Objective Preclinical and genetic epidemiologic studies suggest that modulating cytochrome P450 (CYP)-mediated arachidonic acid metabolism may have therapeutic utility in the management of coronary artery disease (CAD). However, predictors of inter-individual variation in CYP-derived eicosanoid metabolites in CAD patients have not been evaluated to date. Therefore, the primary objective was to identify clinical factors that influence CYP epoxygenase, soluble epoxide hydrolase (sEH), and CYP ω-hydroxylase metabolism in patients with established CAD. Methods Plasma levels of epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs), and 20-hydroxyeicosatetraenoic acid (20-HETE) were quantified by HPLC-MS/MS in a population of patients with stable, angiographically-confirmed CAD (N=82) and healthy volunteers from the local community (N=36). Predictors of CYP epoxygenase, sEH, and CYP ω-hydroxylase metabolic function were evaluated by regression. Results Obesity was significantly associated with low plasma EET levels and 14,15-EET:14,15-DHET ratios. Age, diabetes, and cigarette smoking also were significantly associated with CYP epoxygenase and sEH metabolic activity, while only renin-angiotensin system inhibitor use was associated with CYP ω-hydroxylase metabolic activity. Compared to healthy volunteers, both obese and non-obese CAD patients had significantly higher plasma EETs (P<0.01) and epoxide:diol ratios (P<0.01), whereas no difference in 20-HETE levels was observed (P=NS). Conclusions Collectively, these findings suggest that CYP-mediated eicosanoid metabolism is dysregulated in certain subsets of CAD patients, and demonstrate that biomarkers of CYP epoxygenase and sEH, but not CYP ω-hydroxylase, metabolism are altered in stable CAD patients relative to healthy individuals. Future studies are necessary to determine the therapeutic utility of modulating these pathways in patients with CAD.

Bacterial β-glucuronidase (GUS) enzymes cause drug toxicity by reversing Phase II glucuronidation in the gastrointestinal tract. While many human gut microbial GUS enzymes have been examined with model glucuronide substrates like p-nitrophenol-β-D-glucuronide (pNPG), the GUS orthologs that are most efficient at processing drug-glucuronides remain unclear. Here we present the crystal structures of GUS enzymes from human gut commensals Lactobacillus rhamnosus, Ruminococcus gnavus, and Faecalibacterium prausnitzii that possess an active site loop (Loop 1; L1) analogous to that found in E. coli GUS, which processes drug substrates. We also resolve the structure of the No Loop GUS from Bacteroides dorei. We then compare the pNPG and diclofenac glucuronide processing abilities of a panel of twelve structurally diverse GUS proteins, and find that the new L1 GUS enzymes presented here process small glucuronide substrates inefficiently compared to previously characterized L1 GUS enzymes like E. coli GUS. We further demonstrate that our GUS inhibitors, which are effective against some L1 enzymes, are not potent towards all. Our findings pinpoint active site structural features necessary for the processing of drug-glucuronide substrates and the inhibition of such processing.

Objective Accumulating preclinical and epidemiologic evidence has emerged to suggest that modulation of cytochrome P450 (CYP)-mediated eicosanoid metabolism may be a viable vascular protective therapeutic strategy for the secondary prevention of coronary artery disease (CAD). The functional relationship between CYP-derived eicosanoid metabolite levels and vascular dysfunction in humans with established CAD, however, has not been evaluated. Therefore, we characterized the relationship between inter-individual variation in soluble epoxide hydrolase (sEH) and CYP ω-hydroxylase metabolism and established vascular function phenotypes predictive of prognosis in a cohort of patients with atherosclerotic cardiovascular disease. Methods Plasma epoxyeicosatrienoic acid (EET), dihydroxyeicosatrienoic acid (DHET), and 20-hydroxyeicosatetraenoic acid (20-HETE) levels were quantified by HPLC-MS/MS in 106 patients with stable, angiographically-confirmed CAD. Relationships between biomarkers of CYP-mediated eicosanoid metabolism and vascular function phenotypes were evaluated by Pearson’s correlation. Results A significant inverse association was observed between 20-HETE levels (a biomarker of CYP ω-hydroxylase metabolism) and brachial artery flow-mediated dilation (r = −0.255, p = 0.010). An inverse association was also observed between 14,15-EET:DHET ratios (a biomarker of sEH metabolism) and both monocyte chemoattractant protein-1 levels (r = −0.252, p = 0.009) and a consolidated cellular adhesion molecule ‘score’ reflecting the levels of E-selectin and P-selectin (r = −0.216, p = 0.027). No associations with C-reactive protein or epithelial neutrophil-activating protein-78 levels were observed. Conclusions Collectively, these findings demonstrate that enhanced CYP ω-hydroxylase and sEH metabolic function are associated with more advanced endothelial dysfunction and vascular inflammation, respectively, in patients with established atherosclerotic cardiovascular disease. These findings lay the foundation for future clinical research in this area.

Digital peripheral arterial tonometry (PAT) is an emerging, non-invasive method to assess vascular function. The physiology underlying this phenotype, however, remains unclear. Therefore, we evaluated the relationship between digital PAT and established brachial artery ultrasound measures of vascular function under basal conditions and following reactive hyperemia. Using a cross-sectional study design, digital PAT and brachial artery ultrasound with pulsed wave Doppler were simultaneously completed at baseline and following reactive hyperemia in both individuals with established coronary artery disease (n=99) and healthy volunteers at low cardiovascular disease risk (n=40). Under basal conditions, the digital pulse volume amplitude demonstrated a significant positive correlation with the brachial artery velocity-time integral, that was independent of arterial diameter, in both the healthy volunteer (rs=0.64, P<0.001) and coronary artery disease (rs=0.63, P<0.001) cohorts. Similar positive relationships were observed with baseline brachial artery blood flow velocity and blood flow. In contrast, no relationship between the reactive hyperemia-evoked digital PAT ratio and either brachial artery flow-mediated dilation or shear stress was observed in either cohort (P=NS). In conclusion, these findings demonstrate that digital PAT measures of vascular function more closely reflect basal blood flow in the brachial artery than reactive hyperemia-induced changes in arterial diameter or flow velocity, and the presence of vascular disease does not modify the physiology underlying the digital PAT phenotype.

Adalimumab and Infliximab are recombinant IgG1 monoclonal antibodies (mAbs) that bind and neutralize human tumor necrosis factor alpha (TNFα). TNFα forms a stable homotrimer with unique surface-exposed sites for Adalimumab, Infliximab, and TNF receptor binding. Here, we report the structures of Adalimumab-TNFα and Infliximab-TNFα complexes modeled from negative stain EM and cryo-EM images. EM images reveal complex structures consisting of 1:1, 1:2, 2:2, and 3:2 complexes of Adalimumab-TNFα and Infliximab-TNFα. The 2:2 complex structures of Adalimumab-TNFα and Infliximab-TNFα show diamond-shaped profiles and the 2D class averages reveal distinct orientations of the Fab domains, indicating different binding modes by Adalimumab and Infliximab to TNFα. After separation by size exclusion chromatography and analysis by negative stain EM, the 3:2 complexes of Adalimumab-TNFα or Infliximab-TNFα complexes are more complicated but retain features recognized in the 2:2 complexes. Preliminary cryo-EM analysis of 3:2 Adalimumab-TNFα complex generated a low-resolution density consistent with a TNFα trimer bound with three Fab domains from three individual antibody molecules, while each antibody molecule binds to two molecules of TNFα trimer. The Fc domains are not visible in the reconstruction. These results show the two mAbs form structurally distinct complexes with TNFα.