[CANCER RESEARCH 49, 2374-2378, May 1, 1989]

Specific Inhibitors of Tyrosine-specific Protein Kinases: Properties of

4-Hydroxycinnamamide Derivatives in Vitro

Tadayoshi Shiraishi,' M. Koji Ovada/ Masaaki Tatsuka/ Takashi Yamashita, Kiyoshi Watanabe, andTakeo Kakunaga2

Departments of Oncogene Research [T. S., M. T., T. K.] and Tumor Virology [M. K. O.], Research Institute for Microbial Diseases, Osaka University, Osaka 565, Japan,and Biochemical Research Laboratories, Kanegafuchi Chemical Industry Co., Ltd., Takasago, Hyogo 676, Japan [T. S., T. Y., K. W.]

ABSTRACT

Inhibition by seven synthetic 4-hydroxycinnamamide derivatives, ST271, ST 280, ST 458, ST 494, ST 633, ST 638, and ST 642, of tyrosine-specific protein kinases (tyrosine kinase) of oncogene or proto-oncogeneproducts (pl30gag-v-fps, plOgag-aclin-v-fgr, pp60v-src, pp60c-src)and epidermal growth factor (EGF) receptor kinase were investigated.ST 638 (or-cyano-3-ethoxy-4-hydroxy-5-phenyIthiomethylcinnamaniide)strongly inhibited more of the tyrosine kinases than any of the othercompounds. The susceptibilities of these tyrosine kinases to ST 638increased in the following order: EGF receptor > plOgag-actin-v-fgr >ppoOc-.viY> pl30gag-v-fps, ppfidv-.vn-,with 50% inhibitory concentration

values of 1.1, 4.2, 18, 70, and 87 /¿M,respectively. The phosphorylationof the tyrosine residues in particulate fractions from RR1022 cellsexpressing pp60v-src was inhibited by ST 638 in a dose-dependent way,while it had a negligible effect on the phosphorylations of threonine andserine residues. Kinetic analysis showed that ST 638 competitively inhibited the phosphorylation of an exogenous substrate by the EGF receptorkinase with a K¡of 2.1 MM.ST 638 noncompetitively inhibited autophos-phorylation by EGF receptor kinase. These results indicate that ST 638is a potent and specific inhibitor of tyrosine kinases in vitro, and that itsinhibitory activity is caused by competing with the substrate protein forthe tyrosine kinase binding site.

INTRODUCTION

The activity of tyrosine-specific protein kinase (tyrosine kinase) is associated with several oncogene products of retro-viruses (1-5) and their cellular counterparts (6). Also, somegrowth factor receptors have tyrosine kinase activity that isstimulated by ligand binding (7-9). Many reports suggest thattyrosine kinase activity contributes to the growth of somehuman cancers such as squamous carcinoma (10) and braintumors (11), although nonproliferating systems and differentiated systems (12) also have tyrosine kinase activity. Thus,tyrosine kinase seems to be involved in the control of cellproliferation, carcinogenesis, and cell differentiation and moreinformation is needed to understand its role. Specific inhibitorsof tyrosine kinases are useful for studying the properties andfunctions of tyrosine kinase both in vitro and in vivo. Severalsynthetic compounds containing 4-hydroxycinnamic acid as acommon skeleton are potent inhibitors of the tyrosine kinaseactivity associated with the epidermal growth factor EGF4receptor. These 4-HC derivatives have 50% inhibitory concentrations of 10~'-10~7 M. These compounds have a minimal

effect on such serine-/threonine-protein kinases as cAMP-dependent protein kinase (A kinase), Ca2+/phospholipid-de-

Received 9/6/88; revised 1/6/89; accepted 1/26/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1To whom reprint requests should be addressed.2Supported by a Grant-in-Aid for a special project on Cancer-Bioscience (No.

63614518) from the Ministry of Education, Science and Culture of Japan.Deceased September 21, 1988.

3Present address: Department of Molecular Bioregulation, Institute of Molecular and Cellular Biology for Pharmaceutical University, Yamashina, Kyoto 607,Japan.

4The abbreviations used are: EGF, epidermal growth factor; 4-HC, 4-hydroxycinnamamide; I»'•,„,50% inhibitory concentration; DMEM, Dulbecco's modifiedEagle's minimal essential medium.

pendent protein kinase (C kinase), and casein kinases I and II(13). We investigated the properties of these compounds invitro and showed that they specifically inhibit various tyrosinekinases by competing with the substrate protein. We also founddifferences in susceptibility of various tyrosine kinases to theinhibitors.

MATERIALS AND METHODS

Materials. 4-Hydroxycinnamamide derivatives with the chemicalstructures shown in Fig. 1 were synthesized in this laboratory (14).*The plasma-membrane fractions from A431 cells were prepared asdescribed (IS). Phenylmethylsulfonyl fluoride, dimethyl sulfoxide, andquercetin were obtained from Wako Pure Chemical Industries, Ltd.,Japan; leupeptin from the Peptide Institute, Inc., Japan; protein ASepharose CL4B from Pharmacia Japan; phosphotyrosine, phospho-threonine, phosphoserine, and a-casein from Sigma; [7-"P]ATP (3000

Ci/mmol) was from Amersham Japan.Cell Culture. The following cell lines were used: FSV#9, and NIH/

fgr cells were established by the transformation by fps and fer uncogenesof 3Y1 rat fibroblasts and NIH/3T3 cells, respectively (16, generousgift of Dr. S. A. Aaronson). A human epidermoid carcinoma cell line,A431 (17), was used as the EGF receptor kinase source. FSV#9 andRR1022 cells were grown in DMEM supplemented with 5% fetal calfserum, while A431 and NIH/3T3 cells were cultivated in DMEMsupplemented with 10% FCS. NIH/fgr cells were maintained inDMEM containing 10% calf serum.

Antibody and Antiserum. Monoclonal antibody against the gag protein of avian sarcoma virus was prepared as reported (18). This antibodyspecifically immunoprecipitates p\iOgag-v-fps from the cell lysate ofFSV#9 cells. Antiserum against pp60v-wc (TBRS) was described previously (19). This antiserum cross-reacts with pp60c-src as well aspphOr.vrc. Antiserum against the oncogene product of Gardner-Ra-sheed feline sarcoma virus (plOgag-actin-v-fgr) was obtained fromFischer rats bearing tumors induced by an^rg-transformed rat cell line.

Preparation of Tyrosine Kinases as Immunocomplexes. The oncogeneor proto-oncogene product tyrosine kinases (p\3Qgag-v-fps, pp60v-irc,p7Qgag-actin-v-fgr, and pp60c-src), were immunoprecipitated from celllysates of FSV#9, RR1022, NIH/fgr, and NIH/3T3 cells using theantibody or antisera described above (20), and these immunocomplexeswere used as tyrosine kinase sources for assay of the inhibitory activity.

Preparation of Particulate Fractions from RR1022 Cells. Confluentcultures of RR1022 cells were washed twice with serum-free DMEMand scraped with a rubber policeman into 10 HIMHEPES buffer, pH7.5, containing 0.25 M sucrose, 5 mM EDTA, 50 mM CaCl2-2H2O, 10mM 2-mercaptoethanol, 50 Mg/ml leupeptin, and 25 JIM PMSF. Thecells were collected by centrifugation, suspended in the same buffer,and sonicated. The sonicate was centrifuged at 600 x g for 5 min at4°C.The supernatant fractions were collected and centrifuged at100,000 x g for 30 min at 4"C; the pellets were washed with 20 mM

Tris-HCl buffer, pH 7.5, containing 5 mm MgCl2, 100 MMsodiumvanadate, and 2 mM C'aCI- 211..O. resuspended in the same buffer, andstored at —¿�30°Cin small portions.

Preparation of Partially Purified EGF Receptor Kinase. Partiallypurified EGF receptor kinase was prepared from A431 cells as describedpreviously (21). The purified preparation had 1563 times the specificactivity of the solubilized membrane fractions when a-casein was usedas the substrate.

* Manuscript in preparation.

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SPECIFIC INHIBITORS OF TYROSINE KINASE

iso-Pr CN

ST 271

ST4S8

ST633

ST6«

ST 280

EtO ÇNST 638 HO-^-CH=C XNH2

PhSCH2 ft

Fig. 1. Chemical structures of 4-hydroxycinnamamide derivatives.

Phosphorylation Assay. Protein kinases were mixed with 23 n\ ofkinase buffer (20 mM Tris-HCl, pH 7.5, 5 mivi MgCl2 for ppoOc-srcand pp60c-src; 20 mM Tris-HCl, pH 7.5, 10 mM MnCl2 for p\30gag-v-fps and p70gag-actin-v-fgr; 20 mM Tris-HCl, pH 7.5, 10 mM MnCl2,and 100 ng EOF for EGF receptor) and 2 ßlof [T-32P]ATP (3000 Ci/mmol), and incubated for 15 min at 30°C.The reaction were stoppedby heating in Laemmli's sample buffer (22). After separation of the

reaction products by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis, the gel was dried and autoradiographed. Appropriate bandswere cut from the gel and their radioactivities were measured byCerenkov counting.

Assay of the Tyrosine Kinase Inhibitory Activity. The tyrosine kinaseswere incubated with various concentrations of inhibitors for 30 min at0°C,and then phosphorylation assays were started by the addition of[7-32P]ATP. The products were separated and the radioactivities of

appropriate protein bands were counted as above.Calculation of 1C».The IC5o values were measured from three or

more graphs drawing five points (vertical axis, representing the inhibition percentage and the abscissa, the logarithmic concentrations ofinhibitor) as inhibitor concentrations resulting in a 50% loss of tyrosinekinase activity.

Phosphoamino Acid Analysis. One-dimensional and two-dimensionalanalysis of phosphoamino acids were done as described (20).

RESULTS

Effects of 4-HC Derivatives on Various Tyrosine Kinases. Toinvestigate the inhibition of various tyrosine kinases by the 4-HC derivatives ST 271, 280, 458, 494, 633, 638, and 642, weselected p\30gag-v-fps, p70gag-actin-v-fgr, and pp60v-src astyrosine kinases of activated oncogene products, and pp60c-srcas that of the proto-oncogene product, and EGF receptorkinase. To confirm the specificity of these tyrosine kinaseactivities, the phosphoamino acid compositions of the reactionproducts were analyzed as shown in Fig. 2. The predominantresidue phosphorylated in immunocomplexes of p\30gag-v-fps,p70gag-actin-v-fgr, pp60v-src, and pp60c-src was tyrosine, while

phosphorylation of the threonine or serine residues did notoccur. The same results were obtained with A431 plasma-membrane fractions as previously reported (13). Therefore,these oncogene products are suitable for studying the inhibitionof tyrosine kinase activity by 4-HC derivatives.

We examined the effects of the 4-HC derivatives on thetyrosine kinase activity associated with plQgag-actin-v-fgr. The4-HC derivative's inhibitory effects were also compared withthat of quercetin, an inhibitor of pp60v-src tyrosine kinaseactivity (23) as well as serine-/threonine-protein kinases (13,24). The compound ST 280 markedly decreased the incorporation of the 32P into the M, 70,000 proteins in a dose-depend

ent manner at 1.0 to 25 /¿M-ST 458, 633, and 638 behavedsimilarly. ST 271 and 494 moderately decreased the incorporation of 32Pin this concentration range. Quercetin was a weak

inhibitor (Fig. 3).

The effects of 4-HC derivatives and quercetin on the tyrosinekinase activities of p 130gag-v-fps, pp60v-src, pp6Qc-src, and theEGF receptor were examined. The ICso values of 4-HC derivatives are listed in Table 1. The compounds ST 638 and quercetinmoderately inhibited the p 13Qgag-v-fps kinase activity, whilethe others had weak or no inhibitory activity against the sameenzyme. The compounds ST 271, 280, 638, and quercetinweakly or moderately inhibited the tyrosine kinase activitiesassociated with the RR1022 paniculate fractions. The compounds ST 271, 280, 458, 633, 638, and quercetin potently

P-Ser

P-Thr

P-Tyr

1Fig. 2. One-dimensional phosphoamino acid analysis of proteins phosphoryl

ated with oncogene or proto-oncogene product tyrosine kinases. The immunocomplexes of tyrosine kinases were phosphorylated and phosphoamino acidcompositions of the products were analyzed (20). /, immunoglobulin G heavychain (IgG-H) phosphorylated with the pp60v-src; 2, autophosphorylatedp\30gag-v-fps; J, autophosphorylated plOgag-actin-v-fgr; 4, IgG-H phosphorylated with the pp6Qc-src.

70 K-*. *.

70 K —¿�

Fig. 3. Effects of 4-hydroxycinnamamide derivatives on the tyrosine kinaseactivity of pTOgag-actin-v-fgr. The immunocomplexes prepared from NIH//gr celllysates by specific p7Qgag-actin-v-fgr antisera were incubated in the presence ofvarious concentrations of ST 271, 280, 458, 633, 638, 642, and quercetin. X-rayfilm was exposed at -80°C for 10 h. In A: 1, buffer alone; 2, 5 MMST 271; j, 1

MMST 271. In B: I. 25 MMST 280; 2, 5 MMST 280; 3, 1 MMST 280. In C: 1, 5MMST 458; 2, 1 MMST 458. In D: /, 5 MMST 633; 2, I MMST 633; 3, 0.25 MMST 633. In E: 1, 5 MMST 638; 2, 1 MMST 638. In F: I, 25 MMST 642; 2, 5 MMST 642. In G: I, 25 MMquercetin; 2, 5 MMquercetin; 3, I MMquercetin.

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SPECIFIC INHIBITORS OF TYROSINE KINASE

Table 1 Effects of 4-hydroxycinnamamides on various tyrosine kinasesThe inhibitory activities of 4-HC derivatives against oncogene or proto-oncogene product kinases (p 130gag-v-fps, pp60v-src, p70gag-actin-v-fgr, pp60c-îrc)and

EGF receptor kinase were examined with various concentrations of inhibitors (100, 25, 5, 1, and 0.25 MM).The 1C»values were obtained graphically as described inMaterials and Methods. Each value is the mean of three or more experiments.

ICW(MM)CompoundsST271ST280ST458ST633ST638ST642ST494Quercetinpl30gag-v-fpsFSV#9i">100>100854770>100>10028pp60v-jrcRR1022i97>100>10010587>100>10063P5238>100>10075>100>10024plOgag-actin-v-fgrNIH/fgri238.01.23.24.2>1005090pp60c-srcNIH/fgri4.46.81.05.518»00>1001.2NIH/3T3i1.63.83.44.84.8>100>1003.0EGF

receptorA431PP208.26.51.51.144>1000.40ml.l0.440.440.250.850.85>1000.41

" i, immunocomplexes; p, paniculate fractions; pp. partially purified preparations; in, plasma-membrane fractions. (These data were reproduced from Ref. 13).

inhibited the tyrosine kinase activity associated with eitherpartially purified or plasma membrane-associated EGF receptor, with ICso values ranging from 10~5to 10~7M. Comparison

of the inhibitory susceptibilities of the several tyrosine kinaseswill provide a greater understanding of their functional differences. Although pp60v-src and pp60c-irc are highly homologous in the kinase domain (25), their tyrosine kinase activitiesare very different (26). The susceptibility of these two enzymesto inhibitors was examined. The compounds ST 271, 280,458,633,638, and quercetin markedly decreased the tyrosine kinaseactivity of pp60c-sw in a dose-dependent manner with IC5ovalues of 4.4, 6.8, 1.0, 5.5, 18, and 1.2 MM,respectively (Table1). In contrast, they had a negligible effect on the tyrosinekinase activity of pp60v-src, with IC50values of 97, >100, >100,>100, 87, and 63 MM,respectively. Similar inhibitory effects ofthe compounds were obtained when pp60c-src was preparedfrom NIH/3T3 cells. The susceptibility of pp60c-src to theseinhibitors was 5- to 100-fold greater than that of pp60v-src, as

indicated by the ICso values (Table 1).It was important to know whether tyrosine kinases have

different susceptibilities to these compounds. ST 638's ability

to inhibit these tyrosine kinases increased in the followingorder: EGF receptor > p70gag-actin-v-fgr > pp60c-src >pl30gag-v-fps, pp60v-src with IC50 values of 1.1, 4.2, 18, 70,and 87 MM,respectively. A similar order was observed with ST271, 280, 458, 633, and quercetin.

Furthermore, ST 638 was more potent and widely inhibitorythan the other compounds. ST 642 inhibited only the tyrosinekinase activity of the EGF receptor. ST 458 had its strongestinhibitory effect on pp60c-src but failed to inhibit pp60v-src

kinase (Fig. 4). ST 494 was not an inhibitor, although itsstructure resembled that of ST 638.

Specificity of Inhibitory Activity by 4-HydroxycinnamamideDerivatives. 4-HC derivatives potently inhibits the tyrosinekinase activity of the EGF receptor without affecting eitherserine-/threonine-protein kinase activity (13). This suggeststhat the 4-HC derivatives are specific inhibitors of tyrosinekinases. It is still unclear whether these derivatives specificallyinhibit the tyrosine kinase activity in the presence of otherprotein kinases. To answer this question, we investigated theeffects of ST 271, ST 638, and quercetin on the phosphoaminoacid compositions of RR1022 particulate fractions containingtyrosine-, serine-, and threonine-protein kinases (Table 2). Thetreatment of RR1022 particulate fractions with ST 271 or ST638 inhibited 33-83% of the phosphorylation of tyrosine residues in a dose-dependent manner without affecting the phosphorylation of either serine or threonine. Quercetin increasedthe relative amounts of the phosphotyrosine. These results

53K

8 9 10

53K •¿�•

Fig. 4. Comparison of the susceptibilities of pphOi -\n and pp60c-iir kinaseto inhibition by 4-hydroxycinnamamide derivatives. The cell lysates from RR1022(A ) and NlH/frg cells (B) containing equal amounts of tyrosine kinase activitywere immunoprecipitated with TBRS. The immunocomplexes were phosphoryl-ated, and analyzed as described in "Materials and Methods." Values in parenthe

ses, percentage of inhibition. In A: I, buffer alone (0%); 2, 50 /IM ST 271 (35%);3, 10 MMST 271 (17%); 4, 50 MMST 458 (29%); 5, 10 MMST 458 (0%); 6, 50MMST 633 (30%); 7, 10 MMST 633 (18%). In B: 1, buffer alone (0%); 2, 25 MMST 271 (77%); 3, 10 MMST 271 (56%); 4, 2.5 MMST 271 (46%); 5, 25 MMST458 (92%); 6, 10 MMST 458 (77%); 7, 2.5 MMST 458 (68%); «,25 MMST 458(76%); 9, 10 MMST 633 (60%); JO, 2.5 MMST 633 (36%).

Table 2 Specific inhibition of tyrosine phosphorylation in paniculate fractionsfrom RR1022 cells

Particulate fractions from RR1022 cells were phosphorylated in the presenceof the indicated concentrations of ST 271, ST 638, or quercetin, and precipitatedwith cold trichloroacetic acid. The precipitates were hydrolyzed with HC1 andtwo-dimensional phosphoamino acid analyses were done (20). The values areexpressed as the relative amounts of phosphoamino acids or percentage of controlin parentheses.

Concentration

Relative amounts of phosphoaminoacid (%) (control %)

CompoundsNoneST271ST638

Quercetin(MM)0

2510025

10050P-Tyr6.0(100)

3.1 (52)2.0 (33)3.9 (65)0.9 (15)9.8 (160)P-Thr17.5(100)

18.4(105)13.7 (78)18.1 (103)34.0(194)27.2(155)P-Ser76.5

(100)78.5 (103)84.3(110)78.0(103)65.1 (85)63.0 (82)

indicate that ST 271 and ST 638 are specific inhibitors oftyrosine kinases, and that quercetin is a nonspecific inhibitorof protein kinases.

Kinetics of Inhibition by 4-Hydroxycinnamainide Derivatives.To investigate the manner of inhibition of 4-HC derivatives,competition experiments between ST 638 and either ATP, aphosphate donor, or a-casein, a phosphate acceptor, were done.

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SPECIFIC INHIBITORS OF TYROSINE KINASE

As indicated in Fig. 5, ST 638 was a noncompetitive inhibitorof ATP for EGF receptor au top IK>sphor vialio n and for a-caseinphosphorylation by the EGF receptor kinase. Similar resultswere obtained with ST 633 or with pl30gag-v-fps as a tyrosinekinase (data not shown). ST 638 competitively inhibited thephosphorylation of a-casein by EGF receptor kinase, becauseincreasing the concentration of a-casein in the reaction affectedthe Km of the reaction (Fig. dB). The Km of a-casein for thisreaction was 10 fi\f and the K¡was 2.1 ¿¿M.By contrast, ST 638was unable to compete for the EGF receptor autophosphoryl-ation with a-casein (Fig. 6A). Similar results were obtainedwith ST 633 or with pl30gag-v-fps (data not shown). Theseresults indicate that ST 633 and ST 638 can competitivelyinhibit the phosphorylation of an exogenous substrate by theEGF receptor kinase and p\30gag-v-fps kinase. To exclude thepossibility that nonspecific binding of 4-HC derivatives to a-casein decreased the amount of the derivatives available in thereaction mixture, the concentration of the available ST 638 wasanalyzed by high performance liquid chromatography. Theresults indicated that a-casein did not affect the concentrationof the available ST 638 (data not shown). It was unlikely,therefore, that a-casein's ability to compete with ST 638 arose

from nonspecific binding.

ATP

Fig. 5. Effects of ST 638 on the autophosphorylation reaction and the phosphorylation of exogenous substrate by EGF receptor kinase in the presence ofvarious concentrations of ATP. In A, partially purified EGF receptor preparationwas phosphor ylattd in the presence of various concentrations of ATP with (•)orwithout (O) 5 pM ST 638, and the autophosphorylation of EGF receptor wasanalyzed. In B, a-casein was phosphorylated by partially purified EGF receptorin the presence of various concentrations of ATP with (•)or without (O) 5 fiMST 638, and the phosphorylated a-casein was assayed.

0.1 0.2

a-casein

Fig. 6. Effects of ST 638 on the autophosphorylation and the phosphorylationof exogenous substrate by EGF receptor kinase in the presence of variousconcentrations of a-casein. In ,I. partially purified EGF receptor preparation wasphosphorylated in the presence of various concentrations of a-casein with (•)orwithout (O) 5 /IM ST 638, and the autophosphorylated EGF receptor wasanalyzed. In B, a-casein was phosphorylated by partially purified EGF receptorin the presence of various concentrations of a-casein with (•)or without (O) 5*iMST 638, and phosphorylated a-casein was assayed.

DISCUSSION

In this study, we found that certain 4-hydroxycinnamamidederivatives specifically inhibited tyrosine kinases in vitro. ST638 was a very potent inhibitor of many tyrosine kinasesincluding plOgag-actin-v-fgr, p\30gag-v-fps, pp60v-src, pp60c-src and the EGF receptor. The 4-HC derivatives inhibited theautophosphorylation reaction of EGF receptor, pl3Qgag-v-fps,and plQgag-actin-v-fgr and the phosphorylation of exogenoussubstrate by either p\30gag-v-fps or EGF receptor kinases. The4-HC derivatives also inhibited tyrosine phosphorylation in

RR1022 parlici! laie fractions without affecting the phosphorylation of serine and threonine residues. These results are consistent with our previous results which suggested that 4-HCcompounds inhibit the tyrosine kinase activity of the EGFreceptor but not serine-/threonine-protein kinases such as A-and C-kinases (13). ST 642 specifically inhibited EGF receptorkinase, and ST 458 was the most potent inhibitor against pp60c-src kinase with no inhibition of pp6()v-.vrc kinase. ST 494 had

no activity against any of the tyrosine kinases tested.The compounds ST 633 and 638 seemed to inhibit the EGF

receptor kinase and pl30gag-v-fps kinase by competing for thesame site that the exogenous substrate binds. Kinetic analysisshowed that ST 633 and 638 competitively inhibited the ability

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SPECIFIC INHIBITORS OF TYROS1NE KINASE

of the EGF receptor and p\30gag-v-fps kinases to phosphoryl-ated exogenous substrates. All of the potent tyrosine kinaseinhibitors found in this study have a skeleton of 4-hydroxycin-namic acid, and this structure is similar to that of tyrosineresidues in the substrate protein (13) suggesting that the inhibitory properties of the 4-HC derivatives arise from their structural similarity to the tyrosine residues in the substrate protein.The mechanism by which the 4-HC derivatives inhibit tyrosinekinase appears to be quite different from that of quercetin (23),genistein (27), amiloride (28), and ATP analogues like diaden-osine 5',5'"-P'P4 tetraphosphate (29), all of which inhibittyrosine kinases competitively with respect to ATP (23, 27-29). The 4-HC derivatives are more similar to the phosphoryl-

ation site analogue (30) which is a competitive inhibitor ofsubstrate binding. The kinetic analysis indicate that ST 633 andST 638 inhibit the phosphorylation of exogenous substrate andthe tyrosine kinase autophosphorylation reaction differently,suggesting that the mechanism of these two phosphorylationreactions are distinct from each other. Our results are consistentwith the observations of Weber et al. (31) who reported thatthe phosphorylation of the exogenous substrate by the EGFreceptor kinase was an intermolecular reaction but the autophosphorylation reaction was an intramolecular reaction. Rees-Jones et al. have found that the phosphorylation of the endogenous substrate p 120 and a-casein by the insulin receptor kinasewas competitive, although the autophosphorylation of insulin0-subunit was unaffected by a-casein (32). The 4-HC derivativesare the first reported inhibitors of tyrosine kinase inhibiting thephosphorylation of exogenous substrate and tyrosine kinaseautophosphorylation differently.

The structures of binding sites for either ATP or the proteinsubstrate on pp60v-src kinase are probably quite different fromthose on pp60c-src kinase, although these kinases have highlyhomologous amino acid sequences, especially in their kinasedomains (25). It has been reported that the tyrosine kinaseactivity of pp60v-src is 10-fold greater than that of pp60c-src(26). We found that pp60c-src kinase's susceptibility to inhibition by 4-HC derivatives and quercetin was 5- to 100-foldgreater than that of pp60v-src. In contrast, Barnekow reportedthat diadenosine 5',5'"-P'P4 tetraphosphate inhibited the tyrosine kinase activity of pp60v-src more than that of pp60c-wc(29). ST 633 and 638 are competitive inhibitors with respect tosubstrate protein, and quercetin is a competitive inhibitor withrespect to ATP (23); these results suggest that the structure ofthe ATP binding site and the substrate binding site of pp60c-

src kinase is altered by viral transduction.The 4-HC derivatives are valuable for investigating the prop

erties and function of tyrosine kinase in vitro and in vivo. Theability of the derivatives to specifically inhibit the tyrosinephosphorylation is of special interest since crucial substratesfor tyrosine kinases in intact cells have not been identified.

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1989;49:2374-2378. Cancer Res   Tadayoshi Shiraishi, M. Koji Owada, Masaaki Tatsuka, et al.  

in VitroProperties of 4-Hydroxycinnamamide Derivatives Specific Inhibitors of Tyrosine-specific Protein Kinases:

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