Vol.
171, No.
September
3, 1990 28,
BIOCHEMICAL
AND
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RESEARCH
COMMUNICATIONS
Pages
1990
1087-1092
THE EFFECT OF NEW POTENT SELECTIVE INHIBITORS OF PROTEIN KINASE C ON THE NEUTROPHIL RESPIRATORY BURST B. Twomey,
R. E. Muid,
J.
S. Nixon,* A. D. Sedgwick,* and M. M. Dale
S. E. Wilkinson,*
Departmentof Pharmacology, University CollegeLondon,GowerStreet,London WC1E
6BT,
UK
“ResearchCentre,RocheProductsLtd., BroadwaterRoad,Welwyn GardenCity AL7 3AY, UK Received
August
2, 1990
Summary: New potentinhibitorsof proteinkinaseC werefoundto inhibit proteinkinaseC isolated from rat brainandhumanneutrophils,with alargedegreeof selectivity over CAMP-dependentkinase and Ca2+/calmodulin-dependent kinase. Thesenovel compoundswere potent inhibitors of the fluoride, diCs- and formyl-methionyl-leucyl-phenylalanine-mediated respiratory burstsin intact neutrophils.The opsonizedzymosan-stimulatedburst wasonly marginally affected by the compounds. Theseresultsdiffer from thoseobtainedin studieswith H7 and CI, (which are lesspotent andless specificproteinkinaseC inhibitors)andareconsistentwith thehypothesisthat protein kinaseC has a role in the transductionmechanismfor the neutrophiloxidative burst stimulatedwith fluoride, formyl-methionyl-leucyl-phenylalanineanddiCs. 61990 Academic Press,Inc. It has been proposedthat the signal transductionmechanismsin many cell types involve the breakdownof pho-sphatidylinositol biiphosphate(PIP2)to give inositol trisphosphate(IP3), which increases intracellularCa*+, anddiacylglycerol (DAG) which activatesproteinkinaseC (PKC), and that the two pathwaysfunction synergistically[ 1,2]. Evidencehasbeenput forward which suggests that in the neutrophilthis synergisticinteractionbetweenthe IP3/Ca2’pathwayandthe DAG/PKC pathwayparticipatesin signaltransductionfor &i generation[3,4]. Furthermore,good correlation hasbeenfound, both in kineticsand magnitude,betweenthe DAG concentrationsand superoxide productionrecordedwith variousstimuli [5,6]. Inhibitorsof DAG metabolismwhich would elevate cellular DAG, enhanceOz generationby a numberof agonists[7,8] andthis is alsoconsistentwith amediatorrole for DAG. Thesedataimply that increasedDAG levelsandsubsequent PKC activation areinvolved in thegenerationofsuperoxide.However,acontroversystill existsasto the involvement of PKC in the receptor stimulatedrespiratoryburst. Much of the controversy stemsfrom studies carried out usingdual inhibitors of both PKC and CAMP-dependentprotein kinase(PKA). PKA, whichisactivatedby increasedCAMP, is anegativeregulatorof theoxidative burst [9- 111.Inhibition of both the positive PKC effectsandthe negativePKA effectscouldresultin the ‘no effect’ situation recordedwith the non-specificinhibitors- H7 [ 12,131andCI [ 141.Alternatively, H7 may not inhibit PKC in an intracellularscenarioat the concentrationsusedin theseexperiments[ 151. Abbreviations. 02, superoxide;DAG, diacylglycerol; DiCs, dioctanoylglycerol; fMLP, formylmethionyl-leucyl-phenylalanine;IP3,inositol&phosphate; OZ, opsonizedzymosan;PI, phosphatidylinositol; PIP*, phosphatidylinositolbisphosphate;PKA, CAMP-dependentkinase;PKC, protein kinaseC .
1087
0006-291X&O $1.50 Copyrighr 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Herewe presentdataobtainedwith a seriesof novel potent andselectiveinhibitors of PKC from RocheProductsLtd. [16], showingthat they causean inhibitionofboth isolatedneutrophilPKC (and PKC from other sources)and the neutrophil respiratoryburst stimulatedwith dioctanoylglycerol (diCs)(a direct proteinkinaseC activator), fluoride (aG-proteinactivator) andfmet-leu-phe(fMLP) (a receptoragonist).On the other hand,the oxidative burststimulatedby opsonizedzymosan(OZ) wasonly slightly inhibited.
MATERIAL AND METHODS Inhibition of PKC in vitro Rat brainPKC andhumanneutrophilPKC werepartially purified by ion-exchangechromatography and assayedaccordingto the methoddescribedin [16]. Inhibition of PKA in vitro Bovine heart PKA (Sigma) wasassayedaccordingto the methoddescribedin [ 161.10~1human neutrophilPKA, partiallypurified by ionexchangechromatography,were addedto a reactioncocktail (9Opl)containing0.67mgml-’ histone(type VS, Sigma),11uM CAMP, inhibitor in 10~1DMSO and llnM[y-“P]ATP in 5OmMTris-HCl, 1OmMMgC12pH7.5, After 15minutesincubationat 3O”C, the assayprocedurethereafterwasthat describedfor PKC [ 161. Inhibition of Ca2+/calmodulin-dependentprotein kinasein vitro Ca2+/calmodulm dependentproteinkinase,partially purified from rat brains,wasassayedaccording to the methoddescribedin [ 161. Superoxide production assay Neutrophilswere preparedfrom humanblood by Ficoll-Isopaqueseparationandsuspended in the appropriatebuffer aspreviouslydescribed[ 171.Theneutrophilswereequilibratedfor 20min at 37°C; to those cells which were to be stimulatedwith fMLP, cytochalasin B (Spg ml-‘) was added. Immediately,2~ lo6 cellswerealiqotedto tubescontaininglmg fenicytochrome (horseheart,type III) andinhibitor at the appropriateconcentrationor Tyrode, andeither Tyrode solutionor 75 units superoxidedismutase(bovineblood). Incubationwascontinuedfor afurther 5 min beforebeginning the reactionwith the desiredstimulus.The reactionwasterminatedafter 30 min by the addition of 1mMN-ethylmaleimide.Following centrifugationat 1400gfor 10min at 4°C theabsorbance of the supematantwasreadspectrophotometricrdlyandnmol 05 calculatedaspreviously described[ 171. Resultsfrom eachseparateexperimentwerenormalised,expressingeachvalue for 02 production asa percentageof the maximumcontrol response(i.e. in the absenceof inhibitor) obtainedin that particularexperiment.The meanswith standarderrorsfrom severalexperimentswerecalculatedand displayedgraphically, from which the IC5evalueswereobtained.
RESULTS Compounds2-4 are novel potent PKC inhibitors, structurally relatedto staurosporineand K252a (Fig. l), with rank order of potency 3>4>2 andeachof the threecompoundshasa similarpotency againstboth rat brain and humanneutrophilPKCs.Thesecompoundsexhibited similar inhibitory potency to staurosporine,weremuchmorepotent thanK252a,andmuchmoreselectivethan either staurosporineand K252a, for PKC over both PKA (bovine heart and humanneutrophil) and Ca2+/calmodulin-dependent kinase(Table 1). Thesebis-indolyl maleimideswerepotentinhibitorsof bothdiCa-andfMLP-stimulated02 production with the diCs-inducedburstbeing slightly more sensitiveto inhibition by theseagents(Table 2). As a representativeexample,the dose-inhibitiondatafor compound3 againstthediCaandfMLP 02 responseis shownin Fig. 2a and b, respectively.Both high and low concentrationsof agonist wereusedin eachcase,andit shouldbenotedthat ICsevalueswereidenticalor very similarwhether they wereestimatedfrom the high or low agonistconcentrations.The valuesgiven in Table 2 were 1088
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BIOCHEMICAL
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AND BIOPHYSICAL
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OH iHCH3
K252a
Staurosporine ti
0
0
-
\/I\ -ar,-i
/I) N ‘i’
4 COMPOUND
Ro
0 2 3 4
R3
R1
R7.
R3
6045
H
CH3
7549 8220 8161
(CH2)3NH2
H H H
H CH3
31-
(CHZ)JSC(NH)NH~ (CHZ)JNC(NNO~)NH~
CH3 CH3
Figure 1. Structuresof PKC inhibitors.
obtainedfrom the graphsof the high agonistconcentration.The ICa valuesof thesenovel inhibitors arecomparedwith thoseof K252aandstaurosporine[ 181in table 2. Theseinhibitors wereconsiderablymorepotent in inhibiting the fluoride-mediated02 burst than that inducedby eitherdiC8or fMLP (Table2). K252aandstaurosporine alsogavemuch lower 1% valuesagainstfluoride than againstany of the other stimuli (Table 2) [ 181.This potent inhibitory Table 1 Inhibitor ICw values (PM) against isolated protein kinases rat brain human neu- human neutrophil proCa2+/ calmotrophil protein kinase C tein kinase A dulin dependent kin&ea
rat brain protein kinase C a
bovine heart protein kinase Aa
K252a
0.47
0.20
0.270
0.16
0.30
Staurosporine
0.04
0.01
0.12
0.002
0.02
2
0.08
5.10
0.050
4.20
15
3
0.01
1.50
0.008
n.d.
17
4
0.03
3.30
0.020
n.d.
14
Cl
>l 00
100
n.d.
n.d.
>lOO
Note.I&J values(a)weretakenfromreference
[ 161.
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b fMLP 0
_
I
1 P
no drug
.03
.1
.3
1
3
no drug
.3
1
1
d OZ
r----f
l
3
\ i:
40 20 0 no drug
.003
.Ol
.03
.l
no drug
.3
1
3
COMPOUND 3(/.A4) Figure 2. The effect of a range of compound 3 concentrations on the 02 response generated with 4 stimuli. (a) Dioctanoylglycerol [dies) control at 10pM ( 0 ), with inhibitor ( 02; contro1 at 25pM ( 0 ), with inhibitor at ( b ), n=4. (b) FMet-Leu-Phe (MLP) control at lo- M ( 0 ), with inhibitor ( Cl ); control at lo- M ( 0 ), with inhibitor at ( n ), n=4. (c) Fluoride control at 1OmM (0), with inhibitor (0); control at 18mM ( l ), with inhibitor ( n ), n=4. (d) Opsonized zymosan (OZ) control at 0.3mgjm1, with inhibitor ( Cl ); control at 3mg/ml( 0 ), with inhibitor ( W ), n=4.
effect is illustratedby the dose-inhibitionprofile of compound3 at highandlow fluoride concentrations (Fig. 2~). Compound0 hadlittle effect on fluoride 05 release(Table 2). Thesenovel inhibitorsweremuch lesspotenton the OZ-stimulatedresponse than thoseinducedby the otherstimuli. The dose-inhibitiondatafor compound3 with high and low OZ concentrationsis Table 2 Inhibitor I&J values (pM) with stimulated 02 generation fluoride
diCa
fMLP
oz
K252a
0.045
3.00 2.2a
0.15 0.36a
0.80
Staurosporine
0.006
0.01
0.15 no effectb 0.024’
0.20
2
0.008
0.67
1.97
7.0
3
0.01
0.29
0.95
6.5
4
0.02
0.63
1.50
a10.0
0
>3.0
B10.0
>lO.O
>lO.O
Note: ICso values (a-c) were taken from references [26-281 respectively.
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presentedin Fig. 2d, andclearly showsthe largedecreasein potencyof this compoundin inhibiting OZ-stimulatedo;i generation.However, at concentrationsof compound3 greaterthan 3ph4, the inhibition wasstatisticallysignificantat ~~0.05.Thisprofile isin contrastto that of the lessselective PKC inhibitors, K252a and staurosporine,which were potent inhibitors of the OZ-induced 05 response(Table2) [ 181.
DISCUSSION Thesenovel bis-indolyl maleimideinhibitorsshowconsiderablyimprovedselectivity for PKC over staurosporineandK252aandalsoover H7 andCI, whichareeachreportedto be equipotentagainst both PKC and PKA [12-141.A favourable selectivity of inhibition is crucial in order to make definitive statements concerningthe involvementof PKC in cell-mediatedevents.This isparticularly importantwheninvestigatinga cellularresponse suchastherespiratoryburstwhich hasbeenshown tobenegativelymodulatedbyPKA[ll]and~beenproposedtobemediatedbyaPKC-independent pathway involving possiblya calmodulin-dependent kinase[12, 19,201. For compounds2-4 the rank orderof inhibition againstisolatedneutrophilPKC correlatedwith the orderobservedin the diCg-andfh4LP-stimulatedrespiratoryburst(3>4>2).However,approximately lOO-foldgreaterconcentrationswere requiredfor inhibition in the intact cell. This is not surprising sincethesecompoundsareATP-competitive inhibitors,andintracellularATP concentrationspresent in the neutrophilare very much higherthan thoseusedin the isolatedenzyme assay.This reduced potencyat an intracellularsitetallieswith the observationthat higherinhibitor concentrationswere alsorequiredto antagonisethe TPA-induced phosphorylationof a 47KDa protein in intact human platelets[163.With the stimuli dice and fMLP, the ICsovaluesof compounds2-4 were different from thosepreviously obtainedwith the lessselectivePKC inhibitors,K252a andstaurosporine[ 181 asshownin table 2. Fluoride-mediated02 releasewasmuchmoresensitiveto inhibition by the bis-indolyl maleimides comparedto diCs and fMLP, and this profile wasalso seenwith K252a and staurosporine.One explanationfor theseresultsis that fluoride cancausea decrease in the intracellularATP concentrations [2 l] suchthat the inhibitor concentrationrequiredto block PKC activity is muchreduced. Thesenovel PKCinhibitorsinhibit not only the oxidative burststimulatedby a direct PKC-activator, diCs,but alsothat stimulatedby the G proteinactivator, fluoride, andby the cell surfacereceptor stimulant,fh4LP. The bis-indolyl maleimideswerepoor inhibitor; of the OZ-stimulatedburst, whencomparedwith K252aandstaurosporine.This suggests that PKC playsa minorrole in the OZ-mediatedrespiratory burst.The oxidative burstwith OZ, a phagocyticstimuluswhichinteractswith FcandC3breceptors, could however, require activation of myosinlight chainkinase;both K252aand staurosporineare reportedaspotent inhibitorsof thii enzyme [22,23]. Oneexplanationfor the high potency of all the inhibitorsagainstfluoride and of the differencesin potency against other stimuli involves PKC isoenzymeselectivity. It is now well establishedthat different PKC isoenzymesexist [24] but their selective involvement under different activating conditionshasnot yet beenverified. However, the presenceof at leasttwo PKC isoenzyme~with differentialsensitivity to PKC activatorshasbeenreportedin HMO cells,which areclosely related 1091
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to neutrophils [2S]. The differences seen with the inhibitors against a range of stimuli could be due to their varying potencies against these PKC isoenzymes. In conclusion we report that the neutrophil respiratory burst induced by diC& fMLP and fluoride is potently inhibited by three novel selective PKC inhibitors, It has been reported that DAG is generated both on cell stimulation with fMLP [S] and with fluoride and that the burst is increased when the metabolism of DAG is inhibited [7]. Taken together with our results, this implies that the DAG/PKC pathway has a role in the neutrophil respiratory burst not only when induced by a direct PKC activator but also when induced by receptor stimulation with fMLP and by G protein stimulation with fluoride. REFERENCES 1. Niihizuka, Y. (1984) Nature 308,693-698. Berridge, M.J. and Irvine, R.F. (1984) Nature 312,31S-321. 3. Robinson, J.M., Badwey, J.A., Kamovsky, M.L. and Karnovsky, M.J. ( 1984) B&hem. Biophys. Res. Commun. 122,734-739. 4. Dale, M.M. and Penfield, A. (1984) FEBS Lett. 175,170-172. 5. Rider, L.G. and Niedel, J.E. (1987) J. Biol. Chem. 262,X03-5608. 6. Preiss, J.E., Bell, R.M. and Niedel, J.E. (1987) J. Immunol. 138,1542-1545. 7. Muid, R.E., Penfield, A. and Dale, M.M. (1987) B&hem. Biophys. Res. Commun. 143, 630-637. 8. Gomez-Carnbronero, J., Molski, T.F.P., Becker, E.L. and Sha’afi, RI. ( 1987) Biochem. Biophys. Res. Commun. 148,38-46. 9. Smolen, J.E., Korchak, H.M. and Weissmann, G. (1980) J. Clin. Invest. 65,1077-1085. 10. Fantone, J.C., Marasco, W.A., Elgas, L.J. and Ward, P.A. (1984) Am. J. Path. 115,9-16. 11. DeTogni, P., Cabrini, G. and Di Virgilio, F. (1984) Biochem. J. 224,629-635. 12. Wright, C.D. and Hoffman, M.D. (1986) Biochem. Biophys. Res. Commun. 135,749-755. 13. Seifert, R. and Schachtele, C. (1988) B&hem. Biophys. Res. Commun. 152,585592. 14. Gerard, C., McPhail, L.C., Marfat, A., Stimler-Gerard, N.P., Bass, D.A. and McCall, C.E. (1986) J. Clin. Invest. 77,61-65. 15. Nixon, J.S., Wilkinson, S.E., Davis, P.D., Sedgwick, A.D., Wadsworth, J. and Westmacott, D. (1990) Agents and Actions (in press). 16. Davis, P.D., Hill, C.H., Keech, E., Lawton, G., Nixon, J.S., Sedgwick, A.D., Wadsworth, J., Westmacott, D. and Wilkinson, S.E. (1989) FEBS L&t. 259,61-63. 17. Muid, R.E., Twomey, B. and Dale, M.M. (1988) FEBS L&t. 234,23S-240. 18. Twomey, B., Muid, R.E. and Dale, M.M. (1990) Br. J. Pharmac. 100,819-825. 19. Cooke, E. and Hallett, M.B. (1985) Biochem. J. 232,323-327. 20. Takeshige, K. and Minakami, S. (198 1) Biochem. Biophys. Res. Commun. 99,484-490. 21. Svec, F. (198s) B&him. Biophys. Acta 847,147-154. 22. Nakanishi, S., Yamada, K., Kase, H., Nakamura, S. and Nonomura, Y. (1988). J. Biol. Chem. 263,6215-6219. 23. Watson, S.P., McNally, J., Shipman, L.J. and Godfrey, P.P. (1988) B&hem. J. 249,34S-350. 24. Nishizuka, Y. (1988) Nature 334,661-665. 25. Beh, I., Schmidt, R. and Hecker, E. (1989) FEBS Lett. 249,264-266. 26. Smith, R.J., Justen, J.M. and Sam, L.H. (1988) B&hem. Biophys. Res. Commun. 152, 1497-1503. 27. Sako, T., Tauber, AI., Jeng, A.Y., Yuspa, S.H. and Bhnnberg, P.M. (1989) Cancer Res. 48, 4646-4650. 28. Dewald, B., Thelen, M., Wymann, M.P. and Baggiolini, M. (1989) Biochem. J. 264,879-884. 2.
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171, No.
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3, 1990 28,
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AND
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Pages
1990
1087-1092
THE EFFECT OF NEW POTENT SELECTIVE INHIBITORS OF PROTEIN KINASE C ON THE NEUTROPHIL RESPIRATORY BURST B. Twomey,
R. E. Muid,
J.
S. Nixon,* A. D. Sedgwick,* and M. M. Dale
S. E. Wilkinson,*
Departmentof Pharmacology, University CollegeLondon,GowerStreet,London WC1E
6BT,
UK
“ResearchCentre,RocheProductsLtd., BroadwaterRoad,Welwyn GardenCity AL7 3AY, UK Received
August
2, 1990
Summary: New potentinhibitorsof proteinkinaseC werefoundto inhibit proteinkinaseC isolated from rat brainandhumanneutrophils,with alargedegreeof selectivity over CAMP-dependentkinase and Ca2+/calmodulin-dependent kinase. Thesenovel compoundswere potent inhibitors of the fluoride, diCs- and formyl-methionyl-leucyl-phenylalanine-mediated respiratory burstsin intact neutrophils.The opsonizedzymosan-stimulatedburst wasonly marginally affected by the compounds. Theseresultsdiffer from thoseobtainedin studieswith H7 and CI, (which are lesspotent andless specificproteinkinaseC inhibitors)andareconsistentwith thehypothesisthat protein kinaseC has a role in the transductionmechanismfor the neutrophiloxidative burst stimulatedwith fluoride, formyl-methionyl-leucyl-phenylalanineanddiCs. 61990 Academic Press,Inc. It has been proposedthat the signal transductionmechanismsin many cell types involve the breakdownof pho-sphatidylinositol biiphosphate(PIP2)to give inositol trisphosphate(IP3), which increases intracellularCa*+, anddiacylglycerol (DAG) which activatesproteinkinaseC (PKC), and that the two pathwaysfunction synergistically[ 1,2]. Evidencehasbeenput forward which suggests that in the neutrophilthis synergisticinteractionbetweenthe IP3/Ca2’pathwayandthe DAG/PKC pathwayparticipatesin signaltransductionfor &i generation[3,4]. Furthermore,good correlation hasbeenfound, both in kineticsand magnitude,betweenthe DAG concentrationsand superoxide productionrecordedwith variousstimuli [5,6]. Inhibitorsof DAG metabolismwhich would elevate cellular DAG, enhanceOz generationby a numberof agonists[7,8] andthis is alsoconsistentwith amediatorrole for DAG. Thesedataimply that increasedDAG levelsandsubsequent PKC activation areinvolved in thegenerationofsuperoxide.However,acontroversystill existsasto the involvement of PKC in the receptor stimulatedrespiratoryburst. Much of the controversy stemsfrom studies carried out usingdual inhibitors of both PKC and CAMP-dependentprotein kinase(PKA). PKA, whichisactivatedby increasedCAMP, is anegativeregulatorof theoxidative burst [9- 111.Inhibition of both the positive PKC effectsandthe negativePKA effectscouldresultin the ‘no effect’ situation recordedwith the non-specificinhibitors- H7 [ 12,131andCI [ 141.Alternatively, H7 may not inhibit PKC in an intracellularscenarioat the concentrationsusedin theseexperiments[ 151. Abbreviations. 02, superoxide;DAG, diacylglycerol; DiCs, dioctanoylglycerol; fMLP, formylmethionyl-leucyl-phenylalanine;IP3,inositol&phosphate; OZ, opsonizedzymosan;PI, phosphatidylinositol; PIP*, phosphatidylinositolbisphosphate;PKA, CAMP-dependentkinase;PKC, protein kinaseC .
1087
0006-291X&O $1.50 Copyrighr 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
171, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Herewe presentdataobtainedwith a seriesof novel potent andselectiveinhibitors of PKC from RocheProductsLtd. [16], showingthat they causean inhibitionofboth isolatedneutrophilPKC (and PKC from other sources)and the neutrophil respiratoryburst stimulatedwith dioctanoylglycerol (diCs)(a direct proteinkinaseC activator), fluoride (aG-proteinactivator) andfmet-leu-phe(fMLP) (a receptoragonist).On the other hand,the oxidative burststimulatedby opsonizedzymosan(OZ) wasonly slightly inhibited.
MATERIAL AND METHODS Inhibition of PKC in vitro Rat brainPKC andhumanneutrophilPKC werepartially purified by ion-exchangechromatography and assayedaccordingto the methoddescribedin [16]. Inhibition of PKA in vitro Bovine heart PKA (Sigma) wasassayedaccordingto the methoddescribedin [ 161.10~1human neutrophilPKA, partiallypurified by ionexchangechromatography,were addedto a reactioncocktail (9Opl)containing0.67mgml-’ histone(type VS, Sigma),11uM CAMP, inhibitor in 10~1DMSO and llnM[y-“P]ATP in 5OmMTris-HCl, 1OmMMgC12pH7.5, After 15minutesincubationat 3O”C, the assayprocedurethereafterwasthat describedfor PKC [ 161. Inhibition of Ca2+/calmodulin-dependentprotein kinasein vitro Ca2+/calmodulm dependentproteinkinase,partially purified from rat brains,wasassayedaccording to the methoddescribedin [ 161. Superoxide production assay Neutrophilswere preparedfrom humanblood by Ficoll-Isopaqueseparationandsuspended in the appropriatebuffer aspreviouslydescribed[ 171.Theneutrophilswereequilibratedfor 20min at 37°C; to those cells which were to be stimulatedwith fMLP, cytochalasin B (Spg ml-‘) was added. Immediately,2~ lo6 cellswerealiqotedto tubescontaininglmg fenicytochrome (horseheart,type III) andinhibitor at the appropriateconcentrationor Tyrode, andeither Tyrode solutionor 75 units superoxidedismutase(bovineblood). Incubationwascontinuedfor afurther 5 min beforebeginning the reactionwith the desiredstimulus.The reactionwasterminatedafter 30 min by the addition of 1mMN-ethylmaleimide.Following centrifugationat 1400gfor 10min at 4°C theabsorbance of the supematantwasreadspectrophotometricrdlyandnmol 05 calculatedaspreviously described[ 171. Resultsfrom eachseparateexperimentwerenormalised,expressingeachvalue for 02 production asa percentageof the maximumcontrol response(i.e. in the absenceof inhibitor) obtainedin that particularexperiment.The meanswith standarderrorsfrom severalexperimentswerecalculatedand displayedgraphically, from which the IC5evalueswereobtained.
RESULTS Compounds2-4 are novel potent PKC inhibitors, structurally relatedto staurosporineand K252a (Fig. l), with rank order of potency 3>4>2 andeachof the threecompoundshasa similarpotency againstboth rat brain and humanneutrophilPKCs.Thesecompoundsexhibited similar inhibitory potency to staurosporine,weremuchmorepotent thanK252a,andmuchmoreselectivethan either staurosporineand K252a, for PKC over both PKA (bovine heart and humanneutrophil) and Ca2+/calmodulin-dependent kinase(Table 1). Thesebis-indolyl maleimideswerepotentinhibitorsof bothdiCa-andfMLP-stimulated02 production with the diCs-inducedburstbeing slightly more sensitiveto inhibition by theseagents(Table 2). As a representativeexample,the dose-inhibitiondatafor compound3 againstthediCaandfMLP 02 responseis shownin Fig. 2a and b, respectively.Both high and low concentrationsof agonist wereusedin eachcase,andit shouldbenotedthat ICsevalueswereidenticalor very similarwhether they wereestimatedfrom the high or low agonistconcentrations.The valuesgiven in Table 2 were 1088
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AND BIOPHYSICAL
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OH iHCH3
K252a
Staurosporine ti
0
0
-
\/I\ -ar,-i
/I) N ‘i’
4 COMPOUND
Ro
0 2 3 4
R3
R1
R7.
R3
6045
H
CH3
7549 8220 8161
(CH2)3NH2
H H H
H CH3
31-
(CHZ)JSC(NH)NH~ (CHZ)JNC(NNO~)NH~
CH3 CH3
Figure 1. Structuresof PKC inhibitors.
obtainedfrom the graphsof the high agonistconcentration.The ICa valuesof thesenovel inhibitors arecomparedwith thoseof K252aandstaurosporine[ 181in table 2. Theseinhibitors wereconsiderablymorepotent in inhibiting the fluoride-mediated02 burst than that inducedby eitherdiC8or fMLP (Table2). K252aandstaurosporine alsogavemuch lower 1% valuesagainstfluoride than againstany of the other stimuli (Table 2) [ 181.This potent inhibitory Table 1 Inhibitor ICw values (PM) against isolated protein kinases rat brain human neu- human neutrophil proCa2+/ calmotrophil protein kinase C tein kinase A dulin dependent kin&ea
rat brain protein kinase C a
bovine heart protein kinase Aa
K252a
0.47
0.20
0.270
0.16
0.30
Staurosporine
0.04
0.01
0.12
0.002
0.02
2
0.08
5.10
0.050
4.20
15
3
0.01
1.50
0.008
n.d.
17
4
0.03
3.30
0.020
n.d.
14
Cl
>l 00
100
n.d.
n.d.
>lOO
Note.I&J values(a)weretakenfromreference
[ 161.
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b fMLP 0
_
I
1 P
no drug
.03
.1
.3
1
3
no drug
.3
1
1
d OZ
r----f
l
3
\ i:
40 20 0 no drug
.003
.Ol
.03
.l
no drug
.3
1
3
COMPOUND 3(/.A4) Figure 2. The effect of a range of compound 3 concentrations on the 02 response generated with 4 stimuli. (a) Dioctanoylglycerol [dies) control at 10pM ( 0 ), with inhibitor ( 02; contro1 at 25pM ( 0 ), with inhibitor at ( b ), n=4. (b) FMet-Leu-Phe (MLP) control at lo- M ( 0 ), with inhibitor ( Cl ); control at lo- M ( 0 ), with inhibitor at ( n ), n=4. (c) Fluoride control at 1OmM (0), with inhibitor (0); control at 18mM ( l ), with inhibitor ( n ), n=4. (d) Opsonized zymosan (OZ) control at 0.3mgjm1, with inhibitor ( Cl ); control at 3mg/ml( 0 ), with inhibitor ( W ), n=4.
effect is illustratedby the dose-inhibitionprofile of compound3 at highandlow fluoride concentrations (Fig. 2~). Compound0 hadlittle effect on fluoride 05 release(Table 2). Thesenovel inhibitorsweremuch lesspotenton the OZ-stimulatedresponse than thoseinducedby the otherstimuli. The dose-inhibitiondatafor compound3 with high and low OZ concentrationsis Table 2 Inhibitor I&J values (pM) with stimulated 02 generation fluoride
diCa
fMLP
oz
K252a
0.045
3.00 2.2a
0.15 0.36a
0.80
Staurosporine
0.006
0.01
0.15 no effectb 0.024’
0.20
2
0.008
0.67
1.97
7.0
3
0.01
0.29
0.95
6.5
4
0.02
0.63
1.50
a10.0
0
>3.0
B10.0
>lO.O
>lO.O
Note: ICso values (a-c) were taken from references [26-281 respectively.
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presentedin Fig. 2d, andclearly showsthe largedecreasein potencyof this compoundin inhibiting OZ-stimulatedo;i generation.However, at concentrationsof compound3 greaterthan 3ph4, the inhibition wasstatisticallysignificantat ~~0.05.Thisprofile isin contrastto that of the lessselective PKC inhibitors, K252a and staurosporine,which were potent inhibitors of the OZ-induced 05 response(Table2) [ 181.
DISCUSSION Thesenovel bis-indolyl maleimideinhibitorsshowconsiderablyimprovedselectivity for PKC over staurosporineandK252aandalsoover H7 andCI, whichareeachreportedto be equipotentagainst both PKC and PKA [12-141.A favourable selectivity of inhibition is crucial in order to make definitive statements concerningthe involvementof PKC in cell-mediatedevents.This isparticularly importantwheninvestigatinga cellularresponse suchastherespiratoryburstwhich hasbeenshown tobenegativelymodulatedbyPKA[ll]and~beenproposedtobemediatedbyaPKC-independent pathway involving possiblya calmodulin-dependent kinase[12, 19,201. For compounds2-4 the rank orderof inhibition againstisolatedneutrophilPKC correlatedwith the orderobservedin the diCg-andfh4LP-stimulatedrespiratoryburst(3>4>2).However,approximately lOO-foldgreaterconcentrationswere requiredfor inhibition in the intact cell. This is not surprising sincethesecompoundsareATP-competitive inhibitors,andintracellularATP concentrationspresent in the neutrophilare very much higherthan thoseusedin the isolatedenzyme assay.This reduced potencyat an intracellularsitetallieswith the observationthat higherinhibitor concentrationswere alsorequiredto antagonisethe TPA-induced phosphorylationof a 47KDa protein in intact human platelets[163.With the stimuli dice and fMLP, the ICsovaluesof compounds2-4 were different from thosepreviously obtainedwith the lessselectivePKC inhibitors,K252a andstaurosporine[ 181 asshownin table 2. Fluoride-mediated02 releasewasmuchmoresensitiveto inhibition by the bis-indolyl maleimides comparedto diCs and fMLP, and this profile wasalso seenwith K252a and staurosporine.One explanationfor theseresultsis that fluoride cancausea decrease in the intracellularATP concentrations [2 l] suchthat the inhibitor concentrationrequiredto block PKC activity is muchreduced. Thesenovel PKCinhibitorsinhibit not only the oxidative burststimulatedby a direct PKC-activator, diCs,but alsothat stimulatedby the G proteinactivator, fluoride, andby the cell surfacereceptor stimulant,fh4LP. The bis-indolyl maleimideswerepoor inhibitor; of the OZ-stimulatedburst, whencomparedwith K252aandstaurosporine.This suggests that PKC playsa minorrole in the OZ-mediatedrespiratory burst.The oxidative burstwith OZ, a phagocyticstimuluswhichinteractswith FcandC3breceptors, could however, require activation of myosinlight chainkinase;both K252aand staurosporineare reportedaspotent inhibitorsof thii enzyme [22,23]. Oneexplanationfor the high potency of all the inhibitorsagainstfluoride and of the differencesin potency against other stimuli involves PKC isoenzymeselectivity. It is now well establishedthat different PKC isoenzymesexist [24] but their selective involvement under different activating conditionshasnot yet beenverified. However, the presenceof at leasttwo PKC isoenzyme~with differentialsensitivity to PKC activatorshasbeenreportedin HMO cells,which areclosely related 1091
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to neutrophils [2S]. The differences seen with the inhibitors against a range of stimuli could be due to their varying potencies against these PKC isoenzymes. In conclusion we report that the neutrophil respiratory burst induced by diC& fMLP and fluoride is potently inhibited by three novel selective PKC inhibitors, It has been reported that DAG is generated both on cell stimulation with fMLP [S] and with fluoride and that the burst is increased when the metabolism of DAG is inhibited [7]. Taken together with our results, this implies that the DAG/PKC pathway has a role in the neutrophil respiratory burst not only when induced by a direct PKC activator but also when induced by receptor stimulation with fMLP and by G protein stimulation with fluoride. REFERENCES 1. Niihizuka, Y. (1984) Nature 308,693-698. Berridge, M.J. and Irvine, R.F. (1984) Nature 312,31S-321. 3. Robinson, J.M., Badwey, J.A., Kamovsky, M.L. and Karnovsky, M.J. ( 1984) B&hem. Biophys. Res. Commun. 122,734-739. 4. Dale, M.M. and Penfield, A. (1984) FEBS Lett. 175,170-172. 5. Rider, L.G. and Niedel, J.E. (1987) J. Biol. Chem. 262,X03-5608. 6. Preiss, J.E., Bell, R.M. and Niedel, J.E. (1987) J. Immunol. 138,1542-1545. 7. Muid, R.E., Penfield, A. and Dale, M.M. (1987) B&hem. Biophys. Res. Commun. 143, 630-637. 8. Gomez-Carnbronero, J., Molski, T.F.P., Becker, E.L. and Sha’afi, RI. ( 1987) Biochem. Biophys. Res. Commun. 148,38-46. 9. Smolen, J.E., Korchak, H.M. and Weissmann, G. (1980) J. Clin. Invest. 65,1077-1085. 10. Fantone, J.C., Marasco, W.A., Elgas, L.J. and Ward, P.A. (1984) Am. J. Path. 115,9-16. 11. DeTogni, P., Cabrini, G. and Di Virgilio, F. (1984) Biochem. J. 224,629-635. 12. Wright, C.D. and Hoffman, M.D. (1986) Biochem. Biophys. Res. Commun. 135,749-755. 13. Seifert, R. and Schachtele, C. (1988) B&hem. Biophys. Res. Commun. 152,585592. 14. Gerard, C., McPhail, L.C., Marfat, A., Stimler-Gerard, N.P., Bass, D.A. and McCall, C.E. (1986) J. Clin. Invest. 77,61-65. 15. Nixon, J.S., Wilkinson, S.E., Davis, P.D., Sedgwick, A.D., Wadsworth, J. and Westmacott, D. (1990) Agents and Actions (in press). 16. Davis, P.D., Hill, C.H., Keech, E., Lawton, G., Nixon, J.S., Sedgwick, A.D., Wadsworth, J., Westmacott, D. and Wilkinson, S.E. (1989) FEBS L&t. 259,61-63. 17. Muid, R.E., Twomey, B. and Dale, M.M. (1988) FEBS L&t. 234,23S-240. 18. Twomey, B., Muid, R.E. and Dale, M.M. (1990) Br. J. Pharmac. 100,819-825. 19. Cooke, E. and Hallett, M.B. (1985) Biochem. J. 232,323-327. 20. Takeshige, K. and Minakami, S. (198 1) Biochem. Biophys. Res. Commun. 99,484-490. 21. Svec, F. (198s) B&him. Biophys. Acta 847,147-154. 22. Nakanishi, S., Yamada, K., Kase, H., Nakamura, S. and Nonomura, Y. (1988). J. Biol. Chem. 263,6215-6219. 23. Watson, S.P., McNally, J., Shipman, L.J. and Godfrey, P.P. (1988) B&hem. J. 249,34S-350. 24. Nishizuka, Y. (1988) Nature 334,661-665. 25. Beh, I., Schmidt, R. and Hecker, E. (1989) FEBS Lett. 249,264-266. 26. Smith, R.J., Justen, J.M. and Sam, L.H. (1988) B&hem. Biophys. Res. Commun. 152, 1497-1503. 27. Sako, T., Tauber, AI., Jeng, A.Y., Yuspa, S.H. and Bhnnberg, P.M. (1989) Cancer Res. 48, 4646-4650. 28. Dewald, B., Thelen, M., Wymann, M.P. and Baggiolini, M. (1989) Biochem. J. 264,879-884. 2.
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