- Email: [email protected]
PAF-acether specific binding sites: 1. quantitative SAR study of PAF-acether isosteres
368
TIPS - September 1986
PAF-acether specific binding sites: 1. quantitative SAR study of PAF-acether isosteres J. J. Godfroid and P. Braquet PAF-acether is a phospholipid which mediates a range of biological activities including anaphylaxis and shock. Stereospecific binding sites exist in platelets and lung. In a series of two articles, P. Braquet and J. J. Godfroid describe these sites, their biological control and the medicinal chemistry which has led to the design of specific antagonists. As discussed in Part 1 below, QSAR studies of PAF-acether isosteres have yielded much information about the nature of the binding site itself, and a putative conformation is put forward.
Platelet-activating factor (PAF, PAF-acether, AGEPC) is a phospholipid mediator with a wide spectrum of biological activities 1. It was first described as a soluble component released from rabbit basophils sensitized with immunoglobin E, which induced rabbit platelet aggregation 2. Independently, a PAF-acetherlike substance, isolated from the renal medulla, exhibited a potent hypotensive effect and was termed Antihypertensive Polar Renomedullary Lipid (for a review see Ref. 3). PAF-acether has been identified as 1-O-alkyl2(g)-acetyl-glycero-3-phosphorylcholine (Fig. 1) demonstrated by means of hemisynthesis from natural [choline or ethanolamine] plasmalogens. A short ester function is present in position 2 of the glycerol skeleton. The naturally occurring enantiomer presents the R configuration and is a mixture of C18 and C16 linear alkyl chains in position -1: the platelet stimulating activities are practically the same for both substances. The total synthesis of PAF-acether 4 has allowed an increasingly significant role to be demonstrated for this novel autacoid, mainly in anaphylaxis and shock 1.
(R), but not the s isomer, was effective in stimulating the various PAF-acether responses in vivo and in vitro, in platelet or neutrophils first suggested the involvement of specific receptor(s) 5. Further support was given by the observations that: (1) very low concentrations (usually lower than 0.1 nM) are required for triggering biological effects; (2) specific desensitization occurs after exposure of tissue to PAF-acether; and (3) biological effects are specifically inhibited by PAF-acether antagonists. The existence of PAF-acether specific binding sites was recently confirmed by experiments using [3H]PAF-acether. As seen in Table I, high affinity binding sites have been found in human and rabbit platelets, human neutrophils and human lung membrane. The affinity and number of these binding sites correlate with the tissue and species specificity of the biological effects of PAF-acether;
(R)
CH2- O- R
CH3-C-O-C-
H
13
+/CH3 CH2 - O - P - O - (CH2)2 - N - - CH 3 /\\ -~. O- 0 CH3
Fig. 1. Plate/et Activating Factor (PAFacether) R = nC, sHs7 and nC1eH~, natura/ configuration (R).
human platelets using chromatography of membrane preparation on a sepharose column loaded with PAF-acether/human serum albumin followed by SDS polyacrylamide gel electrophoresis
TABLE I. Occurrence and characteristics of PAF-acether specific binding sites Material Human PMN Human lung tissue
Occurrence of PAF-acether specific binding sites The demonstration that the naturally occurring stereoisomer
Human Human Human Human
J. I. Godfroid is Professor of Organic Chemistry and Pharmacochemistry,Universitd Paris VII, Laboratoire de Pharmacochimie Mol~culaire, 2, Place Jussieu, 75251 Paris cedex 05, France, and P. Braquet is General Manager and Director of Research, Institut Henri Beaufour, 17, Avenue Descartes,92350 Le PlessisRobinson, France.
Rabbit platelet Rat platelet
1986, Elsevier Science Publishers B.V., A m s t e r d a m
rat platelets which do not aggregate to PAF-acether do not possess high affinity binding sites. The PAF-acether receptor in platelet plasma membrane is heat labile and protease-sensitive 6. Furthermore, exposure of platelets to PAF-acether at 37°C for 5 m i n causes desensitization, resulting in a decrease in specific binding and aggregation. Platelets desensitized to PAF-acether are capable of normal response to other agonists such as ADP, collagen, thrombin, calcimycin (A 23187) and arachidonic acid, implying that a specific receptor has been desensitized 7. The lack of correlation between the effects of PAF-acether on platelet aggregation and on several physical properties of pure dipalmitoylphosphatidylcholine bilayer as detected by differential scanning calorimetry suggests that PAFacether receptor sites may not be phospholipids. Attempts to characterize the high affinity PAFacether binding site present in
platelet platelet platelet platelet
Kd (X 10 -9 M) 0.11 +_0.02 0.49 _+0.17 37 _+ 13 1.58 _.+0.36 0.053 __.0.014 ",t 0.7
0.9 __.0.5 -
Number of sites per cell 5.2 __.2.1 x 106 140 __. 7 fmole mg -1 protein 1399 -+ 498 1983 _+391 242 + 64 150-300 (1.61 + 0.34 x 10 TM mg of membrane) 19386 _.+6588 not found
References f c e a b d
a a
* gel-filtered, t calculated from K, (association equilibrium constant) a. Inarrea, P. eta/. (1984) Eur. J. Pharmacol. 105, 309-315; b. Kloprogge, E. and Akkerman, J. W. N. (1984) Biochem. J. 223, 901-909; c. Hwang, S. B. et al. (1983) Biochemistry 22, 4756-4763; d. Hwang, S. B. et al. (1985) Biochem. Biophys. lies. Commun. 126, 972-979; e. Valone, F. H. eta/. (1982) J. Immunol. 129, 1637-1641; f. Valone, F. H. and Goetzl, E. J. (1983) Immunology48, 141-149; g. Valone F. H. Thromb. Ras. (in press).
0165 - 6147/86/$02.00
369
T I P S - September 1986
4.2
2.1
Q
l~l
2.2
1.2
I
o
cAMP level and stimulates hydrolysis of GTP (EDs0 10-9 M; Ref. 9) whereas the non-natural (s) enantiomer is inactive up to 101~M, suggesting that the receptor may be coupled to the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.
1.1
IG I
:
.' -',c - o l -
I
4.1
'
' 4.2
CH2'- O - P - iOi-(CH2)n'- Y
""'l-I
6: o" 'o
i
Fig. 2. Subs#tuents and functional variations used in order to search for agonists and antagonists of PAF-acethar. I. Changes in 1-position: I. 1. fatty chain (high hydrophobicity) necessary for both activities; 1.2. ether function is strictly required for agonistic activities but not for antagonistic activities: cerbamoyl function may be used. 2. Changes in 2 . ~ :2.1. a short ester group (R' = CH~ or C~Hs) is required for both activities; 2.2. ester may be replaced (1) by a short ether function (CH30- for antagonistic activities, C~HsO- for both; (2) methyl carbamoyl group, methyl ketone function for both activities. 3. Changes in 3-position: 3.1. phosphate or phosphonate group are required for agonistic activities; phosphate may be deleted for antagonistic activities (carboxylate or ether functions); 3.2. n = 2 or 3 for agonistic activities, and is greater for antagonistic activities until n = 10; 3.3 Y = methyl ammonium group N+(CH3), N+-CH3 (C~Hs)= or N-methyl-piparidinium, -morpholinium, -pyrrolidinium for better agonists than PAFacether itself. Antagonistic activities are enhanced by other heterocycles such as pyndinium or thiazolidinium group. 4. Changes on glycaryl backbone: 4.1. place isomer (cf. Fig. 4); 4.2. modifications decrease both activities; 4.3. S-enantiomer is inactive.
Structure--activity relationships in agonist series A wide variety of PAF-acether analogues were synthesized in order to: (1) establish the structural requirements for agonistic activities; (2) achieve possible therapeutic effects such as selective antihypertensive activity while eliminating undesirable effects such as platelet aggregation and bronchoconstriction; and (3) search for new antagonists. Fig. 2 presents the different strategies used in the design of new PAF-acether related structures from the original framework. Changes in 1-position The hydrophobicity of the chain is a key point for activity. Thus, the agonistic effects: platelet (washed cells and platelet rich plasma) stimulation, hypotension and thrombo-
of the eluted material, revealed a single protein with an apparent molecular weight of 180 000 (Ref.
PAF-acether-induced signalling process. R-PAF-acether decreases
6).
TABLE II. Structure-activity relationship in PAF-acether series: Influence of the change in the nature of the substitution on C2 with respect to rabbit platelet aggregation (see Refs 11 and 13)
Unfortunately, more detailed biochemical characterization of the PAF-acether receptor has been hampered by the lack of a consistent procedure to solubilize the membrane binding protein and a reliable method to prepare high titre specific antibodies to PAFacether s. The specific binding of PAFacether to platelet plasma membranes is regulated by monovalent and divalent cations and GTP. According to Hwang et al. (see Table I), inhibition of [3H]PAFacether binding is sodium-specific, with an ED5o of 6mM. Li + also inhibits the binding but at a relatively higher concentration (EDs0 = 150 mM). Conversely K +, Cs + and Rb + and the divalent cations Mg 2+, Ca 2+ and Mn 2+ enhance the binding: interestingly, binding of catecholamines to o~adrenergic receptors is enhanced by GTP and Na +, but not by K +. Replacement of H20 by D20 in this incubation induces a total inhibition of PAF-acether-induced aggregation although binding is not affected (see Hwang et al. 1983, Table I). This suggests a role for H + in the early events involved in the
CH, 0 qCH ,,CH,
g+
CH 0 P 0(CH) 'i~M¢
CHz 0 (CH~I~CHj
I C • 0 - 'CH 0C II q I O CH~ 0 P OICH~) NMe, 0 PLATELETAGGREGATION STIMULATION (PRP)
FEATELETAGGREGATIONSTIMULATION (pgp) OH OMe OEI 0 BZ 0 C NH CH H
< 001 010 4 00 100 00
n
0 ~ NH Ph 2 38 100 O0
76 92 0 57 <.001
0 NH NHCOCH, NHCO 0 CH NHC0 0 Bz NHCONHCH, 0NO OSO,CH, CH~COCH, n Pr 4 P, i Bu
001 181 0 gO • 001 0 02 6 2b 0 03 5 65 I ?5 • 001 0 08
Rabbit blood was cctlected on ACD (1 for 9 volumes of blood) and PRP was obtained after centrifugetion at 100 g for 15 min. The platelet number was then adjusted to 3 x 10e cells x m1-1. In order to prepare the washed platelets, PRP was acidified (pH 6.4) with citric acid (0.15 M), then centrifuged at 900 g for 10 min. The supematent was discarded and the pallet was resuspanded in Tyrode's citrate buffer (1 vol. of citrate buffer for 9 vol. Tyrode's pH 6.4). The platelet suspension was centrifuged again (900 g; 10 rain), the supernetant was eliminated and the pellet was r e s u s p ~ in the same buffer. The suspension was then divided into three aliquots. The first was incubated with 0.1 mM acetylsalicylic acid, the second with 0.1 mM ADP and the third with acetylsalicylic acid and ADP used at the same concentrations. After 1 h incubation at room temperature, the three aliquots were centrifuged and resuspencled in a volume of Tyrode's-ACD buffer so as to obtain a pletelet concentration of 21 x I0" cells x ml-L In all exbedments, the reference stimulation (100%) was obtained with CIe PAF-acether.
370
TIPS - September 1986 Refs 11, 13). The main factor for activity appears to be related to the length and the bulk of the substituent with a m a x i m u m Van der Waals radius of 6-7 ,/~ (see Fig. 6).
log R.A.
-1-" -0.5 -
AcO ~ 2 H x
00.S
' '
CH2-OPC
• ~.~-.-~=~:;:"*
-
2-
"'"
"
.',/
• "*,""
3.5 ] ~ , " ,,/"
si
Changes in 3-position The replacement of phosphate group (O-POT-O-) by a phosphonate group (--O-PO2--) does not significantly modify platelet stimulation 14, but its deletion abolishes the activity (Fig. 2; Ref. 15). The distance between the phosphate group and the a m m o n i u m head is critical (Fig. 2). Platelet stimulation significantly decreases from n = 3 to n = 10 (Ref. 16, 29). Certain changes in the nature of the a m m o n i u m group can lead to enhanced agonistic activity in comparison with CIs PAF-acether: e.g. several cyclic analogues such as N-methyl piperidinium, Nmethyl pyrrolidinium isosteres etc. (for example, see Ref. 17).
o
O
THR
- -
•
[] []
I
PRP -= ~ WP . . . . . . .
=:s
~
number of carbon (fatty chain Cl) 0 2 4 6 8 I I J I I
10 I
,,1;1
is 12 I
14 I
16 I
lo 18 I
20 I
22 Ii,
Fig. 3. Agonistic effect in vitro and in vivo of various PAF-acether analogues substituted with different functional group in position 1. The y-axis represents the logarithm of the relative activity of the agonist against C~a PAF-acether [i.e. Iog(EC6o agonist/ECso C~a PAF)]. The x-axis represents the sum of the hydrophobic fragmental constants (Er) calculated from the lipophilicity table./Rekker, R. F. and De Kort, H. M. (1979) Eur. J. Mad. Chem. 14, 479-488]. Maximal activity occurs when :5 < 2r < 8. Platelet sb'mulation: WP: rabbit washed pletelets; PRP: rabbit plasma rich platalets respectively; HYP: hypotension (rat), BRO: bronchoconstriction and THR: thrombocytopenia (guinea-pig). Curves correspond to analogues with x = O - R (R = C4Ha to C2oH~); experimental results may be integrated into such correlations with x = 0 - C6H~ - R' or alkenyloxy. Some data are shown on the diagram and correspond to platelet stimulation with: 0 x = CHs - (CH2)le - C - 0 II
V x = CH3-(CH~)1a-
O z5 x = CH~-(CH~)ls - 0 - CH2 -
cytopenia, but not bronchoconstriction are m a x i m u m for a lipophilicity corresponding to a C16-C18 chain, equivalent to a calculated hydrophobic fragmental constant (Zf) ~ 7.5 (for a review see Ref. 10). The highest bronchoconstrictive activity was found at lower lipophilicity (Zf = 5.90) corresponding to a C14 chain (Fig. 3). All activities in these series are closely correlated 1°. Furthermore, the position of the fatty chain with respect to glyceryl framework and the ether-oxide function, are critical for activity. Phenoxy and alkenoxy analogues of PAF-acether are highly active, but thioether or acyl derivatives are inactive (see Refs 10, 11). Steric effects around the glycerol backbone decrease agonistic activities 12.
Changes in 2-position The ester function is not required for agonistic activity (see Table II). Surprisingly, the methylcarbamate analogue presented the same act-
~ x = CH3-(CH2)~7-S
ivities as PAF-acether; methylketone or ethoxy analogues retained a significant part of PAFacether activity (for example, see
Modifications on the glyceryl backbone Several investigators have reported the synthesis of positional isomers (C1/C2 and C 2 / C 3 ) and their enantiomers (Fig. 4) (see Ref. 11). It would be expected that the spatial arrangement of three glyceryl substituents in PAFacether would be critical for activity. However, this was only partially the case, since only minor overall structural differences exist between the different position isomers. Indeed, only the chirality of the asymmetric centre need be taken into account for activity,
CH 2 - - O - - (CH2).CH3 J(S)
CH 2 --O--(CH2).CH3 J(R)
/c. Ac--O
~ "'H CH2--O--PC HS)
/c.
PC-- O-- CH~. ~'-H O--Ac HR)
CH 2 - - O - - (CH2).CH3 I(R)
/c.. A~--O--C., I
TM O-- PC 2(R)
CH 2 - - O - - (CH~).CH 3 I(S)
pc-o
/c.. L "'"
CH2--O--PC 3(R}
/c. L""
PLACE ISOMER C2/C3
CH 2 - O -- Ac 2(S)
O -- (CH2)~CH3 I(R)
Ac-o-c,~
PAF
O-- (CH2).CH 3 I(S)
pc-o-c,2
/c. L'"H
CH2--O--Ac _3(S)
PLACE ISOMER Cl/C2
Fig. 4. Companson of the R- end s-isomers of PAF-acether with those of its place isomers (PC = phosphorylcholine) It should be emphasized that no matter what oomparison is made between the different isomers, the only difference resides in the displacement of a methylene group, provided that the chirality of C, has been respected.
371
TIPS - S e p t e m b e r 1986 C
-I
/ "~'~o ~ I~ / ~) -|
C
/%,, \\
ELECIRONIC
TRANSFERT
-i
,,,S ) C/
ME.BRA.E TARGET
] --'"~
TARGETMEMBRANE
-t
o....~...c,'"~'~~
MEMBRANE TARGET
--C Fig. 5. Putative role of free electronic doublets of the heteroatom on C~ of PAF-acether glyceryl framework in signal triggenng: effect of electronegativity and mesomerism.
the s-enantiomers of the two positional isomers corresponding to the R-PAF and vice versa (Fig. 4). The loss of activity of both C1/C2 and C 2 / C 3 s-isomers (about 1.5 order of magnitude) may be related to the increase in the length of the substituent at position C2 as discussed above. The length and bulk of the backbone is of importance: acetoxy methylene or methylene incorporated in the glycerol framework significantly decreases platelet aggregation 12.
Conformation of PAF-acether specific membrane binding sites and its modification during cell activation A putative conformation of PAFacether platelet membrane binding sites may be deduced from data obtained with agonists. Agonistic activity decreases when the fatty chain is shortened 1°. Therefore, a lipophilic moiety seems essential for agonistic activity, implying that the long fatty chain of PAF-acether enters deeply into the membrane in a hydrophobic area (e.g. hydrophobic lipid-lipid or lipid-protein interactions). The anchorage of the chain in the membrane and the relative position of the ethoxide function with its environment certainly modify membrane fluidity and membrane activation 11. The significance of an electronic transfer from oxygen doublets of
the ethoxide function to an unknown membrane target is indicated by the low activity of the thioether derivative which has a lower electronegativity (2.5 for sulphur v. 3.5 for oxygen) leading to a reduced availability of the doublets borne by the heteroatom n. Analogues bearing an isosteric group such as CH2 which do not comprise doublets are inactive. A similar result is observed with 1-acyl analogues which possess doublets involved in a mesomerism and which are therefore not available (Fig. 5). The presence of doublets could be made necessary by a possible protonation from the active site. The inhibition of aggregation induced by PAF-acether in D20 (without inhibition of the binding; see Hwang et al. 1983, Table I) supports this hypothesis. As shown in Table II, agonistic activity can be produced with a wide variety of substituents on the carbon 2 of the glyceryl backbone. The main factors which must be taken into account are the length
and the bulk of the moiety (see Fig. 6 for some examples). Agonistic activity is markedly reduced in substituents with large steric hindrance. A similar decrease in activity is also observed with the smallest groups or with C13 and C17 acetal plasmalogens in which C1 and C2 of the glyceryl framework are bound to a long fatty chain via an acetal linkage it. Thus, the C2short chain may participate in the anchorage of PAF-acether on its receptor, leading to a better alignment of the polar head of the mediator with that of membrane phospholipids. This assumption is reinforced by the necessary Rconfiguration generally required for activity. The higher potency of isosteres with various quaternary groups in C3 and the optimal chain length clearly shows the importance of the polar head. The binding of anionic phosphate group to a positively charged moiety (other quaternary group of vicinal phospholipids?) may be needed for agonistic activity.
(P
0
,0 N
"",,, / HYDROPHOBIC AREA
Fig. 6. Spatial representation of three different substituents on C2. (1) The cavity surface areas of the acetate (bottom) propionete (middle) and methylcerbamoyl (top) are close; (2) Their activity on platelet aggregation is also similar.
372
T I P S - S e p t e m b e r 1986
A putative conformation of PAFacether binding site is proposed in Fig. 7, taking into account the above considerations. After binding to its receptor, PAF-acether might indirectly influence the conformation of the unknown target located within the membrane by: (1) an electronic charge transfer from the ethoxide function (see above); (2) modification of the fluidity around the part of the targets included in the bilayer; or
I E = 16 KcJm01e]
(3) disturbing the external protein-phospholipid polar head interactions. After binding to its specific binding sites, PAF-acether may activate the unknown target X (specific receptor protein?). Activation of X may lead to two concomitant events: (1) Hydrolysis of GTP and activation of phospholipase C activity triggering phosphoinositide breakdown with formation of inositol triphosphate [Ins(1,4,5)P3]
t- "" ....... t/,°
-
/
1-
Fig. 7. Putative conformation of platelet PAFacether specific binding site. In Fig. 7,4, the slashed area represents the unknown target which could be triggered by an electronic transfer from the oxygen doublet. The different theoretical energies are presented. The fatty chain fits into the hydrophobic area of the membrane. Fig. 7B, presents the possibility of binding for R (natursI) and s (synthetic) PAF-acether. Note that for s-configuration, the chirslity of Cz does not permit a correct insertion of the autacoid in its binding site. Conversely, the place isomer C~C3 can enter the site, the only difference with PAF-acether resides in the displacement of a methylene group
i
o e-"----®
~,--~0
i~ o.51~,i I
\
IE = 2-5 KcSmole]
....
IE ~ l~~K:"~'l
~,.-
i
7o, I
from C~ to C~.
and diacylglycerol (DG) (Ref. 19), both of which have properties of classical second messengers. Ins(1,4,5)P3 induces the release of calcium from its internal pools leading to increased cytosolic calcium [Ca2+]i. This in turn may open [Ca2+]i-dependent K + channels as observed in PAF-acether-activated mouse macrophages 19. On the other hand, DG is involved in protein kinase C activation leading to subsequent phosphorylation of specific cellular protein which then contributes to various physiological processes (particularly secretion and proliferation). (2) Internalization of the complex PAF-acether specific binding sites by an unknown active or passive process. The binding of PAF-acether to its specific membrane binding sites favours its internalization, either directly, as for other ligand receptor complexes undergoing downregulation 2° or, indirectly, as a consequence of cell activation. Transmembrane movement of PAFacether represents the limiting step of its metabolism as demonstrated in rabbit platelets 21 and explains the subsequent desensitization. PAF-acether is then deactivated in two steps: firstly, PAF-acether is deacetylated by the cytosolic acetylhydrolase into lyso-PAF. Since acetylhydrolase activity is cytosolic, degradation of PAFacether is intracellular. This en-
hydrophobic/ionic interactions
\
~.~)\.
X®/~/0,, --O~
\)r",
%7q,o° X
\ o\
CH2 membrane ..\\~'-'~!R) target ,~--....~ / L~'n .~-~ CH2 .! \ 7
<_
/
~
•
/ ~ ....
• CH 2
%
t oo) \
;
"=-/6.5
,'5-7 ~ -anchorage --/--
I ~)--I I (,
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area
I
I
"
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/\~
I
t /" t. hydrophobic ~ ,~ "~interactions
(,
,
/\~1
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(R) c o n f i g u r a t i o n natural i
f /\-~ (S)
configuration
i
PAF-acether
Place isomer of PAF-acether
373
T I P S - S e p t e m b e r 1986
zymatic system removes specifically the group from the 2-R position. This e n z y m e is present i n the intracellular compartment22, b u t acetyl hydrolase activity has also b e e n detected i n extracellular fluids 23. The properties of the plasma enzyme are similar to those of the enzyme extracted from the cells except that the latter is resistant to the action of proteases 24 a n d is refractory to serine hydrolase inhibitors (L. Touqui, pers. commun.). Acetyl hydrolase differs from phospholipase A2 i n that it cleaves only the short chain at the 2-R position of phospholipids and is Mg 2+ and Ca 2+i n d e p e n d e n t . Interestingly, a positive correlation was found between platelet sensitivity to PAFacether i n different species and its ability to inactivate it. Secondly, as lyso-PAF is lytic a n d detergent, its rapid elimination is crucial for the cell. This is achieved b y acylation of the hydroxyl group i n the 2-R position b y an acyltransferase resulting i n the formation of alkylacyl-glycerylphosphocholine 2s a c o m p o n e n t of the i n n e r layer of the m e m b r a n e 26. The majority of studies with exogenous lyso-PAF show that the p r e d o m i n a n t route of conversion is to alkylacyl-glycerylphosphocholine, a relatively m i n o r a m o u n t b e i n g converted to PAF-acether. This suggests that acyltransferase has a high affinity for lyso-PAF27. Incorporation is mainly catalysed b y a C o A - i n d e p e n d e n t transacylase, which uses phosphatidylcholine as the source of arachidonic acid. Free arachidonic acid is initially incorporated into phosphatidylcholine b y a C o A - d e p e n d e n t acyltransferase and thereafter transferred to lyso-PAF and to other ether lipids b y transacylation28.
Getting the name right Dictionary of Pharmacology by W . C. B o w m a n , A n n e B o w m a n and A l i s o n B o w m a n , B l a c k w e l l Sc,~ntific Publications, 1986. £12.50 (vii + 234 pages) I S B N 0 632 01131 9
In compiling a dictionary of a very specific subject, the question is really not so much 'Where do you start?' b u t more 'Where do you
Reacylation of lyso-PAF is inhibited by calcium (ICs0 of 50-100 I~M; Touqui et al., pets commun.), suggesting that d u r i n g cellular activation an elevation i n Ca 2+ influx may i n h i b i t this enzyme resulting i n the transient accumulation of lyso-PAF thus favouring its utilization b y acetyl transferase (i.e., PAF-acether synthesis). []
[]
[]
Better knowledge of PAFacether specific b i n d i n g site(s) a n d of biochemical events involved in PAF-acether-induced m e m b r a n e signal should enable designing n e w potent antagonists for the future treatment of asthma, thrombosis graft rejection or shock.
Acknowledgement Thanks are due to B. B. Vargaftig and L. Touqui for interesting discussions.
References 1 Vargaftig, B. B., Chignard, M., Benveniste, J., Lefort, I. and Wall, F. (1981) Ann. NY Acad. Sci. 370, 119-137 2 Benveniste, J., Henson, P.M. and Cochrane, C. G. (1972)J. Exp. Med. 136, 1356--1377 3 Muirhead, E. E. and Pitcock,J. A. (1985) ]. Hypertens. 3, 1--8 4 Godfroid, ]. J., Heymans,F., Michel, E., Redeuilh,C., Steiner,E. and Benveniste, J. (1980) FEBS Left. 116, 161-164 5 Vargaftig, B. B. and Benveniste,J. (1983) Trends Pharmacol. Sci. 4, 341-343 6 Valone, F. H. (1984) Immunology 52, 169-174 7 Lalau, Keraly, C. and Benveniste, J. (1982) Br. J. Haematol. 51, 313-322 8 Nishihira, J., Ishibashi, J. and lmai, Y. (1984) ]. Biochem. 95, 1247-1251 9 Avdonin,P. V., Svitina-Ulitina,I. V. and Kulikov, V. I. (1985) Biochem. Biophys. Res. Commun. 131, 307-313 10 Godfroid, I. J., Broquet, C., Jouquey, J., Lebbarn, M., Heymans, F., Steiner, E., Michel, E., Coeffier, E., Fichelle,F. and
stop?'.
In
this the Bowman tribe describe very precisely where they draw their limits for inclusions. Drug classes are included b u t drug names are not. Physiological and biochemical terms are culled, b u t only w h e n the compilers feel they have become part of essential pharmacological parlance. The preferences and prejudices of the compilers come Dictionary
the
preface
to
of Pharmacology,
Worcel, M. 1. Med. Chem. (in press) 11 Braquet,P. and Godfroid,J. ]. in Platelet activating Factor (Snyder, F., ed.), Plenum, (in press) 12 Wissner, A., Schaub, R. E., Sum, P. E., Kohler,C. A. and Goldstein,B. M. (1985) J. Med. Chem. 28, 1181-1187 13 Hadvary, P., Cassal, ]. M., Hirth, G., Barner, R. and Beaumgartner, H.P. (1986) in Proceedings of the Platelet Activating
Factor
Symposium
(Benveniste,J. and Arnoux,B., eds), pp. 57, Elsevier 14 Moschidis, M. C., Demopoulos, C. A. and Kritikou, L. G. (1983) Chem. Phys. Lipids 33, 87-92 15 Heymans,F., Borrel,M. C., Broquet, C., Lefort,]. and Godfroid,]. J. (1985)J. Med. Chem. 28, 1094-1096 16 Tokumura, A., Homma, H. and Hanahan, D. J. (1985)]. Biol. Chem. 260, 12710-12714 17 Ohno, N., Fujita, K., Nakai, H., Kobayashi,S., Yamashita,M., Inove, K. and Nojima, S. (1983) in Proceedings of the Platelet Activating Factor Symposium
(Benveniste,J. and Arnoux,B., eds), p. 9, Elsevier 18 Lapetina,E. G. (1982)]. Biol. Chem. 257, 7314-7317
19 Garay, R. and Braquet, P. Biochem. Pharmacol. (in press) 20 Schlessinger, J. (1980) Trends Biochem. Sci. 5, 210-214 21 Lachachi, H., Plantavid, M., Simon, M. F., Chap, H., Braquet, P. and Douste Blazy, L. (1985) Biochem. Biophys. Res. Commun. 132, 460-466 22 Blank,M. L., Lee, T., Fitzgerald,V. and Snyder, F. (1981) J. Biol. Chem. 256, 175-178 23 Farr, R. S., Cox, C. P., Wardlow, M. L. and Jorgensen, R. (1980) Clin. Immunol. Immunopathol. 15, 318-330 24 Blank, M. L., Hall, M. N., Cress, E. A. and Snyder, F. (1983) Biochem. Biophys. Res. Commun. 113, 666--671 25 Cabot, M. C., Blank,M. L., Welsh, C. l., Horan, M. l., Cress, E. A. and Snyder,F. (1982) Life Sci. 31, 2891-2898 26 Touqui, L., Jacquemin,C., Dumarey,C. and Vargaftig, B.B. (1985) Biochem. Biophys. Acta 833, 111-118 27 Chilton, F. H., O'Flaherty, J.T., Ellis, J. M., Swendsen,C. L. and Wykle,R. L. (1983) J. Biol. Chem. 258(12),7268-7271 28 Kramer, R. M. and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811 29 Wissner, A., Schaub, R. E., Sum, P. E., Kohler,C. A. and Goldstein,B. M. (1986) J. Med. Chem. 29, 328-333 through to a small extent, but this is forgivable considering the potential for personal influence. I am sure that the major aim of this dictionary is to clarify terms that have tended to lose precision through overuse (and careless use). It is surprising then that enzymes are not denoted according to their IUB recommended nomenclature or Enzyme Commission numbers. However, in general, pharmacological definitions are admirably precise. In some entries key references are also given taking the reader to original m e a n i n g s and definitions
TIPS - September 1986
PAF-acether specific binding sites: 1. quantitative SAR study of PAF-acether isosteres J. J. Godfroid and P. Braquet PAF-acether is a phospholipid which mediates a range of biological activities including anaphylaxis and shock. Stereospecific binding sites exist in platelets and lung. In a series of two articles, P. Braquet and J. J. Godfroid describe these sites, their biological control and the medicinal chemistry which has led to the design of specific antagonists. As discussed in Part 1 below, QSAR studies of PAF-acether isosteres have yielded much information about the nature of the binding site itself, and a putative conformation is put forward.
Platelet-activating factor (PAF, PAF-acether, AGEPC) is a phospholipid mediator with a wide spectrum of biological activities 1. It was first described as a soluble component released from rabbit basophils sensitized with immunoglobin E, which induced rabbit platelet aggregation 2. Independently, a PAF-acetherlike substance, isolated from the renal medulla, exhibited a potent hypotensive effect and was termed Antihypertensive Polar Renomedullary Lipid (for a review see Ref. 3). PAF-acether has been identified as 1-O-alkyl2(g)-acetyl-glycero-3-phosphorylcholine (Fig. 1) demonstrated by means of hemisynthesis from natural [choline or ethanolamine] plasmalogens. A short ester function is present in position 2 of the glycerol skeleton. The naturally occurring enantiomer presents the R configuration and is a mixture of C18 and C16 linear alkyl chains in position -1: the platelet stimulating activities are practically the same for both substances. The total synthesis of PAF-acether 4 has allowed an increasingly significant role to be demonstrated for this novel autacoid, mainly in anaphylaxis and shock 1.
(R), but not the s isomer, was effective in stimulating the various PAF-acether responses in vivo and in vitro, in platelet or neutrophils first suggested the involvement of specific receptor(s) 5. Further support was given by the observations that: (1) very low concentrations (usually lower than 0.1 nM) are required for triggering biological effects; (2) specific desensitization occurs after exposure of tissue to PAF-acether; and (3) biological effects are specifically inhibited by PAF-acether antagonists. The existence of PAF-acether specific binding sites was recently confirmed by experiments using [3H]PAF-acether. As seen in Table I, high affinity binding sites have been found in human and rabbit platelets, human neutrophils and human lung membrane. The affinity and number of these binding sites correlate with the tissue and species specificity of the biological effects of PAF-acether;
(R)
CH2- O- R
CH3-C-O-C-
H
13
+/CH3 CH2 - O - P - O - (CH2)2 - N - - CH 3 /\\ -~. O- 0 CH3
Fig. 1. Plate/et Activating Factor (PAFacether) R = nC, sHs7 and nC1eH~, natura/ configuration (R).
human platelets using chromatography of membrane preparation on a sepharose column loaded with PAF-acether/human serum albumin followed by SDS polyacrylamide gel electrophoresis
TABLE I. Occurrence and characteristics of PAF-acether specific binding sites Material Human PMN Human lung tissue
Occurrence of PAF-acether specific binding sites The demonstration that the naturally occurring stereoisomer
Human Human Human Human
J. I. Godfroid is Professor of Organic Chemistry and Pharmacochemistry,Universitd Paris VII, Laboratoire de Pharmacochimie Mol~culaire, 2, Place Jussieu, 75251 Paris cedex 05, France, and P. Braquet is General Manager and Director of Research, Institut Henri Beaufour, 17, Avenue Descartes,92350 Le PlessisRobinson, France.
Rabbit platelet Rat platelet
1986, Elsevier Science Publishers B.V., A m s t e r d a m
rat platelets which do not aggregate to PAF-acether do not possess high affinity binding sites. The PAF-acether receptor in platelet plasma membrane is heat labile and protease-sensitive 6. Furthermore, exposure of platelets to PAF-acether at 37°C for 5 m i n causes desensitization, resulting in a decrease in specific binding and aggregation. Platelets desensitized to PAF-acether are capable of normal response to other agonists such as ADP, collagen, thrombin, calcimycin (A 23187) and arachidonic acid, implying that a specific receptor has been desensitized 7. The lack of correlation between the effects of PAF-acether on platelet aggregation and on several physical properties of pure dipalmitoylphosphatidylcholine bilayer as detected by differential scanning calorimetry suggests that PAFacether receptor sites may not be phospholipids. Attempts to characterize the high affinity PAFacether binding site present in
platelet platelet platelet platelet
Kd (X 10 -9 M) 0.11 +_0.02 0.49 _+0.17 37 _+ 13 1.58 _.+0.36 0.053 __.0.014 ",t 0.7
0.9 __.0.5 -
Number of sites per cell 5.2 __.2.1 x 106 140 __. 7 fmole mg -1 protein 1399 -+ 498 1983 _+391 242 + 64 150-300 (1.61 + 0.34 x 10 TM mg of membrane) 19386 _.+6588 not found
References f c e a b d
a a
* gel-filtered, t calculated from K, (association equilibrium constant) a. Inarrea, P. eta/. (1984) Eur. J. Pharmacol. 105, 309-315; b. Kloprogge, E. and Akkerman, J. W. N. (1984) Biochem. J. 223, 901-909; c. Hwang, S. B. et al. (1983) Biochemistry 22, 4756-4763; d. Hwang, S. B. et al. (1985) Biochem. Biophys. lies. Commun. 126, 972-979; e. Valone, F. H. eta/. (1982) J. Immunol. 129, 1637-1641; f. Valone, F. H. and Goetzl, E. J. (1983) Immunology48, 141-149; g. Valone F. H. Thromb. Ras. (in press).
0165 - 6147/86/$02.00
369
T I P S - September 1986
4.2
2.1
Q
l~l
2.2
1.2
I
o
cAMP level and stimulates hydrolysis of GTP (EDs0 10-9 M; Ref. 9) whereas the non-natural (s) enantiomer is inactive up to 101~M, suggesting that the receptor may be coupled to the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.
1.1
IG I
:
.' -',c - o l -
I
4.1
'
' 4.2
CH2'- O - P - iOi-(CH2)n'- Y
""'l-I
6: o" 'o
i
Fig. 2. Subs#tuents and functional variations used in order to search for agonists and antagonists of PAF-acethar. I. Changes in 1-position: I. 1. fatty chain (high hydrophobicity) necessary for both activities; 1.2. ether function is strictly required for agonistic activities but not for antagonistic activities: cerbamoyl function may be used. 2. Changes in 2 . ~ :2.1. a short ester group (R' = CH~ or C~Hs) is required for both activities; 2.2. ester may be replaced (1) by a short ether function (CH30- for antagonistic activities, C~HsO- for both; (2) methyl carbamoyl group, methyl ketone function for both activities. 3. Changes in 3-position: 3.1. phosphate or phosphonate group are required for agonistic activities; phosphate may be deleted for antagonistic activities (carboxylate or ether functions); 3.2. n = 2 or 3 for agonistic activities, and is greater for antagonistic activities until n = 10; 3.3 Y = methyl ammonium group N+(CH3), N+-CH3 (C~Hs)= or N-methyl-piparidinium, -morpholinium, -pyrrolidinium for better agonists than PAFacether itself. Antagonistic activities are enhanced by other heterocycles such as pyndinium or thiazolidinium group. 4. Changes on glycaryl backbone: 4.1. place isomer (cf. Fig. 4); 4.2. modifications decrease both activities; 4.3. S-enantiomer is inactive.
Structure--activity relationships in agonist series A wide variety of PAF-acether analogues were synthesized in order to: (1) establish the structural requirements for agonistic activities; (2) achieve possible therapeutic effects such as selective antihypertensive activity while eliminating undesirable effects such as platelet aggregation and bronchoconstriction; and (3) search for new antagonists. Fig. 2 presents the different strategies used in the design of new PAF-acether related structures from the original framework. Changes in 1-position The hydrophobicity of the chain is a key point for activity. Thus, the agonistic effects: platelet (washed cells and platelet rich plasma) stimulation, hypotension and thrombo-
of the eluted material, revealed a single protein with an apparent molecular weight of 180 000 (Ref.
PAF-acether-induced signalling process. R-PAF-acether decreases
6).
TABLE II. Structure-activity relationship in PAF-acether series: Influence of the change in the nature of the substitution on C2 with respect to rabbit platelet aggregation (see Refs 11 and 13)
Unfortunately, more detailed biochemical characterization of the PAF-acether receptor has been hampered by the lack of a consistent procedure to solubilize the membrane binding protein and a reliable method to prepare high titre specific antibodies to PAFacether s. The specific binding of PAFacether to platelet plasma membranes is regulated by monovalent and divalent cations and GTP. According to Hwang et al. (see Table I), inhibition of [3H]PAFacether binding is sodium-specific, with an ED5o of 6mM. Li + also inhibits the binding but at a relatively higher concentration (EDs0 = 150 mM). Conversely K +, Cs + and Rb + and the divalent cations Mg 2+, Ca 2+ and Mn 2+ enhance the binding: interestingly, binding of catecholamines to o~adrenergic receptors is enhanced by GTP and Na +, but not by K +. Replacement of H20 by D20 in this incubation induces a total inhibition of PAF-acether-induced aggregation although binding is not affected (see Hwang et al. 1983, Table I). This suggests a role for H + in the early events involved in the
CH, 0 qCH ,,CH,
g+
CH 0 P 0(CH) 'i~M¢
CHz 0 (CH~I~CHj
I C • 0 - 'CH 0C II q I O CH~ 0 P OICH~) NMe, 0 PLATELETAGGREGATION STIMULATION (PRP)
FEATELETAGGREGATIONSTIMULATION (pgp) OH OMe OEI 0 BZ 0 C NH CH H
< 001 010 4 00 100 00
n
0 ~ NH Ph 2 38 100 O0
76 92 0 57 <.001
0 NH NHCOCH, NHCO 0 CH NHC0 0 Bz NHCONHCH, 0NO OSO,CH, CH~COCH, n Pr 4 P, i Bu
001 181 0 gO • 001 0 02 6 2b 0 03 5 65 I ?5 • 001 0 08
Rabbit blood was cctlected on ACD (1 for 9 volumes of blood) and PRP was obtained after centrifugetion at 100 g for 15 min. The platelet number was then adjusted to 3 x 10e cells x m1-1. In order to prepare the washed platelets, PRP was acidified (pH 6.4) with citric acid (0.15 M), then centrifuged at 900 g for 10 min. The supematent was discarded and the pallet was resuspanded in Tyrode's citrate buffer (1 vol. of citrate buffer for 9 vol. Tyrode's pH 6.4). The platelet suspension was centrifuged again (900 g; 10 rain), the supernetant was eliminated and the pellet was r e s u s p ~ in the same buffer. The suspension was then divided into three aliquots. The first was incubated with 0.1 mM acetylsalicylic acid, the second with 0.1 mM ADP and the third with acetylsalicylic acid and ADP used at the same concentrations. After 1 h incubation at room temperature, the three aliquots were centrifuged and resuspencled in a volume of Tyrode's-ACD buffer so as to obtain a pletelet concentration of 21 x I0" cells x ml-L In all exbedments, the reference stimulation (100%) was obtained with CIe PAF-acether.
370
TIPS - September 1986 Refs 11, 13). The main factor for activity appears to be related to the length and the bulk of the substituent with a m a x i m u m Van der Waals radius of 6-7 ,/~ (see Fig. 6).
log R.A.
-1-" -0.5 -
AcO ~ 2 H x
00.S
' '
CH2-OPC
• ~.~-.-~=~:;:"*
-
2-
"'"
"
.',/
• "*,""
3.5 ] ~ , " ,,/"
si
Changes in 3-position The replacement of phosphate group (O-POT-O-) by a phosphonate group (--O-PO2--) does not significantly modify platelet stimulation 14, but its deletion abolishes the activity (Fig. 2; Ref. 15). The distance between the phosphate group and the a m m o n i u m head is critical (Fig. 2). Platelet stimulation significantly decreases from n = 3 to n = 10 (Ref. 16, 29). Certain changes in the nature of the a m m o n i u m group can lead to enhanced agonistic activity in comparison with CIs PAF-acether: e.g. several cyclic analogues such as N-methyl piperidinium, Nmethyl pyrrolidinium isosteres etc. (for example, see Ref. 17).
o
O
THR
- -
•
[] []
I
PRP -= ~ WP . . . . . . .
=:s
~
number of carbon (fatty chain Cl) 0 2 4 6 8 I I J I I
10 I
,,1;1
is 12 I
14 I
16 I
lo 18 I
20 I
22 Ii,
Fig. 3. Agonistic effect in vitro and in vivo of various PAF-acether analogues substituted with different functional group in position 1. The y-axis represents the logarithm of the relative activity of the agonist against C~a PAF-acether [i.e. Iog(EC6o agonist/ECso C~a PAF)]. The x-axis represents the sum of the hydrophobic fragmental constants (Er) calculated from the lipophilicity table./Rekker, R. F. and De Kort, H. M. (1979) Eur. J. Mad. Chem. 14, 479-488]. Maximal activity occurs when :5 < 2r < 8. Platelet sb'mulation: WP: rabbit washed pletelets; PRP: rabbit plasma rich platalets respectively; HYP: hypotension (rat), BRO: bronchoconstriction and THR: thrombocytopenia (guinea-pig). Curves correspond to analogues with x = O - R (R = C4Ha to C2oH~); experimental results may be integrated into such correlations with x = 0 - C6H~ - R' or alkenyloxy. Some data are shown on the diagram and correspond to platelet stimulation with: 0 x = CHs - (CH2)le - C - 0 II
V x = CH3-(CH~)1a-
O z5 x = CH~-(CH~)ls - 0 - CH2 -
cytopenia, but not bronchoconstriction are m a x i m u m for a lipophilicity corresponding to a C16-C18 chain, equivalent to a calculated hydrophobic fragmental constant (Zf) ~ 7.5 (for a review see Ref. 10). The highest bronchoconstrictive activity was found at lower lipophilicity (Zf = 5.90) corresponding to a C14 chain (Fig. 3). All activities in these series are closely correlated 1°. Furthermore, the position of the fatty chain with respect to glyceryl framework and the ether-oxide function, are critical for activity. Phenoxy and alkenoxy analogues of PAF-acether are highly active, but thioether or acyl derivatives are inactive (see Refs 10, 11). Steric effects around the glycerol backbone decrease agonistic activities 12.
Changes in 2-position The ester function is not required for agonistic activity (see Table II). Surprisingly, the methylcarbamate analogue presented the same act-
~ x = CH3-(CH2)~7-S
ivities as PAF-acether; methylketone or ethoxy analogues retained a significant part of PAFacether activity (for example, see
Modifications on the glyceryl backbone Several investigators have reported the synthesis of positional isomers (C1/C2 and C 2 / C 3 ) and their enantiomers (Fig. 4) (see Ref. 11). It would be expected that the spatial arrangement of three glyceryl substituents in PAFacether would be critical for activity. However, this was only partially the case, since only minor overall structural differences exist between the different position isomers. Indeed, only the chirality of the asymmetric centre need be taken into account for activity,
CH 2 - - O - - (CH2).CH3 J(S)
CH 2 --O--(CH2).CH3 J(R)
/c. Ac--O
~ "'H CH2--O--PC HS)
/c.
PC-- O-- CH~. ~'-H O--Ac HR)
CH 2 - - O - - (CH2).CH3 I(R)
/c.. A~--O--C., I
TM O-- PC 2(R)
CH 2 - - O - - (CH~).CH 3 I(S)
pc-o
/c.. L "'"
CH2--O--PC 3(R}
/c. L""
PLACE ISOMER C2/C3
CH 2 - O -- Ac 2(S)
O -- (CH2)~CH3 I(R)
Ac-o-c,~
PAF
O-- (CH2).CH 3 I(S)
pc-o-c,2
/c. L'"H
CH2--O--Ac _3(S)
PLACE ISOMER Cl/C2
Fig. 4. Companson of the R- end s-isomers of PAF-acether with those of its place isomers (PC = phosphorylcholine) It should be emphasized that no matter what oomparison is made between the different isomers, the only difference resides in the displacement of a methylene group, provided that the chirality of C, has been respected.
371
TIPS - S e p t e m b e r 1986 C
-I
/ "~'~o ~ I~ / ~) -|
C
/%,, \\
ELECIRONIC
TRANSFERT
-i
,,,S ) C/
ME.BRA.E TARGET
] --'"~
TARGETMEMBRANE
-t
o....~...c,'"~'~~
MEMBRANE TARGET
--C Fig. 5. Putative role of free electronic doublets of the heteroatom on C~ of PAF-acether glyceryl framework in signal triggenng: effect of electronegativity and mesomerism.
the s-enantiomers of the two positional isomers corresponding to the R-PAF and vice versa (Fig. 4). The loss of activity of both C1/C2 and C 2 / C 3 s-isomers (about 1.5 order of magnitude) may be related to the increase in the length of the substituent at position C2 as discussed above. The length and bulk of the backbone is of importance: acetoxy methylene or methylene incorporated in the glycerol framework significantly decreases platelet aggregation 12.
Conformation of PAF-acether specific membrane binding sites and its modification during cell activation A putative conformation of PAFacether platelet membrane binding sites may be deduced from data obtained with agonists. Agonistic activity decreases when the fatty chain is shortened 1°. Therefore, a lipophilic moiety seems essential for agonistic activity, implying that the long fatty chain of PAF-acether enters deeply into the membrane in a hydrophobic area (e.g. hydrophobic lipid-lipid or lipid-protein interactions). The anchorage of the chain in the membrane and the relative position of the ethoxide function with its environment certainly modify membrane fluidity and membrane activation 11. The significance of an electronic transfer from oxygen doublets of
the ethoxide function to an unknown membrane target is indicated by the low activity of the thioether derivative which has a lower electronegativity (2.5 for sulphur v. 3.5 for oxygen) leading to a reduced availability of the doublets borne by the heteroatom n. Analogues bearing an isosteric group such as CH2 which do not comprise doublets are inactive. A similar result is observed with 1-acyl analogues which possess doublets involved in a mesomerism and which are therefore not available (Fig. 5). The presence of doublets could be made necessary by a possible protonation from the active site. The inhibition of aggregation induced by PAF-acether in D20 (without inhibition of the binding; see Hwang et al. 1983, Table I) supports this hypothesis. As shown in Table II, agonistic activity can be produced with a wide variety of substituents on the carbon 2 of the glyceryl backbone. The main factors which must be taken into account are the length
and the bulk of the moiety (see Fig. 6 for some examples). Agonistic activity is markedly reduced in substituents with large steric hindrance. A similar decrease in activity is also observed with the smallest groups or with C13 and C17 acetal plasmalogens in which C1 and C2 of the glyceryl framework are bound to a long fatty chain via an acetal linkage it. Thus, the C2short chain may participate in the anchorage of PAF-acether on its receptor, leading to a better alignment of the polar head of the mediator with that of membrane phospholipids. This assumption is reinforced by the necessary Rconfiguration generally required for activity. The higher potency of isosteres with various quaternary groups in C3 and the optimal chain length clearly shows the importance of the polar head. The binding of anionic phosphate group to a positively charged moiety (other quaternary group of vicinal phospholipids?) may be needed for agonistic activity.
(P
0
,0 N
"",,, / HYDROPHOBIC AREA
Fig. 6. Spatial representation of three different substituents on C2. (1) The cavity surface areas of the acetate (bottom) propionete (middle) and methylcerbamoyl (top) are close; (2) Their activity on platelet aggregation is also similar.
372
T I P S - S e p t e m b e r 1986
A putative conformation of PAFacether binding site is proposed in Fig. 7, taking into account the above considerations. After binding to its receptor, PAF-acether might indirectly influence the conformation of the unknown target located within the membrane by: (1) an electronic charge transfer from the ethoxide function (see above); (2) modification of the fluidity around the part of the targets included in the bilayer; or
I E = 16 KcJm01e]
(3) disturbing the external protein-phospholipid polar head interactions. After binding to its specific binding sites, PAF-acether may activate the unknown target X (specific receptor protein?). Activation of X may lead to two concomitant events: (1) Hydrolysis of GTP and activation of phospholipase C activity triggering phosphoinositide breakdown with formation of inositol triphosphate [Ins(1,4,5)P3]
t- "" ....... t/,°
-
/
1-
Fig. 7. Putative conformation of platelet PAFacether specific binding site. In Fig. 7,4, the slashed area represents the unknown target which could be triggered by an electronic transfer from the oxygen doublet. The different theoretical energies are presented. The fatty chain fits into the hydrophobic area of the membrane. Fig. 7B, presents the possibility of binding for R (natursI) and s (synthetic) PAF-acether. Note that for s-configuration, the chirslity of Cz does not permit a correct insertion of the autacoid in its binding site. Conversely, the place isomer C~C3 can enter the site, the only difference with PAF-acether resides in the displacement of a methylene group
i
o e-"----®
~,--~0
i~ o.51~,i I
\
IE = 2-5 KcSmole]
....
IE ~ l~~K:"~'l
~,.-
i
7o, I
from C~ to C~.
and diacylglycerol (DG) (Ref. 19), both of which have properties of classical second messengers. Ins(1,4,5)P3 induces the release of calcium from its internal pools leading to increased cytosolic calcium [Ca2+]i. This in turn may open [Ca2+]i-dependent K + channels as observed in PAF-acether-activated mouse macrophages 19. On the other hand, DG is involved in protein kinase C activation leading to subsequent phosphorylation of specific cellular protein which then contributes to various physiological processes (particularly secretion and proliferation). (2) Internalization of the complex PAF-acether specific binding sites by an unknown active or passive process. The binding of PAF-acether to its specific membrane binding sites favours its internalization, either directly, as for other ligand receptor complexes undergoing downregulation 2° or, indirectly, as a consequence of cell activation. Transmembrane movement of PAFacether represents the limiting step of its metabolism as demonstrated in rabbit platelets 21 and explains the subsequent desensitization. PAF-acether is then deactivated in two steps: firstly, PAF-acether is deacetylated by the cytosolic acetylhydrolase into lyso-PAF. Since acetylhydrolase activity is cytosolic, degradation of PAFacether is intracellular. This en-
hydrophobic/ionic interactions
\
~.~)\.
X®/~/0,, --O~
\)r",
%7q,o° X
\ o\
CH2 membrane ..\\~'-'~!R) target ,~--....~ / L~'n .~-~ CH2 .! \ 7
<_
/
~
•
/ ~ ....
• CH 2
%
t oo) \
;
"=-/6.5
,'5-7 ~ -anchorage --/--
I ~)--I I (,
I(s)
area
I
I
"
f
I
/\~
I
t /" t. hydrophobic ~ ,~ "~interactions
(,
,
/\~1
(S) configurationsynthetic
(R) c o n f i g u r a t i o n natural i
f /\-~ (S)
configuration
i
PAF-acether
Place isomer of PAF-acether
373
T I P S - S e p t e m b e r 1986
zymatic system removes specifically the group from the 2-R position. This e n z y m e is present i n the intracellular compartment22, b u t acetyl hydrolase activity has also b e e n detected i n extracellular fluids 23. The properties of the plasma enzyme are similar to those of the enzyme extracted from the cells except that the latter is resistant to the action of proteases 24 a n d is refractory to serine hydrolase inhibitors (L. Touqui, pers. commun.). Acetyl hydrolase differs from phospholipase A2 i n that it cleaves only the short chain at the 2-R position of phospholipids and is Mg 2+ and Ca 2+i n d e p e n d e n t . Interestingly, a positive correlation was found between platelet sensitivity to PAFacether i n different species and its ability to inactivate it. Secondly, as lyso-PAF is lytic a n d detergent, its rapid elimination is crucial for the cell. This is achieved b y acylation of the hydroxyl group i n the 2-R position b y an acyltransferase resulting i n the formation of alkylacyl-glycerylphosphocholine 2s a c o m p o n e n t of the i n n e r layer of the m e m b r a n e 26. The majority of studies with exogenous lyso-PAF show that the p r e d o m i n a n t route of conversion is to alkylacyl-glycerylphosphocholine, a relatively m i n o r a m o u n t b e i n g converted to PAF-acether. This suggests that acyltransferase has a high affinity for lyso-PAF27. Incorporation is mainly catalysed b y a C o A - i n d e p e n d e n t transacylase, which uses phosphatidylcholine as the source of arachidonic acid. Free arachidonic acid is initially incorporated into phosphatidylcholine b y a C o A - d e p e n d e n t acyltransferase and thereafter transferred to lyso-PAF and to other ether lipids b y transacylation28.
Getting the name right Dictionary of Pharmacology by W . C. B o w m a n , A n n e B o w m a n and A l i s o n B o w m a n , B l a c k w e l l Sc,~ntific Publications, 1986. £12.50 (vii + 234 pages) I S B N 0 632 01131 9
In compiling a dictionary of a very specific subject, the question is really not so much 'Where do you start?' b u t more 'Where do you
Reacylation of lyso-PAF is inhibited by calcium (ICs0 of 50-100 I~M; Touqui et al., pets commun.), suggesting that d u r i n g cellular activation an elevation i n Ca 2+ influx may i n h i b i t this enzyme resulting i n the transient accumulation of lyso-PAF thus favouring its utilization b y acetyl transferase (i.e., PAF-acether synthesis). []
[]
[]
Better knowledge of PAFacether specific b i n d i n g site(s) a n d of biochemical events involved in PAF-acether-induced m e m b r a n e signal should enable designing n e w potent antagonists for the future treatment of asthma, thrombosis graft rejection or shock.
Acknowledgement Thanks are due to B. B. Vargaftig and L. Touqui for interesting discussions.
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stop?'.
In
this the Bowman tribe describe very precisely where they draw their limits for inclusions. Drug classes are included b u t drug names are not. Physiological and biochemical terms are culled, b u t only w h e n the compilers feel they have become part of essential pharmacological parlance. The preferences and prejudices of the compilers come Dictionary
the
preface
to
of Pharmacology,
Worcel, M. 1. Med. Chem. (in press) 11 Braquet,P. and Godfroid,J. ]. in Platelet activating Factor (Snyder, F., ed.), Plenum, (in press) 12 Wissner, A., Schaub, R. E., Sum, P. E., Kohler,C. A. and Goldstein,B. M. (1985) J. Med. Chem. 28, 1181-1187 13 Hadvary, P., Cassal, ]. M., Hirth, G., Barner, R. and Beaumgartner, H.P. (1986) in Proceedings of the Platelet Activating
Factor
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(Benveniste,J. and Arnoux,B., eds), pp. 57, Elsevier 14 Moschidis, M. C., Demopoulos, C. A. and Kritikou, L. G. (1983) Chem. Phys. Lipids 33, 87-92 15 Heymans,F., Borrel,M. C., Broquet, C., Lefort,]. and Godfroid,]. J. (1985)J. Med. Chem. 28, 1094-1096 16 Tokumura, A., Homma, H. and Hanahan, D. J. (1985)]. Biol. Chem. 260, 12710-12714 17 Ohno, N., Fujita, K., Nakai, H., Kobayashi,S., Yamashita,M., Inove, K. and Nojima, S. (1983) in Proceedings of the Platelet Activating Factor Symposium
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19 Garay, R. and Braquet, P. Biochem. Pharmacol. (in press) 20 Schlessinger, J. (1980) Trends Biochem. Sci. 5, 210-214 21 Lachachi, H., Plantavid, M., Simon, M. F., Chap, H., Braquet, P. and Douste Blazy, L. (1985) Biochem. Biophys. Res. Commun. 132, 460-466 22 Blank,M. L., Lee, T., Fitzgerald,V. and Snyder, F. (1981) J. Biol. Chem. 256, 175-178 23 Farr, R. S., Cox, C. P., Wardlow, M. L. and Jorgensen, R. (1980) Clin. Immunol. Immunopathol. 15, 318-330 24 Blank, M. L., Hall, M. N., Cress, E. A. and Snyder, F. (1983) Biochem. Biophys. Res. Commun. 113, 666--671 25 Cabot, M. C., Blank,M. L., Welsh, C. l., Horan, M. l., Cress, E. A. and Snyder,F. (1982) Life Sci. 31, 2891-2898 26 Touqui, L., Jacquemin,C., Dumarey,C. and Vargaftig, B.B. (1985) Biochem. Biophys. Acta 833, 111-118 27 Chilton, F. H., O'Flaherty, J.T., Ellis, J. M., Swendsen,C. L. and Wykle,R. L. (1983) J. Biol. Chem. 258(12),7268-7271 28 Kramer, R. M. and Deykin, D. (1983) J. Biol. Chem. 258, 13806-13811 29 Wissner, A., Schaub, R. E., Sum, P. E., Kohler,C. A. and Goldstein,B. M. (1986) J. Med. Chem. 29, 328-333 through to a small extent, but this is forgivable considering the potential for personal influence. I am sure that the major aim of this dictionary is to clarify terms that have tended to lose precision through overuse (and careless use). It is surprising then that enzymes are not denoted according to their IUB recommended nomenclature or Enzyme Commission numbers. However, in general, pharmacological definitions are admirably precise. In some entries key references are also given taking the reader to original m e a n i n g s and definitions