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

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

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

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

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