Mechanism of inhibition for N6022, a first-in-class drug targeting S-nitrosoglutathione reductase.

N6022 is a novel, first-in-class drug with potent inhibitory activity against S-nitrosoglutathione reductase (GSNOR), an enzyme important in the metabolism of S-nitrosoglutathione (GSNO) and in the maintenance of nitric oxide (NO) homeostasis. Inhibition of GSNOR by N6022 and related compounds has shown safety and efficacy in animal models of asthma, chronic obstructive pulmonary disease, and inflammatory bowel disease [Sun, X., et al. (2011) ACS Med. Chem. Lett. 2, 402-406]. N6022 is currently in early phase clinical studies in humans. We show here that N6022 is a tight-binding, specific, and fully reversible inhibitor of GSNOR with an IC(50) of 8 nM and a K(i) of 2.5 nM. We accounted for the fact that the NAD(+)- and NADH-dependent oxidation and reduction reactions, catalyzed by GSNOR are bisubstrate in nature in our calculations. N6022 binds in the GSNO substrate binding pocket like a competitive inhibitor, although in kinetic assays it behaves with a mixed uncompetitive mode of inhibition (MOI) toward the GSNO substrate and a mixed competitive MOI toward the formaldehyde adduct, S-hydroxymethylglutathione (HMGSH). N6022 is uncompetitive with cofactors NAD(+) and NADH. The potency, specificity, and MOI of related GSNOR inhibitor compounds are also reported.

Greenhouse gas fluxes from turfgrass systems: Species, growth rate, clipping management, and environmental effects.

Turfgrass systems can be an important source or sink for greenhouse gases (GHG), including carbon dioxide (CO2 ), nitrous oxide (N2 O), and methane (CH4 ). Further research is required in turfgrass systems; therefore, our objectives were to evaluate the effects of turfgrass species, growth rate, clipping management, and environmental conditions on GHG emissions. Greenhouse gas fluxes were measured in two separate field experiments in West Lafayette, IN. Experiment 1 investigated GHG flux in three cool-season (C3 ) and two warm-season (C4 ) turfgrass species during two growing seasons. Experiment 2 investigated fluxes in two C3 cultivars with varying growth rates and under different clipping management regimes. The C3 turfgrasses had the highest mean CO2 flux rates ranging from 0.373 to 0.431 g CO2 -C m-2 h-1 compared with 0.273 to 0.361 g CO2 -C m-2 h-1 for C4 turfgrasses. Mean hourly N2 O flux rates ranged from 43.3 to 50.9 µg N2 O-N m-2 h-1 for C3 compared with 11.1 to 14.4 µg N2 O-N m-2 h-1 for C4 turfgrasses. Methane flux was more variable across time, but overall C4 turfgrasses were more likely to be a CH4 source, whereas C3 turfgrasses were often a CH4 sink. Growth rate and grass clipping management treatments had negligible impact on measured GHG flux. The differences in management practices specific to C3 and C4 turfgrasses had the largest impact on GHG flux. Results indicate the impact and importance of turfgrass species selection on GHG flux and also provide more information on our overall understanding on carbon and nitrogen cycling in urban soils.

Significant changes in mitral valve leaflet matrix composition and turnover with tachycardia-induced cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) involves significant remodeling of the left ventricular-mitral valve (MV) complex, but little is known regarding the remodeling of the mitral leaflets. The aim of this study was to assess changes in matrix composition and turnover in MV leaflets with DCM. METHODS AND RESULTS: Radiopaque markers were implanted in 24 sheep to delineate the MV; 10 sheep underwent tachycardia-induced cardiomyopathy (TIC), whereas 14 sheep remained as controls. Biplane videofluoroscopy was performed before and after TIC. Immunohistochemistry was performed on leaflet cross-sections taken from the septal, lateral, anterior, and posterior commissures attachment segments. Staining intensity was quantified within each attachment segment and leaflet region (basal, mid-leaflet, and free edge). Mitral regurgitation increased from 0.2+/-0.4 before TIC to 2.2+/-0.9 after TIC (P<0.0002). TIC leaflets demonstrated significant remodeling compared to controls, including greater cell density and loss of leaflet layered structure (all P<0.05). Collagen and elastic fiber turnover was greater in TIC, as was the myofibroblast phenotype (all P<0.05). Compositional differences between TIC and control leaflets were heterogeneous by annular segment and leaflet region, and related to regional changes in leaflet segment length with TIC. CONCLUSIONS: This study shows that the MV leaflets are significantly remodeled in DCM with changes in leaflet composition, structure, and valve cell phenotype. Understanding how alterations in leaflet mechanics, such as those induced by DCM, drive cell-mediated remodeling of the extracellular matrix will be important in developing future treatment strategies.

An acid dissociation bridging ELISA for detection of antibodies directed against therapeutic proteins in the presence of antigen.

Bridging Enzyme-Linked Immunosorbent Assay (ELISA) is commonly used in detection of antibodies directed against therapeutic proteins. Advantages of the bridging ELISA include the capability to detect antibodies regardless of their isotype or the species of origin. However, detection of antibodies can be difficult, if not impossible, in the presence of high levels of the antigen in the sample matrix. This protocol describes a bridging ELISA that uses a covalently coupled high density antigen surface combined with an acid dissociation step to allow for antibody detection in the presence of antigen in human serum.

Cellular and Extracellular Matrix Basis for Heterogeneity in Mitral Annular Contraction.

PURPOSE: Regional heterogeneity in mitral annular contraction, which is generally ascribed to the fibrous vs. muscular annular composition, ensures proper leaflet motion and timing of coaptation. It is unknown whether the fibroblast-like cells in the annulus modulate this heterogeneity, even though valvular interstitial cells (VICs) can be mechanically "activated." METHODS: Fourteen sheep underwent implantation of radiopaque markers around the mitral annulus defining four segments: septal (SEPT), lateral (LAT), and anterior (ANT-C) and posterior (POST-C) commissures. Segmental annular contraction was calculated using biplane videofluoroscopy. Immunohistochemistry of annular cross sections assessed regional matrix content, matrix turnover, and cell phenotype. Micropipette aspiration measured the Young's modulus of the leaflets adjacent to the myocardial border. RESULTS: Whereas SEPT contained more collagen I and III, LAT demonstrated more collagen and elastin turnover as shown by greater decorin, lysyl oxidase, and matrix metalloprotease (MMP)-13 and smooth muscle alpha-actin (SMaA). This greater matrix turnover paralleled greater annular contraction in LAT vs. SEPT (22.5% vs. 4.1%). Similarly, POST-C had more SMaA and MMP13 than ANT-C, consistent with greater annular contraction in POST-C (18.8% vs. 11.1%). Interestingly, POST-C had the greatest effective modulus, significantly higher than LAT. CONCLUSIONS: These data suggest that matrix turnover by activated VICs relates to annular motion heterogeneity, maintains steady-state mechanical properties in the annulus, and could be a therapeutic target when annular motion is impaired. Conversely, alterations in this heterogeneous annular contraction, whether through disease or secondary to ring annuloplasty, could disrupt this normal pattern of cell-mediated matrix remodeling and further adversely impact mitral valve function.

Heterogeneity of Mitral Leaflet Matrix Composition and Turnover Correlates with Regional Leaflet Strain.

To determine how extracellular matrix and contractile valvular cells contribute to the heterogeneous motion and strain across the mitral valve (MV) during the cardiac cycle, regional MV material properties, matrix composition, matrix turnover, and cell phenotype were related to regional leaflet strain. Radiopaque markers were implanted into 14 sheep to delineate the septal (SEPT), lateral (LAT), and anterior and posterior commissural leaflets (ANT-C, POST-C). Videofluoroscopy imaging was used to calculate radial and circumferential strains. Mechanical properties were assessed using uniaxial tensile testing and micropipette aspiration. Matrix composition and cell phenotypes were immunohistochemically evaluated within each leaflet region [basal leaflet (BL), mid-leaflet (ML), and free edge]. SEPT-BL segments were stiffer and stronger than other valve tissues, while LAT segments demonstrated more extensibility and strain. Collagens I and III in SEPT were greater than in LAT, although LAT showed greater collagen turnover [matrix metalloprotease (MMP)-13, lysyl oxidase] and cell activation [smooth muscle alpha-actin (SMaA), and non-muscle myosin (NMM)]. MMP13, NMM, and SMaA were strongly correlated with each other, as well as with radial and circumferential strains in both SEPT and LAT. SMaA and MMP13 in POST-C ML was greater than ANT-C, corresponding to greater radial strains in POST-C. This work directly relates leaflet strain, material properties, and matrix turnover, and suggests a role for myofibroblasts in the heterogeneity of leaflet composition and strain. New approaches to MV repair techniques and ring design should preserve this normal coupling between leaflet composition and motion.

Pharmacological inhibition of S-nitrosoglutathione reductase improves endothelial vasodilatory function in rats in vivo.

Nitric oxide (NO) exerts a wide range of cellular effects in the cardiovascular system. NO is short lived, but S-nitrosoglutathione (GSNO) functions as a stable intracellular bioavailable NO pool. Accordingly, increased levels can facilitate NO-mediated processes, and conversely, catabolism of GSNO by the regulatory enzyme GSNO reductase (GSNOR) can impair these processes. Because dysregulated GSNOR can interfere with processes relevant to cardiovascular health, it follows that inhibition of GSNOR may be beneficial. However, the effect of GSNOR inhibition on vascular activity is unknown. To study the effects of GSNOR inhibition on endothelial function, we treated rats with a small-molecule inhibitor of GSNOR (N6338) that has vasodilatory effects on isolated aortic rings and assessed effects on arterial flow-mediated dilation (FMD), an NO-dependent process. GSNOR inhibition with a single intravenous dose of N6338 preserved FMD (15.3 ± 5.4 vs. 14.2 ± 6.3%, P = nonsignificant) under partial NO synthase inhibition that normally reduces FMD by roughly 50% (14.1 ± 2.9 vs. 7.6 ± 4.4%, P < 0.05). In hypertensive rats, daily oral administration of N6338 for 14 days reduced blood pressure (170.0 ± 5.3/122.7 ± 6.4 vs. 203.8 ± 1.9/143.7 ± 7.5 mmHg for vehicle, P < 0.001) and vascular resistance index (1.5 ± 0.4 vs. 3.2 ± 1.0 mmHg · min · l(-1) for vehicle, P < 0.001), and restored FMD from an initially impaired state (7.4 ± 1.7%, day 0) to a level (13.0 ± 3.1%, day 14, P < 0.001) similar to that observed in normotensive rats. N6338 also reversed the pathological kidney changes exhibited by the hypertensive rats. GSNOR inhibition preserves FMD under conditions of impaired NO production and protects against both microvascular and conduit artery dysfunction in a model of hypertension.

Programmable bio-nanochip technology for the diagnosis of cardiovascular disease at the point-of-care.

Cardiovascular disease remains the leading cause of death in the world and continues to serve as the major contributor to healthcare costs. Likewise, there is an ever-increasing need and demand for novel and more efficient diagnostic tools for the early detection of cardiovascular disease, especially at the point-of-care (POC). This article reviews the programmable bio-nanochip (P-BNC) system, a new medical microdevice approach with the capacity to deliver both high performance and reduced cost. This fully integrated, total analysis system leverages microelectronic components, microfabrication techniques, and nanotechnology to noninvasively measure multiple cardiac biomarkers in complex fluids, such as saliva, while offering diagnostic accuracy equal to laboratory-confined reference methods. This article profiles the P-BNC approach, describes its performance in real-world testing of clinical samples, and summarizes new opportunities for medical microdevices in the field of cardiac diagnostics.

Pharmacokinetics and safety of a fully human hepatocyte growth factor antibody, AMG 102, in cynomolgus monkeys.

PURPOSE: AMG 102, a fully human monoclonal antibody that binds to hepatocyte growth factor (HGF), is a potential cancer therapeutic agent because of its ability to disrupt HGF/c-Met signaling pathways which have been implicated in most tumor types. To support a phase 1 study, the pharmacokinetic and safety profile of AMG 102 was assessed in cynomolgus monkeys. MATERIALS AND METHODS: Serum concentration-time data from single- (i.v. and s.c.) and repeated-dose (i.v.) studies of up to 13 weeks were used for pharmacokinetic analysis. Safety was assessed in a single-dose safety pharmacology study with i.v. doses of 0 (vehicle), 25, 100, or 300 mg/kg and a 4-week toxicity study with once weekly i.v. doses of 0 (vehicle), 5, 25, or 100 mg/kg. RESULTS: AMG 102 exhibited linear pharmacokinetics over a 600-fold dose range (0.5 to 300 mg/kg) with a mean terminal half-life of 5.6 days after i.v. dosing. Clearance and volume of distribution at steady state were 1.22 ml/h and 198.3 ml, respectively. Estimated bioavailability was 72% for s.c. administration. Antibody response to AMG 102 was observed in a small percentage of monkeys. No treatment-related cardiovascular, respiratory, or CNS changes were observed. Administration of AMG 102 for 4 weeks was well tolerated at doses up to 100 mg/kg. Potential treatment-related effects were limited to minimal/moderate gastric mucosa hemorrhage in the mid- and high-dose groups. CONCLUSIONS: The nonclinical pharmacokinetic and safety profile of AMG 102 effectively supports clinical investigation.