex. Peptides 18(10) 1635–1639, 1997.—Endomorphin-1 (Tyr-Pro-Trp-Phe-NH₂) and endo-morphin-2 (Tyr-Pro-Phe-Phe-NH₂) were previously isolated from bovine brain. Both peptides showed the greatest selectivity and affinity for the mu opiate receptor of any endogenous substance found to date and may serve as natural ligands for the mu-opiate receptor. We have purified them from the fronto-parietal cortex of human brain tissue by solid phase extraction and high performance liquid chromatography. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against endomorphin-1. The isolated endomorphins showed full biological activity. The tetrapeptides were found in human brain in much higher amounts than in bovine frontal cortex.     

The occurrence and fine structural characterization of microbodies inEuglena gracilis

Summary The fine structure of an organelle morphologically similar to microbodies found in higher plants and animals was studied in cells ofEuglena gracilis fixed simultaneously in glutaraldehyde and osmium tetroxide. These organelles were 0.4 to 0.8 microns in diameter, bounded by a single membrane, and frequently observed in close spatial association with both endoplasmic reticulum and mitochondria. Their finely granular matrices frequently contained membranous cores. Though these organelles were relatively abundant in acetate- and ethanolgrown cells, they were rarely observed in glucose-grown cells, an indication that they play the same role in the metabolism of 2-carbon substrates as do glyoxysomes in higher plants. The presence of these organelles, assumed to be microbodies, is also of considerable interest since catalase, an enzyme characteristic of microbodies from a variety of sources, was not detected.This work was supported in part by grant GY 3804 from the National Science Foundation to L.B.G.     

Proficiency testing of conventional drug susceptibility tests of Mycobacterium tuberculosis

Proficiency testing of indirect drug susceptibility tests of Mycobacterium tuberculosis was begun in 1985 by the Laboratory Centre for Disease Control (LCDC) with the participation of Provincial Public Health Laboratories in Canada. Comparable sets of 60 cultures of Mycobacterium tuberculosis representing 30 strains were distributed by LCDC to the participating laboratories to be tested for drug susceptibility against isoniazid, streptomycin, rifampin, and ethambutol using conventional methodologies. Intralaboratory agreement values determined by comparing results obtained on sets of duplicate cultures were high and were found to vary little from drug to drug and from laboratory to laboratory. Interlaboratory agreement was determined by comparing results reported by participating laboratories to those obtained by the Reference Laboratory. Agreement percentages were found to be lower for drug-resistant cultures than for drug-susceptible cultures. The reliability of drug susceptibility testing results was higher for isoniazid and rifampin, than for ethambutol and streptomycin. This study shows that the higher subsidiary drug concentrations do not compare well with main drug concentrations, especially in the case of streptomycin and ethambutol. The significance of the higher subsidiary concentrations in in vitro susceptibility testing is therefore in need of clarification. The proficiency testing results obtained in this study compare favorably with those reported in other developed countries despite the fact that a variety of testing procedures are used throughout the country.     

Release of [3H]noradrenaline by α1-adrenoceptor agonists

Mouse isolated vas deferens preincubated with [³-H]noradrenaline was superfused and the effect of ₁-adrenoceptor agonists was studied on the release of total radioactivity ([³H]noradrenaline +³H-metabolites) and [³H]noradrenaline. Reverse phase high pressure liquid chromatography (HPLC) combined with scintillation spectrometry was used to separate [³H]noradrenaline from its metabolites. Among the ₁-adrenoceptor agonists (1-phenylephrine, ST-587(2-(2-chloro-5-trifluoromethyl phenylimino)-imidazole), (–)-amidephrine, methoxamine, cirazoline and l-noradrenaline) studied l-phenylephrine, ST-587 and l-noradrenaline were capable of releasing³H-noradrenaline. The effect of noradrenaline was stereospecific. As determined by HPLC combined with scintillation spectrometry the release of total radioactivity in response to l-noradrenaline is mainly due to [³H]noradrenaline. It is suggested that l-noradrenaline, l-phenylephrine, and ST-587 in addition to their direct effect on different receptors they also have indirect action through the release of noradrenaline which might be partly involved in the pharmacological responses. The mechanisms whereby l-noradrenaline and l-phenylephrine release noradrenaline would appear to involve a saturable Ca-independent and a cocaine and temperature sensitive process. On the basis of our findings among the ₁-adrenoceptor agonist studied (–)-amidephrine, methoxamine and cirazoline is a better choice than l-phenylephrine or ST-587 for selective stimulation of postjunctional ₁-adrenoceptor, they do not release noradrenaline.     

Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

The photosystem II core complex (TSF-IIa) is composed of polypeptides of molecular weight 54-, 47-, 42-, and 30 kilodaltons (kD) and cytochrome b-559. After treatment with trypsin or α-chymotrypsin for 20 hours, the TSF-IIa particles still retained their photochemical activity and the light-induced cytochrome b-559 signal, although all of the polypeptides of the complexes, except the 30 kD unit were extensively degraded. Proteolytic treatment decreased the apparent molecular weight of the complex from 250,000 to 100,000 daltons as determined by gel filtration, and also decreased the protein to chlorophyll ratio by 40%. Chlorophyll a appeared to be associated with the 47- and 42 kD polypeptides. Proteolysis of the complex produced a single chlorophyll a band with a slightly higher electrophoretic mobility. This band was not equivalent to the 30 kD polypeptide. Proteolysis also reduced the sensitivity of the TSF-IIa particles to 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), but did not completely abolish it.