Crystallization and preliminary X‐ray diffraction analysis of eukaryotic α2‐macroglobulin family members modified by methylamine,proteases and glycosidases

α₂‐Macroglobulin (α₂M) has many functions in vertebrate physiology. To understand the basis of such functions, high‐resolution structural models of its conformations and complexes with interacting partners are required. In an attempt to grow crystals that diffract to high or medium resolution, we isolated native human α₂M (hα₂M) and its counterpart from chicken egg white (ovostatin) from natural sources. We developed specific purification protocols, and modified the purified proteins either by deglycosylation or by conversion to their induced forms. Native proteins yielded macroscopically disordered crystals or crystals only diffracting to very low resolution (>20 Å), respectively. Optimization of native hα₂M crystals by varying chemical conditions was unsuccessful, while dehydration of native ovostatin crystals improved diffraction only slightly (10 Å). Moreover, treatment with several glycosidases hindered crystallization. Both proteins formed spherulites that were unsuitable for X‐ray analysis, owing to a reduction of protein stability or an increase in sample heterogeneity. In contrast, transforming the native proteins to their induced forms by reaction either with methylamine or with peptidases (thermolysin and chymotrypsin) rendered well‐shaped crystals routinely diffracting below 7 Å in a reproducible manner.     

Effects of fat on glucose uptake and utilization in patients with non-insulin-dependent diabetes.

It was the aim of this study to determine whether FFA inhibit insulin-stimulated whole body glucose uptake and utilization in patients with non-insulin-dependent diabetes. We performed five types of isoglycemic (approximately 11mM) clamps: (a) with insulin; (b) with insulin plus fat/heparin; (c) with insulin plus glycerol; (d) with saline; (e) with saline plus fat/heparin and two types of euglycemic (approximately 5mM) clamps: (a) with insulin; (b) with insulin plus fat/heparin. During these studies, we determined rates of glucose uptake, glycolysis (both with 3[3H] glucose), glycogen synthesis (determined as glucose uptake minus glycolysis), carbohydrate oxidation (by indirect calorimetry) and nonoxidative glycolysis (determined as glycolysis minus carbohydrate oxidation). Fat/heparin infusion did not affect basal glucose uptake, but inhibited total stimulated (insulin stimulated plus basal) glucose uptake by 40-50% in isoglycemic and in euglycemic patients at plasma FFA concentration of approximately 950 and approximately 550 microM, respectively. In isoglycemic patients, the 40-50% inhibition of total stimulated glucose uptake was due to near complete inhibition of the insulin-stimulated part of glucose uptake. Proportional inhibition of glucose uptake, glycogen synthesis, and glycolysis suggested a major FFA-mediated defect involving glucose transport and/or phosphorylation. In summary, fat produced proportional inhibitions of insulin-stimulated glucose uptake and of intracellular glucose utilization. We conclude, that physiologically elevated levels of FFa could potentially be responsible for a large part of the peripheral insulin resistance in patients with non-insulin-dependent diabetes mellitus.     

Clinical predictors and efficacy of antitachycardia pacing in patients with implantable cardioverter defibrillators: the importance of the patient's sex

The ICD has become accepted as primary therapy for malignant ventricular arrhythmias. The incorporation of antitachycardia pacing into ICDs has provided a better tolerated alternative to shocks but has the potential disadvantage of delaying definitive therapy. Accordingly, we sought to delineate the characteristics of patients likely to experience unsuccessful termination of pacing and to identify ineffective pacing strategies. Of 519 patients who received ICDs, 11 clinical and tachycardia characteristics in the 162 who received antitachycardia pacing therapy for sustained ventricular arrhythmias were evaluated. Tachycardia episodes were grouped according to outcome of pacing (successful, unsuccessful, acceleration). Of 1,946 episodes, 1,502 (77.2%) were successfully reverted with pacing, 322 (16.5%) were unsuccessful, and 121 (6.2%) were accelerated. Antitachycardia pacing was less successful in women, patients with a history of myocardial infarction, those with more severe left ventricular dysfunction, those who received antiarrhythmic drugs, and those programmed to ramp pacing. Tachycardia acceleration was inversely related to tachycardia cycle length and was more frequent in patients programmed to more aggressive ramp pacing protocols. Women had an almost threefold incidence of tachycardia acceleration compared with men (14% vs 5%, P < 0.001). Antitachycardia pacing is generally successful in terminating ventricular tachycardia and has a low incidence of tachycardia acceleration. Caution should be used with rapid tachycardias and aggressive ramp pacing protocols because of an increased risk of acceleration. Antitachycardia pacing appears less successful and has a higher incidence of complications in women.     

Preclinical Characterization of the Antiviral Activity of SCH 900518 (Narlaprevir), a Novel Mechanism-Based Inhibitor of Hepatitis C Virus NS3 Protease

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*_i) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC₉₀) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5× EC₉₀ of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Despite recent advances, the eradication of chronic HCV infection remains challenging. Approximately 50% of patients infected with the most prevalent form of the virus (genotype 1) fail to respond to treatment with the current standard of care of pegylated alpha interferon in combination with ribavirin (4, 13). Thus, there is a great unmet medical need for the development of new agents with activity against HCV to improve the sustained virological response (SVR). The HCV genome is translated as a single polyprotein precursor which is processed by cellular and viral proteases into structural proteins (the C, E1, and E2 proteins), followed by nonstructural (NS) proteins (the p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B proteins). The NS3 protein is multifunctional, with the N-terminal region (amino acids 1 to 181) encoding a serine protease and the remaining polypeptide encoding a helicase. The NS3 protease noncovalently binds to the cofactor NS4A, which contributes to the formation and stability of the active site and helps to anchor the NS3/NS4A complex to the membrane. The NS3 protease is responsible for the processing of the HCV polyprotein at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (for a review, see reference 19).The NS3 protease is essential for viral replication (6) and represents an important target for antiviral therapy. The clinical efficacies of NS3 protease inhibitors were first demonstrated with compounds targeting the enzyme active site in monotherapy and in combination with pegylated interferon: BILN 2061, a macrocyclic tripeptide in a short monotherapy trial (8), and boceprevir (SCH 503034) and telaprevir (VX-950), both of which are ketoamide derivatives (21, 22, 27, 28). Mutations conferring resistance to these small-molecule inhibitors have been identified in the NS3 protease domain by selection of replicon cells in the presence of compounds; many of the mutations selected in vitro have also been detected in patients during clinical trials of boceprevir and telaprevir (9, 11, 21, 25, 27, 28). Although encouraging clinical results have been achieved with these first-generation protease inhibitors, additional improvements in inhibitor potency and pharmacokinetic properties offers opportunities to increase drug coverage and to further increase the SVR.The combination of medicinal chemistry and structure-based design has led to the synthesis of a new compound, SCH 900518 (narlaprevir; Fig. ​Fig.1)1) (1), with improved antiviral activity in the replicon system compared with the activities of boceprevir (12) and telaprevir (16, 17). The characterization of its inhibition mechanism and in vitro resistance profile is presented in this report.Open in a separate windowFIG. 1.Structure of SCH 900518. P1 to P4 refer to different side chains.     

Molecular analysis of HCV type 1 to 5 envelope gene: application to investigations of posttransfusion transmission of HCV

BACKGROUND: Until 1990, HCV infection was common in transfused patients, resulting in more than 200,000 cases of posttransfusion hepatitis C in France alone. A molecular method that permits the investigation of posttransfusion hepatitis C infections is presented. STUDY DESIGN AND METHODS: Viral sequences in the envelope region of HCV were obtained for 12 pairs of blood recipients and their respective blood donors. The HCV strains studied belonged to types 1 (subtypes 1a and 1b), 2, 3, 4, and 5. Genetic distances and mutation rates were determined, and sequences were submitted to phylogenetic analysis along with sequences retrieved from nucleotide databases. RESULTS: Pairwise distances in the donor-recipient pairs were found to be less than 0.05 mutation per site, which corresponds to a mutation rate ranging from 0.6 x 10(-3) to 2.1 x 10(-3) per site per year. Sequences obtained from the 12 donor-recipient pairs clustered in 12 monophyletic nests. CONCLUSION: The genetic analysis of the envelope region of HCV can be used for the forensic evaluation of virus transmission. It permits the refutation of a link between blood transfusion and HCV transmission, rather than proof of the existence of such a link.