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Dimethandrolone undecanoate (DMAU), an oral investigational male hormonal contraceptive, is a prodrug that is rapidly converted to its active metabolite, dimethandrolone (DMA). Poor and variable oral bioavailability of DMA after DMAU dosing is a critical challenge to develop it as an oral drug. The objective of our study was to elucidate the mechanisms of variable pharmacokinetics of DMA. We first identified DMA metabolites formed in vitro and in vivo in human hepatocyte incubation and serum samples following oral DMAU administration in men, respectively. The metabolite identification study revealed two metabolites, DMA-glucuronide (DMA-G; major) and the androstenedione analog of DMA (minor), in the hepatocyte incubations. After oral DMAU administration, only DMA-G was detected in serum, which was >100-fold compared with DMA levels, supporting glucuronidation as the major elimination mechanism for DMA. Next, 13 clinically relevant UDP-glucuronosyltransferase (UGT) enzymes were tested for their involvement in DMA-G formation, which revealed a major role of UDP-glucuronosyltransferase 2B17 (UGT2B17) isoform with a smaller contribution of UGT1A9 in DMA-G formation. These data were confirmed by dramatically higher DMA glucuronidation rates (>200- and sevenfold) in the high versus the null UGT2B17-expressing human intestinal and liver microsomes, respectively. Since human UGT2B17 is a highly variable enzyme with a 20%–80% gene deletion frequency, the in vitro data suggest a major role of UGT2B17 polymorphism on the first-pass metabolism of DMA. Further, considering DMA is a selective and sensitive UGT2B17 substrate, it could be used as a clinical probe of UGT2B17 activity. SIGNIFICANCE STATEMENT: Dimethandrolone (DMA) is an active metabolite of dimethandrolone undecanoate (DMAU), an investigational male hormonal contraceptive. Previous studies have indicated poor and inconsistent bioavailability of DMAU following oral administration. This study found that UDP-glucuronosyltransferase 2B17–mediated high intestinal first-pass metabolism is the key mechanism of variable DMA bioavailability.

The purpose of this study was to examine sex-based trends in incidence of elective abdominal aortic aneurysm (AAA), ruptured AAA, ruptured AAA repair and AAA-related mortality.A retrospective analysis of patients presenting with AAA from 2005-2015 was conducted. Rates of elective AAA repair, ruptured AAA, ruptured AAA repair, and mortality were obtained from linking provincial administrative data using medical services insurance billing number. The age-adjusted incidence of elective AAA repair, overall rate of ruptured AAA, ruptured AAA repair, and AAA-related mortality was calculated for each sex based on Canadian census estimates, adjusted to the Canadian standard population. Weighted linear regression was performed to analyze trends in incidence over time.1986 elective AAA repairs were identified, of which 1098 were repaired open and 898 endovascular AAA repair (EVAR). 570 ruptured AAAs were identified, of which 295 (52%) were repaired: 259 open and 36 EVAR. The proportion of ruptured AAA that was repaired did not change over time (p=0.54). The proportion repairs performed using EVAR increased significantly in both elective (p0.001) and rupture repairs (p0.001). During the study period, 662 patients died of AAA-associated mortality. The average incidence of elective AAA repair in men was 29.3 (95% CI: 27.8 to 30.8) per 100,000 and decreased over time (p=0.04), whereas the average incidence in women was 9.2 [8.3 to 10.0] and stable (p=0.07). The incidence of open elective AAA repair was 10.5 [9.9-11.1] with a decreasing trend over time (p0.001) and EVAR was 9.0 (8.5-9.6) with an increasing trend over time (p0.001). A decreasing trend of overall ruptured AAA (5.4 [5.0-5.9], p0.001), ruptured AAA repair (2.9 [2.5-3.2], p=0.02), and of AAA-related mortality (6.2 [5.8-6.8], p0.001) was found, with consistent trends in both sexes. The incidence of open ruptured AAA repair decreased over time (p=0.001) whereas the incidence of ruptured EVAR remained stable (p=0.23).The incidence of elective AAA repair is decreasing in males but not females, whereas the incidence of rupture has decreased in both sexes. This has translated into reduced incidence of AAA-related mortality. Increased adoption of EVAR for ruptured AAA should continue these trends.

The long and challenging drug development process begins with discovery biology for the selection of an appropriate target for a specific indication. Target is a broad term that can be applied to a range of biological entities such as proteins, genes, and ribonucleic acids (RNAs). Although there are numerous databases available for mining biological entities, publicly available searchable, downloadable databases to aid in target selection for a specific disease or indication (e.g., developing contraceptives and infertility treatments) are limited. We report the development of the Contraceptive and Infertility Target DataBase (https://www.citdbase.org), which provides investigators an interface to mine existing transcriptomic and proteomic resources to identify high-quality contraceptive/infertility targets. The development of similar databases is applicable to the identification of targets for other diseases and conditions.

Ruptured abdominal aortic aneurysms (RAAAs) are surgical emergencies that require immediate and expert treatment. It has been unclear whether presentation during evenings and weekends, when "on call" teams are primarily responsible for patient care, is associated with worse outcomes. Our objective was to evaluate the outcomes of patients presenting with RAAAs after-hours vs during the workday.A retrospective cohort study of all RAAAs in Nova Scotia between 2005 and 2015 was performed through linkage of administrative databases. Patients who had presented to the hospital with RAAAs during the workday (Monday through Friday, 6 am to 6 pm) were compared with those who had presented after-hours (6 pm to 6 am during the week and on weekends). The baseline and operative characteristics were identified for all patients through the available databases and a review of the medical records. Mortality before surgery, 30-day mortality, and operative mortality were compared between groups using multivariable logistic regression, adjusting for factors clinically significant on univariable analysis.A total of 390 patients with RAAAs were identified from 2005 to 2015, of whom 205 (53%) had presented during the workday and 185 (47%) after-hours. The overall chance of survival (OCS) was 45% overall, 49% if admitted to hospital, and 64% if surgery had been performed. During the workday, the OCS was 43% overall, 48% if admitted to hospital, and 67% if surgery had been performed. After-hours, the OCS was 46% overall, 49% if admitted to hospital, and 61% if surgery had been performed. Mortality before surgery was increased for patients who had presented to the hospital during the workday compared with after-hours (36% vs 26%; P = .04). The 30-day mortality (57% vs 54%; P = .62), rates of operative management (63% vs 72%; P = .06), and operative mortality (33% vs 39%; P = .33) were similar between the workday and after-hours groups (57% vs 54%; P = .06). After adjusting for significant clinical variables, the patients who had presented with RAAAs after-hours had had a similar odds of dying before surgery (odds ratio [OR], 0.64; 95% confidence interval [CI], 0.41-1.03), operative management (OR, 1.47; 95% CI, 0.93-2.31), 30-day mortality (OR, 0.98; 95% CI, 0.63-1.51), and operative mortality (OR, 1.33; 95% CI, 0.78-2.26). In the subgroup of patients presenting to a hospital with endovascular capabilities, patients presenting after-hours had had similar odds of 30-day mortality (OR, 1.07; 95% CI, 0.57-2.02), and operative mortality (OR, 1.14; 95% CI, 0.58-2.23).We found that patients presenting to the hospital with RAAAs after-hours did not have increased adjusted odds of mortality before surgery, operative management, 30-day mortality, or operative mortality.

BackgroundThe advent of new methods of male contraception would increase contraceptive options for men and women and advance male contraceptive agency. Pharmaceutical R&D for male contraception has been dormant since the 1990s. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) has supported a contraceptive development program since 1969 and supports most ongoing hormonal male contraceptive development. Nonhormonal methods are in earlier stages of development.ContentSeveral hormonal male contraceptive agents have entered clinical trials. Novel single agent products being evaluated include dimethandrolone undecanoate, 11β-methyl-nortestosterone dodecylcarbonate, and 7α-methyl-19-nortestosterone. A contraceptive efficacy trial of Nestorone®/testosterone gel is underway. Potential nonhormonal methods are at preclinical stages of development. Many nonhormonal male contraceptive targets that affect sperm production, sperm function, or sperm transport have been identified.SummaryNICHD supports development of reversible male contraceptive agents. Other organizations such as the World Health Organization, the Population Council, and the Male Contraception Initiative are pursuing male contraceptive development, but industry involvement remains limited.

Objective The objective of this study was to evaluate the safety and effectiveness of single ProGlide use per bilateral access site for endovascular aneurysm repair (EVAR) of abdominal aortic aneurysms. Methods A retrospective cohort study was performed for all elective percutaneous EVARs from November 2015 to December 2017 at the QEII Health Sciences Centre (Halifax, Nova Scotia, Canada). Exposure of interest was number of ProGlides used per access site, dichotomized into bilateral single ProGlide closure vs nonsingle ProGlide closure on at least one femoral arteriotomy. Outcomes included Valve Academic Research Consortium (VARC)-2 and Bleeding Academic Research Consortium (BARC) criteria. Groups were compared with Fisher exact test, analysis of variance, or Wilcoxon rank sum, as appropriate. Logistic regression was used to compare the effect of single ProGlide use on VARC-2 and BARC criteria. Results A total of 131 cases were included, of which 116 had bilateral single ProGlide use for access closure. Baseline characteristics including comorbidities and smoking status were compared between groups. Groups were similar for all characteristics except smoking status, with an increased proportion of former smokers in the nonsingle ProGlide group. There were 119 (90.8%) patients who had single ProGlide use on the right femoral artery and 121 (92.4%) on the left; 16 (12.2%) patients had ProGlide deployment issues. Median maximal right and left femoral sheath diameters were 16F (interquartile range [IQR], 16F-18F) and 14F (IQR, 14F-16F), respectively. Median length of stay was 1 day (IQR, 1-1 day). VARC-2 criteria occurred in 8 of 131 (6.11%) patients, 6 of 116 (5.17%) with bilateral single ProGlides and 2 of 15 (13.3%) with nonsingle ProGlides. BARC criteria occurred in 6 of 131 (4.58%) patients, 5 of 116 (4.31%) with bilateral single ProGlides and 1 of 15 (6.67%) with nonsingle ProGlides. Single ProGlide use was not associated with a difference in VARC-2 (odds ratio, 0.35; 95% confidence interval, 0.64-1.94) or BARC (odds ratio, 0.63; 95% confidence interval, 0.07-6.79) criteria. No patients developed pseudoaneurysms or required repeated intervention for bleeding. Conclusions Single ProGlide use per vascular access site in patients undergoing EVAR is a safe and effective method for access closure with sheath diameters up to and including 16F.

Neutrophils are rapidly recruited from the peripheral blood to the inflammatory site to initiate inflammatory response against pathogenic infections. The process to recruit neutrophils must be properly regulated since the abnormal accumulation of neutrophils can cause organ damage and dysfunction. The acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a common cause of respiratory failure that is characterized by the infiltration of neutrophils and epithelial integrity disruption. Indeed, recent studies suggest a pathogenic role of neutrophils in the clinic severity of the coronavirus disease 2019 (COVID-19) ARDS. The chemokine CXCL1, which is rapidly induced by inflammatory stimuli, plays a key role in neutrophil influx during lung inflammation. The molecular basis ofCxcl1induction is not fully understood. Here we report that TET1, a member of the ten eleven translocation (TET) methylcytosine dioxygenase protein family, displays a striking specificity in the regulation of gene expression in macrophages. RNA sequencing (RNA-seq) analysis showed thatTet1disruption significantly altered the expression of only 48 genes that includeCxcl1and several other genes known to be important for cell migration and trafficking in bone marrow derived macrophages (BMDMs) in response to LPS stimulation. TET1 regulates the induction ofCxcl1by facilitating the DNA demethylation of theCxcl1promoter. InTet1−/−mice, the induction ofCxcl1was suppressed, resulting in defective neutrophil recruitment to the lung during LPS-induced acute lung injury. Our results identify a novel epigenetic mechanism that selectively controlsCxcl1induction and neutrophil recruitment during acute lung injury.Key PointsTET1 has a striking specificity in macrophage gene regulation and controlsCxcl1induction by inflammatory stimuli via DNA demethylationNeutrophil recruitment is defective inTet1deficient mice during acute lung injury

Several radiolabeled folic acid conjugates have been developed for targeted imaging and therapy. However, the therapeutic concept with radiolabeled folate conjugates has not yet been applied to cli...

BACKGROUNDDevelopment of new methods of male contraception would address an unmet need for men to control their fertility and could increase contraceptive options for women. Pharmaceutical research and development for male contraception was active in the 1990s but has been virtually abandoned. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) has supported a contraceptive development program since 1969 and supports the majority of hormonal male contraceptive development. Nonhormonal methods are also in development but are at earlier stages.CONTENTSeveral hormonal male contraceptive agents have entered clinical trials. Single-agent products being evaluated include dimethandrolone undecanoate, 11β-methyl-nortestosterone dodecyl carbonate, and 7α-methyl-19-nortestosterone. A contraceptive efficacy trial of Nestorone® gel and testosterone gel in a single application will begin in 2018. Potential nonhormonal methods are at preclinical stages of development. Many nonhormonal male contraceptive targets that affect either sperm production or sperm function have been identified. Targeted pathways include the retinoic acid pathway, bromodomain and extraterminal proteins, and pathways for Sertoli cell–germ cell adhesion or sperm motility. Druggable targets include CatSper, the sperm Na+/K+-exchanger, TSSK, HIPK4, EPPIN, and ADAMs family proteins. Development of a procedure to reversibly block the vas deferens (initially developed in India in the 1980s) is undergoing early stage research in the US under the trade name Vasalgel™.SUMMARYNICHD has supported the development of reversible male contraceptive agents. Other organizations such as the World Health Organization and the Population Council are pursuing male contraceptive development, but industry involvement remains dormant.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a mainstay analytical technique in pharmaceutical research and development and clinical diagnosis due to several advantages including excellent selectivity, specificity, and high sensitivity. LC-MS/MS has become the method of choice for steroids analysis due to its fast analytical time and improved specificity yet has a challenge in the separation and measurement of isomers with the same product ions. Here we describe a high-sensitivity LC/LC-MS/MS method that combines chiral chromatography and reverse-phase chromatography (LC/LC) along with MS/MS to rapidly separate and quantify steroid isomers of 11ß-methyl-19-nortestosterone (11ß-MNT) and endogenous testosterone in serum.

Clouds and aerosols increase the fraction of global solar irradiance that is diffuse light. This phenomenon is known to increase the photosynthetic light use efficiency (LUE) of closed-canopy vegetation by redistributing photosynthetic photon flux density (400–700 nm) from saturated, sunlit leaves at the top of the canopy, to shaded leaves deeper in the canopy. We combined a process-based carbon cycle model with 10 years of eddy covariance carbon flux measurements and other ancillary data sets to assess 1) how this LUE enhancement influences interannual variation in carbon uptake, and 2) how errors in modeling diffuse fraction affect predictions of carbon uptake. Modeled annual gross primary productivity (GPP) increased by ≈0.94% when observed levels of diffuse fraction were increased by 0.01 (holding total irradiance constant). The sensitivity of GPP to increases in diffuse fraction was highest when the diffuse fraction was low to begin with, and lowest when the diffuse fraction was already high. Diffuse fraction also explained significantly more of the interannual variability of modeled net ecosystem exchange (NEE), than did total irradiance. Two tested radiation partitioning models yielded over- and underestimates of diffuse fraction at our site, which propagated to over- and underestimates of annual NEE, respectively. Our findings highlight the importance of incorporating LUE enhancement under diffuse light into models of global primary production, and improving models of diffuse fraction.

Several radiolabeled folic acid conjugates have been developed for targeted imaging and therapy. However, the therapeutic concept with radiolabeled folate conjugates has not yet been applied to clinical applications owing to the high renal absorbed dose. The effectiveness of targeted radionuclide therapy (TRT) depends primarily on the absorbed dose rate and on the total absorbed dose delivered to the tumor and to normal tissue. Owing to various limitations associated with organ level dosimetry, voxel-based dosimetry has become essential for the assessment of a more accurate absorbed dose during TRT. In this study, we synthesized iron oxide nanoparticle (IONP)-conjugated radiolabeled folate (

CONTEXT: 11β-Methyl-19-nortestosterone-17β-dodecylcarbonate (11β-MNTDC) is an orally bioavailable prodrug of 11β-methyl-19-nortestosterone (11β-MNT) with androgenic and progestational activity. OBJECTIVES: (i) Quantify 11β-MNT binding to androgen and progesterone receptors. (ii) Evaluate safety, tolerability, and serum gonadotropin and testosterone suppression by 11β-MNTDC in men. DESIGN AND SETTING: (i) In vitro receptor binding and transactivation studies and (ii) randomized, double-blind, placebo-controlled single-dose, dose-escalating phase I study at two academic medical centers. PARTICIPANTS AND INTERVENTION: Twelve healthy male volunteers were randomized (five active, one placebo) to escalating single oral doses (100, 200, 400, and 800 mg) of 11β-MNTDC or placebo given with or without food. MAIN OUTCOME MEASURES: (i) In vitro 11β-MNT/11β-MNTDC human receptor binding and transactivation and (ii) safety and tolerability, pharmacokinetics, and quantification of serum gonadotropin and testosterone concentrations for 24 hours following dosing. RESULTS: 11β-MNT avidly binds and activates human androgen and progesterone receptors, but 11β-MNTDC has minimal activity. Single oral doses of 11β-MNTDC were well tolerated without serious adverse events. Administration of 11β-MNTDC with food markedly increased average 11β-MNTDC and 11β-MNT serum concentrations (P < 0.001 for all doses) compared with fasting with a significant dose-related effect on average serum drug concentrations (P < 0.0001). The 200-, 400-, and 800-mg doses significantly suppressed average serum testosterone concentrations (P < 0.05). CONCLUSIONS: A single, oral dose of 11β-MNTDC up to 800 mg administered with food is safe and well tolerated in healthy men. The active drug 11β-MNT has androgenic and progestational activity, rapidly suppresses serum testosterone, and is a promising candidate for an effective once-daily oral male hormonal contraceptive.

Objective To compare postoperative morphine equivalent intake after open abdominal aortic aneurysm (AAA) repair among analgesic modalities: systemic analgesia (SA) only with no regional anesthesia, surgically positioned paravertebral catheter (PVC), and thoracic epidural analgesia (TEA). Methods This retrospective cohort study included patients undergoing elective open AAA at the Queen Elizabeth II Health Science Center, Halifax, Nova Scotia. Demographics, morphine equivalents, methods of analgesia administration, and outcomes data were collected on all patients from 2005 to 2016. Total morphine equivalent (MEQ) on postoperative days (PODs) 1, 2, and 3 were compared among patients with SA, PVC, and TEA. A multivariable zero-inflated log-linear regression was used to determine the association between analgesic modality and MEQ. Multivariable logistic regression models were used to determine associations between analgesic modality and postoperative pain, rates of discharge from intensive care within 1 day and opioid-related adverse events. Results The study cohort included 355 patients: 177 retroperitoneal and 178 transperitoneal repairs; 173 patients underwent SA, 117 PVC, and 65 TEA. On POD1, median MEQs were 984 (interquartile range [IQR], 342-1525) for SA, 89 (33-246) for PVC, and 49 (0-90) for TEA. On POD2, the median MEQs were 105 (IQR, 57-210) for SA, 45 (15-99) for PVC, and 30 (0-64) for TEA. On POD3, the median MEQs were 45 (IQR, 15-120) for SA, 30 (0-60) for PVC, and 10 (0-45) for TEA. On multivariable log-linear regression, compared with SA, PVC and TEA were associated with increased odds of receiving no opioids on POD1 (odds ratio [OR], 66.85; 95% confidence interval [CI], 17.49-255.57; and OR, 214.68; 95% CI, 60.20-766.38; respectively), POD 2 (OR, 6.97; 95% CI, 3.61-13.46; and OR, 28.73; 95% CI, 15.68-52.62; respectively), and POD 3 (OR, 3.93; 95% CI, 2.72-5.67; and OR, 4.68; 95% CI, 3.20-6.86; respectively). If patients did receive opioids, compared with SA, PVC and TEA were associated with decreased consumption on POD1 (RR, 0.22; 95% CI, 0.18-0.27; and RR, 0.16; 95% CI, 0.12-0.20; respectively), POD2 (RR, 0.50; 95% CI, 0.42-0.58; and RR, 0.46; 95% CI, 0.37-0.56; respectively), and POD3 (RR, 0.78; 95% CI, 0.66-0.93; and RR, 0.76; 95% CI, 0.63-0.93; respectively). Compared with SA, PVC was associated with earlier discharge from intensive care (OR, 2.75; 95% CI, 1.17-6.45) and TEA was not (OR, 1.12; 95% CI, 0.56-2.2). Compared with TEA, PVC was not associated with increased rate of opioid-related adverse events (OR, 0.44; 95% CI, 0.08-2.44). Conclusions PVC and TEA are associated with decreased MEQ compared with SA. PVC is associated with earlier discharge from intensive care compared with SA and similar rates of opioid-related adverse events compared with TEA. Paravertebral analgesia appears to be a safe and effective analgesic modality in patients undergoing retroperitoneal approach for abdominal aneurysm repair.

Paravertebral catheters are a well-established analgesic modality in thoracic surgery but have not been described in abdominal aortic surgery. We describe a simple, safe, and effective technique of paravertebral catheter insertion by the operative surgeon after a retroperitoneal abdominal aortic aneurysm repair. Once the aneurysm repair is complete, an extrapleural plane between the parietal pleura and the twelfth rib is created through blunt dissection. A catheter is advanced into the space percutaneously under direct vision, and a continuous infusion of local anesthetic is administered. Paravertebral catheters typically remain in place for 3 to 5 days and provide excellent postoperative non-narcotic analgesia.

Objective.Large vessel uptake on positron emission tomography/computerized tomography (PET/CT) supports the diagnosis of giant cell arteritis (GCA). Its value, however, in patients without arteritis on temporal artery biopsy and in those receiving glucocorticoids remains unknown. We compared PET/CT results in GCA patients with positive (TAB+) and negative temporal artery biopsies (TAB−), and controls.Methods.Patients with new clinically diagnosed GCA starting treatment with glucocorticoids underwent temporal artery biopsy and PET/CT. Using a visual semiquantitative approach, 18F-fluorodeoxyglucose (FDG) uptake was scored in 8 vascular territories and summed overall to give a total score in patients and matched controls.Results.Twenty-eight patients with GCA and 28 controls were enrolled. Eighteen patients with GCA were TAB+. Mean PET/CT scores after an average of 11.9 days of prednisone were higher in patients with GCA compared to controls, for both total uptake (10.34 ± 2.72 vs 7.73 ± 2.56; p = 0.001), and in 6 of 8 specific vascular territories. PET/CT scores were similar between TAB+ and TAB− patients with GCA. The optimal cutoff for distinguishing GCA cases from controls was a total PET/CT score of ≥ 9, with an area under the receiver-operating characteristic curve of 0.75, sensitivity 71.4%, and specificity 64.3%. Among patients with GCA, these measures correlated with greater total PET/CT scores: systemic symptoms (p = 0.015), lower hemoglobin (p = 0.009), and higher platelet count (p = 0.008).Conclusion.Vascular FDG uptake scores were increased in most patients with GCA despite exposure to prednisone; however, the sensitivity and specificity of PET/CT in this setting were lower than those previously reported.

The discovery of BMS-605339 (35), a tripeptidic inhibitor of the NS3/4A enzyme, is described. This compound incorporates a cyclopropylacylsulfonamide moiety that was designed to improve the potency of carboxylic acid prototypes through the introduction of favorable nonbonding interactions within the S1' site of the protease. The identification of 35 was enabled through the optimization and balance of critical properties including potency and pharmacokinetics (PK). This was achieved through modulation of the P2* subsite of the inhibitor which identified the isoquinoline ring system as a key template for improving PK properties with further optimization achieved through functionalization. A methoxy moiety at the C6 position of this isoquinoline ring system proved to be optimal with respect to potency and PK, thus providing the clinical compound 35 which demonstrated antiviral activity in HCV-infected patients.

Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with K i values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC 50 s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC 50 , 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC 50 , >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC 50 s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensive in vitro genotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

Trifluoroacetic acid (TFA) is a common reagent in both solid-phase and solution peptide synthesis. It is used for the deprotection and/or cleavage of the synthesized peptide from the resin. The use of TFA under these standardized conditions is thought to be sufficiently mild, thereby preventing degradation of the desired product. However, peptides of the general structure R1-(N-alkyl X1)-X2-R2 are hydrolyzed by standard TFA solid-phase peptide synthesis (SPPS) cleavage/deprotection conditions providing fragments R1-(N-alkyl X1)-OH and H-X2-R2. The fragmentation is observed during a TFA cleavage both from the resin and in solution. The hydrolysis is proposed to proceed via an oxazolone-like intermediate in which equilibration of the chiral center of the N-alkylated residue occurs. This mechanism is supported by H/D exchange as observed MS and NMR in conjunction with HPLC.

A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.

Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al. (2003) Proc. Natl. Acad. Sci. 100, 757-762). Nonphosphorylated TSP9 is associated with the membrane, whereas, after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of nonphosphorylated TSP9 both in aqueous solution and in membrane mimetic micelles. The results show that both wild type nonphosphorylated TSP9 and a triple-mutant (T46E + T53E + T60E) mimic of the triphosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle-induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal alpha-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for nonphosphorylated TSP9 in SDS micelles indicated that the molecule is highly flexible with the highest order in the N-terminal alpha-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain the selective dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function.

The recent discovery of the first "self-sacrifice" mechanism for bacterial resistance to the enediyne antitumor antibiotics, where enediyne-induced proteolysis of the resistance protein CalC inactivates both the highly reactive metabolite and the resistance protein, revealed yet another ingenious bacterial mechanism for controlling reactive metabolites. As reported herein, the first 3D structures of CalC and CalC in complex with calicheamicin (CLM) divulge CalC to be a member of the steroidogenic acute regulatory protein (StAR)-related transfer (START) domain superfamily. In contrast to previous studies of proteins known to bind DNA-damaging natural products ( e.g ., bleomycins, mitomycins, and nine-membered chromoprotein enediynes), this is the first demonstrated involvement of a START domain fold. Consistent with the CalC self-sacrifice mechanism, CLM in complex with CalC is positioned for direct hydrogen abstraction from Gly113 to initiate the oxidative proteolysis-based resistance mechanism. These structural studies also illuminate, for the first time, a small DNA-binding region within CalC that may serve to localize CalC to the enediyne target (DNA). Given the role of START domains in nuclear/cytosolic transport and translocation, this structural study also may implicate START domains as post-endocytotic intracellular chaperones for enediyne-based therapeutics such as MyloTarg.

Roadblock/LC7 is a member of a class of dynein light chains involved in regulating the function of the dynein complex. We have determined the three-dimensional structure of isoform 1 of the mouse Roadblock/LC7 cytoplasmic dynein light chain (robl1_mouse) by NMR spectroscopy. In contrast to a previously reported NMR structure of the human homolog with 96% sequence identity (PDB 1TGQ), which showed the protein as a monomer, our results indicate clearly that robl1 exists as a symmetric homodimer. The two beta3-strands pair with each other and form a continuous ten-stranded beta-sheet. The 25-residue alpha2-helix from one subunit packs antiparallel to that of the other subunit on the face of the beta-sheet. Zipper-like hydrophobic contacts between the two helices serve to stabilize the dimer. Through an NMR titration experiment, we localized the site on robl1_mouse that interacts with the 40 residue peptide spanning residues 243 through 282 of IC74-1_rat. These results provide physical evidence for a symmetrical interaction between dimeric robl1 and the two molecules of IC74-1 in the dynein complex.

The structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein, was determined by NMR spectroscopy. The structure is dominated by a beta-barrel sandwich. A two-stranded anti-parallel beta-sheet, which seals off one end of the beta-barrel, is flanked by two flexible loops rich in acidic amino acids. Although this fold often provides a ligand binding site, the structure did not reveal an appreciable cavity inside the beta-barrel. The three-dimensional structure of At3g04780.1-des15 provides an entry point for understanding its functional role and those of its mammalian homologs.

We have identified a mu-selective opioid receptor agonist without a cationic amino group in the molecule from libraries of bicyclic beta-turn peptidomimetics. The biologically active conformation of the lead is proposed to mimic an endomorphin type III 4 --1 beta-turn conformation.

Treatment of rat pituitary GH4C1 cells with estradiol, insulin, and epidermal growth factor induces secretory granule accumulation, PRL storage, and stabilization of ICA512, a membrane protein associated with secretory granules. In these investigations we found that the same treatment induced accumulation over 2-fold of other proteins in the secretory pathway, including synaptosomal-associated protein of 25 kDa (SNAP-25), synaptotagmin III, synaptobrevin, synaptophysin, and cyclophilin B, and did not affect accumulation of others, including synaptotagmin I, calnexin, and glucose-regulated protein 94. The induction of proteins was not a coordinate event, because epidermal growth factor alone maximally stimulated SNAP-25 accumulation, but not that of synaptotagmin III. Induction of SNAP-25 accumulation occurred without an increase in its synthesis, and induction of cyclophilin B occurred without an increase in its messenger RNA accumulation, suggesting that accumulation may be caused by stabilization of the proteins. SNAP-25 immunofluorescence was located in the cytoplasm and on the plasma membrane and sometimes was heavily concentrated in protrusions from the cell surface, especially in hormone-treated cells. Frequenin immunofluorescence was also sometimes concentrated in intense patches, but did not colocalize with SNAP-25. Growth hormone and prolactin immunofluorescence was not found in the protrusions and sometimes did not colocalize with each other when they were present in the same cell. Hormone treatment of GH4C1 cells therefore induces accumulation of specific proteins in all parts of the secretory pathway and causes morphological changes in addition to accumulating secretory granules.

Familial isolated GH deficiency type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32–71-GH, a protein that cannot fold normally. The mechanism by which this mutant suppresses the secretion of wild-type GH encoded by the normal allele is not known. Coexpression of del32–71-GH with wild-type human GH in transient transfections of the neuroendocrine cell lines GH4C1 and AtT20 suppressed accumulation of wild-type GH. The suppression of wild-type GH accumulation by del32–71-GH was a posttranslational effect on wild-type GH caused by decreased stability, rather than decreased synthesis, of wild-type GH. Coexpression of del32–71-GH with human PRL did not suppress accumulation of PRL, indicating that there was not a general suppression of secretory pathway function. Accumulation of del32–71-GH protein was not necessary for the suppression of wild-type GH, because del32–71-GH did not accumulate in the neuroendocrine cell line...

Treatment with 1 nM estradiol, 300 nM insulin, and 5 nM epidermal growth factor induces secretory granule accumulation and prolactin storage in GH4C1 rat pituitary tumor cells. The same triple treatment induced more than 6-fold accumulation of both the precursor (100 kDa, pro-ICA512) and the mature forms (60-70 kDa, ICA512 transmembrane fragment) of ICA512, a receptor protein tyrosine phosphatase-like protein that is preferentially localized in secretory granule membranes. Accumulation of ICA512 resembles that of prolactin storage, for the combination of all three, estradiol, insulin, and epidermal growth factor, gave the greatest induction, which was maximal at 4 days. This effect was specific, as the levels of the small GTP-binding protein Rab3, which is also associated with secretory granule membranes, were unaffected by the triple hormone/growth factor treatment. Increased transcription and translation of ICA512 could only partially account for its 6-fold accumulation, as ICA512 messenger RNA and ICA512 synthesis levels were 1.8 +/- 0.35- and 1.6 +/- 0.17-fold in triple treated GH4C1 cells compared with those in untreated cells, respectively. Pulse-chase procedures showed that pro-ICA512 was more stable in treated cells. These results indicate that the enlargement of the secretory granule compartment results in the stabilization of ICA512 and raise the possibility that trafficking of secretory granules may affect ICA512's function and vice versa.

Monte Carlo calculations show a classical γ-turn in a family of metabolites known as the malformins. This led to the synthesis of epimeric seven-membered ring γ-turn mimetics starting from leucine. NMR temperature coefficient studies were also performed.

The preparation of sterically hindered secondary amino esters from the reaction of N-protected α-amino aldehydes and 3-, or 2-oxo esters with α-amino esters by reductive amination is described. The resulting amino esters were converted to the β-lactam or acylated to form N-acyl secondary amides.

Syntheses of optically active ether-linked β-lactams and aza-proline analogues via 4-bromo-2-hydroxybutanoic acid esters derived from (S)- or (R)-malic acid are described. From these intermediates peptide secondary structure mimetics can be synthesized.

Traditionally, CD has been used extensively for peptides in secondary structure analysis. In recent years, NMR chemical shifts and nuclear Overhauser enhancements have been widely used in conjunction with CD to assess the secondary structures of peptides and proteins; however, there are many instances where the estimation of secondary structure contents differs significantly between the two methods. In order to elucidate the perceived differences between the two methods, secondary structure estimations by CD and 1H NMR chemical shifts were compared for over 50 peptides. The linear peptides investigated were largely unstructured, approximately 15-50 residues in size, and lacked stable tertiary conformation. These peptides were studied in different solvent systems including water, alcohol-water, micelles and urea. A strong correlation exists for secondary structure assessment by CD and NMR chemical shifts; however, an interesting trend of higher estimation of helical contents by NMR was observed for peptide fragments from globular proteins studied in water. This may be a result of associative properties of these peptides in water. Additionally, a new method of quantitating secondary structure contents based on 1H NMR chemical shifts is reported.

Attempts to enhance the efficacy of our previously reported CD4 CDR2-like (residues 40-45) mimetic 1 by incorporation of the critical guanidine residue Arg-59 of CD4 are described.

Background To support clinical development of HCV non-structural protein (NS) 3 protease inhibitors (PIs), phenotypic monitoring of patient isolates is a prerequisite for understanding the emergence of resistance. HCV isolates typically fail to replicate in cell culture, necessitating the use of alternative phenotyping methods. Methods An NS3 protease chimeric replicon system was developed to monitor the phenotype of clinical isolates. The transfer of NS3 protease domain sequences from HCV-infected patients to the background of genotype (Gt) 1a-H77c, 1b-Con1 and 2a-JFH-1 lab strain replicons adapted to high-level cell culture replication was investigated. Results NS3 protease sequences derived from HCV Gt 1a or Gt 1b infected patients were transferred into Gt 1a and 1b replicons, respectively. Replication was detected for 20% of Gt 1a and 75% of Gt 1b sequences. Incorporation of known cell culture adaptive change NS3-E176G improved replication of Gt 1b but not of Gt 1a sequences. Transfer of Gt 1a clinical sequences into the Gt 1b background enhanced replication and allowed phenotypic analysis of all sequences. A correlation was observed between clinical isolate sequence polymorphisms and reduced susceptibility to NS3 PI. In mixed populations containing known NS3 PI resistance changes NS3-R155K or D168E/V, sensitivity of resistance detection was ≥10%. Conclusions An HCV replicon capable of supporting phenotypic characterization of patient-derived HCV NS3 protease sequences was developed. Pre-existence of amino acid changes associated with NS3 PI resistance highlights the need for combination therapies in the treatment of HCV.

The three-dimensional solution structure of the DNA binding domain (DBD) of the retinoid X receptor alpha (RXR alpha) was determined by nuclear magnetic resonance spectroscopy. The two zinc fingers of the RXR DBD fold to form a single structural domain that consists of two perpendicularly oriented helices and that resembles the corresponding regions of the glucocorticoid and estrogen receptors (GR and ER, respectively). However, in contrast to the DBDs of the GR and ER, the RXR DBD contains an additional helix immediately after the second zinc finger. This third helix mediates both protein-protein and protein-DNA interactions required for cooperative, dimeric binding of the RXR DBD to DNA. Identification of the third helix in the RXR DBD thus defines a structural feature required for selective dimerization of the RXR on hormone response elements composed of half-sites (5'-AGGTCA-3') arranged as tandem repeats.

A library of the urea type 6,6-bicyclic β-turn mimetics was conveniently prepared by using solid-phase chemistry with diversity at the i position in good yield and purity. The solution structure of the urea type scaffold was shown to mimic a type I β-turn structure.