Shiga-like toxin I (SLT-I) is a virulence factor of Escherichia coli strains that cause disease in humans. Like other members of the Shiga toxin family, it consists of an enzymatic (A) subunit and five copies of a binding subunit (the B-pentamer). The B-pentamer binds to a specific glycolipid, globotriaosylceramide (Gb3), on the surface of target cells and thereby plays a crucial role in the entry of the toxin. Here we present the crystal structure at 2.8 A resolution of the SLT-I B-pentamer complexed with an analogue of the Gb3 trisaccharide. The structure reveals a surprising density of binding sites, with three trisaccharide molecules bound to each B-subunit monomer of 69 residues. All 15 trisaccharides bind to one side of the B-pentamer, providing further evidence that this side faces the cell membrane. The structural model is consistent with data from site-directed mutagenesis and binding of carbohydrate analogues, and allows the rational design of therapeutic Gb3 analogues that block the attachment of toxin to cells.

The development of HIV integrase (IN) strand transfer inhibitors (INSTIs) and our understanding of viral resistance to these molecules have been hampered by a paucity of available structural data. We recently reported cocrystal structures of the prototype foamy virus (PFV) intasome with raltegravir and elvitegravir, establishing the general INSTI binding mode. We now present an expanded set of cocrystal structures containing PFV intasomes complexed with firstand second-generation INSTIs at resolutions of up to 2.5 Å. Importantly, the improved resolution allowed us to refine the complete coordination spheres of the catalytic metal cations within the INSTIbound intasome active site. We show that like the Q148H/G140S and N155H HIV-1 IN variants, the analogous S217H and N224H PFV INs display reduced sensitivity to raltegravir in vitro. Crystal structures of the mutant PFV intasomes in INSTI-free and -bound forms revealed that the amino acid substitutions necessitate considerable conformational rearrangements within the IN active site to accommodate an INSTI, thus explaining their adverse effects on raltegravir antiviral activity. Furthermore, our structures predict physical proximity and an interaction between HIV-1 IN mutant residues His148 and Ser/Ala140, rationalizing the coevolution of Q148H and G140S/ A mutations in drug-resistant viral strains.

HDM2 binds to an alpha-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their alpha-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

To begin to examine the structural basis for the deposition of soluble A beta amyloid peptide onto senile plaques in Alzheimer's disease, we have prepared A beta congeners and measured their activity in an in vitro plaque growth assay. The N-terminal fragment, A beta (1-28)-OH, was inactive at all pH values tested. While the central fragment, A beta (10-35)-NH2, and the full length peptide, A beta (1-40)-OH, were inactive below pH 4, both were active (plaque competent) between pH 5 and 9. The active and inactive fragments were studied by nuclear magnetic resonance spectroscopy in water at submillimolar concentrations at pH 2.1 and 5.6. Changes in chemical shifts, coupling constants, and nuclear Overhauser enhancements indicate a pH dependent folding transition in A beta (10-35)-NH2 as it becomes active. The conformation of the active fragment is not helical, and preliminary data indicate the presence of several turns and at least two short strands. In contrast, the inactive fragment A beta (1-28)-OH did not undergo a similar folding transition. Earlier nuclear magnetic resonance studies of amyloid peptides in fluorinated alcohols or detergent micelles at low pH described a helical conformation and proposed a helix to sheet transition in plaque formation; the present study demonstrates that no such conformations are present in water under conditions where the peptides can adhere to authentic amyloid plaques.

TMC435 is a small-molecule inhibitor of the NS3/4A serine protease of hepatitis C virus (HCV) currently in phase 2 development. The in vitro resistance profile of TMC435 was characterized by selection experiments with HCV genotype 1 replicon cells and the genotype 2a JFH-1 system. In 80% (86/109) of the sequences from genotype 1 replicon cells analyzed, a mutation at NS3 residue D168 was observed, with changes to V or A being the most frequent. Mutations at NS3 positions 43, 80, 155, and 156, alone or in combination, were also identified. A transient replicon assay confirmed the relevance of these positions for TMC435 inhibitory activity. The change in the 50% effective concentrations (EC 50 s) observed for replicons with mutations at position 168 ranged from <10-fold for those with the D168G or D168N mutation to ϳ2,000-fold for those with the D168V or D168I mutation, compared to the EC 50 for the wild type. Of the positions identified, mutations at residue Q80 had the least impact on the activity of TMC435 (<10-fold change in EC 50 s), while greater effects were observed for some replicons with mutations at positions 43, 155, and 156. TMC435 remained active against replicons with the specific mutations observed after in vitro or in vivo exposure to telaprevir or boceprevir, including most replicons with changes at positions 36, 54, and 170 (<3-fold change in EC 50 s). Replicons carrying mutations affecting the activity of TMC435 remained fully susceptible to alpha interferon and NS5A and NS5B inhibitors. Finally, combinations of TMC435 with alpha interferon and NS5B polymerase inhibitors prevented the formation of drug-resistant replicon colonies.Hepatitis C is a blood-borne infection that can ultimately result in severe liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma (7). The chronic nature of the disease and the significant possibility of long-term liver damage have led to the current global health burden, with an estimated 180 million people being infected, of whom 130 million are chronic hepatitis C virus (HCV) carriers (54).The current standard-of-care therapy for HCV-infected patients consists of a combination of weekly injected pegylated alpha interferon (Peg-IFN-␣) and twice-daily oral ribavirin. Treatment of HCV genotype 1-infected patients with this regimen for 48 weeks has a limited success rate (a 40 to 50% sustained virological response [SVR]) and is associated with a wide range of side effects, including flu-like symptoms, anemia, and depression, leading to treatment discontinuation in a significant proportion of patients (31, 48). Therefore, specifically targeted antiviral therapies for hepatitis C (STAT-C) have been a major focus of drug discovery efforts. Treatments with several NS3/4A protease inhibitors and NS5A and NS5B polymerase inhibitors, alone or in combination with Peg-IFN-␣-ribavirin, have recently shown encouraging results in clinical trials (17,36).HCV NS3 is an essential, bifunctional, multidomain protein that possesses protease and RNA helicase activiti...

The performance of several commercially available docking programs is compared in the context of virtual screening. Five different protein targets are used, each with several known ligands. The simulated screening deck comprised 1000 molecules from a cleansed version of the MDL drug data report and 49 known ligands. For many of the known ligands, crystal structures of the relevant protein-ligand complexes were available. We attempted to run experiments with each docking method that were as similar as possible. For a given docking method, hit rates were improved versus what would be expected for random selection for most protein targets. However, the ability to prioritize known ligands on the basis of docking poses that resemble known crystal structures is both method- and target-dependent.

The RAR-related orphan receptor gamma t (RORγt) is a nuclear receptor required for generating IL-17-producing CD4 + Th17 T cells, which are essential in host defense and may play key pathogenic roles in autoimmune diseases. Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol and lipid metabolism. Here, we describe the identification of several naturally occurring oxysterols as RORγt agonists. The most potent and selective activator for RORγt is 7β, 27-dihydroxycholesterol (7β, 27-OHC). We show that these oxysterols reverse the inhibitory effect of an RORγt antagonist, ursolic acid, in RORγ-or RORγt-dependent cell-based reporter assays. These ligands bind directly to recombinant RORγ ligand binding domain (LBD), promote recruitment of a coactivator peptide, and reduce binding of a corepressor peptide to RORγ LBD. In primary cells, 7β, 27-OHC and 7α, 27-OHC enhance the differentiation of murine and human IL-17-producing Th17 cells in an RORγt-dependent manner. Importantly, we showed that Th17, but not Th1 cells, preferentially produce these two oxysterols. In vivo, administration of 7β, 27-OHC in mice enhanced IL-17 production. Mice deficient in CYP27A1, a key enzyme in generating these oxysterols, showed significant reduction of IL-17-producing cells, including CD4+ and γδ + T cells, similar to the deficiency observed in RORγt knockout mice. Our results reveal a previously unknown mechanism for selected oxysterols as immune modulators and a direct role for CYP27A1 in generating these RORγt agonist ligands, which we propose as RORγt endogenous ligands, driving both innate and adaptive IL-17-dependent immune responses.

Hepatitis C virus is a blood-borne infection and the leading cause of chronic liver disease (including cirrhosis and cancer) and liver transplantation. Since the identification of HCV in 1989, there has been an extensive effort to identify and improve treatment options. An important milestone was reached in 2011 with the approval of the first-generation HCV NS3/4A protease inhibitors. However, new therapies are needed to improve cure rates, shorten treatment duration, and improve tolerability. Here we summarize the extensive medicinal chemistry effort to develop novel P2 cyclopentane macrocyclic inhibitors guided by HCV NS3 protease assays, the cellular replicon system, structure-based design, and a panel of DMPK assays. The selection of compound 29 (simeprevir, TMC435) as clinical candidate was based on its excellent biological, PK, and safety pharmacology profile. Compound 29 has recently been approved for treatment of chronic HCV infection in combination with pegylated interferon-α and ribavirin in Japan, Canada, and USA.