We have used a structure-based drug design approach to identify small molecule inhibitors of the hepatitis C virus (HCV) NS3⅐4A protease as potential candidates for new anti-HCV therapies. VX-950 is a potent NS3⅐4A protease inhibitor that was recently selected as a clinical development candidate for hepatitis C treatment. In this report, we describe in vitro resistance studies using a subgenomic replicon system to compare VX-950 with another HCV NS3⅐4A protease inhibitor, BILN 2061, for which the Phase I clinical trial results were reported recently. Distinct drug-resistant substitutions of a single amino acid were identified in the HCV NS3 serine protease domain for both inhibitors. The resistance conferred by these mutations was confirmed by characterization of the mutant enzymes and replicon cells that contain the single amino acid substitutions. It is estimated that 170 million patients worldwide and about 1% of the population in developed countries are chronically infected with hepatitis C virus (HCV) 1 (1). The majority of acute HCV infections become chronic, some of which progress toward liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care is pegylated interferon ␣ in combination with ribavirin, which has a sustained viral response rate of 40 -50% in genotype 1 HCV-infected patients, which accounts for the majority of the hepatitis C population in the United States and Japan, and of 80 -90% in patients infected with genotype 2 or 3 HCV (4, 5) (for a review, see Ref. 6). Thus, more effective therapeutic drugs with fewer side effects and shorter treatment durations are needed for patients infected with HCV.HCV is an enveloped, single-stranded RNA virus with a 9.6-kb positive-polarity genome, which encodes a polyprotein precursor of about 3,000 amino acids. The HCV polyprotein is proteolytically processed by cellular and HCV proteases into at least 10 distinct products, in the order of NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 7). NS3 serine protease and helicase as well as NS5B RNA-dependent RNA polymerase are believed to be components of a replication complex responsible for viral RNA replication and have been shown to be essential for the HCV replication in chimpanzees (8). These HCV enzymes have been the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 9). However, successful discovery of a new HCV-specific drug candidate has been hampered by the lack of a robust, reproducible infectious virus cell culture system. The development of a HCV replicon system by Lohmann et al. (10) and subsequent optimization by several laboratories (11, 12) has enabled quantitative evaluation of the antiviral potency of HCV inhibitors.The HCV NS3⅐4A protease is responsible for cleavage at four sites within the HCV polyprotein to generate the N termini of the NS4A, NS4B, NS5A, and NS5B proteins (13-17). It has been shown that the central region (amino acids 21-30) of the 54-residue NS4A protein is essentia...

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These structures span all three caspase subgroups, and provide a basis for inferring substrate and inhibitor binding, as well as selectivity for the entire caspase family. This information will influence the design of selective caspase inhibitors to further elucidate the role of caspases in biology and hopefully lead to the design of therapeutic agents to treat caspase-mediated diseases, such as rheumatoid arthritis, certain neurogenerative diseases and stroke.

Chronic hepatitis C has become one of the most common liver diseases and is estimated to affect 170 million patients worldwide and ϳ1% of the population in developed countries (1). In many patients, hepatitis C virus (HCV) 2 infection leads to liver cirrhosis or hepatocellular carcinoma (2, 3). The current standard of care, a 48-week treatment with pegylated interferon (IFN)-␣ in combination with ribavirin, has a sustained viral response rate of 40 -50% in the difficult-to-treat genotype 1 HCV-infected patients (Refs. 4 and 5; for a review, see Refs. 6 and 7), which accounts for the majority of the hepatitis C patient population in the developed countries. A more effective treatment with fewer side effects and shorter treatment durations is urgently needed for HCVinfected patients.HCV is an enveloped virus containing a single-stranded, positive polarity RNA that encodes a polyprotein precursor of ϳ3000 amino acids. The HCV polyprotein is proteolytically processed by cellular and viral proteases into at least 10 distinct products in the order of NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH (for a review, see Ref. 8). The structural proteins are processed by host signal peptidases, whereas the nonstructural (NS) proteins are processed by two virally encoded proteases, the NS2⅐3 and NS3⅐4A proteases. The NS2⅐3 protease is responsible for the cleavage between the NS2 and NS3 proteins, whereas the NS3⅐4A serine protease is responsible for the release of the remaining four nonstructural proteins, NS4A, NS4B, NS5A, and NS5B (9 -13). The essentiality of the NS3⅐4A serine protease for viral replication has been demonstrated by the nonproductive infection following liver inoculation of chimpanzees with a genomic HCV RNA containing a mutation in the NS3 protease active site (14). It has been shown that the central region (amino acids 21-30) of the 54-residue NS4A protein is essential and sufficient for the enhancement of the proteolytic activity of the NS3 serine protease (15-19). The central region of NS4A forms a tight heterodimer with the NS3 protein (18), for which the first x-ray crystal structure was solved in 1996 (20). The NS3⅐4A serine protease has been one of the major targets for the development of HCV-specific therapeutics during the past decade (for a review, see Ref. 21). VX-950, a potent, small molecule, selective inhibitor of the HCV NS3⅐4A serine protease, was discovered using structurebased drug design techniques (22). Clinical proof of concept for HCV protease inhibitors (PIs) has been demonstrated by Boehringer Ingelheim and Vertex Pharmaceuticals Inc. using BILN 2061 (23) and VX-950, 3 respectively. Both compounds reduced HCV viral load in patients by ϳ2-3 log 10 in the first 3 days of dosing. In some patients treated with VX-950, the HCV viral load dropped by Ͼ4 log 10 to below the limit of detection (Ͻ10 IU/ml) during 14 days of dosing. 3Because of the error-prone nature of the viral reverse transcriptase of retroviruses or the RNA-dependent RNA polymerase of RNA viruses, drug resistance frequen...

The discovery of new antibacterial agents with novel mechanisms of action is necessary to overcome the problem of bacterial resistance that affects all currently used classes of antibiotics. Bacterial DNA gyrase and topoisomerase IV are well-characterized clinically validated targets of the fluoroquinolone antibiotics which exert their antibacterial activity through inhibition of the catalytic subunits. Inhibition of these targets through interaction with their ATP sites has been less clinically successful. The discovery and characterization of a new class of low molecular weight, synthetic inhibitors of gyrase and topoisomerase IV that bind to the ATP sites are presented. The benzimidazole ureas are dual targeting inhibitors of both enzymes and possess potent antibacterial activity against a wide spectrum of relevant pathogens responsible for hospital- and community-acquired infections. The discovery and optimization of this novel class of antibacterials by the use of structure-guided design, modeling, and structure-activity relationships are described. Data are presented for enzyme inhibition, antibacterial activity, and in vivo efficacy by oral and intravenous administration in two rodent infection models.

RhoA, a ubiquitous intracellular GTPase, mediates cytoskeletal responses to extracellular signals. A 2.1 A resolution crystal structure of the human RhoA-GDP complex shows unique stereochemistry in the switch I region, which results in a novel mode of Mg2+ binding.

Topoisomerase IV and DNA gyrase are related bacterial type II topoisomerases that utilize the free energy from ATP hydrolysis to catalyze topological changes in the bacterial genome. The essential function of DNA gyrase is the introduction of negative DNA supercoils into the genome, whereas the essential function of topoisomerase IV is to decatenate daughter chromosomes following replication. Here, we report the crystal structures of a 43-kDa N-terminal fragment of Escherichia coli topoisomerase IV ParE subunit complexed with adenylyl-imidodiphosphate at 2.0-Å resolution and a 24-kDa N-terminal fragment of the ParE subunit complexed with novobiocin at 2.1-Å resolution. The solved ParE structures are strikingly similar to the known gyrase B (GyrB) subunit structures. We also identified single-position equivalent amino acid residues in ParE (M74) and in GyrB (I78) that, when exchanged, increased the potency of novobiocin against topoisomerase IV by nearly 20-fold (to 12 nM). The corresponding exchange in gyrase (I78 M) yielded a 20-fold decrease in the potency of novobiocin (to 1.0 M). These data offer an explanation for the observation that novobiocin is significantly less potent against topoisomerase IV than against DNA gyrase. Additionally, the enzyme kinetic parameters were affected. In gyrase, the ATP K m increased Ϸ5-fold and the V max decreased Ϸ30%. In contrast, the topoisomerase IV ATP K m decreased by a factor of 6, and the V max increased Ϸ2-fold from the wild-type values. These data demonstrate that the ParE M74 and GyrB I78 side chains impart opposite effects on the enzyme's substrate affinity and catalytic efficiency.Type II topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. Bacterial genomes encode two type II topoisomerases, DNA gyrase and topoisomerase IV (TopoIV), that function in DNA replication. DNA gyrase is unique in coupling the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. In the absence of the ATP substrate, DNA gyrase can relax negatively supercoiled plasmid DNA. These activities result from the enzyme's ability to wrap (Ϸ150 bp) DNA (23, 31) around itself upon binding the DNA substrate. This DNA wrapping preferentially presents the T-segment (transported DNA segment) to the gyrase-DNA complex so that the introduction of negative supercoils is the primary outcome. In contrast, TopoIV and other eukaryotic type II topoisomerases only bind a Ϸ30-bp region of DNA (20,35). TopoIV utilizes the energy of ATP hydrolysis to decatenate newly replicated chromosomal DNA but also has the ability to relax positive and negative DNA supercoils in an ATP-dependent manner (8,43).In prokaryotes, these type II topoisomerases are composed of two subunits. In Escherichia coli, the gyrase subunits are named A and B and the corresponding TopoIV subunits are named C and E. For each enzyme, these subunits combine into a heterotetrameric (gyrase, A 2 B 2 ; and TopoIV, C 2 E 2 ) complex to form the functional enzymes. I...

The platelet-activating factor PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid first messenger active in general cell activation, fertilization, inflammatory and allergic reactions, asthma, HIV pathogenesis, carcinogenesis, and apoptosis. There is substantial evidence that PAF is involved in intracellular signalling, but the pathways are poorly understood. Inactivation of PAF is carried out by specific intra- and extracellular acetylhydrolases (PAF-AHs), a subfamily of phospholipases A2 that remove the sn-2 acetyl group. Mammalian brain contains at least three intracellular isoforms, of which PAF-AH(Ib) is the best characterized. This isoform contains a heterodimer of two homologous catalytic subunits alpha1 and alpha2, each of relative molecular mass 26K, and a non-catalytic 45K beta-subunit, a homologue of the beta-subunit of trimeric G proteins. We now report the crystal structure of the bovine alpha1 subunit of PAF-AH(Ib) at 1.7 A resolution in complex with a reaction product, acetate. The tertiary fold of this protein is closely reminiscent of that found in p21(ras) and other GTPases. The active site is made up of a trypsin-like triad of Ser 47, His 195 and Asp 192. Thus, the intact PAF-AH(Ib) molecule is an unusual G-protein-like (alpha1/alpha2)beta trimer.