Optimization of 2-piperidin-4-yl-acetamides as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists: Designing out hERG inhibition

Herein, we disclose the discovery and optimization of 2-piperidin-4-yl-acetamide derivatives as MCH-R1 antagonists. Structural investigation of piperidin-4-yl-amide and piperidin-4-yl-ureas identified 2-piperidin-4-yl-acetamide-based MCH-R1 antagonists with outstanding in vivo efficacy but flawed with high affinity towards the hERG potassium channel. While existing hERG SAR information was employed to discover highly potent MCH-R1 antagonists with minimized hERG inhibition, additional hurdles prevented their subsequent clinical exploration.     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.     

Modulation of the cell-mediated immune function by interferon α, β or γ can partially reverse the immunosuppression induced by human T-cell leukemia virus I in human cord blood cultures

Summary Infection with human T-cell leukemia virus type I (HTLV-I) is associated in vitro and in vivo with a remarkable depression of cell-mediated immune functions. In the present report it is shown that early events following virus-induced suppression of the cell-mediated immune response of freshly isolated cord blood mononuclear cells (CBL) infected with HTLV-I can be partially counteracted by treatment with interferons , or (IFN). All three types of IFN exerted a protective effect on CBL cultures exposed to the virus. This resulted in: (a) a reduced number of virus-positive cells until 4 weeks of culture; (b) delay in the clonal expansion of infected cells (IFN and ); (c) increased natural killer cell activity of CBL, 1 week post-infection (p.i.), mediated by IFN; (d) increase of allospecific recognition of infecting and priming HTLV-I donor MT-2 cells by CBL in a cytotoxic-T-lymphocyte-like response, mediated by IFN and particularly by IFN; (e) phenotype distribution of CBL subpopulations, tested 4 days p.i., more similar to that of non-infected CBL cultures.In contrast, the overall CBL proliferation, that is profoundly depressed during the first week p.i., was not restored by IFN treatments, suggesting that boosting of the cell-mediated killing induced by IFN might involve the maturation of undifferentiated precursor cells rather than stimulation of their proliferation. The improvement of the efficiency of the antiviral immune response induced by treatment with IFN is likely to contribute to the clearance of virus-positive cells during the early phase of infection. This would provide experimental evidence to support an immunopharmacological approach contributing to the conversion of HTLV-I carriers from positive to negative.     

Rat liver lowMr phosphotyrosine protein phosphatase isoenzymes: Purification and amino acid sequences

Two lowM _r phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as lowM _r acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH₂-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40–73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function.     

A mite survey in the dumpsters for urban solid rubbish

Summary The presence of house dust mites and storage mites in dumpsters was investigated in 3 different areas of Venice hinterland. The aim of this study was to find a relationship between some reported respiratory symptoms among 6 workers who were responsible for moving the dumpsters to the truck and the bio-aerosol released from such operation. These symptoms were closely related to the work and after allergological evaluation we found sensitisation to house dust mites in all 6 employees. The mine exposure in the workplace was assessed by a standardized sampling and analysis of dust obtained after brushing the inside surfaces of dumpsters. In 50% of the specimens (10/20) we found mites belonging to the following families: Pyroglyphidae (Dermatophagoides spp.), Acaridae (Acarus siro) and a lower amount of Cheyletidae (Cheyletus spp.). An indirect test (guanine test) was also performed and gave positivity in 16 dust samples (80%). The insides of dumpsters showed to be an optimal environment for mites survival and reproduction because of cracks and grooves on the walls, the high humidity level and the presence of mould. These mites probably come from dwelling places and other environments (markets, slaughters-houses, farms, etc.). This suggests that mites allergens can be released during rubbish discharging from the dumpsters and represent a possible risk for the employees.     

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.     

Oxidative destruction of DNA by the adriamycin-iron complex

The 2:1 adriamycin-Fe(III) complex is able to bind to DNA and to catalyze its oxidative destruction. The binding of the drug-metal complex to DNA is indicated by characteristic spectral changes which are different from those seen with adriamycin intercalation and by the propensity of the drug-metal complex to precipitate DNA. Furthermore, intercalated adriamycin appears not to be available for iron binding. The resulting ternary complex is quite stable: it is not disrupted by incubation in the presence of EDTA and can be isolated by using Sephadex G-50 column chromatography. Disruption of the ternary complex requires vigorous conditions (extraction with phenol at 60 degrees C). The adriamycin-iron complex in free solution has the capacity to catalyze the reduction of oxygen by thiols. The DNA-bound drug-metal complex preserves this capacity over a wide range of complex/DNA ratios. As a consequence of this thiol-dependent oxygen reduction, DNA is cleaved. This thiol-dependent DNA cleavage has been shown to require hydrogen peroxide as an intermediate product. These results have led us to propose that the thiol-dependent DNA cleavage reaction has two stages involving (1) reduction of oxygen leading to hydrogen peroxide and then (2) peroxide-dependent DNA cleavage. An unusual property of this reaction is that the cleavage is not random but gives rise to a defined 2300 base pair fragment.     

A gene controlling fetal hemoglobin expression in adults is not linked to the non-alpha globin cluster.

The possible linkage between a gene causing heterocellular hereditary persistence of fetal hemoglobin (HPFH) and human non-alpha globin loci has been studied in a large Sardinian family. In this family a homozygous beta o-thalassemic patient was found, with an unusually mild form of this disease, which was ascribed to the co-existence of a gene causing heterocellular HPFH. DNA polymorphisms in the non-alpha globin cluster were analyzed by restriction enzyme digestion with HincII, HindIII and BamHI and with epsilon-, gamma-and beta-globin probes; the pattern of inheritance of these polymorphisms indicates that the HPFH gene is transmitted with one beta o-thalassemic gene in a single instance, with the second beta o-thalassemic gene in three instances and with a normal beta-globin gene in two cases. These data indicate that this HPFH gene is not linked to the non-alpha globin gene cluster, in contrast to previous observations with different HPFH genes, and suggest that this gene might code for diffusible substances acting, directly or indirectly, on gamma-globin gene expression.