Preclinical characterization of the antiviral activity of SCH 900518 (narlaprevir), a novel mechanism-based inhibitor of hepatitis C virus NS3 protease.

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*(i)) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC(90)) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5x EC(90) of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.

Iridoid biosynthesis in staphylinid rove beetles (Coleoptera: Staphylinidae, Philonthinae).

The biosynthesis of chrysomelidial and plagiodial was studied in the rove beetle subtribe Philonthina (Staphylinidae). Glandular homogenates were found to convert synthetic (2E,6E)-[trideuteromethyl-5,5-(2)H(5)]octa-2,6-diene-1,8-diol (10) into nor-chrysomelidial (14) and nor-plagiodial (13). The overall transformation requires; i) oxidation of the substrate at C(1) and C(8), ii) cyclization of the resulting dialdehyde to nor-plagiodial followed by iii) isomerization to give nor-chrysomelidial. The oxidase requires molecular oxygen as a cofactor and operates with removal of the pro-R hydrogen from C(1) and C(8) of synthetic (1R,8R,2E,6E)-[1,8-(2)H(2)]-2,6-dimethyl-octa-2,6-diene-1,8-diol (15), producing a dialdehyde along with H(2)O(2). Unlike enzymes from iridoid-producing leaf beetle larvae, the Philonthus enzyme is able to oxidize saturated substrates such as citronellol. Crude protein extracts prepared from Philonthus glands by ammonium sulfate precipitation, were found to produce hydrogen peroxide at a rate of 0.085+/-0.003 ng H(2)O(2) (ng protein)(-1) hr(-1) with nerol as an oxidase substrate. The cyclase operates with opposite stereochemistry to the enzyme(s) from Phaedon cochleariae and other herbivorous leaf beetles, specifically removing the C(5)-H(R) hydrogen atom from (4R,5S,2E,6E)-[4,5-(2)H(2)]-2-methyl-octa-2,6-diene-1,8-diol (17). These findings have enabled us to construct a detailed account of iridoid biosynthesis in rove beetles, which resembles the biosynthetic route in leaf beetle larvae, but exhibits distinct stereochemical differences.

An object relations perspective on couples group therapy.

This article presents a couples group therapy treatment approach that uses analytic object relations concepts such as transference/countertransference, projective identification, containment, and the holding environment. Object relations theory is seen as the most useful theory with which to view couple interaction because it is based on a two-person psychology and focuses on the impact of relational systems on the development of the person. This includes the idea that the person grows within the attachment to another person. Group therapy is seen as a more effective treatment approach because the group is a resilient holding environment that provides an avenue in which projective identifications can be understood and contained, power can be redefined, isolation of the couple can be decreased, and the couple's responses can become more versatile. The model described is illustrated with clinical vignettes from an open-ended couples group.

Race, youth violence, and the changing jurisprudence of waiver.

This article analyzes the legal history and jurisprudential theory of legislative offense-exclusion and prosecutorial waiver laws over the past quarter-century. Initially concerns about racial discrimination and civil rights motivated the Supreme Court in Kent v. United States to require due process in judicial waiver hearings. Offense-exclusion and "direct file" laws evolved and expanded in direct reaction to Kent as lawmakers sought simple and expedient alternatives to judicial waiver hearings. The "just deserts" sentencing movement of the 1970s, which advocated determinate and presumptive offense-based sentences, provided a conceptual alternative to judicial discretion and a jurisprudential rationale for offense exclusion laws. Research on delinquent and criminal careers in the 1970s, which initially promised empirically grounded selective incapacitation sentencing strategies, provided another conceptual foundation for offense-based waiver laws that focused on youths' prior records. Finally, offense exclusion provided a politically attractive strategy for "get tough" public officials who proposed to "crack down" on "baby boom" increases in youth crime. The jurisprudential shift in sentencing emphases from considerations of the offender to characteristics of the offense relocated waiver and sentencing discretion from judges to prosecutors. By the early 1990s, as a result of political "crack-downs" on youth crime, the scope of excluded offense legislation increased substantially, became overly inclusive and excessively rigid, and exhibited many of the negative features associated with mandatory sentencing laws.

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.

Identification and analysis of bacterial protein secretion inhibitors utilizing a SecA-LacZ reporter fusion system.

Protein secretion is an essential process for bacterial growth, yet there are few if any antimicrobial agents which inhibit secretion. An in vivo, high-throughput screen to detect secretion inhibitors was developed based on the translational autoregulation of one of the central protein components, SecA. The assay makes use of a SecA-LacZ fusion reporter construct in Escherichia coli which is induced when secretion is perturbed. Several compounds, including two natural product extracts, which had the ability to induce the reporter fusion were identified and the MICs of these compounds for Staphylococcus aureus strain MN8 were found to be < or =128 microg/ml. Enzyme-linked immunosorbent assay, Western blotting, and immunoprecipitation techniques were used to analyze the affects of these compounds on protein secretion. Six representative compounds presented here appear to be bona fide secretion inhibitors but were found to have deleterious effects on membranes. It was concluded that, while the method described here for identifying inhibitors of secretion is valid, screens such as this, which are directed against the membrane-bound portion of a pathway, may preferentially identify compounds which affect membrane integrity.