410

Blochimicu

et Biophysicu

Acta, 1042 (1990) 410-412

Elsevier

BBALIP

BBA Report

50285

Inhibition

of phosphatidylinositol-specific phospholipase by phosphonate substrate analogues

M.S. Shashidhar, Instriute

Johannes

of Molecular

Biology

J. Volwerk, John F.W. Keana and 0. Hayes Griffith and Department

of Chumstry,

(Received

Key words:

Phospholipase

C

3 August

C, phosphatidylinositol-specific;

lJnruet5t.y

of Oregon,

Eugene, OR (U.S.A.)

1989)

Inhibition;

Phosphonate;

Substrate

analog;

(Bacillus

cereus)

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositolspecific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids.

Phospholipase C specific for inositol phospholipids (PI-PLC) has been the subject of intense investigations in recent years [l-8]. This ubiquitous enzyme functions as a signal amplifier in the inositol phospholipid-dependent transmembrane signal transduction of eukaryotic cells by generating the intracellular second messengers, diacylglycerol and inositol phosphates in response to the binding of hormones, growth factors, neurotransmitters and other agonists to specific receptors on the external surface of the cell [9,10]. PI-PLC activity is also found in the culture media of several bacteria, including Bacillus cereus [ 11 ,121, Bacillus thuringiensis [l 1,131, Stuphylococcus aureus [14] and Clostridium nouyi [15]. The bacterial enzyme is of interest because of its ability to cleave the glycosyl-phosphatidylinositol moieties which constitute the membrane anchors of a novel and rapidly expanding class of membrane proteins [16-181. Several eukaryotic (reviewed in Ref. 1) and two microbial (Refs. 19 and 20) PI-PLC’s have been recently cloned and sequenced, but to date there is little information available regarding the mechanism of action of these important enzymes. Here, we report the inhibition of phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from B. cereus by three phosphonate-containing analogues (l-3) of phosphatidylinositol.

Correspondence: 0. Hayes Griffith, Institute of Molecular Umversity of Oregon, Eugene, OR 97403, U.S.A. 0005.2760/90/$03.50

“, 1990 Elsevier Science Publishers

Biology,

B.V. (Biomedical

The design and preparation of specific, high-affinity inhibitors has proven to be a valuable tool in the study of enzyme function and mechanism (for example, see Refs. 21-23). Inhibitory substrate analogues, incorporated into affinity matrices, can provide a means for facile isolation [24] and/or localization [25] of target proteins. Inhibitor 2, specifically designed with this application in mind, contains a carboxylate functional group suitable for matrix attachment. In addition, enzyme inhibitors also are of medical interest because of their potential as pharmacological agents (for example, see [26-281). The structures of phosphonate analogues l-3, along with that of PI (for comparison) are shown below *. In these new inhibitors, the PI-PLC-hydrolysable phosphodiester bond of PI (see arrow) has been replaced by the chemically stable phosphonate linkage, while a simpler hydrophobic group replaces the diacylglycerol moiety found in natural PI. The phosphonate analogues l-3 were obtained in good yield by coupling of the corresponding phosphonic acids with rucemic-2,3,4,5,6penta-0benzyl-myo-inositol, followed by deprotection and conversion to the free acid by ion-exchange. Racemic-2,3,4,5,6-Penta-0-benzyl-myo-inositol, was

* Phosphonates l-3 were prepared in racemic form. Separation of the optical isomers of the penta-protected inositol intermediate has been described [29] and is currently being used to prepared D- and L-myo-inositol phosphonate analogues. Division)

411

Phosphatidylinositol

(PI)

R, = R2 = fatty alkyl chain

l,R=H-

2, R = H&CH2)5CHi3, R =

CH3(CH2),+

synthesized following a route modified from the one described [30], which resulted in a product completely free of contaminating isomers. Details of the synthesis of the penta-protected inositol, phosphonates 1-3 and complete analytical data for all final products and intermediates are reported elsewhere [31].

1

I

-INoe1mL

_

20 -

0

4

8 INHIBITOR

12 CONCENTRATION

16

20

(mM)

Fig. 1. Inhibition of phosphatidylinositol-specific phospholipase C from B. cereus by phosphonates 1-3 and myo- and epi-inositol as a function of concentration. Activity assays were performed as described 112.131. except that all components of the assay mixture, including added inhibitors. were premixed to ensure complete solubilization before PI-PLC was added. The final concentrations in the assay mixture (0.5 ml total volume) were: sodium borate/HCI (pH 7.5) 40 mM; sodium deoxycholate, 0.16% (3.9 mM); phosphatidylinositol, 2 mM; and inhibitor as indicated. The reaction was initiated by addition of enzyme to a final concentration of 15 ng/ml. Pure phospholipase C was prepared from the culture supernatant of B. cereus as reported earlier (121. Activity measurements were reproducible within 5-88.

Because of the heterogeneous nature of the assay systems used, the inhibition of lipolytic enzymes acting on lipid aggregates has been the subject of considerable debate in the literature, pancreatic and snake venom phospholipases A z representing a case in point (for example, see Refs. 32-36). For phospholipase A,, mixed-micellar systems containing excess detergent have been adopted as the most ideally suited for inhibition studies [37-391. However, even with these systems it is generally difficult to show conclusively that inhibition is a specific effect, i.e., competition between inhibitor and substrate molecules for the enzyme active site [32]. According to Jain and co-workers [32,40], conclusive evidence for competitive inhibition of lipolytic enzymes can only be obtained under ‘scooting conditions’, i.e., with the enzyme essentially, irreversibly bound to the lipid/water interface. Such conditions are known for phospholipase A, [32,40], but have not yet been described for other lipolytic enzymes, including PI-PLC. We therefore, selected a mixed-micellar assay system containing the detergent sodium deoxycholate * to evaluate the inhibition of B. cereus PI-PLC by phosphonate analogues 1- 3. Graphs illustrating the inhibition of PI-PLC by phosphonates 1-3 and the effects of myo- and epi-inositol are shown in Fig. 1. All three phosphonates inhibited the PI-PLC activity in a concentration-dependent manner. Inhibition was observed with analogues 2 and 3, which most likely partition at least partly into the micellar lipid phase, but also with the water-soluble analogue 1. The effect of phosphonate 1 together with that of myo-inositol (see below) indicates that inhibition is not restricted to molecules present at the lipid/water interface. This can be understood if the enzyme itself is in dynamic equilibrium between two states, bound to the micellar lipid/water interface and free in aqueous solution. The active site of the enzyme is thus exposed both to the aqueous inhibitor and the micellar substrate, permitting competition between these species. Palmitoyl phosphonate 3, however, is clearly the most potent inhibitor. It is also the most lipophylic of the three analogues and this may contribute to its efficacy as an inhibitor. Phosphonate 3 is a close analogue of l-acyllysophosphatidylinositol, a known substrate of B. cereus PI-PLC [41]. Inhibition thus appears to be more effective as the structure of the phosphonate inhibitor tends towards that of the substrate. This is consistent with the notion that competition at the enzyme active site is the

* We observe optimal activity for B. cereus PI-PLC with the detergent deoxycholate as used in the standard assay 112). With Triton X-100, for example. the activity is less than 10% of that observed with deoxycholate. The relative activities in different detergents, however. are dependent on other factors including buffer composition, and further study is required.

412 main mechanism underlying the inhibitory action of these molecules. For comparison, we also investigated the inhibition by myo-inositol and one of its isomers, epi-inositol. Myo-Inositol inhibited the PI-PLC activity, whereas epi-inositol consistently showed a small stimulation * of the PI-PLC activity (Fig. 1). Since epi-inositol differs from myo-inositol by inversion of a single hydroxyl group only, the lack of inhibition by this inositol isomer suggests that the active site of B. cereus PI-PLC contains a binding pocket for the myo-inositol moiety with strict geometric constraints. This would be consistent with the high specificity for the polar headgroup of the substrate observed for this enzyme. The data presented here indicate that phosphonate analogues of phosphatidylinositol show promise as inhibitors of phosphatidylinositol-specific phospholipases C and may provide generally useful tools for the study of phospholipase C and other proteins interacting with inositol phospholipids. These studies were supported by US Public Health Service Grants GM 25698 and GM 27137. References 1 Rhee, S.G., Suh, P.-G., Ryu, S.-H. and Lee, S.Y. (1984) Science 244, 5466550. 2 Bloomquist, B.T., Shortridge, R.D., Schneuwly, S., Perdew, M., Monte]], C., Steller, H., Rubin, G. and Pak, W.L. (1988) Cell 54, 1233133. 3 Hereld, D., Hart, G.W. and Englund, P.T. (1988) Proc. Nat]. Acad. Sci. USA 85, 8914-8918. 4 Katan, M., Kriz, R.W., Totty, N., Meldrum, E.. Aldape, R.A., Knopf, J.L. and Parker, P.J. (1988) Cell 54, 171-177. 5 Stahl, M.L., Ferenz, C.R., Kelleher, K.L., Kriz. R.W. and Knopf, J.L. (1988) Nature 332, 269-272. 6 Suh. P.-G., Ryu, S.H., Moon, K.H., Suh, H.W. and Rhee, SC. (1988) Cell 54, 161-169. 7 Suh, P.-G., Ryu, S.H. Moon, K.H., Suh, H.W. and Rhee, SC. (1988) Proc. Nat]. Acad. Sci. USA 85, 5419-5423. 8 Bennett, CF., Balcarek, J.M., Varrichio, A. and Crooke, S.T. (1988) Nature 334, 268-270. 9 Berridge, M.G. (1986) in Receptor Biochemistry and Methodology, Vol. 7, pp. 25-47, Alan Liss, New York. 10 Berridge, M.J. (1987) Biochim. Biophys. Acta 907, 33-45.

* A stimulatory effect was also observed with other compounds including scyllo-inositol, glucose, sucrose, Tris. The nature of this phenomenon is unknown, involve an effect on the mixed-micellar substrate.

polyhydroxy glycerol and but it may

11 Ikezawa, H. and Taguchi, R. (1981) Methods Enzymol. 71. 731-741. 12 Volwerk, J.J., Wetherwax, P.B., Evans, L.M., Kuppe, A. and Griffith, O.H. (1989) J. Cell. Biochem. 39, 315-325. 13 Volwerk, J.J., Koke, J.A., Wetherwax, P.B. and Griffith. O.H. (1989) FEMS Microbiology Lett., in press. 14 Low, M.G. (1981) Methods Enzymol. 71, 741-746. 15 Taguchi. R. and Ikezawa, H. (1978) Arch. Biochem. Biophys. 186, 1966201. 16 Ferguson, M.A.J. and Williams, A.F. (1988) Annu. Rev. Biochem. 57, 285-320. 17 Low, M.G. and Saltiel, A.R. (1988) Science 239, 2688275. 18 Low, M.G.. Stiernberg, J.. Waneck, G.L.. Flavell, R.A. and Kincade, P.W. (1988) FEBS Lett. 82, 143-146. 19 Henner, D.J., Yang, M., Chen, E., Hellmis, R.. Rodriguez, H. and Low, M.G. (1988) Nucleic Acids Res. 16, 10383. 20 Kuppe. A.. Evans, L.M., McMilIen, D.A. and Griffith, O.H. (1989) J. Bacterial., in press. 21 Kraut, J. (1988) Science 242, 533-540. 22 Walsh, C. (1979) Enzymatic Reaction Mechanisms, pp. 123-131. W.H. Freeman & Co., New York. 23 Dixon, M. and Webb, E.C. (1979) Enzymes, pp. 3322467. Longman, London. 24 Cuatrecasas, P. and Anfinsen, C.B. (1971) Methods Enzymol. 22. 345-378. 25 Mrsny, R.J., Birrell, G.B., Volwerk, J.J., Widdicombe, J.H. and Griffith, O.H. (1987) European J. Cell Biol. 45, 200-208. 26 Stark, G.R. and Bartlett, P.A. (1983) Pharmac. Ther. 23, 45-78. 27 Porter, C.C. (1970) Chemical Mechanisms of Drug Action, C.C. Thomas, Springfield, IL. 28 Voicu, V. and Olinescu, R. (1977) Enzymatic Mechanisms in Pharmacodynamics, Ch. 2-4, Abacus Press, Tunbridge Wells. 29 Lin, G. and Tsai, M.-D. (1989) J. Am. Chem. Sot. 111, 3099-3101. 30 Gigg, R. and Warren, CD. (1969) J. Chem. Sot. (C) 2367-2371. 31 Shashidhar, MS., Keana, J.F.W., Volwerk, J.J. and Griffith, O.H. (1989) Chem. Phys. Lipids, in press. 32 Jain, M.K. and Berg, O.G. (1989) Biochim. Biophys. Acta 1002, 1277156. 33 Dennis, E.A. (1983) in The Enzymes, Vol. 16, Ch. 9, Academic Press, New York. 34 Volwerk. J.J. and De Haas, G.H. (1982) in Lipid-Protein Interactions, Vol. 1 (Jost. P.C., Griffith, O.H., eds.), pp. 69-141, Wiley, New York. 35 Verger, R. (1980) Methods Enzymol. 64A, 340-392. 36 Verger, R. and De Haas, G.H. (1976) Annu. Rev. Biophys. Bioeng. 5, 77-117. R. and Verger, R. 37 De Haas, G.H., Van Oort, M.G., Dijkman, (1989) Biochem. Sot. Trans. 17, 2744276. 38 Yuan, W. and Gelb, M.H. (1988) J. Am. Chem. Sot. 116. 266552666. 39 Dennis, E.A. (1987) Drug Dev. Res. 10, 205-220. 28, 40 Jain, M.K., Yuan, W. and Gelb, M.H. (1989) Biochemistry 4135-4139. 41 Sundler, R., Alberts, A.W. and Vagelos, P.R. (1978) J. Biol. Chem. 253. 4175-4179.

Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues.

Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bac...
329KB Sizes 0 Downloads 0 Views