Inhibition of the plasma glycosylphosphatidylinositol-specific phospholipase D by synthetic analogs of lipid A and phosphatidic acid

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), a plasma e nzyme with extensive sequence similarity to integrin alpha subunits, is inh ibited by micromolar concentrations of lipid A, phosphatidic acid (PA) and lysophosphatidic acid (M. G. Low and K.-S. Huang, J. Biol. Chem, 268, 8480- 8490, 1993), In this study we have explored the mechanism of inhibition usi ng synthetic analogs of lipid A, and PA. Monosaccharide analogs of lipid A, which varied in the number and position of the phosphate groups, the type of acyl group, and its linkage to the glucosamine ring, were tested for the ir ability to inhibit GPI-PLD, A compound (SDZ 880.431) containing 3-aza-gl ucosamine 1,4-diphosphate as the polar headgroup was identified which had a potency (IC, similar to 1 mu M) similar to natural lipid A preparations. R emoval of either phosphate residue increased the IC50 markedly. Analogs of PA such as (7-nitro-2-1,3-benzoxadiazo-4-yl)amino-PA, ceramide l-phosphate, and hexadecyl phosphate had similar to IC50 values ranging from 1 to 5 mu M, indicating that considerable variation in the structure of the hydrophob ic groups was permissible. Inhibition of GPI-PLD by long-chain PA could not be blocked by high concentrations of glycerol 1-phosphate or dibutyryl PA. These results indicate that the hydrophobic groups do not have a passive r ole in inhibition but are directly involved in the binding interaction with GPI-PLD, We propose that this diverse group of inhibitors all bind to a co mmon site on GPI-PLD, the central hydrophobic cavity predicted by the beta- propeller model for integrin alpha subunits and GPI-PLD. (C) 1999 Academic Press.