Fatty acid glycolates are nanomolar inhibitors of small cell lung carcinoma peptidylglycine a-amidating monooxygenase, with binding affinities orders of magnitude stronger than those of the analogous thioesters, the corresponding substrate glycine derivatives or the natural substrate procalcitonin. Peptidylglycine a-amidating monooxygenase (PAM) is a bifunctional enzyme, which catalyses the final step in the biosynthesis of C-terminal peptide amides, being oxidative cleavage of their corresponding glycine-extended precursors (Scheme 1).1–5 The peptidylglycine a-hydroxylating monooxygenase (PHM) and peptidylamidoglycolate lyase (PAL) subunits respectively induce the formation and reaction of the intermediate a-hydroxylated glycine derivatives. Amidated peptides produced in this manner include a wide variety of mammalian peptide hormones and have been implicated in a broad range of pathological conditions, such as asthma,6 inflammation7 and cancer.8 PAM is also responsible for the biosynthesis of non-peptide amides including fatty acid amides such as oleamide, which affects sleep regulation.9 The important role of PAM combined with the fact that amidated peptide hormones are often thousands of times more active than their glycine-extended precursors has led to significant interest in its regulation.10–25 trans-4-Phenylbut-3-enoic acid is an irreversible, mechanismbased inhibitor of PAM.10–12 It is reported to have a KI value of 96 nM against PAM of prostate cancer cells13 and is effective in vivo in reducing serum PAM activity14 as well as inducing antiinflammatory and analgesic effects.15,16 Other compounds of this class show at best micromolar activity as PAM substrates and inhibitors,17 as is the case with several other compound types,18–22 and only homocysteine derivatives that act by binding to active site copper in the PHM subunit are known to show potency down to 10 nM.23 A variety of substrate analogues having a glycolate instead of a glycine are reported to have KI or IC50 values26 against PAM ranging from 40–580 mM.24,25 Of these, the fatty acid derivative 1a was found to be marginally the most potent, prompting us to use it as the basis for further structure–activity-relationship studies with other fatty acid glycolates as well as with thioesters. Our earlier work25 had used only Xenopus laevis (frog) PAM but in this study we also evaluated the compounds using PAM secreted into the media and extracted from cultured human cancer cell lines. We were specifically interested in identifying more potent and selective PAM inhibitors, more potent as they would be expected to be physiologically active at lower doses, and more selective for sub-types of PAM associated with particular disease states, so as to avoid complications associated with generic inhibition of PAM. Accordingly, we focused on human cancer cell lines where high levels of PAM have been reported. We now report that the glycolate 1a inhibits the enzyme extracted from H889 small cell lung carcinoma cells with an IC50 value of 50 nM. This represents an increase in binding affinity of almost three orders of magnitude over that seen for the glycolate 1a with frog PAM, whereas very little increase is seen with either the corresponding thioester 1b or substrate 1c. The binding affinity of the glycolate 1a (IC50 50 nM) with H889 PAM is also 2–3 orders of magnitude greater than that of the corresponding thioester 1b (IC50 7 mM) and substrate 1c (KM,app 200 mM), and the glycine-extended precursor of the natural growth hormone calcitonin (KM,app 300 mM). In addition, the glycolate 1a shows much greater potency against PAM extracted from mammalian cells than that secreted into the cell media. Similar patterns of reactivity are seen with the glycolates 2a, 3a and 5c, the thioesters 2b and 3b, and the glycine derivatives 2c and 3c, establishing that glycolates are peculiarly potent and selective inhibitors of human cancer cellular PAM.