Synthesis and evaluation of thiazepines as interleukin-1beta converting enzyme (ICE) inhibitors

A series of monocyclic thiazepine inhibitors of interleukin-1beta converting enzyme (ICE) were synthesized in eight steps from commercially available intermediates. In vitro biological evaluation showed the thiazepines to be moderately potent ICE inhibitors, with the most active compound exhibiting an IC50 value of 30 nM in an enzyme inhibition assay. Compounds of this class possessed good selectivity against the related enzymes caspase-3 and caspase-8.     

The role of oxygen-increased respirator in humans ascending to high altitude

ABSTRACT: BACKGROUND: Acute mountain sickness is common for people who live in low altitude areas ascending to the high altitude. Many instruments have been developed to treat mild cases of AMS. However, long-lasting and portable anti-hypoxia equipment for individual is not yet available. METHODS: Oxygen-increased respirator (OIR) has been designed to reduce the risk of acute mountain sickness in acute exposure to low air pressure. It can increase the density of oxygen by increasing total atmospheric pressure in a mask. Male subjects were screened, and eighty-eight were qualified to perform the experiments. The subjects were divided into 5 groups and were involved in some of the tests at 4 different altitudes (Group 1, 2: 3700 m Group 3,4,5: 4000 m, 4700 m, 5380 m) with and without OIR. These tests include heart rate, saturation of peripheral oxygen (SpO2), malondialdehyde (MDA), superoxide dismutase (SOD), blood lactate (BLA) and PWC (physical work capacity) -170. RESULTS: The results showed that higher SpO2, lower heart rate (except during exercise) and better recovery of heart rate were observed from all the subjects 'with OIR' compared with 'without OIR' (P < 0.05). Moreover, compared with 'without OIR', subjects 'with OIR' in Group 1 had lower concentrations of MDA and BLA, and a higher concentration of SOD (P < 0.05), while subjects 'with OIR' in Group 2 showed better physical capacity (measured by the PWC-170) (P < 0.05). The additonal experiment conducted in a hypobaric chamber (simulating 4,000 m) showed that the partial pressure of oxygen in blood and arterial oxygen saturation were higher 'with OIR' than 'without OIR' (P < 0.05). CONCLUSIONS: We suggested that OIR may play a useful role in protecting people ascending to high altitude before acclimatization.     

Disruption of the OCH1 and MNN1 genes decrease N-glycosylation on glycoprotein expressed in Kluyveromyces lactis

Glycoproteins secreted by the yeast Kluyveromyces lactis are usually modified by the addition at asparagines-linked glycosylation sites of heterogeneous mannan residues. The secreted glycoproteins in K. lactis that become hypermannosylated will bear a non-human glycosylation pattern and can adversely affect the half-life, tissue distribution and immunogenicity of a therapeutic protein. Here, we describe engineering a K. lactis strain to produce non-hypermannosylated glycoprotein, decreasing the outer-chain mannose residues of N-linked oligosaccharides. We investigated and developed the method of two-step homologous recombination to knockout the OCH1 gene, encoding α1,6-mannosyltransferase and MNN1 gene, which is homologue of Saccharomyces cerevisiae MNN1, encoding a putative α1,3-mannosyltransferase. We found the Kloch1 mutant strain has a defect in hyperglycosylation, inability in adding mannose to the core oligosaccharide. The N-linked oligosaccharides assembled on a secretory glycoprotein, HSA/GM–CSF in Kloch1 mutant, contained oligosaccharide Man_13–14GlcNAc₂, and in Kloch1 mnn1 mutant, contained oligosaccharide Man_9–11GlcNAc₂, whereas those in the wild-type strain, consisted of oligosaccharides with heterogeneous sizes, Man_>30GlcNAc₂. Taken together, these results indicated that KlOch1p plays a key role in the outer-chain mannosylation of N-linked oligosaccharides in K. lactis. The KlMnn1p, was proved to be certain contribution to the outer hypermannosylation, most possibly encodes α1,3-mannosyltransferase. Therefore, the Kloch1 and Kloch1 mnn1 mutants can be used as a foundational host to produce glycoproteins lacking the outer-chain hypermannoses and further maybe applicable to be a promising system for yeast therapeutic protein production.     

Inheritance of susceptibility to induction of nephroblastomas in the Noble rat

Noble (Nb) strain rats are susceptible to nephroblastoma induction with transplacental exposure to direct-acting alkylating agent N-nitrosoethylurea (ENU), while F344 strain rats are highly resistant. To study the inheritance of susceptibility to induction of these embryonal renal tumors, fetal Nb and F344 rats and F1, F2 and reciprocal backcross hybrids were exposed transplacentally to ENU once on day 18 of gestation. Nephroblastomas developed in 53% of Nb offspring with no apparent gender difference, while no nephroblastomas developed in inbred F344 offspring. F1 and F2 hybrid offspring had intermediate responses, 28% and 30%, respectively. Nephroblastoma incidence in the offspring of F1 hybrids backcrossed to the susceptible strain Nb was 46%, while that in F1 hybrids backcrossed to resistant strain F344 was much lower (16%). Carcinogenic susceptibility is therefore consistent with the involvement of one major autosomal locus; the operation of a gene dosage effect; and a lack of simple Mendelian dominance for either susceptibility or resistance. Since established Wilms tumor-associated suppressor genes, Wt1 and Wtx, were not mutated in normal or neoplastic tissues, genomic profiling was performed on isolated Nb and F344 metanephric progenitors to identify possible predisposing factors to nephroblastoma induction. Genes preferentially elevated in expression in Nb rat progenitors included Wnt target genes Epidermal growth factor receptor, Inhibitor of DNA binding 2, and Jagged1, which were further increased in nephroblastomas. These studies demonstrate the value of this model for genetic analysis of nephroblastoma development and implicate both the Wnt and Notch pathways in its pathogenesis.     

Development of prophylactic recombinant HPV58-attenuated Shigella live vector vaccine and evaluation of its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model

To develop a prophylactic recombinant HPV58L1-attenuated Shigella live vector vaccine and evaluate its protective efficacy and immunogenicity in the guinea pig keratoconjunctivitis model, the HPV58L1 gene was cloned into vector pUCmt, and then subcloned into the suicide vector pCVD442. The recombinant plasmid pCVD442-HPV58L1 was introduced into attenuated Shigella （sf301：AvirG） with the helper plasmid PRK2013 by filter mating. The positive colonies were harvested and confirmed by polymerase chain reaction. The expression of the HPV58L1 protein with a molecular weight of 60 kDa was confirmed by western blot. The ability of the interested protein to self-assemble into virus-like particles was identified by transmission electron microscope, and murine erythrocyte hemagglutination assay. The guinea pig keratoconjunctivitis model was used to evaluate the protective efficacy and immunogenicity of the vaccine. Animal experiments showed that there was no keratoconjunctivitis occurred in the immunized group （HPV58-attenuated Shigella）, and the serum levels of anti-HPV58Ll-IgG and -IgA were obviously increased （P 〈 0.05）, but the anti-sf301 LPS-IgG just slightly increased （P〉 0.05）. Enzyme-linked immunosorbent spot assay showed that HPV58Ll-specific IgA-antibody-secreting cells （ASC） and IgG-ASC of spleen and lymph nodes were also obviously increased （P 〈 0.01）. In this study, a recombi- nant HPV58Ll-attenuated Shigella live vector vaccine was successfully constructed, and it could induce strong humoral immune responses in the immunized animals, and induce protective antibody production.     

Synthesis and evaluation of tricyclic pyrrolopyrimidinones as dipeptide mimetics: inhibition of interleukin-1beta-converting enzyme

The application of a tricyclic pyrrolopyrimidinone scaffold for the synthesis of peptidomimetic inhibitors of interleukin-1beta-converting enzyme (ICE) is reported. The synthesis of the tricyclic scaffold and conversion of it to a variety of target ICE inhibitors were accomplished in 4-5 steps. In vitro biological evaluation of the tricyclic pyrrolopyrimidinones revealed fair to good ICE inhibitors, with the most active compound exhibiting an IC50 of 14 nM in a caspase-1 enzyme binding assay.     

Synthesis of 9-(2-beta-C-methyl-beta-d-ribofuranosyl)-6-substituted purine derivatives as inhibitors of HCV RNA replication

A series of 9-(2'-beta-C-methyl-beta-d-ribofuranosyl)-6-substituted purine derivatives were synthesized as potential inhibitors of HCV RNA replication. Their inhibitory activities in a cell based HCV replicon assay were reported. A prodrug approach was used to further improve the potency of these compounds by increasing the intracellular levels of 5'-monophosphate metabolites. These nucleotide prodrugs showed much improved inhibitory activities of HCV RNA replication.     

Temperature differentially affects encounter and docking thermodynamics of antibody--antigen association

Using BIACORE SPR, we have examined the mechanism of temperature effects on the binding kinetics of two closely related antibody Fabs (H10 and H26) which recognize coincident epitopes on hen egg-white lysozyme (HEL), and whose association and dissociation kinetics are best described by the two-step conformational change model which we interpret as molecular encounter and docking. Time-course series data obtained at a series of six temperatures (6, 10, 15, 25, 30 and 37 degrees C) showed that temperature differentially affects the rate constants of the encounter and docking steps. Docking is more temperature-sensitive than the encounter step, and energetically less favorable at higher temperatures. At elevated temperatures, the time required for docking is longer and the apparent increase in off-rate reflects the greater proportion of the molecules failing to dock and remaining in the less stable encounter state. As a consequence, distribution of free energy change between the encounter and docking steps is altered. At physiological temperature (37 degrees C) the docking step of the H26 complex is energetically unfavorable and most complexes essentially do not dock. There is a significant decrease in total free energy change of the H26 complex at higher temperatures. Elevated temperature changes the rate-limiting step of H26--HEL association from the encounter to the docking step, but not that of H10--HEL. Our results indicate that the mechanism by which elevated temperature reduces the affinities of antigen--antibody complexes is to decrease the net docking rate, and/or stability of the docked complex; at higher temperatures, a smaller proportion of the complexes actually anneal to a more stable docked state. This mechanism may have broad applicability to other receptor--ligand complexes.     

Interleukin-2 and interleukin-7 augment the cytolytic activity and expand the antitumor killing spectrum of α CD3-induced activated killer cells: potential use in the immunotherapy of non-immunogenic tumors

 This study investigates the effect of different cytokines on the growth and antitumor activity of the α CD3-induced killer cells CD3-AK, and the potential of the use of CD3-AK cells in cancer immunotherapy. Eight cytokines were tested. Only three (interleukin-2, -4 and -7) were able to support the growth of CD3-AK cells, which selectively killed different tumor targets of diversified origin. Culturing in interleukin-4 (IL-4) or IL-7 alone could maintain the growth of CD3-AK cells for 6 – 8 days. Only IL-2 could maintain long-term growth, but further addition of IL-4 exerted an inhibitory effect, which terminated the cell growth in 2 weeks. In contrast, despite the fact that IL-7 inhibited the proliferation of CD3-AK cells cultured in IL-2, as determined by [³H]thymidine uptake, the recovery of viable cells was not reduced. In 10 days, CD3-AK cells cultured in IL-2 alone or IL-2 plus IL-7 increased 160- or 176-fold respectively. There is an inverse relationship between the in vitro growth ability and Fas expression on the CD3-AK cells. Further, IL-7 increased the cytolytic activity of the CD3-AK cells two- to threefold. CD3-AK cells could be maintained in IL-2 or IL-2 plus IL-7 for 60 – 240 days or more. The long-term-cultured CD3-AK cells not only possessed a high level of cytolytic activity, but also showed a wide spectrum of killing with different tumor targets; the normally “resistant” targets, such as EL-4 lymphoma, fibrosarcoma, or melanoma, became susceptible. When the in vivo antitumor activity of the CD3-AK cells against a non-immunogenic tumor, EL-4, was tested by tumor-neutralization experiments, we found that only the long-term-cultured cells gave significant protection, with those maintained in both IL-2 and IL-7 giving the highest degree of protection. Thus, these long-term-cultured CD3-AK cells may have the potential to be used for immunotherapy of a variety of tumors whatever their immunogenicity. Received: 7 August 1996/accepted: 10 October 1996     

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.