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 oncogenicity of the L858R mutant form of the epidermal growth factor receptor (EGFR) in non-small-cell lung cancer is thought to be due to the constitutive activation of its kinase domain. The selectivity of the marketed drugs gefitinib and erlotinib for L858R mutant is attributed to their specific recognition of the active kinase and to weaker ATP binding by L858R EGFR. We present crystal structures showing that neither L858R nor the drug-resistant L858R+T790M EGFR kinase domain is in the constitutively active conformation. Additional co-crystal structures show that gefitinib and dacomitinib, an irreversible anilinoquinazoline derivative currently in clinical development, may not be conformation specific for the active state of the enzyme. Structural data further reveal the potential mode of recognition of one of the autophosphorylation sites in the C-terminal tail, Tyr-1016, by the kinase domain. Biochemical and biophysical evidence suggest that the oncogenic mutations impact the conformational dynamics of the enzyme.

Summary Patients with non-small cell lung cancer (NSCLC) that have kinase-activating epidermal growth factor receptor (EGFR) mutations are highly responsive to first- and second-generation EGFR inhibitors. However, these patients often relapse due to a secondary, drug-resistant mutation in EGFR where the gatekeeper threonine is converted to methionine (T790M). Several third-generation EGFR inhibitors have been developed that irreversibly inactivate T790M-EGFR while sparing wild-type EGFR, thus reducing epithelium-based toxicities. Using chemical proteomics, we show here that individual T790M-EGFR inhibitors exhibit strikingly distinct off-target profiles in human cells. The FDA-approved drug osimertinib (AZD9291), in particular, was found to covalently modify cathepsins in cell and animal models, which correlated with lysosomal accumulation of the drug. Our findings thus show how chemical proteomics can be used to differentiate covalent kinase inhibitors based on global selectivity profiles in living systems and identify specific off-targets of these inhibitors that may impact drug activity and safety.

A novel microfermentation and scale-up platform for parallel protein production in Escherichia coli is described. The vertical shaker device Vertiga, which generates low-volume high density (A(600) approximately 20) Escherichia coli cultures in 96-position deep-well plates without auxiliary oxygen supplementation, has been coupled to a new disposable shake flask design, the Ultra Yield flask, that allows for equally high cell culture densities to be obtained. The Ultra Yield flask, which accommodates up to 1 l in culture volume, has a baffled base and a more vertical wall construction compared to traditional shake flask designs. Experimental data is presented demonstrating that the Ultra Yield flask generates, on average, an equivalent amount of recombinant protein per unit cell culture density as do traditional shake flask designs but at a substantially greater amount per unit volume. The combination of Vertiga and the Ultra Yield flask provides a convenient and scalable low-cost solution to parallel protein production in Escherichia coli.

HIV-1 reverse transcriptase (RT) RNase H (HIV-RH) is a key target of anti-AIDS drugs. Metal-chelating compounds are an important class of chemicals in pharmacological drug discovery, especially in relation to HIV-RT and the highly-related HIV-integrase. The correlation between the metal-chelating properties and enzyme activities of the metal chelators is always of high scientific interest, as an understanding of this may accelerate the rational optimization of this class of inhibitors. Our NMR data show that Mg 2+ and Ca 2+ bind specifically to the active site of the RNase H domain and two Mg 2+ ions sequentially bind one molecule of RNase H. We also demonstrate here, using saturated and unsaturated tricyclic N-hydroxypyridones designed to block the active site, that the primary binding sites and affinities of divalent metal ions are correlated with the structures of the chelating motifs. Chemical shift perturbation studies of protein/metal-ion/compound ternary complexes also indicate that divalent metal ions play important roles for the specific interaction of the compounds with the RNase H active site.