Active-site-directed specific competitive inhibitors of phospholipase A2: novel transition-state analogs

Abstract

More than 100 amphiphilic phosphoesters, possible tetrahedral transition-state analogues capable of coordinating to the calcium ion at the active site of phospholipase A2, were designed, synthesized, and tested as inhibitors for the hydrolysis of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol vesicles in the scooting mode. This assay system permits the study of structurally diverse inhibitors with phospholipase A2S from different sources, and it is not perturbed by factors that change the quality of the interface. As a prototype, 1-hexadecyl-3-trifluoroethylglycero-2-phosphomethanol (MJ33) was investigated in detail. Only the (S)-(+) analogue of MJ33 is inhibitory, and it is as effective as the sn-2 phosphonate or the sn-2 amide analogues of sn-3 phospholipids. The inhibitory potencies of the various phosphoesters depended strongly on the stereochemical and structural features, and the mole fractions of inhibitors required for 50% inhibition, X1(50), ranged from more than 1 to less than 0.001 mole fraction. The affinity of certain inhibitors for enzymes from different sources differed by more than 200-fold. The inhibitors protected the catalytic site residue His-48 from alkylation in the presence of calcium but not barium as expected if the formation of the EI complex is supported only by calcium. The equilibrium dissociation constant for the inhibitor bound to the enzyme at the interface was correlated with the XI(50) values, which were different if the inhibition was monitored in the pseudo-zero-order or the first-order region of the progress curve. These results show that the inhibitors described here interfered only with the catalytic turnover by phospholipase A2's bound to the interface, their binding to the enzyme occurred through calcium, and the inhibitors did not have any effect on the dissociation of the enzyme bound to the interface.

Crystal Structure of Human Group X Secreted Phospholipase A2

“…b The inhibitor concentrations that produce half-activity of DMPM hydrolysis, IC 50 , are reported in micromolar for Zn 2ϩ and MPD. For the other inhibitors, which are assumed to be completely partitioned in the DMPM interface, X I 50 is the mole fraction of the inhibitor that gives half-activity (18). The reported values of X I 50 are reproducible to within 10% of each value.…”

“…We also gain insight into structure-based design of inhibitors that are selective for this sPLA 2 . Attempts were made to obtain the structure with and without the active-site inhibitor 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33) (18). Two crystal forms were obtained that led to independent structures of hGX, both without bound inhibitor.…”

Arginine 53 is involved in head‐group specificity of the active site of porcine pancreatic phospholipase A2

“…The observed differences up to sevenfold in the Z values of the MyrAholPCho inhibitor are comparable to those of 1/K (compare Tables 3 and 4), suggesting that Z = SIK and K , S S. An exact estimation of K, cannot be given since knowledge of the rate constants in the micellar interface, used here, is not available. For a bilayer system, where the individual rate constants are available, Jain et al (1991) calculated a k", for Pam,GroPCho of 100 moll100 mol for porcine pancreatic PLA,. Surface dilution has been used by Dennis (1973) in an attempt to determine Ir, for N. nuju nuju venom PLA,.…”

A Novel Nontoxic Inhibitor of the Activation of NADPH Oxidase Reduces Reactive Oxygen Species Production in Mouse Lung

“…The compound is lipid soluble and somewhat soluble in aqueous media with a critical micelle concentration of approximately 10 mM. This agent has been shown to have specificity for PLA 2 , specifically pancreatic (type 1B) PLA 2 and Prdx6 PLA 2 , but does not inhibit cytosolic (type IV) PLA 2 , phospholipases C/D, or other lipases (Jain et al, 1991a;Fisher et al, 1992;Gelb et al, 1994;Kim et al, 1997). MJ33 is a chemically nonreactive compound that mimics the tetrahedral transition state of PLA 2 substrates and inhibits activity through its binding to the PLA 2 protein (Gelb et al, 1994).…”