Synthesis, Structure−Activity Relationships, and the Effect of Polyethylene Glycol on Inhibitors of Phosphatidylinositol-Specific Phospholipase C fromBacillus cereus

Abstract

Substrate analog inhibitors of Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) were synthesized and screened for their suitability to map the active site region of the enzyme by protein crystallography. Analogs of the natural substrate phosphatidylinositol (PI) were designed to examine the importance of the lipid portion and the inositol phosphate head group for binding to the enzyme. The synthetic compounds contained pentyl, hexyl, or hexanoyl and octyl lipid chains at the sn-1 and sn-2 positions of the glycerol backbone and phosphonoinositol, phosphonic acid, methyl phosphonate, phosphatidic acid, or methyl phosphate at the sn-3 position. The most hydrophobic compound, dioctyl methyl phosphate 14, was also the best inhibitor with an IC50 of 12 microM. In a series of dihexyl lipids, compounds with phosphonoinositol head groups inhibited more strongly than those that do not contain inositol but are otherwise identical. Compound 29, a short-chain lipid with a phosphonoinositol head group, was found to be a competitive inhibitor and the most potent in this series with an IC50 of 18 microM (Ki = 14 microM). Analogs with dihexyl chains were better inhibitors than those with dihexanoyl chains, presumably because the ether-linked lipids are more hydrophobic than the ester-linked lipids. No appreciable difference in inhibition was found between a phosphonoinositol lipid and the corresponding difluorophosphonoinositol lipid. Inositols and inositol derivatives that do not contain lipid moieties show IC50s about 3 orders of magnitude above those of the short-chain lipids. In this group, glucosaminyl(alpha 1-->6)-D-myo-inositol inhibited more strongly than myo-inositol, which in turn is a better inhibitor than inositol phosphate. The addition of polyethylene glycol (PEG-600) resulted in a marked decrease in inhibition by the short-chain lipids, but had little effect on the water-soluble head group analogs. This is accounted for in terms of solubilization of the amphipathic inhibitors by PEG. Since PEG is required in the crystallization, these data indicate that the best strategy for obtaining enzyme inhibitor complexes is to start by cocrystallizing PI-PLC with the head group analogs. The next step is to synthetically add the shortest possible hydrophobic moieties to the analogs and cocrystallize these with the enzyme. This strategy may be applicable to other lipolytic enzymes.

NMR structure of the bovine prion protein isolated from healthy calf brains

“…Expression and purification of Bacillus cereus PI‐PLC in E. coli B. cereus PI‐PLC was expressed and purified as previously described (Ryan et al , 1996).…”

Allosteric Interactions within Subsites of a Monomeric Enzyme: Kinetics of Fluorogenic Substrates of PI-Specific Phospholipase C

“…The fluorogenic substrates butyl-FLIP and methyl-FLIP were prepared as previously described (Zaikova et al, 2001). PI-PLC from B. cereus was prepared by the method of Ryan et al (1996) and is available from Molecular Probes (Eugene, OR). DiC 6 PC was obtained from Avanti Polar Lipids (Alabaster, AL).…”

Cysteine-less glycosylphosphatidylinositol-specific phospholipase C is inhibited competitively by a thiol reagent: evidence for glyco-mimicry by p-chloromercuriphenylsulphonate

“…PI-PLC binds to both the inositol and the glycan moieties of the substrate [36,37], and a GPI is a better substrate for PI-PLC than is PI [38][39][40]. Scission of the phosphodiester bond at the inositol 1-position is influenced by glycan recognition.…”