THE JOURNAL OF BIOLOGXXL CHEMISTRY Vol. 265, No. 5, Issue of February 15, pp. 2657-2664,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U. S. A.

The Interaction of Phospholipase A2 with Phospholipid Analogues and Inhibitors*

(Received for publication, July 17, 1989)

Lin Yu$, Raymond A. Deems& Joseph HajduQ, and Edward A. Dennis*7 From the $Department of Chemistry, University of California at San Diego, La Jolla, California 92093 and the SDepartment of Chemistry, California State University, Northridge, California 91330

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja nuja) phospholipase AZ and its substrate. Special em- phasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase AZ inhibitor. A six-step syn- thesis of this compound, 1-palmitylthio-2-palmitoyl- amino-l,2-dideoxy-sn-glycero-3-phosphorylethanol- amine (thioether amide-PE), was developed. Other an- alogues studied included 1-palmitylthio-2-palmitoyl- amino-l,2-dideox-sn-gylycero-3-phosphorylcholine, l-palmityl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine,l-palmityl- thio-2([(tetradecyloxy)carbonyl]amino}-l,2-dideoxy- sn-glycero-3-phosphorylcholine, 1-palmitoyl-2([(oct- adecylylamino)carbonyl]amino)-2-deoxy-sn-glyc- ero-3-phosphorylcholine, and sphingomyelin. Inhibi- tion studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of in- hibition. The best inhibitor was the thioether amide- PE which had an ICsO of 0.45 PM. In contrast, sphin- gomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase AZ inhibition led to the following con- clusions about phospholipid-phospholipase AZ interac- tions: (i) sn-2 amide analogues bind tighter than natu- ral phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor ana- logues containing the ethanolamine polar head group appear to be more potent inhibitors than those contain- ing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospho- lipase AZ is activated by choline-containing phospho- lipids. Thus, choline-containing non-hydrolyzable an- alogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom en- zyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydropho- bicity of the sn-1 functional group. (iv) The cr-methyl-

* Financial support for this work was provided by National Science Foundation Grant DMB 88-17392 and National Institutes of Health Grant GM-20501. A preliminary report of the synthesis and inhibition by the thioether amide-PE was given by L. Yu and E. A. Dennis at the American Chemical Society 198th National Meeting, September 11, 1989, Miami, FL (Abstr. 50). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

ll To whom correspondence should be addressed.

ene group of the m-2 acyl chain is important for the binding of phospholipids to the catalytic site but not to the activator site.

Phospholipase A* is a lipolytic enzyme that specifically hydrolyzes the sn-2 ester bond of phospholipids (1). The enzyme is widespread in nature and exists in both extracel- lular and intracellular forms (l-3). In addition to the obvious role it plays in phospholipid catabolism, the enzyme is also believed to be involved in a series of vital regulatory processes via its ability to release arachidonic acid for the subsequent biosynthesis of eicosanoids (4-6). Eicosanoids are implicated in the pathophysiology of many diseases, especially those involving inflammation and allergy (5-7). Due to the biolog- ical importance of phospholipase A?, there is considerable current interest in understanding the enzyme’s mechanism and how its activity is controlled in vivo.

To date the most extensively studied phospholipase APs are the extracellular ones obtained from mammalian pancreas and snake venom. The studies of these enzymes have served as models for the studies of the less abundant, more difficult to obtain intracellular enzymes. An important aspect of such studies has been the use of inhibitors to probe substrate/ enzyme interactions. Inhibitors can provide valuable infor- mation about how phospholipids interact with phospholipase AZ, about which enzyme residues are involved, and about the enzyme’s general mechanism of action.

A growing number of compounds have been found that inhibit these phospholipase A2s. The best characterized inhib- itor isp-bromophenacylbromine, an irreversible inhibitor that specifically modifies the active site histidine residue (8, 9). Other covalent inhibitors are the natural product manoalide (10, 11) and its synthetic analogue, manoalogue (12), which have been shown to covalently modify lysine residues. While helping to identify active site residues, irreversible inhibitors, especially those that do not resemble the substrate, offer little information about the nature of substrate binding. Informa- tion about binding is more readily obtained from reversible inhibitors. By comparing the binding affinities of a closely related series of inhibitors, one can draw inferences about what structural features in the phospholipid are important for binding. This information can be correlated with sequence and x-ray crystal data to help delineate the active site inter- actions of the enzyme and its mechanistic details.

To this end, we have previously synthesized several phos- pholipid analogues containing a sn-2 amide (13-16) and, in a preliminary communication (17), have reported on an ether amide phospholipid analogue which is a reversible inhibitor. We have now extended these earlier studies (17) to include a series of substrate analogues that allow us to correlate phos- pholipid structure to inhibitory potency. We have systemati-

2657

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2658 Phospholipase Az Inhibitors

tally studied the effect of substituting an amide group for the sn-2 ester, of changing the polar head group, of varying the hydrophobicity of the sn-1 functional group, and of varying the composition of the sn-2 acyl group. The results of these studies have led to a better understanding of the structural features of the phospholipid molecule that are important for binding and to the development of a very potent reversible phospholipase A2 inhibitor.

EXPERIMENTAL PROCEDURES

Materials

Phospholipase A* was purified to homogeneity from cobra venom (Naja naja naja) as described previously (18). 4,4’-Dithiodipyridine, palmitoyl chloride, and decanoyl chloride were purchased from Ald- rich. Triton X-100 was obtained from Sigma. l,Z-Dipalmitoyl-sn- glycero-3-phosphorylcholine (DPPC)’ and sphingomyelin were ob- tained from Avanti Polar Lipids, Inc. 1-Palmitylthio-Z-palmitoylam- ino-1,2-dideoxy-sn-glycero-3-phosphorylcholine (thioether amide- PC), 1-palmityl-2-palmitoylamino-2-deoxy-sn-glycero-3-phosphoryl- choline (ether amide-PC), l-palmitoyl-2-palmitoylamino-2-deoxy-sn- glycero-3-phosphorylcholine (ester amide-PC), 1-palmitylthio-2- { [(tetradecyloxy)carbonyl]aminol-1,2-dideoxy-2-deoxy-sn-glycero-3- phosphorylcholine (carbamoyl-PC), and 1-palmitoyl-2-l [(octadecy- lamino)carbonyl]aminoJ-2-deoxy-sn-glycero-3-phosphorylcholine (alkylureido-PC) were synthesized as described previously (13-16). 3- Trityl-1,2-dideoxy-1,2-(thiocarbonyldithio)-sn-glycerol was prepared according to the method of Hendrickson et al. (19). Choline p- toluenesulfonate was prepared as described by Brockerhoff and Ay- engar (20). D-Serine methyl ester hydrochloride was prepared as described by Greenstein and Winitz (21). 2-Phthalimidoethylphos- phoryl dichloride was prepared by the procedure of Rosenthal (22). Chloroform and benzene were dried by distillation over phosphorus pentoxide. Phosphorus oxychloride was freshly distilled prior to use. All other solvents were reagent grade or better. Silica Gel 60 (230- 400 mesh, Merck) was purchased from Aldrich. The ion exchanger, Rexyn I-300, was obtained from Fisher.

General Methods

Thin-layer chromatography (TLC) was carried out on Analtech Silica Gel G-250 glass plates. General detection was by exposing to iodine vapor or spraying the plates with 2 M sulfuric acid, followed by charring on a hot plate. Trityl compounds were visualized by their bright yellow color after gently warming the sprayed plate. Phospho- lipids were detected with molybdate spray (23). Flash chromatography was performed as described by Still et al. (24) using Silica Gel 60 (230-400 mesh, Merck).

One-dimensional proton NMR spectra and two-dimensional ho- monuclear J-correlation (‘H COSY) spectra were taken on an exten- sively modified Varian 360 MHz NMR spectrometer, using tetrame- thyl-silane as an internal standard. Carbon 13 NMR and phosphorus 31 NMR spectra were obtained at 50 and 81 MHz, respectively, on a 200-MHz Nicolet NMC-NB-200 spectrometer. Trimethyl phosphine (5%) was used as an external standard for the ‘iP spectra. Optical rotation was measured with a Perkin-Elmer model 141 polarimeter. FAD mass spectra were obtained at the University of California Riverside mass spectrometry facility.

1 The abbreviations used are: DPPC, 1,2-dipalmitoyl-sn-glycero-3- phosphorylcholine; thioether amide-PC, l-palmitylthio-2-palmitoyl- amino-1,2-dideoxy-sn-glycero-3-phosphorylcholine; ether amide- PC, l-palmityl-2-palmitoylamino-2-deoxy-sn-glycero-3-phosphoryl choline; ester amide-PC, l-palmitoyl-2-palmitoylamino-2-deoxy-sn- glycero-3-phosphorylcholine; carbamoyl-PC, l-palmitylthio-2-{[(te- tradecvloxv)carbonvllaminol-1,2-dideoxy-sn-glycero-3-phosphoryl- choline; alkylureido-PC, l-palmitoyl-2{ [(bctadecylylamino)carbonyl] amino]-2-deoxv-sn-zlvcero-3-phosphorylcholine; thioetheramide-PE, l-palmitylthio:2-pai~itoylan;ino-i,2-dideoxy-sn-glycero-3-phospho- rylethanolamine; thio-PC 1,2-bis(decanoylthio)-1,2-dideoxy-sn-glyc- ero-3-phosphorylcholine; thio-PE, 1,2-bis(decanoylthio)-1,2dideoxy- sn-glycero-3-phosphorylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine.

Inhibition Studies

The inhibitory potency of the phospholipid analogues was evalu- ated by determining the analog&concentration required to decrease the hvdrolvtic activitv of the nhosnholipase AZ bv 50% (I&O). The effect-of various phospholipid analogties-on cobra venom phospholi- pase AZ was determined using the well defined Triton X-lOO/phos- pholipid mixed micelle system (25). In this study, thio-PC and thio- PE were used as substrate.

Enzyme Assay

The phospholipase A) activity toward thio-PC and thio-PE was determined spectrophotometrically at 324 nm by measuring the change in absorption due to the reaction of the product’s free thiol group with 4,4-dithiobispyridine. The mixed micelles of phospholipid, phospholipid analogue, and Triton X-100 were prepared fresh daily by one of the following two methods: (i) A mixture of the desired amounts of phospholipid and lipid analogue in a chloroform solution was dried under a stream of nitrogen and then in uacuo. Triton X- 100, in standard assay buffer, was then added and the mixture was sonicated until a clear solution was obtained. (ii) Alternatively, the micelles of phospholipid and Triton X-100, and the micelles of the phospholipid analogue and Triton X-100, were prepared separately. Aliquots of these solutions were combined to obtain the proper concentration of the inhibitor in the micelles. The mixture was again sonicated prior to assay.

The standard assay mixture consisted of 0.5 mM of either thio-PC or thio-PE substrate, 4.25 mM Triton X-100, 25 mM Tris buffer (pH 8.5), 0.1 M KCl, and 10 mM CaClz. The enzyme solution (10 ng/pl) contained 4 mM Triton X-100 in the Tris buffer. The cuvette, con- taining 0.3 ml of assay mixture and 5 ~1 of 4,4’-dithiobispyridine (50 mM in ethanol), was placed in the compartment of a Perkin-Elmer model 552 spectrophotometer thermostated at 30 “C and balanced against a blank containing water only. After incubation of the sub- strate for 2 min at 30 “C, phospholipase A, (5 pl containing 50 ng of protein) was added to initiate the reaction. The average of duplicates is reported.

Synthesis of 1,2-Bis(decanoylthio)-1,2-dideoxy-sn-glycero-3- phosphorylcholine (Thio-PC)

l-Trityl-2,3-dimercapto-I-propanol 121-A tetrahydrofuran solu- tion of 3-trityl-1,2-dideoxy-1,2-(thiocarbonyldithio)-sn-glycerol 1 (4.98 g, 12.2 mmol) was reduced by LiAlH, according to the procedure of Hendrickson et al. (19). The crude mercaptan was dissolved in 25 ml of acetone and applied to a 250-g column of silicic acid in hexanes/ acetone (5O:l). Prior to packing the column, silicic acid was deoxy- genated by vacuum and stored under argon. The column was eluted with 1200 ml of hexanes/acetone (50~1). The chromatography was monitored by TLC developed with hexanes/acetone (7:l). A clear oil was obtained (3.64 g, 81% yield), which gave a single spot on TLC (RF= 0.65). ‘H NMR (CDC&) 6 2.91 (d, 2H), 3.03 (m, 2H), 3.24 (m, lH), 3.39 (m, 2H), 7.30 and 7.43 ppm (m, 15H).

3-Trityl-,1,2-bis(decanoylthio)-1,2-dideoxy-sn-glycerol [3]-Deca- noyl chloride (5.72 g, 30.0 mmol) in 10 ml of hexanes was slowly added to a solution of 1-trityl-2,3-dimercapto-l-propanol 2 (3.64 g, 9.9 mmol) in 40 ml of hexanes and 6 ml of dry pyridine. After the solution was stirred at room temperature for 7 h, 100 ml of benzene were added and the mixture was washed subsequently with water, 0.5 M NH,OH in methanol/water (3:1), and methanol/water (1:l). The organic phase was dried over anhydrous MaSO and evaporated in “a&o. A clear oil was obtained (6.59 g, 99%-yield), which showed a single spot on TLC in hexanes/acetone (7:l) (RF = 0.75). ‘H NMR (CDCl,) d 0.876 (t, 6H), 1.251 (s, 24H), 1.638 (m, 4H), 2.454 (t, 2H), 2.522 (t, 2H), 3.203 (m, 2H), 3.314 (m, 2H), 3.872 (m, lH), 7.294 and 7.417 ppm (m, 15H).

1,2-Bis(caprylthio)-1,2-dideoxy-sn-glycero-3-phosphorylcholine [~/--TO a solution of l-trityl-1,2-bis(caprylthio)-1,2-dideoxy-sn- glycerol 3 (6.59 g, 9.76 mmol) in 150 ml of CHCl, was added a 14% BFs (5 ml, 10.33 mmol) solution in methanol. The reaction was stirred at room temperature and monitored by TLC in hexanes/ethyl acetate (81) (R, = 0.35 for detritvlated product). The reaction went to completion in 2 h. The product mixture was washed three times with ice water. The organic phase was dried over anhydrous Na&O, and evaporated in uacuo. This product showed a major spot on TLC in hexanes/ethyl acetate (8:l). This viscous liquid was used directly in the next step without further purification.

The crude detritylated compound 4 was dissolved in 130 ml of dry

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Phospholipase A2 Inhibitors 2659

CHCl, containing 1.0 ml of dry pyridine (12.5 mmol). A solution of freshly distilled POCI, (1.8 g, 11.7 mmol) in 30 ml of dry CHCI, was added to the solution of compound 4. The reaction was stirred under argon at 50 “C for 1 h. The disappearance of the starting material was followed by TLC in hexanes/acetone (7:l) (RF= 0.45). After the reaction was complete, the mixture was cooled to room temperature. Choline p-toluenesulfonate (3.64 g, 9.73 mmol) and 3.0 ml of dry pyridine were added. The mixture was stirred for 3 h at room temperature. The reaction mixture was washed with two portions of water; methanol was added to break the emulsion. The chloroform solution was evaporated in uocuo. The crude product was dissolved in 50 ml of CHC13/MeOH/H20 (65:25:4) and passed through an ion exchange column of Rexyn I-300 which was preequilibrated with the same solvent. The column was monitored by TLC in chloroform/ methanol/water/concentrated ammonia (95:35:5.5:2) (RF= 0.64). The eluted solution was evaporated in IJUCUO and lyophilized. The solid was dissolved in 30 ml of chloroform/methanol/water (65:25:4) and purified on a silica gel column (200 g) using the same solvent mixture. After lvonhilization from drv benzene, a white solid (1.40 g. 24% vield from compound 3) was obtained. IR (thin film, chloroform): 3350 (br), 2960 (sh), 2930 (s), 2860 (s), 1470 (s), 1690 (s), 1250 (m), 1090 (m), cm-‘. ‘H NMR (CDCl,/D,O 125:l) 6 0.879 (t, 6H), 1.285 (S, 24H), 1.628 (m, 4H), 2.542 (t, 4H), 3.083 (m, lH), 3.252 (S, QH), 3.453 (m, lH), 3.675 (m, 2H), 3.846 (m, 2H), 3.997 (m, lH), 4.274 ppm (m, 2H); 3’P NMR gave only one peak at -0.460 ppm; i3C NMR spectra showed that only thiol ester exist in the product (198.0 ppm). [a]$ = 12.6 (c 9.557 mM, CHC&).

C2sHB706NPS2 (M + H+)

Calculated: 598.3364 Found: 598.3384

Synthesis of 1,2-Bis(decanoylthio)-1,2-dideoxy-sn-glycero-3 phosphorylethanolamine (Thio-PE)

Thio-PC (150 mg, 0.25 mmol) was transformed into thio-PE by the action of phospholipase D according to the procedure of Comfur- ius and Zwaal (26). The crude product was purified bv flash chroma- tography using chloroform/methanol/water (65:25:1) as eluting sol- vents. This thio-PE was obtained in 91% vield (127 me. 0.23 mmol) as a white powder. ‘H NMR (CDClJCDiOD Q:l) 6 0.879 (t, 6H); 1.260 (S 24H), 1.632 (m, 4H), 2.156 (br, 4H), 2.535 (t, 4H), 3.132 (dd, lH), 3,207 (m, 2H), 3.387 (dd, lH), 3.860 (m, 2H), 4.061 (m, lH), 4.145 (m, 2H); 31P NMR showed only one peak at -0.137 ppm.

G,H,,06NPS, (M + H+)

Calculated: 556.2895 Found: 556.2892

Synthesis of I-Palmitylthio-2-palmitoylnmino-1,2-dideoxy-sn-glycero- 3-phosphorylethanolamine (121

N-Palmitoyl-D-serine Methyl Ester [7]-n-Serine methyl ester hydrochloride (5.0 g, 32.1 mmol) was suspended in 250 ml of chloro- form containing triethylamine (3.25 g, 32.1 mmol) and I-N,N-di- methyl-aminopyridine (3.92 g, 32.1 mmol). To this mixture was added palmitoyl chloride (8.82 g, 32.1 mmol) dropwise with stirring. The solution was incubated at room temperature for 24 h. One liter of water was then added to precipitate the product. The white solid was washed several times with water, filtered, and finally flash chromat- ographed on silica gel with chloroform/ethyl acetate (9:l) to give pure product 7 (10.9 g, 95%). ‘H NMR (CDClJ d 0.879 (t, 3H), 1.254 (s, 24H), 1.669 (m, 2H), 2.265 (t, 2H), 3.79 (s, 3H), 3.966 (dd, 2H), 4.701 (m, lH), 6.527 (b, 1H).

S-Palmityl-N-palmitoyl-D-cysteine Methyl Ester [91-Compound 7 (8.0 g, 22.3 mmol) was dissolved in 40 ml of chloroform and cooled in an ice bath. Pyridine (7.0 ml, 86 mmol) was then added, followed by the addition of p-toluenesulfonyl chloride (12.8 g, 67.0 mmol) in small portions with constant stirring. The reaction was completed in 6 h (monitored by TLC). Chloroform (100 ml) and water (10 ml) were added and the organic layer was washed successively with water, 2 N HCl, 5% NaHC03, and water, and then dried over Na,SOa. The solvent was removed under reduced pressure and the crude tosylate 8 was used directly in next reaction without purification.

A mixture of crude compound 8, hexadecyl mercaptan (9.10 g, 35.2 mmol), and sodium methoxide (1.9 g, 35 mmol) in 150 ml of methanol was refluxed overnight. The solvent was removed in vacuum and the solid was then washed with water several times. Finally, the crude

product was crystallized from acetone/hexanes to yield (5.3 g, 41%) of compound 9. ‘H NMR (CDCl3) 6 0.880 (t, 6H), 1.255 (s, 50H), 1.549 (m, 2H), 1.649 (m, 2H), 2.246 (t, 2H), 2.492 (t, 2H), 2.986 (m, 2H), 3.766 (s, 3H), 4.830 (m, lH), 6.293 (d, 1H).

1 -Palmitylthio-2-palmitoylamido-1,2-dideoxy-sn-glycerol [IO]-To a refluxing mixture of compound 9 (0.55 g, 0.92 mmol) and LiBH, (33 mg, 1.5 mmol) in 40 ml of dry ether was added 0.061 ml of MeOH over 10 min and refluxed for an additional 30 min. The mixture was cooled in an ice bath and 1 N HCl was added to quence the reaction. The mixture was diluted with water and extracted with chloroform. The combined extracts were dried over anhydrous sodium sulfate, and the chloroform was evaporated under vacuum. The product showed only one spot on TLC (RF 0.5 in hexanes/acetone 7:3). Comnound 10 was obtained in 99% yield (0.52 a). ‘H NMR (CDCln) 6 0.880 (t, 6H), 1.255 (s, 50H), 1.60s(m, 4H), 2.219 (t, 2H), 2.567 (t, 2H), 2.706 (dd, 2H), 3.780 (dd, 2H), 4.008 (m, lH), 6.0.% (d, W.

l-Palmitylthio-2-palmitoylamido-1,2-dideox.y-sn-g~~cero-3-~hos- phor$etha~olamine )lZ]--A mixture of compound 10 (0.5 g,-0.877 mmol), 2-phthalimidoethylphosphoryl dichloride (3.2 g, 10 mmol), and pyridine (1.6 g, 20 mmol) was stirred at room temperature for 24 h. The solvent was evaporated and the residue was dissolved in 100 ml of ether and stirred for another 24 h. The ether was removed under vacuum and the crude product was dissolved in 100 ml of chloroform and washed with 30 ml of water. The chloroform was removed, and the white solid was dissolved in 100 ml of absolute ethanol. To this solution was added 15 ml of 10% hydrazine in ethanol in small portions with cooling, and the solution was then refluxed for 2 h. After the solution was cooled, 10 ml of 20% aqueous HCl was added and the solution allowed to stand at room temperature for another 2 h. The ethanol was removed under vacuum and the residue was dissolved in 150 ml of chloroform/methanol (21). The solution was washed with 30 and 20 ml of 1% NaCl. The crude product was concentrated from the organic layer and purified by flash chromatog- raphy on silica gel with chloroform/methanol/water (65:25:1) as eluting solvent. The yield of compound 12 was 182 mg (30%). ‘H NMR (C!DC& and CD,OD): d 0.879 (t, 6H), 1.252 (s, 5OH), 1.583 (br, 4H), 2.224 (t, 2H), 2.561 (t, 2H), 2.708 (br, ZH), 3.133 (br, 2H), 3.899 (br, lH), 4.076 (br, 2H), 4.129 (br, 2H).

Ca,H,sO,N,PS (M + H’)

Calculated: 693.5369 Found: 693.5378

RESULTS

Chemical Synthesis-The thio-PC was synthesized by a modified version of the procedure of Hendrickson et al. (19). As shown in Scheme 1, the basic synthetic strategy of this modified method is the same as that of the original paper. The disadvantages of the previous procedure is (a) the diffi- culty of purifying the intermediate, compound 3, and the product, thio-PC, and (b) the acyl migration and ester hy- drolysis of compound 4 that occur during its purification by silica gel column chromatography. The low yield of the origi- nal method can be overcome by the following modifications. Originally, compound 3 was made through the direct acylation of the crude reduction product of compound 1. Instead of the laborious purification of compound 3, compound 2 was puri-

LiAIH4 0 - HS l-l- Rk

81% CHflTr

1 2 3

8 EiF$MeOH 0

R6.S CH20H 24%

4 5

SCHEME 1. Synthesis of 1,2-bis(decanoylthio)-1,2-dideoxy- sn-glycerol-3-phosphorylcholine.

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2660 Phospholipase AZ Inhibitors

fied first and then acylated with decanoyl chloride. This modified procedure afforded pure compound 3 in quantitative yields. Because of the potential acyl migration and hydrolysis of compound 4, it was directly phosphorylated without puri- fication. Purification of compound 5 does not involve acyl migration or hydrolysis. Finally, phosphatidylcholine was pu- rified by ion exchange and silica gel column chromatography rather than by high pressure liquid chromatography as in the original paper (27). The overall yield of the modified method was twice that of the original one (19 uersu.s 9%).

The synthesis of the thioether amide-PE is outlined in Scheme 2. This approach provides an alternate synthetic pathway to that employed by Bhatia et al. (16). The synthesis is based on the selective acylation of the amino group of D- serine methyl ester and the subsequent selective reduction, with LiBH+ of the ester group. The chirality of the optically active a-carbon in D-serine provided the asymmetric center in thioether amide-PE. Reaction of one equivalent of palmi- toy1 chloride with D-serine methyl ester at room temperature afforded compound 7 in quantitative yield after workup. By activating the hydroxyl group of compound 7 with tosyl chloride, the thioether was introduced by refluxing the meth- anol solution of tosylate and hexadecyl mercaptan. The re- duction of compound 9 with a mild reducing reagent, LiBH4, led to the formation of the cysteine derivative 10 in essen- tially quantitative yield. Finally, the phosphorylethanolamine moiety was introduced by treating compound 10 with 2- phthalimidoethylphosphoryl dichloride (22).

Kinetic Studies-There are two important factors that must be taken into account when studying phospholipases. The first is that the activity of these enzymes critically depends on the physicochemical structure of the phospholipid sub- strate; any molecule which disturbs the structure, i.e. the lipid-water interface, could influence phospholipase activity. It is important to choose an assay system that minimizes this secondary effect. We, therefore, used the well defined Triton X-lOO/phospholipid mixed micelle system (25, 28). These mixed micelles are composed predominately of the nonionic detergent Triton X-100. The presence of small amounts of substrate and phospholipid analogue have negligible effect on the surface environment and micelle structure (29). Thus, the observed inhibition of phospholipase AZ activity by phospho- lipid analogs is presumably due to the direct interaction of the enzyme with the inhibitors and not due to indirect effects of the inhibitors on the substrate micelles.

The second factor is that any enzyme acting on an aggre- gated substrate can exhibit surface dilution kinetics. This phenomenon is observed when the enzymatic activity depends on both the bulk and the surface concentration of substrate

RCOWDMAP RgHN 95%

CH2SR REHN-CH

LiSH@eOH CH2SR

. Rt!HN+H PhtCH2CH20POC12/E13N .

c oohh 93% ~HSH 9 10

0 WWz

0 RkHN

r Rt!HN

t H20P03CH2CH2Phl H20P03CH$H2NH3

11 12

Ts = p-Toluenesulfonyl

Pht = Phthalimido

SCHEME 2. Synthesis of 1-palmitylthio-1,2-dideoxy-sn-glyc- erol-3-phosphorylethanolamine.

(28, 30, 31). While the addition of enzymatically inert com- pounds to the interface does not dilute the substrate’s bulk concentration, it does dilute its surface concentration. The effect of this surface dilution on the phospholipase AZ activity is shown in Fig. 1. Triton X-100 acts as an inert non-hydro- lyzable diluent for the cobra venom enzyme (32). Increasing the Triton X-100 concentration dilutes the substrate’s surface concentration resulting in a decrease in phospholipase AZ activity. Plotting enzyme activity verws Triton X-100 con- centration would yield a curve that is very similar to the one that would be produced if Triton X-100 were only a compet- itive inhibitor. Thus, this Triton X-100 curve is the critical control for these studies. If the compound being tested is truly inert and does not interact with the enzyme at all, it would still surface dilute the substrate and produce a curve identical to the Triton X-100 curve. Therefore, it is the deviation from this curve that determines whether a compound is an activa- tor, an inhibitor, or neither. Surface dilution ceases to be a problem if the inhibitor is very potent and its concentration in the interface is low enough to not dilute the substrate’s surface concentration significantly.

For the structure-activity analysis, the compounds were grouped into four categories according to their structural relationships. In group A, the importance of the amide group at the sn-2 position of a phospholipid was studied by compar- ing the effect of dipalmitoyl-PC and the amide analogue 15 on phospholipase AZ hydrolysis of thio-PC. In group B (com- pounds 12, 13), the effect of the head group was studied by replacing the phosphorylcholine with phosphorylethanola- mine. In group C (compounds 13, 14, 15, and sphingomye- lin), the analogues were modified systematically at the sn-1 position with an ester, an ether, or a thioether group; in addition, the natural phospholipid, sphingomyelin, was also studied. In group D (compounds 16, 17), the molecules were modified by replacing the ester group at the m-2 position with either carbamoyl or alkylureido groups.

The replacement of the sn-2 ester by an amide has a significant effect on the interaction of the enzyme with its substrate. This effect was evaluated by comparing the inhi- bition of thio-PC hydrolysis by the compounds in group A, DPPC (which should theoretically be a competing substrate) and compound 15. Compound 15 differs from DPPC only in the sn-2 functional group. While DPPC is hydrolyzed by the enzyme, its hydrolysis is not detected in the thio assay because the lysophospholipid produced does not contain a free sulfhy- dry1 group and, thus, can not react with the chromogenic

01 ’ I 0 4 a 12

[ADDITIVE] (mM)

FIG. 1. Effect of the substitution of the an-2 ester by an amide on the interaction of the phospholipase Az and substrate analogues. Dipalmitoyl-PC (0); ester amide-PC (0). Triton X-100 (A) is used to illustrate the surface dilution effect. Thio-PC concen- _-.---- &ion is 0.5 mM and Triton X-100 is 4.0 mM.

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Phospholipase A2 Inhibitors 2661

reagent. Since DPPC binds to the enzyme but its hydrolysis is not detected, one would expect it to look like a competitive inhibitor in the thio assay. However, DPPC did not show any inhibition at concentrations up to 10 mM. By comparing it to the Triton X-100 control, DPPC clearly activates the hydrol- ysis of thio-PC. On the other hand, compound 15 clearly shows a significant inhibition. Due to the small amount of compound 15 used in the assay, the surface dilution effect caused by it is negligible. Therefore, compound 15 is a potent inhibitor having an I& of 0.156 mM.

Replacing the phosphorylcholine polar head group by phos- phorylethanolamine, group B, also effects the potency of phospholipase Aa inhibition. As shown in Fig. 2, the half- maximal inhibition of thio-PC hydrolysis by thioether amide- PE was 0.45 pM, which is 4 times lower than the thioether amide-PC (2 gM). The inhibition of PE hydrolysis was also measured as shown in Fig. 3. The I& of thioether amide-PE was about 6.5 pM. The thioether amide-PC did not follow a simple inhibition curve; thus, an IC& could not be determined. In both of these experiments, the concentration of inhibitor was negligible compared to the concentration of surfactant and the total surface, and, thus, the surface dilution effect was also negligible.

Since the thioether amide-PE was the most potent inhibi- tor, we chose it to test whether the inhibition was reversible. The enzyme and inhibitor were incubated together and then diluted 200-fold into an assay. We found that the effect of the inhibitor could be overcome by dilution or by a high substrate concentration. We also measured a time course of inhibition and found that it was not time dependent. Therefore, we conclude that the inhibition is reversible for thioether amide- PE. Since the other compounds are closely related to the thioether amide-PE, we assume that their inhibition is also reversible.

[INHIBITOR] (PM)

FIG. 2. Inhibition of phospholipase Az hydrolysis of thio- PC/Triton X-100 mixed micelles by thioether amide-PE (0) and thioether amide-PC (A). Substrate concentration is 0.5 mM.

- *Od ‘;

m 60 E

-'g 40y \

; ‘1ky.j > 0 50 100 150 200 250

[INHIBITOR] (PM)

FIG. 3. Inhibition of phospholipase AZ hydrolysis of thio- PE/Triton X-100 mixed micelles by thioether amide-PE (0) and thioether amide-PC (A). Substrate concentration is 0.5 mM.

In group C, the hydrophobicity of the an-1 functional group was varied. As shown in Table I, the potency of inhibition increased with increasing hydrophobicity. The most potent inhibitor in this series was the thioether amide-PC 13 which has the most hydrophobic group, the thioether, at the sn-1 position. Its inhibition of thio-PC hydrolysis is shown in Fig. 2, and the I& was 2 pM. On the other hand, sphingomyelin, which has the most hydrophilic group at the sn-1 position, affects thio-PC hydrolysis in a complex manner, Fig. 4. Ap- parently, two competing phenomena are involved. Sphingo- myelin activates phospholipase AZ hydrolysis of phosphati- dylcholine and, at high concentrations, dilutes the substrate surface concentration. In Fig. 4, the effect of surface dilution on the enzymatic activity is illustrated by substituting Triton X-100 for sphingomyelin. Taking into account surface dilu- tion, by comparing the sphingomyelin curve to the Triton X- 100 curve, it is clear that sphingomyelin activates the hydrol- ysis of thio-PC but the observed inhibition is due solely to surface dilution. Sphingomyelin does not appear to inhibit the enzyme directly.

In group D, the binding specificity of the substrate ana- logues was altered by replacing the a-methylene group in the sn-2 chain. As shown in Table I, the analogues in group D are not inhibitors of phospholipase AZ at all. Taking into account surface dilution, compound 16 shows no inhibition of the thio-PC hydrolysis. Although compound 16 differs from com- pound 13 only in the LY position of the sn-2 fatty chain, the former does not affect the activity of phospholipase AZ, while the latter is an excellent inhibitor. Compound 17 did not show any inhibition, even at the highest concentration (5 mM) used, while compound 15 is a potent inhibitor having an IC& of 0.156 mM. These results imply that the cu-methylene group of the sn-2 acyl chain is required for phospholipid binding to the catalytic site of phospholipase AZ. Although the com- pounds in group D did not inhibit PC hydrolysis, they were excellent activators of PE hydrolysis. Fig. 5 shows that com- pound 17 has no effect on the hydrolysis of thio-PC but activates the hydrolysis of thio-PE. In other experiments, we have also shown that all of the other PC analogues were also activators of PE hydrolysis, even though they inhibit the hydrolysis of PC.

DISCUSSION

“Surface Dilution Kinetics” and Inhibition-All enzymes that act on lipid interfaces can potentially be affected by the phenomenon of surface dilution. This effect is particularly troublesome when studying reversible inhibition because many of these inhibitors are amphipathic molecules that will intercalate into the interface and dilute the surface concen- tration of the substrate. This dilution can be mistaken for competitive inhibition and must be taken into account if the interaction of these molecules with the enzyme is to be de- tected. There are several ways to deal with this phenomenon.

The first method applies if the inhibitor is very potent. In this case, its surface concentration will be negligible, and thus, the surface dilution that it causes will also be negligible. This was found to be the case with several of the inhibitors in this study. A second method of decreasing the inhibitor’s surface dilution effect is to dilute both the substrate and the inhibitor with a large amount of some inert non-hydrolyzable com- pound. Under these conditions the change in surface area caused by changing the inhibitor concentration is reduced simply because the total surface area is increased. In some cases, the concentration of substrate can be kept constant by balancing changes in the inhibitor concentration with changes in the inert diluent. This is difficult to do when working with

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2662 Phospholipme Az Inhibitors

TABLE I Inhibition of phospholipase Az by phospholipid analogues

All inhibition studies were carried out in mixed micelles of thio-PC (0.5 mM) and Triton X-100 (4.0 mM). ND, no inhibition was detected even at millimolar concentrations.

Group Compound

A Dipalmitoyl-PC

B Thioether amide-PE 12

B, C Thioether amide-PC 13

C Ether amide-PC 14

A, C Ester amide-PC 15

C Sphingomyelin

D Carbamoyl-PC 16

D Alkylurido-PC 17

vesicles but is more tractable in a mixed micelle system. The Triton X-lOO/phospholipid mixed micelle system is one such system that works for the cobra venom phospholipase AZ. A second advantage of the mixed micelle system is that the effect of the inhibitor on interface and the surrounding lipids is also reduced.

The third strategy is to actually measure the dilution effect directly. We have employed this technique by using Triton X-100 as a diluent and measuring activity as a function of surface concentration of substrate. All of our inhibition pro- files were compared to this curve. One could also calculate its effect if a suitable kinetic model were available. In any case, surface dilution must be taken into account if false inhibition results are to be avoided.

Potent Phospholipase Az Inhibitors-The design of inhibi- tors based on non-hydrolyzable phospholipid analogues has proven to be an effective means by which tight binding inhibitors of phospholipase A2 can be obtained. The replace-

ND

CH,OPC

CH,SR

R!HN I --TH

CH,OPE

CHzSR

.EHN I 7-H

CHZOPC

CH,OPC

CHzOPC

0 HOCHCH=CHR

R!HN ’ H

I CH,OPC

0.00045

0.002

0.038

0.156

ND

ND

CH,OOCR

RHNiHNlH

CH,OPC

ND

ment of the ester moiety by the corresponding amide function at the m-2 position abolishes the enzymatic activity of phos- pholipase AZ (13, 17, 33, 34) and also increases the affinity of the analogues for the enzyme. The judicious substitution of other groups on the amide analogues has increased their potency. The 0.45 j.tM IC& of thioether amide-PE 12 is one of the tightest phospholipase AZ inhibitors found to date. In addition to finding this potent inhibitor, these studies have pinpointed four aspects of enzyme/lipid interaction that are important for substrate binding: (i) The introduction of an amide moiety in the m-2 position significantly increased the binding of the phospholipid derivative to the catalytic site. (ii) Increasing the hydrophobicity of the functional group in the an-1 position increases the affinity between the phospho- lipid molecule and the enzyme. (iii) The cY-methylene group of the m-2 fatty chain plays an important role in differen- tiating the binding of phospholipid to the two functional sites of the cobra venom phospholipase AZ. (iv) The ethanolamine-

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Phospholipase A2 Inhibitors 2663

hydrophobic residues: Phe-5, Ile-9, Phe-22, Ala-102 and -103, Phe-106, and the disulfide bridge between Cys-29 and -45. These residues form a hydrophobic binding pocket at the catalytic site. By increasing the hydrophobicity of the an-1 functional group, the hydrophobic interaction between the enzyme and substrate analogue appears to be enhanced.

> A.

.A--

[ADDITIVE] (mM)

FIG. 4. Effect of sphingomyelin (0) on the hydrolysis of thio-PC catalyzed by phospholipase AZ. The surface dilution effects is also shown by substituting Triton X-100 (A) for sphingo- myelin. Thio-PC concentration is 0.5 mM.

The “Dual Phospholipid Model” and Inhibition-Points iii and iv, the effects of the a-methylene group and the head group, can be explained by the Dual Phospholipid Model. The Dual Phospholipid Model was developed for the cobra venom phospholipase Az which has two distinct functional sites: an activator site and a catalytic site. The activator site specifically requires a phosphorylcholine group to activate the enzyme while the catalytic site hydrolyzes phospholipids with either a phosphorylcholine or phosphorylethanolamine head group (29,41,42). We have previously shown that this effect is due to a direct interaction between the activator and the enzyme and is not due to some general effect on the interface. Thus, this enzyme interacts with two phospholipid molecules; whether this takes place at two distinct physical sites or in one large site that binds two phospholipids is not known.

0.0 0.4 0.8 1.2

[ALKYLUREIDO PC] (mM)

FIG. 5. Effect of alkylurido-PC 16 on the phospholipase AS activity using thio-PC (A) or thio-PE (0) as substrate. Substrate concentration is 0.5 mM and Triton X-100 is 4.0 mM.

A manifestation of this phenomenon was found in the inhibition of phosphatidylethanolamine hydrolysis by thioether amide-PC as shown in Fig. 3. This unusual inhibi- tion curve is produced by the two competing phenomena: activation and inhibition. Because of the phosphorylcholine moiety, the thioether amide-PC can bind to the activator site and activate thio-PE hydrolysis. This effect predominates at low inhibitor concentration and is responsible for the increase in activity in this region. As the thioether amide-PC concen- tration increases, it begins to compete with the substrate for the catalytic site. At high concentrations, the inhibition pre- dominates and significant overall inhibition is observed. This implies that the binding of the thioether-PC is tighter to the activator site than to the catalytic site. The replacement of the ry-methylene group, compounds 16 and 17, yields ana- logues that are very poor inhibitors but good activators. Ap- parently the a-methylene group is required for the binding to the catalytic site but not to the activator site. These two results indicate that the two functional sites have different environments and are probably two physically different sites.

containing inhibitor is more potent than the choline-contain- ing analogues.

Regarding point i, the high affinity of the nonhydrolyzable isosteric phospholipid derivatives can be explained on the basis of hydrogen bonding. Chemical modification (8, 9) and x-ray crystallographic studies of related phospholipase A+ (35-37) have demonstrated the importance of a catalytic histidine residue. In addition, the three-dimensional structure of bovine pancreatic phospholipase Az indicates that there is a tightly bound water molecule lying in the plane of the imidazole ring. The amide proton could form a hydrogen bond with this water molecule inside the active site. Formation of such a hvdroeen bond would increase the bindina affinitv of the analogue to the enzyme. The hydrogen binding character- istics and the orientation of the carbonyl oxygen of the normal substrate could preclude such an effect. A more definitive analysis of this hypothesis awaits the x-ray structure of the enzyme with substrate bound in the catalytic site.

An aspect of the activation process yet to be investigated is whether PC activates its own hydrolysis? While the activation of PE hydrolysis was easily demonstrated, the activation of PC hydrolysis was much more difficult to test. Because PC acts as both substrate and activator, it was not possible to ascertain the non-activated rate of PC hydrolysis. We have now made use of the fact that sphingomyelin is a more efficacious activator than PC. PC activates PE hydrolysis by 7-lo-fold; sphingomyelin activates it by 20-fold (42). If PC hydrolysis is activatable, the addition of sphingomyelin should produce an additional activation over and above that of PC itself. When surface dilution effects are taken into account, thio-PC hydrolysis is clearly activated as shown in Fig. 3. Thus, PC hydrolysis can be activated by a more potent activator. In addition, there appears to be no inhibition by sphingomyelin. This would seem to indicate that sphingo- myelin’s binding to the catalytic site is much weaker than the substrate’s. These results clearly demonstrate that PC not only activates PE hydrolysis but also its own.

The correlation between hydrophobicity of the sn-1 func- tional group and the binding affinity, point ii, is consistent with the observation that the substrate binding pocket of the catalytic site of phospholipase A2 is hydrophobic. X-ray crys- tallographic studies (38-40) have revealed that the interior wall of the catalytic site is covered with highly conserved,

In conclusion, the studies presented in this article demon- strate the importance of taking into account surface dilution when studying enzymes that act on lipid interfaces. They have also pinpointed four structural features involved in the interaction of phospholipase A, and its substrate. First, the amide analogues bind tighter than natural phospholipids.

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2664 Phospholipase Az Inhibitors

Second, the phosphorylethanolamine-containing analogues 17. Davidson, F. F., Hajdu, J., and Dennis, E. A. (1986) Biochem. appear to hind to the phospholipase AP tighter than those Biophys. Res. Commun. 13’7,587-592

containing phosphorylcholine head groups but the activation 18. Hazlett, T. L., and Dennis, E. A. (1985) Toxicon 23,457-466

caused by the PC compounds must be taken into account. 19. Hendrickson, H. S., Hendrickson, E. K., and Dybuig, R. H. (1983)

J. Lipid Res. 24, 1532-1537 Third, the hydrophobicity of the sn-1 group has a significant 20. Brockerhoff, H., and Ayengar, N. K. N. (1979) Lipids 14,88-89 effect on the binding of phospholipid to the enzyme. The more 21. Greenstein, J. P., and Wintz, M. (1961) in Chmlstry of the Amino

hydrophobic the ~a-1 functional group, the tighter the hinting Acids, pp. 925-928, John Wiley & Sons, New York

of phospholipid. Fourth, the a-methylene group of the w-2 22. Rosenthal, A. F. (1975) Methods Enzymol. 35~. 429-529 23. Ryn, E. K., and MacCoss, M. (1979) J. Lipid Res. 20, 561-563

fatty chain is essential for the binding of the analogue to the 24. Still, W. C., Kahn, M., and Mitra, A. (1978) J. Org. Chem. 43,

catalytic site but not to the activator site. Further studies are 2923-2925

in progress to develop a kinetic model for this inhibition. 25. Dennis, E. A. (1973) Arch. Biochem. Biophys. 158,485-493 26. Comfurius. P.. and Zwaal. R. F. A. (1977) Biochim. Bio~hvs. Acta

1.

2.

3. 4. 5.

6. 7. 8.

9.

10.

Il.

12.

13.

14.

15.

16.

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Physiol. Biochem. Pharmacol. 91, 91-203 van den Bosch, H. (1980) Biochim. Biophys. Acta 604,191-246 Dennis, E. A. (1987) Bio/Technology 5, 1294-1300 Samuelson, B., Duhlen, S. E., Lindgren, J. A., Rouzer, C. R., and

Serhan, C. N. (1987) Science 237, 1171-1176 Samuelsson, B. (1987) Drug 33, 2-9 Smith, W. (1989) Biochem. J. 259, 315-324 Roberts, M. F., Deems, R. A., Mincey, T. C., and Dennis, E. A.

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L Yu, R A Deems, J Hajdu and E A DennisThe interaction of phospholipase A2 with phospholipid analogues and inhibitors.

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