During replication of hepatitis C virus (HCV), the final steps of polyprotein processing are performed by a viral proteinase located in the N-terminal one-third of nonstructural protein 3. The structure of NS3 proteinase from HCV BK strain was determined by X-ray crystallography at 2.4 angstrom resolution. NS3P folds as a trypsin-like proteinase with two beta barrels and a catalytic triad of His-57, Asp-81, Ser-139. The structure has a substrate-binding site consistent with the cleavage specificity of the enzyme. Novel features include a structural zinc-binding site and a long N-terminus that interacts with neighboring molecules by binding to a hydrophobic surface patch.

Human rhinoviruses, the most important etiologic agents of the common cold, are messenger-active single-stranded monocistronic RNA viruses that have evolved a highly complex cascade of proteolytic processing events to control viral gene expression and replication. Most maturation cleavages within the precursor polyprotein are mediated by rhinovirus 3C protease (or its immediate precursor, 3CD), a cysteine protease with a trypsin-like polypeptide fold. Highresolution crystal structures of the enzyme from three viral serotypes have been used for the design and elaboration of 3C protease inhibitors representing different structural and chemical classes. Inhibitors having ␣,␤-unsaturated carbonyl groups combined with peptidyl-binding elements specific for 3C protease undergo a Michael reaction mediated by nucleophilic addition of the enzyme's catalytic Cys-147, resulting in covalent-bond formation and irreversible inactivation of the viral protease. Direct inhibition of 3C proteolytic activity in virally infected cells treated with these compounds can be inferred from dose-dependent accumulations of viral precursor polyproteins as determined by SDS͞PAGE analysis of radiolabeled proteins. Cocrystal-structure-assisted optimization of 3C-protease-directed Michael acceptors has yielded molecules having extremely rapid in vitro inactivation of the viral protease, potent antiviral activity against multiple rhinovirus serotypes and low cellular toxicity. Recently, one compound in this series, AG7088, has entered clinical trials.

Hepatitis C virus (HCV) is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses (23). It is responsible for persistent infections in humans, with associated risk of chronic liver diseases, including cirrhosis and hepatocellular carcinoma. Nearly 3% of the global population is chronically infected with HCV, and there are no clinically proven vaccines. Antiviral therapeutic agents are at an early stage of clinical evaluation, and standard treatments (interferon and ribavirin combinations) are associated with suboptimal response rates and/or high incidence of side effects. Complicating the discovery of new therapies is the highly complex and incompletely understood nature of the viral life cycle. The HCV genome consists of a single strand of RNA of about 9,600 nucleotides encoding a polypeptide precursor of about 3,000 amino acids (26). Co-and posttranslational proteolytic cleavage of this precursor by cellular and viral enzymes yields structural proteins involved in viral assembly, along with nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which are required for membrane-associated RNA replication (14).Nonstructural protein NS5A is a critical component of HCV replication and is involved in several cellular processes, such as interferon resistance (3, 13) and apoptotic regulation (9). It is a phosphoprotein of 447 residues with three domains (35), and while no clear enzymatic functions have been assigned, it appears to function through interactions with other HCV proteins and host cell factors (17). Domain I (residues 1 to 213) contains a zinc-binding motif (35) and an amphipathic N-terminal helix which promotes membrane association (4, 12, 30), possibly through specific interaction of the helix with target membrane proteins (8). Domain II (residues 250 to 342) has regulatory functions, such as interactions with protein kinase PKR and PI3K (13), as well as NS5B (32); contains the interferon sensitivity-determining region (13); and appears to lack major elements of secondary structure (22). Recent studies have demonstrated that domain III (residues 356 to 447) plays a critical role in infectious virion assembly but not in RNA replication (1,34) and that the former role is modulated by phosphorylation within the domain (33). High-throughput screening of small-molecule inhibitors using HCV replicon cell systems has identified NS5A as a promising therapeutic target (31).A crystal structure of domain I lacking the amphipathic helix and spanning residues 25 to 215 showed two subdomains and a homodimeric association and was interpreted as having a potential role in RNA binding (36). Although specific binding to domain I was not described, RNA binding to full-length NS5A has been reported, using, for example, the 3Ј nontranslated region of HCV (15). Efforts in our laboratory to study the structure of NS5A have yielded an alternative arrangement of the domain I homodimer (residues 33 to 202) that differs substantially from that previously described. The observation that the NS5A do...

The original model of Eco RI endonuclease (1) proved refractory to crystallographic refinement, whereby model coordinates were adjusted to optimize the fit to the observed diffraction data (the best R factor for the original model is 0.25). We The new data yielded a multiple isomorphous replacement (MIR) electron density map that suggests a different connectivity between elements of secondary structure in the protein (3). A model based on that connectivity has refined robustly by the X-PLOR and Konnert-Hendrickson methods (4) to an R factor of 0.20 (no ordered solvent has been included in this model, nor have coordinates for the first 16 amino acid residues, which appear disordered) (5). The root-mean-square deviations in bond distance, angle distance, and 1-4 dihedral distance are 0.016 A, 0.031 A, and 0.031 A,

Summary Novel inhibitors are needed to counteract the rapid emergence of drug-resistant HIV variants. HIV-1 reverse transcriptase (RT) has both DNA polymerase and RNase H (RNH) enzymatic activities, but approved drugs that inhibit RT target the polymerase. Inhibitors that act against new targets, like RNH, would be effective against all of the current drug-resistant variants. Here, we present 2.80 Å and 2.04 Å resolution crystal structures of an RNH inhibitor, β-thujaplicinol, bound at the RNH active site of both HIV-1 RT and an isolated RNH domain. β-thujaplicinol chelates two divalent metal ions at the RNH active site. We provide biochemical evidence that β-thujaplicinol is a slow-binding RNH inhibitor with non-competitive kinetics and suggest that it forms a tropylium ion that interacts favorably with RT and the RNA:DNA substrate.

The design, synthesis, and biological evaluation of reversible, nonpeptidic inhibitors of human rhinovirus (HRV) 3C protease (3CP) are reported. A novel series of 2,3-dioxindoles (isatins) were designed that utilized a combination of protein structure-based drug design, molecular modeling, and structure-activity relationship (SAR). The C-2 carbonyl of isatin was envisioned to react in the active site of HRV 3CP with the cysteine responsible for catalytic proteolysis, thus forming a stabilized transition state mimic. Molecular-modeling experiments using the apo crystal structure of human rhinovirus-serotype 14 (HRV-14) 3CP and a peptide substrate model allowed us to design recognition features into the P1 and P2 subsites, respectively, from the 5- and 1-positions of isatin. Attempts to optimize recognition properties in the P1 subsite using SAR at the 5-position were performed. In addition, a series of ab initio calculations were carried out on several 5-substituted isatins to investigate the stability of sulfide adducts at C-3. The inhibitors were prepared by general synthetic methods, starting with commercially available 5-substituted isatins in nearly every case. All compounds were tested for inhibition of purified HRV-14 3CP. Compounds 8, 14, and 19 were found to have excellent selectivity for HRV-14 3CP compared to other proteolytic enzymes, including chymotrypsin and cathepsin B. Selected compounds were assayed for antiviral activity against HRV-14-infected HI-HeLa cells. A 2.8 A cocrystal structure of derivative 19 covalently bound to human rhinovirus-serotype 2 (HRV-2) 3CP was solved and revealed that the isatin was situated in essentially the same conformation as modeled.

We report collection of 2.5 A resolution X-ray diffraction data from newly grown crystals of the rare 'small unit cell' form of the long snake neurotoxin, alpha-bungarotoxin. The previous model of the molecule has been rebuilt, and refined using least-square methods to a crystallographic residual of 0.24 at 2.5 A resolution. alpha-Bungarotoxin's crystal structure is compared with the crystal structures of two other snake neurotoxins (cobratoxin and erabutoxin), and with its solution structure inferred from spectroscopic studies. Significant differences include less beta-sheet in bungarotoxin's crystal structure than in solution, or in the crystal structures of other neurotoxins, and an unusual orientation in the crystal of the invariant tryptophan. The functional, binding surface of bungarotoxin is described; it consists primarily of hydrophobic and hydrogen-bonding groups and only a few charged side-chains. The structure is compared with experimental binding parameters for neurotoxins.

The conventional protein kinase C isoform, PKCII, is a signaling kinase activated during the hyperglycemic state and has been associated with the development of microvascular abnormalities associated with diabetes. PKCII, therefore, has been identified as a therapeutic target where inhibitors of its kinase activity are being pursued for treatment of microvascular-related diabetic complications. In this report, we describe the crystal structure of the catalytic domain of PKCbetaII complexed with an inhibitor at 2.6 A resolution. The kinase domain of PKCbetaII was cleaved and purified from full-length PKCbetaII expressed in baculovirus-infected insect cells. The overall kinase domain structure follows the classical bilobal fold and is in its fully activated conformation with three well-defined phosphorylated residues: Thr-500, Thr-641, and Ser-660. Different from the crystal structures of nonconventional PKC isoforms, the C-terminus of the PKCbetaII catalytic domain is almost fully ordered and features a novel alpha helix in the turn motif. An ATP-competitive inhibitor, 2-methyl-1H-indol-3-yl-BIM-1, was crystallized with the PKCbetaII catalytic domain as a dimer of two enzyme-inhibitor complexes. The bound inhibitor adopts a nonplanar conformation in the ATP-binding site, with the kinase domain taking on an intermediate, open conformation. This PKCbetaII-inhibitor complex represents the first structural description of any conventional PKC kinase domain. Given the pathogenic role of PKCbetaII in the development of diabetic complications, this structure can serve as a template for the rational design of inhibitors as potential therapeutic agents.