Activated Ketones as Inhibitors of Intracellular Ca2+-Dependent and Ca2+-Independent Phospholipase A2

• Published in: Journal of the American Chemical Society Vol. 118; no. 24; pp. 5519 - 5525
• Main Authors: Conde-Frieboes, Kilian, Reynolds, Laure J, Lio, Yi-Ching, Hale, Michael R, Wasserman, Harry H, Dennis, Edward A
• Format: Journal Article
• Language: English
• Published: American Chemical Society 19.06.1996
• DOI: 10.1021/ja953553w
• ISSN: 0002-7863

Studies are reported on two different types of activated ketones as inhibitors of two important intracellular phospholipase A2s (PLA2):  the group IV 85 kDa Ca2+-dependent phospholipase A2 (cPLA2) and the P388D1 Ca2+-independent phospholipase A2 (iPLA2). In a mixed micelle assay, we observed that the reaction progress curve of cPLA2 in the presence of a trifluoromethyl ketone (TFMK) is linear at pH 7.4, while at pH 9.0 it is nonlinear and slows with time. An investigation of this discrepancy demonstrated that the TFMKs are slow, tight-binding inhibitors of the cPLA2 at both pH's, that the rate of dissociation of the enzyme−inhibitor complex is the same at both pH's, but that the rate of association of enzyme and inhibitor is slower at pH 7.4 than at pH 9.0. A novel group of activated ketone inhibitors has been synthesized that contain a fatty acyl tricarbonyl. These compounds also inhibit the cPLA2 in the mixed micelle assay. The inhibition of cPLA2 by the tricarbonyls is readily reversible upon dilution and does not involve slow binding. For both types of inhibitor, no preference for fatty acid chain was observed as the palmityl analogs inhibited as well as the arachidonoyl analogs, despite the fact that the cPLA2 shows a strong preference for arachidonoyl-containing phospholipid substrates over palmitoyl-containing substrates. With the iPLA2, the inhibition by TFMKs is reversible and does not involve slow or tight binding. The tricarbonyls also inhibited the iPLA2, but were less potent than the TFMKs. Unlike the cPLA2, the iPLA2 does exhibit a fatty acid preference as the palmityl analogs of both compounds inhibit better than the arachidonoyl analogs. The palmityl TFMK displayed a 10-fold lower IC50 at pH 9.0 than at pH 7.5, whereas the potency of the tricarbonyl was unchanged in this range. Thus, the TFMKs inhibit both the cPLA2 and the iPLA2, but the mechanism of inhibition of the two enzymes appears to be quite different. The tricarbonyls also inhibited both enzymes, but in both cases in a reversible manner and as such appear to be poorer inhibitors than the TFMKs.