Tigecycline is a novel glycylcycline antibiotic that possesses broad-spectrum activity against many clinically relevant species of bacterial pathogens. The mechanism of action of tigecycline was delineated using functional, biophysical, and molecular modeling experiments in this study. Functional assays showed that tigecycline specifically inhibits bacterial protein synthesis with potency 3-and 20-fold greater than that of minocycline and tetracycline, respectively. Biophysical analyses demonstrated that isolated ribosomes bind tigecycline, minocycline, and tetracycline with dissociation constant values of 10 ؊8 , 10 ؊7 , and >10 ؊6 M, respectively. A molecular model of tigecycline bound to the ribosome was generated with the aid of a 3.40-angstrom resolution X-ray diffraction structure of the 30S ribosomal subunit from Thermus thermophilus. This model places tigecycline in the A site of the 30S subunit and involves substantial interactions with residues of H34 of the ribosomal subunit. These interactions were not observed in a model of tetracycline binding. Modeling data were consistent with the biochemical and biophysical data generated in this and other recent studies and suggested that tigecycline binds to bacterial ribosomes in a novel way that allows it to overcome tetracycline resistance due to ribosomal protection.

The three-dimensional structure of the catalytic domain of stromelysin-1 complexed with an N-carboxyl alkyl inhibitor has been determined by NMR methods. The global fold consists of three helices, a five stranded beta-sheet and a methionine located in a turn near the catalytic histidines, classifying stromelysin-1 as a metzincin. Stromelysin-1 is unique in having two independent zinc binding sites: a catalytic site and a structural site. The inhibitor binds in an extended conformation. The S1' subsite is a deep hydrophobic pocket, whereas S2' appears shallow and S3' open.

HCV-796 selectively inhibits hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. In hepatoma cells containing a genotype 1b HCV replicon, HCV-796 reduced HCV RNA levels by 3 to 4 log 10 HCV copies/g total RNA (the concentration of the compound that inhibited 50% of the HCV RNA level was 9 nM). Cells bearing replicon variants with reduced susceptibility to HCV-796 were generated in the presence of HCV-796, followed by G418 selection. Sequence analysis of the NS5B gene derived from the replicon variants revealed several amino acid changes within 5 Å of the drug-binding pocket. Specifically, mutations were observed at Leu314, Cys316, Ile363, Ser365, and Met414 of NS5B, which directly interact with HCV-796. The impacts of the amino acid substitutions on viral fitness and drug susceptibility were examined in recombinant replicons and NS5B enzymes with the single-amino-acid mutations. The replicon variants were 10-to 1,000-fold less efficient in forming colonies in cells than the wild-type replicon; the S365L variant failed to establish a stable cell line. Other variants (L314F, I363V, and M414V) had four-to ninefold-lower steady-state HCV RNA levels. Reduced binding affinity with HCV-796 was demonstrated in an enzyme harboring the C316Y mutation. The effects of these resistance mutations were structurally rationalized using X-ray crystallography data. While different levels of resistance to HCV-796 were observed in the replicon and enzyme variants, these variants retained their susceptibilities to pegylated interferon, ribavirin, and other HCV-specific inhibitors. The combined virological, biochemical, biophysical, and structural approaches revealed the mechanism of resistance in the variants selected by the potent polymerase inhibitor HCV-796.Hepatitis C virus (HCV) is an enveloped, positive-sense, single-stranded RNA virus of approximately 9.6 kb that possesses an RNA-dependent RNA polymerase (RdRp), NS5B. Like that in many RNA viruses, this RNA replicase lacks a proofreading mechanism. The mutation rate of the HCV RdRp is estimated to be 10

Anti-hepatitis C virus (HCV) drug development has been challenged by a lack of experience with inhibitors inclusive of in vitro, animal model, and clinical study. This manuscript outlines activity and correlation across such a spectrum of models and into clinical trials with a novel selective nonstructural protein 5B (NS5B) polymerase inhibitor, HCV796. Enzyme assays yielded median inhibitory concentration ( H epatitis C virus (HCV) infects an estimated 170 million people across the world, 1 with substantial risk of severe chronic liver disease, cirrhosis, and hepatocellular cancer. 2,3 Standard therapy with pegylated interferon and ribavirin clears virus in only half of those treated, is associated with significant treatment-related morbidity, and is cost-prohibitive in much of the world. 4,5 HCV is the leading indication for liver transplantation across the developed world. Despite intensive effort, no new drugs have been approved for therapy in the last decade. More effective and less toxic anti-HCV therapeutics as well as preventive vaccines are greatly needed. A major challenge with development of anti-HCV drugs has been the lack of in vitro, animal model, and clinical experience with inhibitors. Although correlation exists for interferon, there are limited data for molecules with novel mechanisms. The goal of the current work was to delineate the activity of a selective nonstructural protein 5B (NS5B) polymerase inhibitor in vitro by enzyme assay and in the replicon, in vivo in the chimeric mouse model, and in patients infected with HCV.Abbreviations: EC 50 , half maximal effective concentration; hAAT, Human ␣-1 antitrypsin; HCV, hepatitis C virus; IC 50 , median inhibitory concentration; IFN, interferon; NS5B, nonstructural protein 5B; pegylated interferon; RdRp, SCID, severe combined immunodeficiency; uPA, urokinase plasminogen activator. From

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Some of the conformational changes required for infectivity and involved in the control of capsid stability and neurovirulence in mice may occur in the vicinity of the fivefold axis of the poliovirus, where there are significant structural differences among the three poliovirus serotypes in the surface exposed loops of VP1 (BC, DE, and HI). A surface depression is located at the fivefold axis of PV2L that is not present in the other two poliovirus serotypes. The observed interaction of RNA with VP4 supports the observation that loss of VP4 ultimately leads to the loss of viral RNA. A model is proposed that suggests dual involvement of the virion fivefold and pseudo-threefold axes in receptor-mediated initiation of infection by picornaviruses.

Tigecycline is a broad-spectrum glycylcycline antibiotic with activity against not only susceptible grampositive and gram-negative pathogens but also strains that are resistant to many other antibiotics. In the process of determining quality control (QC) limits for the American Type Culture Collection reference strains for tigecycline, a number of inconsistencies in MICs were encountered which appeared to be related to the age of the Mueller-Hinton broth (MHB) medium used in the MIC testing. The objective of this study was to determine the cause of the discrepant MIC results between fresh and aged MHB. The MICs of tigecycline were determined in MHB that was either prepared fresh (<12 h old), prepared and stored at 4°C, stored at room temperature, stored anaerobically, or supplemented with the biocatalytic oxygen-reducing reagent Oxyrase. When tested in fresh media, tigecycline was 2 to 3 dilutions more active against the CLSI-recommended QC strains compared to aged media (MICs of 0.03 to 0.25 and 0.12 to 0.5 g/ml, respectively). Media aged under anaerobic conditions prior to testing or supplemented with Oxyrase resulted in MICs similar to those obtained in fresh medium (MICs of 0.03 to 0.12 and 0.03 to 0.25 g/ml, respectively). Time-kill kinetics demonstrated a >3 log 10 difference in viable growth when tigecycline was tested in fresh or Oxyrase-supplemented MHB compared to aged MHB. High-pressure liquid chromatography analysis revealed the accumulation of an early peak (oxidative by-product of tigecycline) to be 3.5% in fresh media and 25.1% in aged media after 24 h and that addition of Oxyrase prevented the accumulation of this oxidized by-product. These results suggested that the activity of tigecycline was affected by the amount of dissolved oxygen in the media. The use of fresh MHB or supplementation with Oxyrase resulted in a more standardized test method for performing MIC tests with tigecycline.Tigecycline (GAR-936) is the 9-t-butylglycylamido derivative of minocycline and is the first in class of the "glycylcycline" antibiotics to be developed (17). Tigecycline acts by inhibition of protein translation in bacteria by binding to the 30S ribosomal subunit and blocking entry of amino-acyl tRNA molecules into the A site of the ribosome (2). This prevents incorporation of amino acid residues into elongating peptide chains. Unlike the classical tetracyclines, tigecycline is not affected by any of the known tetracycline resistance determinants (15). Tigecycline demonstrates activity against a broad range of gram-positive, gram-negative aerobic, anaerobic, and "atypical" antibiotic-susceptible and -resistant bacteria (3, 5-10, 14, 16, 18).In the course of development of tigecycline, several activities were undertaken in an attempt to establish the quality control (QC) limits for the Clinical Laboratory Standards Institute (CLSI; formerly NCCLS)-recommended American Type Culture Collection (ATCC) strains to be used for MIC testing with tigecycline. As the development of tigecycline moved toward clinical studies,...

A novel series of HCV NS5B RNA-dependent RNA polymerase inhibitors containing a pyrano[3,4-b]indole scaffold is described leading to the discovery of compound 16, a highly potent and selective inhibitor that is active in the replicon system.