Covalent Inhibitors of Human Monoacylglycerol Lipase: Ligand-Assisted Characterization of the Catalytic Site by Mass Spectrometry and Mutational Analysis

SummaryThe active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser129 by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve ∼80% enzyme inhibition. Although Cys215 and/or Cys249 mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys249 is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs

Novel Electrophilic and Photoaffinity Covalent Probes for Mapping the Cannabinoid 1 Receptor Allosteric Site(s)

ACKNOWLEDGMENTS The work was supported by National Institutes of Health grants DA027113 and EY024717 to G.A.T. and DA09158 to A.M. A portion of this work was submitted in 2011 by A. Kulkarni in partial fulfillment of M.S. degree requirements from Northeastern University, Boston, MA.Peer reviewedPublisher PD

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

Therapeutic Efficacy of an ω-3-Fatty Acid-Containing 17-β Estradiol Nano-Delivery System against Experimental Atherosclerosis.

Atherosclerosis and its consequences remain prevalent clinical challenges throughout the world. Initiation and progression of atherosclerosis involves a complex, dynamic interplay among inflammation, hyperlipidemia, and endothelial dysfunction. A multicomponent treatment approach targeted for delivery within diseased vessels could prove beneficial in treating atherosclerosis. This study was undertaken to evaluate the multimodal effects of a novel ω-3-fatty acid-rich, 17-β-estradiol (17-βE)-loaded, CREKA-peptide-modified nanoemulsion system on experimental atherosclerosis. In vitro treatment of cultured human aortic endothelial cells (ECs) with the 17-βE-loaded, CREKA-peptide-modified nanoemulsion system increased cellular nitrate/nitrite, indicating improved nitric oxide formation. In vivo, systemic administration of this nanoemulsion system to apolipoprotein-E knock out (ApoE-/-) mice fed a high-fat diet significantly improved multiple parameters related to the etiology and development of occlusive atherosclerotic vasculopathy: lesion area, circulating plasma lipid levels, and expression of aortic-wall inflammatory markers. These salutary effects were attributed selectively to the 17-βE and/or ω-3 polyunsaturated fatty acid components of the nano-delivery system. At therapeutic doses, the 17-βE-loaded, CREKA-peptide modified nanoemulsion system appeared to be biocompatible in that it elicited no apparent adverse/toxic effects, as indexed by body weight, plasma alanine aminotransferase/aspartate aminotransferase levels, and liver and kidney histopathology. The study demonstrates the therapeutic potential of a novel, 17-βE-loaded, CREKA-peptide-modified nanoemulsion system against atherosclerosis in a multimodal fashion by reducing lesion size, lowering the levels of circulating plasma lipids and decreasing the gene expression of inflammatory markers associated with the disease

<i>In vivo</i> safety profile evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system.

<p>(A) Body weights of control mice fed a high-fat diet and mice fed a high-fat diet and treated with either the blank nanoemulsion, 17-βE in solution, or the 17-βE-loaded CREKA-peptide-modified nanoemulsion. (n = 8 independent animals per group). (B) Histology of liver and kidney tissues isolated from the control (untreated) and specified treatment groups. (n = 6 independent animals per group).</p

Characterization of the uptake of the 17-βE-loaded CREKA-peptide-modified nanoemulsion system and nitric oxide production by human aortic ECs in culture.

<p>(A) Uptake and distribution of the rhodamine-123 dye labeled CREKA-peptide modified nanoemulsion system in cultured human aortic ECs. (B) Transmission electron microscopy of CREKA-peptide modified 17-βE loaded nanoemulsion formulation. The nanoemulsion is spherical with diameter of ~150nm. Scale bar is 500 nm. (C) Effect of 17-βE solution and nanoemulsion on nitric oxide production (nitrate plus nitrite) by cultured human aortic ECs. Data reported as mean ± S.D. for n = 4 independent samples. The asterisk represents statistical significance (<i>P</i> < 0.05) between untreated control cells and the different solution and CREKA-peptide-modified nanoemulsion-based treatments indicated.</p

<i>In vivo</i> histological evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system on plaque elastin and smooth muscle cell content (A) Elastin staining of the aortic valves and plaque elastin analysis quantified using Image J (n = 4 independent animals per group).

<p>There was no significant difference in the elastin content across treatment groups. (B) SMA staining of the aortic valves and plaque SMC content analysis quantified using Image J (n = 4 independent animals per group). There was no significant difference in the elastin content across treatment groups.</p

<i>In vivo</i> histological evaluation of systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system on plaque size and lipid content.

<p>(A) H & E staining of the aortic valves and plaque area analysis quantified using Image J (n = 4 independent animals per group). The asterisk represents statistical significance between the untreated treatment group and the different 17-βE solution, 17-βE nanoemulsion and blank nanoemulsion treatment groups (<i>P</i> < 0.05). (B) Oil-red-O staining of the aortic valves and plaque lipid analysis quantified using Image J (n = 4 independent animals per group). The asterisk represents statistical significance between the untreated treatment group and the different 17-βE solution, 17-βE nanoemulsion and blank nanoemulsion treatment groups (<i>P</i> < 0.05).</p

Systemic administration of 17-βE as a solution and a CREKA-peptide-modified nanoemulsion system alters atherosclerosis-related gene expression within the aorta and improves plasma lipid profile.

<p>(A) The effect of 17-βE in solution, 17-βE in nanoemulsion, and the blank nanoemulsion on relative gene expression in ApoE^-/- mice receiving a high-fat diet measured relative to gene expression in wild-type mice. The asterisk indicates statistical significance (<i>P</i> < 0.05) relative to the baseline gene expression measured in the wild-type mice. (n = 4 independent animals per group). (B) Total plasma cholesterol levels measured in the untreated and the different treatment groups at the beginning and end of the efficacy study. Values are represented as means ± SD (n = 6 independent animals per group). The asterisk represents significance between the untreated animals and the different treatment groups (<i>P</i> < 0.05). (C) Total plasma triglyceride levels measured in the untreated and the different treatment groups at the beginning and end of the efficacy study. Values are represented as means ± SD (n = 6 independent animals per group). The asterisk represents significance between the untreated animals and the different treatment groups (<i>P</i> < 0.05).</p