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In this, the second of two Letters, the optimization of the pyrrolidine-5,5-trans-lactam template (exemplified by 1a) as a mechanism-based inhibitor of hepatitis C NS3/4A protease is described. “Right Box” analysis of cassette dosing screening pharmacokinetic data was used to rapidly categorize the compounds. GW0014 (compound 4d) emerged as the compound displaying an optimal balance of biochemical and replicon potency, along with low i.v. clearance in the dog.

In this, the first of two Letters, we describe how a P3/P4 urea linking unit was used to greatly enhance the biochemical and replicon potency of inhibitors based upon the pyrrolidine-5,5-trans-lactam template. Compound 7b demonstrated a 100 nM IC50 in the replicon cell-based surrogate HCV assay.

In this, the second of two letters, we describe the elaboration of the pyrrolidine-5,5-trans-lactam template to delineate the requirements for optimal substitution of the pyrrolidine and lactam nitrogen atoms. Central to the strategy is the use of rapid iterative synthesis in conjunction with X-ray crystal structure determination of ligand−protein complexes.

In this, the first of two letters, we outline the use of the pyrrolidine-5,5-trans-lactam template to design small, neutral, mechanism-based inhibitors of hepatitis C NS3/4A protease. The hitherto unreported reaction of the acyl iminium ion precursor 4 with dialkyl-substituted silyl ketene acetals (e.g., 8b) is described. Compound 12b, with a spirocyclobutyl P1 substituent and a cyclopropylacyl substituent on the lactam nitrogen, has a kobs/I of 400 M^-1 s^-1 and demonstrates activity in a replicon cell-based surrogate HCV assay.

Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.

ABSTRACTA study of immunopathology in the central nervous system (CNS) during infection with a virulent strain of Murray Valley encephalitis virus (MVE) in weanling Swiss mice following peripheral inoculation is presented. It has previously been shown that virus enters the murine CNS 4 days after peripheral inoculation, spreads to the anterior olfactory nucleus, the pyriform cortex, and the hippocampal formation at 5 days postinfection (p.i.), and then spreads throughout the cerebral cortex, caudate putamen, thalamus, and brain stem between 6 and 9 days p.i. (P. C. McMinn, L. Dalgarno, and R. C. Weir, Virology 220:414–423, 1996). Here we show that the encephalitis which develops in MVE-infected mice from 5 days p.i. is associated with the development of a neutrophil inflammatory response in perivascular regions and in the CNS parenchyma. Infiltration of neutrophils into the CNS was preceded by increased expression of tumor necrosis factor alpha and the neutrophil-attracting chemokine N51/KC within the CNS. Depletion of neutrophils with a cytotoxic monoclonal antibody (RB6-8C5) resulted in prolonged survival and decreased mortality in MVE-infected mice. In addition, neutrophil infiltration and disease onset correlated with expression of the enzyme-inducible nitric oxide synthase (iNOS) within the CNS. Inhibition of iNOS by aminoguanidine resulted in prolonged survival and decreased mortality in MVE-infected mice. This study provides strong support for the hypothesis that Murray Valley encephalitis is primarily an immunopathological disease.

Closed adaptive sequential procedures were introduced by Bechhofer and Kulkarni (1982) as a new method for selecting the best of t ([image omitted] 2) Bernoulli populations. We modify this approach to obtain two procedures for selecting the best of t objects in a curtailed Round Robin-type paired-comparison experiment. Objects are paired sequentially and the experiment is stopped as soon as one object has achieved a number of preferences that no other object can equal (weak curtailment) or surpass (strong curtailment) if the tournament were run to completion. Ties for first place are broken at random, Weak curtailment clearly selects the same object as the complete Round Robin, with generally substantially fewer comparisons. Strong curtailment effects a further appreciable reduction in the average number of comparisons needed. It is shown that the probabilities of selecting a particular object are the same for weak and strong curtailment if the Bradley-Terry model holds, but generally not otherwise. Some comparisons are made with Knockout tournaments. Extensions to the selection of more than one object are also discussed.

Summary It has been shown that the capacitance, thickness and composition of black lipid films may depend strongly on the hydrocarbon solvent used in their formation. By the use of n-hexadecane, films have been formed which contain effectively no solvent and which are comparable to the leaflets of the mesomorphic phase of the pure lipid. These films have capacitances of ca. 0.6 µF/cm2 and hydrocarbon thicknesses of ca. 31 Å. Thinner black films of higher capacitances are also described.

A key intermediate 14 for the synthesis of lactacystin 1 has been constructed in four steps and 33% overall yield. The key steps involve cyclization of a suitably functionalized glutamic acid derivative and concomitant alkylation of the resulting β,β-diketoester system, C-acylation of the cyclic α-amidoketone 9, and decarboxylbenzylation of 12. Alkylation of a related β,β-diketoester 5 was additionally achieved with several electrophiles.