Mass of tryptic peptide containing Cys126 of hNAAA after covalent modification.

<p>T10-β peptides identified in the tryptic digest of untreated (control) and AM6701 or N-Cbz-serine β-lactone treated hNAAA samples.</p

MALDI-TOF MS/MS analysis of the hNAAA tryptic peptide T10-β after covalent modification.

<p>Tandem MALDI-TOF MS/MS spectra of the T10-β peptide (sequence: CTSIVAQDSR) demonstrates covalent modification of Cys126 by both AM6701 (Panel (A)) and <i>N-</i>Cbz-serine β-lactone (Panel (B)).</p

Representation of the active site of hNAAA after treatment with <i>N-</i>Cbz-serine β-lactone.

<p>Homology model illustrates acylated catalytic nucleophile Cys126 after treatment with <i>N-</i>Cbz-serine β-lactone.</p

Putative mechanism of inhibition of hNAAA for three compounds studied.

<p>Panel (A). Reversible inhibition of hNAAA by AM9023. Panel (B). Irreversible inhibition of hNAAA by AM6701 via thiocarbamylation of Cys126. Panel (C). Irreversible inhibition of hNAAA by <i>N-</i>Cbz-serine β-lactone most likely proceeds via route 2.</p

Potencies of hNAAA inhibitors.

<p>The <i>k</i>_inact and <i>K</i>_I values for the covalent inhibitors were obtained as described in the Experimental Procedures. The IC₅₀ values were calculated after 2 hours preincubation of the enzyme and inhibitor before addition of the substrate. Values are averages ± SD of three independent experiments.</p

Binding Site Characterization of AM1336, a Novel Covalent Inverse Agonist at Human Cannabinoid 2 Receptor, Using Mass Spectrometric Analysis

Cannabinoid 2 receptor (CB2R), a Class-A G-protein coupled receptor (GPCR), is a promising drug target under a wide array of pathological conditions. Rational drug design has been hindered due to our poor understanding of the structural features involved in ligand binding. Binding of a high-affinity biarylpyrazole inverse agonist AM1336 to a library of the human CB2 receptor (hCB2R) cysteine-substituted mutants provided indirect evidence that two cysteines in transmembrane helix-7 (H7) were critical for the covalent attachment. We used proteomics analysis of the hCB2R with bound AM1336 to directly identify peptides with covalently attached ligand and applied in silico modeling for visualization of the ligand–receptor interactions. The hCB2R, with affinity tags (FlaghCB2His6), was produced in a baculovirus–insect cell expression system and purified as a functional receptor using immunoaffinity chromatography. Using mass spectrometry-based bottom-up proteomic analysis of the hCB2R-AM1336, we identified a peptide with AM1336 attached to the cysteine C284(7.38) in H7. The hCB2R homology model in lipid bilayer accommodated covalent attachment of AM1336 to C284(7.38), supporting both biochemical and mass spectrometric data. This work consolidates proteomics data and in silico modeling and integrates with our ligand-assisted protein structure (LAPS) experimental paradigm to assist in structure-based design of cannabinoid antagonist/inverse agonists

Representation of the active site of hNAAA after treatment with AM6701.

<p>Homology model illustrates thiocarbamylation of catalytic nucleophile Cys126 after treatment with AM6701.</p

Concentration dependent inhibition of purified hNAAA by three compounds.

<p>hNAAA was incubated with the compounds AM6701 (squares), <i>N-</i>Cbz-serine β-lactone (circles), and AM9023 (diamonds) for two hours in order to reach full inhibition before measuring activity. Panel (A). A radioactivity-based assay with [¹⁴C] PEA as substrate. Panel (B). A fluorescence-based assay with PAMCA as substrate. Representative curves are displayed.</p

Active-Site Inhibitors Modulate the Dynamic Properties of Human Monoacylglycerol Lipase: A Hydrogen Exchange Mass Spectrometry Study

Human monoacylglycerol lipase (hMGL) regulates endocannabinoid signaling primarily by deactivating the lipid messenger 2-arachidonoylglycerol. Agents that carbamylate hMGLs catalytic Ser¹²² constitute a leading class of therapeutically promising hMGL inhibitors. We have applied peptide-level hydrogen/deuterium exchange mass spectrometry to characterize hMGL’s conformational responses to two potent carbamylating inhibitors, AM6580 (irreversible) and AM6701 (slowly reversible). A dynamic, solvent-exposed lid domain is characteristic of hMGL’s solution conformation. Both hMGL inhibitors restricted backbone enzyme motility in the active-site region and increased substrate binding-pocket solvent exposure. Covalent reaction of AM6580 with hMGL generates a bulkier carbamylated Ser¹²² residue as compared to the more discrete Ser¹²² modification by AM6701, a difference reflected in AM6580’s more pronounced effect upon hMGL conformation. We demonstrate that structurally distinct carbamylating hMGL inhibitors generate particular conformational ensembles characterized by region-specific hMGL dynamics. By demonstrating the distinctive influences of two hMGL inhibitors on enzyme conformation, this study furthers our understanding at the molecular level of the dynamic features of hMGL interaction with small-molecule ligands

Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH, we show that 5-(4-hydroxyphenyl)­pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs