Structure-activity relationship studies on 1-[2-(4-Phenylphenoxy)ethyl]pyrrolidine (SC-22716), a potent inhibitor of leukotriene A(4) (LTA(4)) hydrolase.

Leukotriene B(4) (LTB(4)) is a pro-inflammatory mediator that has been implicated in the pathogenesis of a number of diseases including inflammatory bowel disease (IBD) and psoriasis. Since the action of LTA(4) hydrolase is the rate-limiting step for LTB(4) production, this enzyme represents an attractive pharmacological target for the suppression of LTB(4) production. From an in-house screening program, SC-22716 (1, 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine) was identified as a potent inhibitor of LTA(4) hydrolase. Structure-activity relationship (SAR) studies around this structural class resulted in the identification of a number of novel, potent inhibitors of LTA(4) hydrolase, several of which demonstrated good oral activity in a mouse ex vivo whole blood assay.

Characterization of the cytoprotective action of peroxynitrite decomposition catalysts.

The formation of the powerful oxidant peroxynitrite (PN) from the reaction of superoxide anion with nitric oxide has been shown to be a kinetically favored reaction contributing to cellular injury and death at sites of tissue inflammation. The PN molecule is highly reactive causing lipid peroxidation as well as nitration of both free and protein-bound tyrosine. We present evidence for the pharmacological manipulation of PN with decomposition catalysts capable of converting it to nitrate. In target cells challenged with exogenously added synthetic PN, a series of metalloporphyrin catalysts (5,10,15,20-tetrakis(2,4,6-trimethyl-3, 3-disulfonatophenyl)porphyrinato iron (III) (FeTMPS); 5,10,15, 20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III) (FeTPPS); 5,10, 15,20-tetrakis(N-methyl-4'-pyridyl)porphyrinato iron (III) (FeTMPyP)) provided protection against PN-mediated injury with EC50 values for each compound 30-50-fold below the final concentration of PN added. Cytoprotection was correlated with a reduction in the level of measurable nitrotyrosine. In addition, we found our catalysts to be cytoprotective against endogenously generated PN in endotoxin-stimulated RAW 264.7 cells as well as in dissociated cultures of hippocampal neurons and glia that had been exposed to cytokines. Our studies thus provide compelling evidence for the involvement of peroxynitrite in cytokine-mediated cellular injury and suggest the therapeutic potential of PN decomposition catalysts in reducing cellular damage at sites of inflammation.

The herpesvirus protease: mechanistic studies and discovery of inhibitors of the human cytomegalovirus protease.

The herpesvirus protease is a recently identified enzyme which is essential for viral replication. It is found in all herpesviruses and offers a new molecular target for therapeutic intervention. Its genomic structure has recently been described and consists of a large open reading frame which encodes a fusion protein containing an amino-terminal protease domain in-frame with a carboxyl-terminal "assembly protein-like" domain. Auto-processing releases the amino-terminal protease as a maturational enzyme. The herpesvirus protease has been characterized as a novel serine protease. Four surface accessible sulfhydryl groups have been identified in the human cytomegalovirus (HCMV) protease. Utilizing a fluorogenic DABCYL-EDANS substrate assay, directed screening has identified a class of sulfhydryl-modifying benzimidazolylmethyl sulfoxides which inhibits recombinant HCMV protease. Site-directed mutagenesis studies suggest oxidative modification of surface-accessible HCMV protease Cys138 (and possibly Cys161) by this class of inhibitors. The benzimidazolylmethyl sulfoxide 1 inhibits HCMV protease (IC50 = 1.9 microM), exhibits selectivity vs. mammalian serine proteases, and exhibits antiviral activity in an HCMV infected cell culture assay.

Characterization and comparison of avian and murine helper cell lines for production of replication-defective retroviruses for avian transformation.

Several approaches were taken to identify improved helper cell lines for the production of replication-defective avian retroviral vectors for avian transformation. Both QT6 and D17 cells were engineered to become helper cell lines for the production of reticuloendotheliosis virus vectors. The results showed that the majority of lines from the D17, QT6, and D17C3 cells produced titers in the 10(2) to 10(3) cfu/mL range, with one QT6 line producing 10(5) cfu/mL. This high producer line was relatively free of helper virus when restricted to low passage. An amphotropic murine cell line produced a 6- to 10-fold higher amount of virus and had a comparable higher titer on chicken cells, suggesting possible application to avian transformation.

Transfer and expression of the bacterial NPT-II gene in chick embryos using a Schmidt-Ruppin retrovirus vector.

In an effort to introduce foreign genes into chickens, the bacterial neomycin phosphotransferase (NPT-II) gene was cloned into an infectious avian retroviral vector derived from the Schmidt-Ruppin A strain of RSV. The NPT-II gene was stable in the vector during passage in vitro and infected cells were resistant to G418. Fertilized chicken embryos were inoculated with the recombinant virus on day 0 and screened on day 20 for the NPT-II gene in blood cell DNA. Approximately 12% of the embryos were positive for the NPT-II gene. Screening of DNA from the brain, muscle, liver and foot of the positive embryos indicated that the NPT-II gene copy number could vary in a single embryo. However, some embryos had nearly equal NPT-II copy number in each tissue examined. To determine the expression of the bacterial gene, tissue extracts from the positive embryos were assayed for NPT-II activity. The results indicated that NPT-II activity varied depending on the tissue, with activity being highest in muscle and foot regardless of NPT-II gene copy number.

Limbic encephalitis after inhalation of a murine coronavirus.

The spread of a neurotropic coronavirus, mouse hepatitis virus strain A59, in the mouse central nervous system was studied after intranasal inoculation. Mouse hepatitis virus strain A59 spread during the 3- to 5-day postinoculation period, through the olfactory pathway into the limbic system. Coronavirus particles were detected in the limbic system by electron microscopy. The combination of temporal propagation through an anatomical-physiological central nervous system pathway and anatomical restriction of viral infection suggests that specific interneuronal transport is important in spread of the virus. This experimental system may represent a model for diseases associated with human coronaviruses (common cold viruses) and/or the human limbic system.

Coronavirus mouse hepatitis virus (MHV)-A59 causes a persistent, productive infection in primary glial cell cultures.

MHV-A59 causes a chronic demyelinating disease in mice which is accompanied by persistence of viral genome in white matter. As part of the investigation into the mechanism of viral persistence, infection of glial cells, probable targets for chronic infection, was studied by the use of mixed glial, enriched oligodendrocyte and enriched astrocyte cultures. Following MHV-A59 infection in vitro, approximately 10% of oligodendrocytes and 30% of astrocytes expressed viral antigens in the absence of overt cytopathic effect. All cultures released infectious virus for the lifetime of the cultures, for at least 45 days in the case of mixed glial cultures. Cultures derived from previously infected mice were similar to those infected in vitro with respect to percentage of cells expressing viral antigen and levels of infectious virus produced. These results show (1) that glial cells are early sites of infection in vivo as well as sites of infection in vitro cultures, and (2) that glial cells support a non-lytic but productive infection in vitro and thus may contribute to viral persistence in vivo.

The organ tropism of mouse hepatitis virus A59 in mice is dependent on dose and route of inoculation.

The organ tropism of MHV-A59, a murine coronavirus, was studied in 4-6 week-old C57BL/6 mice inoculated by different routes and with various amounts of virus. MHV-A59 caused hepatitis after intracerebral and intraperitoneal inoculation (two clearly artificial routes) and also after intranasal and intragastric inoculation (two routes more likely to mimic naturally acquired infection). For each route, the severity of hepatitis was dependent on the amount of virus inoculated. Significantly higher doses were needed to cause hepatitis by the intranasal or intragastric routes. We have shown previously that mice inoculated intracerebrally with MHV-A59 develop mild meningoencephalitis followed by chronic central nervous system (CNS) disease, characterized by primary demyelination (1). We extend these results here to show that acute CNS disease can be produced also by intranasal and intragastric inoculation, although much larger doses are needed as compared to intracerebral inoculation. Thus induction of demyelination, not only by the intracerebral route but also by the intranasal route, provides a useful model system to study virus-induced demyelination.

Infection of the basal ganglia by a murine coronavirus.

The coronavirus, mouse hepatitis virus strain A59 (MHV-A59), causes mild encephalitis and chronic demyelination. Immunohistochemical techniques showed that MHV-A59-infected C57BL/6 mice contained dense deposits of viral antigen in the subthalamic nucleus and substantia nigra, with fewer signs of infection in other regions of the brain. The animals showed extra- and intracellular vacuolation, neuronal loss, and gliosis in the subthalamic-nigral region. Such localization is unprecedented among known viral encephalitides of humans and other species. This infection by a member of a viral class capable of causing both encephalitis and persistent infection in several species may be related to postencephalitic parkinsonism.

Persistence of mouse hepatitis virus A59 RNA in a slow virus demyelinating infection in mice as detected by in situ hybridization.

Mouse hepatitis virus strain A59 produces chronic central nervous system demyelination in rodents. As late as 6 months after intracerebral inoculation of mice 4 to 6 weeks old, when infectious virus cannot be recovered and viral antigens cannot be detected in the central nervous systems and livers of these animals, primary demyelination is still evident. Using cloned virus-specific DNAs and the highly sensitive and specific technique of in situ hybridization, we have detected low levels of mouse hepatitis virus A59 RNA in the central nervous systems and livers of mice 10 months after inoculation. We suggest that viral persistence may play a role in mouse hepatitis virus A59-induced chronic demyelination.