Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.

The imidazolinones, a new chemical class of herbicides, were shown to be uncompetitive inhibitors of acetohydroxyacid synthase from corn. This is the first common enzyme in the biosynthetic pathway for valine, leucine, and isoleucine. The K, for the imidazolinones tested ranged from 2 to 12 micromolar. These results may explain the mechanism of action of these new herbicides.The imidazolinones are a new chemical class of herbicides discovered and being developed by American Cyanamid Company (see structures in Table I). These herbicides kill both monocotyledonous and dicotyledonous species, and selectivity is achieved by differential metabolism of the compounds to nonherbicidal forms (7, 9). Death of the whole plant can require several weeks. The meristematic tissues die first, followed by slow necrosis of the mature tissue.The precise mechanism of action of many herbicides are unknown. The imidazolinones are somewhat unusual in that a biochemical pathway has been identified which is inhibited by these herbicides. The imidazolinones interfere with the biosynthesis of the branched chain amino acids valine, leucine, and isoleucine (10). Corn tissue grown in suspension culture and treated with AC 243,997 (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]nicotinic acid) showed changes in the free amino acid levels (10). Most of the amino acids either increased or remained constant, but the levels of valine, leucine, and isoleucine decreased. Exogenously supplying these three amino acids to corn tissue cultures and seedlings prevented the phytotoxic effects of AC 243,997 (10). All three of these amino acids were necessary for maximal protection to occur.There are four enzymes that catalyze biosynthetic steps leading to valine, leucine, and isoleucine. Acetohydroxyacid synthase (EC 4.1.3.18) is the first enzyme in the biosynthesis ofvaline and leucine and the second enzyme in the biosynthesis of isoleucine. Acetohydroxyacid synthase is feedback inhibited cooperatively by valine and leucine in higher plants (4). Thus, it is a key controlling point for the levels of the branched chain amino acids and is a prime site for inhibition by a xenobiotic. The imidazolinones were found to be potent inhibitors of acetohydroxyacid synthase and this may explain why the levels ofvaline, leucine, and isoleucine decreased after treatment with the imidazolinones.MATERIALS AND METHODS Chemicals. Analytical grade imidazolinones were synthesized at the American Cyanamid Agricultural Research Center, Princeton, NJ. Acetoin was obtained from Eastman Kodak.Plant Material. Corn (Zea mays L. Pioneer var 3547) seeds were planted in sand and watered with 0.2 mm CaC12. The plants were grown in darkness at 28°C for 3 d. The roots and shoots from the etiolated seedlings were used.Acetohydroxyacid Synthase Assay. The procedure used was a modification ofone used by B. J. Miflin (5). Roots and coleoptiles of the corn seedlings were homogenized in an extraction buffer (50 mm K-phosphate buffer (pH 7.5),5 mM EDTA, 5 mm MgCl2, 1 mM valine, 1 mM leu...

This study shows that besides the residues in the flap and residues 79-81 in the S1 substrate-binding pocket which undergo conformational changes upon inhibitor binding, residues 29 and 30 can also adapt their conformation to fit certain inhibitors. Conformational flexibility of the HIV protease plays an important role in inhibitor binding.

The crystal structure of HIV-2 protease in complex with a reduced amide Inhibitor

Effects of the imidazolinone herbicide AC243 997 [2-(5-isopropyl)-5-methyl-4-oxo-2-imidazolin-2-yl) nicotinic acid] on the growth of corn (Zea mays L. ‘Black Mexican Sweet’) cell suspension cultures were characterized. The herbicide caused half maximal growth inhibition at concentrations in the range of 10 nM to 30 nM. Significant reductions in protein synthesis were observed following treatment of cells with 10 μM herbicide. Analysis of free amino acid pool sizes indicated that levels of the biosyn-thetically related amino acids leucine and valine were substantially decreased in cells exposed to 10 μM herbicide. Supplementation of suspension culture growth media with leucine and valine plus isoleucine (1 mM each) reversed growth inhibitory effects of AC243 997 at levels ranging from 10 nM to 1 mM. The results suggest a specific interaction of this imidazolinone herbicide with leucine, valine, and isoleucine metabolism.