Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase

Abstract

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.

Rapid detection, discovery, and identification of post‐translationally myristoylated proteins during apoptosis using a bio‐orthogonal azidomyristate analog

“…We found that, like [ 3 H]myristate, the incorporation of azidomyristate into immunoprecipitated ctPAK2 was abolished in the presence of the NMT inhibitor HMA (19) (Fig. 3A), suggesting that the azidomyristate is recognized as a substrate by the highly selective NMT (5).…”

“…3A), suggesting that the azidomyristate is recognized as a substrate by the highly selective NMT (5). Of note, when incorporated into cells, HMA is metabolically activated to 2-hydroxymyristoyl-CoA, a specific inhibitor of NMT that does not inhibit protein palmitoylation (17)(18)(19). While preventing the incorporation of [ 3 H]myristate or azidomyristate into ctPAK2 in cells induced to undergo apoptosis, HMA did not impair caspase 3-mediated PAK2 cleavage on STS treatment (data not shown).…”

Inhibition of Protein Palmitoylation, Raft Localization, and T Cell Signaling by 2-Bromopalmitate and Polyunsaturated Fatty Acids

“…3B). Furthermore, 2-hydroxymyristate, a known inhibitor of myristoylation (41,42), inhibits membrane localization of Fyn and Fyn (16)-eGFP to a greater extent than 2-bromopalmitate (Fig. 2B).…”

SOS3 Function in Plant Salt Tolerance Requires N-Myristoylation and Calcium Binding

“…To determine whether protein N-myristoylation might be important for plant salt tolerance, we first tested the effect of HMA, a competitive inhibitor of N-myristoyltransferase (Paige et al, 1990;Nadler et al, 1993;Galbiati et al, 1996). One-week-old Arabidopsis seedlings were treated with 1 mM HMA for 1 day and then transferred to grow on a regular nutrient medium or on a medium supplemented with 100 mM NaCl.…”