1rph#{243}stins:tyrosine kin*i)Iockcks as novel antiproliferative agents and dissectors of signal transduction A7EXMWZR

LEVITZII

1)epartment of Biological Cbem,str Jemsakni Jerusalem 91904, IsraeL

The Alexapder

ABSTRACT Protein tyrosine kinases (PTKs) are members of a growing family of oncoproteins and protooncoproteins that play a pivotal role in normal and abnormal proliferative processes. This hallmark identifies these unique proteins as potential targets for antiproliferative therapy This review discusses the current status of PTK inhibitors, with special emphasis on tyrphostins as antiproliferative agents and as potential drugs for cancers, leukemias, psoriasis, and restenosis as well as other proliferative conditions. The development of tyrphostins as selective signal blockers can be viewed as a first step toward the development of “smart” cocktails as antiproliferative agents. Each of these custom-made cocktails will be aimed at proliferative conditions whose transduction pathways can be characterized by molecular tools. The review also discusses the use of F1’K blockers as tools to study signal transduction processes in which protein tyrosine kinases are implicated. Levitzki, A. Tyrphostines: tyrosine kinase blockers as novel antiproliferative agents and dissectors of signal transduction FASEBJ. 6: 3275-3282; 1992. Key Words:

protein

tyrosine

kinase

PTK

blockers

TYROSINE KINASES (PTKs)’ play a key role in normal cell division and abnormal cell proliferation. Enhanced PTK activity has been associated with proliferative diseases such as cancer (1), atherosclerosis (2), and probably psoriasis (3, 4). A few examples that demonstrate the direct correlation of increased PTK activity and a particular pathological condition are: 1) In mammary carcinoma and ovarian carcinoma, the amplification of the neu/HER2/c-erbB2 proto-oncogene has been found to be the hallmark of the disease in 30% of the cases. The degree of amplification of the proto-oncogene and the overexpression of its protein product were found to correlate with the severity of disease and poor prognosis (5). 2) In human chronic myleogenous leukemia, the enhanced tyrosine kinase activity that underlies the disease is a consequence of the activation of the cellular c-abl proto-oncogene resulting from its fusion with the bcr element from another chromosome (6). 3) The release of platelet-derived growth factor (PDGF) by platelets at damaged surfaces of endothelium layering the internal surface of blood vessels results in stimulation of PDGF receptor on vascular smooth muscles cells. Stimulation of the PDGF receptor leads to their enhanced proliferation and consequently to the generation of atherosclerotic plaques (2). 4) Overexpression of the transforming growth factor a (TGFa) is believed by some to play a key role in the etiology of psoriasis in humans (3, 4). The role of PTKs in cellular proliferation suggests that specific PTK inhibitors could, in principle, have therapeutic potential. The potential use of PTK blockers as antiproliferPROTEIN

Slibennan

Institute

of Life Sciences,

The Hebrew

University

of

ative agents was recognized as early as 1981, when quercetin (Fig. 1) was suggested as a PTK blocker (7-9). Oncoproteins and proto-oncoproteins in general, and PTKs in particular, are likely to be more selective targets for chemotherapy than inhibitors of DNA or RNA synthesis or of cytoskeletondisrupting agents because they represent a subclass of proteins whose activities are essential for cell proliferation and greatly amplified in proliferative diseases. Thus the development of blockers of oncoproteins and proto-oncoprotemns is expected to lead to more focused therapies. This does not mean that such inhibition will be completely devoid of toxic side effects, as PTKs are involved in normal proliferative processes and metabolic signals. To mention a few: 1) The receptor for insulin is a PTK involved mainly in the regulation of metabolic processes rather than growth. Thus the stimulation of metabolic processes such as glycogen synthesis, glucose uptake, and antilypolysis all depend on insulin receptor tyrosine kinase activity. 2) T cell activation by mitogens is also mediated by tyrosine kinase activation. 3) The differentiation response to nerve growth factor (NGF) is mediated by the phosphorylation of trk, the high-affinity receptor for NGF. 4) Thrombin-induced platelet aggregation is mediated through tyrosine kinases of the src family. These examples, as well as others that may be further elucidated in the future, make the task of developing PTK inhibitors as antiproliferative drugs a challenging one. However, prospects are good because PTKs, like other enzymes, are rather specific in the cellular signaling events in which they participate. This, in principle, should allow the development of selective PTK blockers, minimize side effects, and obtain an optimal therapeutic index. Signal interceptors of PTK oncoproteins are likely to be a valuable addition to existing cytotoxic chemicals, radiation, and biological therapies. This principle applies to other signal interceptors such as prospective blockers of transcription factors, p21 Hras farnesylation inhibitors, or low molecular weight compounds that may force mutant p53 to attain its wild-type conformation and function as a wild-type protein.

THE

SEARCH

FOR

The search for PTK sine phosphorylation pounds that inhibit

PTK

BLOCKERS

blockers began very soon after the tyroreaction was discovered. Natural comphosphorylation on tyrosine have been

‘Abbreviations: PTKs, protein tyrosine kinases; PDGF, plateletderived growth factor; TGFa, transforming growth factor a; SAR, structure-activity relationship; BMN, benzylidenemalononitrile; GAP, GTPase-activating protein; EGF, epidermal growth factor; PLCy, phospholipase C’y; PAF, platelet activation factor; NGF, nerve growth factor; tBocTyrAm, tBoc-tyrosine-aminomalonic acid; InsR, insulin receptor kinase.

3275 0892-6638/92/0006-3275/501.50. © FASEB ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 17, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe

ment” type (17). According nucleophile, which is activated kinase, attacks the P7 of ATP tack, yielding a phosphorylated

Quercetin

Genistein H

HO.

OH

OH

#{231}O

.NyH

OH

OH

L1,LCOOH

Erbstatln

OH

Lavendustin A

I

j8 Br

Herblmycln

to this mechanism, the by a basic side chain of the by a direct nucleophiic atsubstrate and ADP with no phosphorylated intermediate. Furthermore, detailed kinetic analysis demonstrates that the EGF receptor kinase reaction follows a sequential Bi-Bi Rapid Equilibrium Random mechanism (18). These findings support the view that one can design blockers that interact with either the substrate site or the ATP subsite, or both. Our ongoing synthetic program to generate potent and selective PTK blockers, which we have named “tyrphostins’ follows a few guidelines (19-21), which are: 1) To use straightforward synthetic procedures in order to facilitate structure-activity relationship (SAR) studies, allowing the synthesis of a number of blockers of the same family and allowing for upscaling of the synthesis. 2) To aim for compounds that compete with the substrate subsite, with minimal influence on ATP binding. 3) To aim for compounds that inhibit PTKs, with weak or no inhibition of ser/thr kinases. 4) To prepare compounds selective for the key PTKs we decided to study. These include: EGFR kinase (HERI), HER2/neu kinase, pp6O C and abl kinases. 5) To control the solubility of the compounds, such that the PTK blockers can traverse the cell membrane and can therefore be tested in intact cells and experimental animals. Small synthetic peptides containing tyrosine as potential inhibitors have serious drawbacks, mainly conformational instability, inferior pharmacokinetic properties such as poor permeation through cell membranes, poor absorption through the intestinal tract, and sensitivity to pmteolytic enzymes. We have shown previously that small and rather hydrophobic tyrosine or phenylalanine-containing peptides can block insulin action on fat cells by inhibiting insulin receptor tyrosine kinase activity. The effective concentration needed was in the millimolar range (22). These findings led us to design small molecules based on the structure of the smallest of the natural PTK blocker described, erbstatin (12), and on the structure of the tyrosyl residue. The finding that erbstatin (23) and small tyrosine-containing peptides (22) are both competitive inhibitors of tyrosine kinases suggested to us that the p-hydroxyphenyl moiety of the tyrosyl residue of a PTK substrate and the conjugated double bond of erbstatin could be excellent leads for novel PTK blockers (19-21).

A

(+)

Aeroplyslnln-1

Figure 1. Some naturally occurring PTK inhibitors. The compounds depicted inhibit a number of PTKs, including growth factor receptors and cellular PTKs. Although no pattern of specificity has been found, no systematic attempt was made to determine the selectivity, if any, of these compounds. found and a few have proved to be potent P1’K blockers. The first blocker discovered was the natural product quercetin, a flavone (7-9) (Fig. 1). Quercetin, however, also inhibits cAMP-dependent kinase, protein kinase C, and other ATPrequiring enzymes. This is probably why this compound is highly cytotoxic and therefore of little clinical potential. Recently, synthetic derivatives of quercetin were found to be more selective toward PTKs (10), and therefore renewed interest in quercetin derivatives can be expected. The isoflavone genistein (Fig. 1) has been shown to be more selective than quercetin toward PTKs (11). Other naturally occurring compounds such as erbstatin (12), herbimycin A (13), lavendustin A (14), and (+) aeroplysinin-1 (15) (Fig. 1) were also reported to be potent PTK blockers. These compounds, in contrast to quercetin and like genistein, are more selective inhibitors of PTKs and therefore represent potential leads for the design of novel antiproliferative agents. Controversy, however, prevails concerning the selectivity of erbstatin. Bishop et al. (16) claim that erbstatin inhibits protein kinase C with a K1 value of 11 sM. These authors also claim that erbstatin is competitive with respect to ATP and noncompetitive against the substrate. The pattern of specificity of these natural compounds is only now beginning to emerge, since many laboratories have begun using them extensively. We therefore thought that one should try and design PTK blockers on a rational basis and develop a long-range synthetic program that would allow us to generate many compounds and establish structure activity relationships. In order to approach rational design of PTK blockers one needs to consider what is known about the mechanism of the PTK reaction. Kinetic/mechanistic studies of kinases employing ATP analogs bearing chiral P7 suggest a ‘direct displacement” mechanism rather than a ‘double displace-

DESIGN

AND

SYNTHESIS

OF

TYRPHOSTINS

Erbstatin is limited in structural elements that permit modifications intended to improve selectivity, and by itself is rather unstable. We incorporated elements of erbstatin and tyrosine, thus generating hundreds of benzylidenemalononitrile (BMN) compounds, which proved to be effective protein tyrosine kinase inhibitors. We have named this family of synthetic PTK blockers (as well as other similar families) tyrphostins (19-2 1). The relatively low inhibitory activity of the tyrphostin 4-hydroxybenzene malononitrile (IC50 = 560 LM, a = H, fi = H, n = 1; Fig. 2) prompted us to in-

(HO)n Figure

2. The general formula

of benzenemalononitrile

tyrphostins.

LEVITZKI The FASEB lournal 3276 Vol. 6 November 1992 ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 17, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe

troduce more hydroxyls on the aromatic ring and other substituents at positions a and 13. Indeed, improved potency against the EGFR kinase could be achieved (20, 21; unpublished data). We also demonstrated the importance of the cis cyano group for biological activity of tyrphostins (20, 21). Rigid tyrphostin analogs were prepared to probe the conformational requirements of tyrphostins as PTK inhibitors. Some of these analogs were found to be very potent, thus defining the active conformation of the blocker (21). We were also able to improve the potency of the tyrphostins by introducing amide and keto functions in the a position (Fig. 2). More recently we have developed other tyrphostins based on lavendustin A and S-aryl benzenemalononitriles (Fig. 3). Some of these new tyrphostins were found to be highly potent both in vitro and in intact cells (A. Levitzki et al., unpublished results). Assuming that the transition state of tyrosyl phosphorylation by PTK has a tyrosyl residue attached through its phenolic hydroxyl to an ATP, as depicted in Fig. 4, one can design bisubstrate tyrphostin blockers. Such an inhibitor is expected to bind to the active site with an affinity constant that is the product of the two affinity constants for the two separate moieties (24). Based on the tentative transition state shown in Fig. 4, we designed and synthesized a series of bisubstrate inhibitors that possess a quinoline moiety as the ATP analog (Fig. 5). The Isoquinoline moiety has already been used successfully as an ATP mimic (25). Indeed, the quinoline-tyrphostin bisubstrate blocker inhibits EGFR kinase much better than each half separately, although the affinity is not yet high enough to begin biological experiments. The IC50 of the compound for the inhibition of EGFR autophosphorylation (Fig. 5) is 6 iM compared with 30 iM of the parent tyrphostin and a nonmeasurable high value for the quinoline sulfonamide moiety (> > 1 mM). The antiproliferative activity of the compound is IC50 = 3 tiM. Because the compound blocks serum-induced proliferation with similar potency, it seems that the antiproliferative activity is mediated through a PTK downstream of the growth factor receptor (P. Atadja, M. Chorev, and A. Levitzki, unpublished results). Attempts are currently being made to improve the juxtapositioning of the tyrphostin and the quinoline moieties in order to achieve enhanced affinities and selectivity toward the EGF receptor.

SELECTIVITY

OF

TYRPHOSTINS

IN VITRO

Most

tyrphostins are 100- to 104-fold more potent in inhibitP1’Ks than PKA, PKC, or Ca2/calmodulin-dependent kinases (20, 21, 26; and our unpublished data). As tyrphostins are relatively new, we know little about their exact specificity vis-#{224}-vis different PTKs. However, we have acing

OH0

0

r r0 r .

0-

0

0-

0

.Adenosine

-

0-

NH

Figure FTKs.

4. Tentative transition

state of tyrosine phosphorylation

by

cumulated enough information on a fair number of tyrphostins to make the observation that tyrphostins can easily discriminate between the insulin receptor kinase and the EGFR kinase in vitro (19). Certain tyrphostins do not discriminate between abi kinase and EGF receptor kinase, whereas others exhibit two orders of magnitude difference in affinity between these two PTKs (27). More strikingly, some tyrphostins even discriminate between the closely related EGF receptor (HER!) and the neu/ErbB2 (HER2) gene product, which are 80% homologous in the kinase domain (21; N. Osherov and A. Levitzki, unpublished results). We also observed that some tyrphostins discriminate between pl4Ocab1, pl85, and p210 in vitro, although all these abi kinases, differing only in their NH2-terminal sequences, possess identical catalytic domains (27). These results suggest that abi proteins may possess different substrate specificities and/or affinities toward intracellular target substrates. When examined in detail, the three abi proteins also possess different affinities toward synthetic substrates and ATP (27). It seems that some tyrphostins interact with residues within the tyrosine kinase domain, which may be common among many P1’Ks, thus probing similar structural elements. These are the rather nonspecific tyrphostins with ‘relaxed” specificity. Genistein, which is used quite extensively, also seems to block many PTKs (11, 28-35), thus belonging to the family of nonselective PTK blockers. Similar findings are accumulating with regard to herbimycm A (13). It is likely that a directed synthesis of PTK blockers based on genistein or herbimycin may lead to more selective PTK blockers, as with tyrphostin and lavendustin derivatives (27). Our findings with tyrphostins are quite encouraging because they strengthen the basic hypothesis that even though numerous PTKs have identical intracellular target substrates, such as GTPase-activating protein (GAP) or phospholipase Cy (PLC-y), they differ from each other both in their affinities toward these identical putative substrates and in having more specialized substrates. Related is the ob-

OH

SAI S-aryl

tyrphostins

Figure 3. S-aryl

TVDPi-lCTlMC.

Lavendustin-derived tyrphostins

TVQ(IJr

and lavendustin-derived

I(Ip.JAcI

RI(Yi(FPc

blockers PTK blockers.

Figure

5. A bisubstrate

quinoline

tyrphostin.

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servation that a few tyrphostins are much more efficacious in inhibiting EGF-dependent cell proliferation than EGF receptor kinase (21; N. Osherov and A. Levitzki, unpublished results). This could be due to the accumulation of these tyrphostins in the cell or to their inhibitory action on unknown PTKs downstream in the EGF signaling pathway (36). This phenomenon is currently under investigation in our laboratory. Similar results were obtained with thiazolidine dione PTK blockers (37) (Fig. 6). One interesting result is that genistein, which blocks EGF receptor kinase (30), was also found to block topoisomerase 11(28). Some tyrphostins were also recently found to block topoisomerase I and II (J. Pierre, J. Markovits, and A. Levitzki, unpublished results). The chemical intermediate in the topoisomerase reaction is the covalent link between the DNA phosphate and a tyrosyl residue of the enzyme (Fig. 7). Thus it is not surprising that some PTK blockers may have more than one mechanism of action. It is probably not unreasonable to pursue drugs that affect both P1’Ks and topoisomerases as antiproliferative agents in that the two inhibitory activities may act synergistically with each other. Indeed, tyrphostin AG213(RG 50864), which is a potent blocker of EGFR kinase and some other V1’Ks, has been claimed to possess antiproliferative effects that may not be related to its EGFR kinase inhibitory activity (36).

MODE

More

detailed

kinetic

analysis

revealed

that

tyr-

phostins can be divided into four classes: 1) competitive inhibitors vis-#{224}-vis the substrate as well as against ATP; 2) ‘mixed” competitive with respect to both substrate and ATP (in this case the inhibitor diminishes the affinity toward both ATP and substrate, but they can still bind); 3) competitive against ATP and mixed competitive vis-#{224}-vis the substrate; and 4) competitive vis-#{224}-vis the substrate and mixed competitive or noncompetitive with respect to ATP (I. Posner, M. Engel, and A. Levitzki, unpublished results). The fact that different tyrphostins exhibit different modes of inhibition of EGF receptor kinase suggests that the active site of PTKs may be quite extended, being able to accommodate small molecules in a large variety of configurations. This should not be surprising because the enzyme acts on high molecular weight protein substrates, and thus small molecules may bind in different modes to the enzyme active site. Only future work will tell whether the mode of action of tyrphostin on different PTKs is similar or different.

CH3 N

S -

Figure

CH2

0-

DNA Figure

7. Phosphorylation on the topoisomerase DNA as a reaction intermediate.

Preliminary results in this which is a “mixed” competitive

OF INHIBITION

All the natural FFK blockers (Fig. 1) except erbstatin were found to be ATP competitors. Erbstatin was claimed to be competitive against the substrate and noncompetitive against ATP (23). Our analysis, however, of the kinetic data indicates that erbstatin is a mixed competitive inhibitor of ATP and of the substrate (I. Posner, M. Engel and A. Levitzki, unpublished results). Tyrphostins were initially designed to compete with the substrate subsite and not with the ATP subsite (19). Indeed, initial kinetic analysis suggested that the compounds are competitive against the substrate.

TOP OISOM ERASE

6. Thiazolidine-Diones

NQ

P1’K blockers.

R

direction inhibitor

tyrosine

residue by

show that with respect

AG213,

to ATP in EGFR kinase (I. Posner et al., unpublished results), is noncompetitive against ATP in abi kinase (27). Our current synthetic efforts are directed toward high-affinity blockers with minimal effect on the ATP subsite. Irrespective of their mode of inhibition, all the PTK blockers except quercetin are 100to

10,000-fold

less

potent

against

ser/thr

kinases.

In

this

respect, the tyrphostins, which are ATP competitors, resemble the unrelated PTK blockers such as lavendustin A (14) and its derivatives (27, 38; A. Gazit et al., unpublished results), which have been shown to be ATP competitive. Recently, however, we were able to modify the lavendustin A nucleus and generate compounds that are competitive with the substrate in EGFR kinase (our unpublished data). Genistein is also an ATP competitor with a good margin of selectivity in favor of PTKs compared with ser/thr kinases (11). These observations demonstrate that the ATP binding domain in PTKs differs markedly from the ATP site in other kinases. Although selectivity of tyrphostins as ATP competitors can be achieved, this feature is undesirable because the high intracellular concentration of ATP renders competition against ATP ineffective in intact cells or in vivo. Indeed, potent tyrphostins that compete with ATP with K1 values in the 50 nM range block EGF-dependent cell growth at IC50 = 10 to 15 &M (19-21; N. Osherov et al., unpublished results). Similar results were obtained by Shiraishi et al. (39) with a PTK blocker, ST638, which is structurally related to tyrphostins. We have recently shown that when the intracellular concentration of ATP is artificially decreased by treatment with deoxyglucose and NaN3, the ATP-competitive tyrphostins become more potent in intact cells (N. Osherov and A. Levitzki, unpublished results).

BIOLOGICAL BLOCKERS Antiproliferative

EFFECTS

OF

TYROSINE

KINASE

effects

Tyrphostins as well as other PTK blockers were examined in many biological systems. Tyrphostins have been found to be successful antiproliferative agents and to be largely nontoxic

3278 Vol. 6 November 1992 The FASEB lournal IEVIT7KI ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 17, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe

to cells grown in culture. Thus, tyrphostins (19, 21, 29, 40-43) and erbstatin (44, 45) block PLCy activation by EGF as well as DNA synthesis. Another PTK blocker of the tyrphostin type was also reported to block EGF-stimulated phosphorylation of intracellular substrates in intact cells (39). Signaling in T lymphocyte cells was also reported to be blocked by PTK blockers, including members of the tyrphostin family (32, 46). The potency of tyrphostins to block

degree of cytoskeleton organization and the degree of tyrosine phosphorylation, especially of cytoskeleton proteins (55). Umezawa et al. (56) reported that erbstatin is also capable of reversing the pp60’-transformed NRK cells. Similar results were obtained with herbimycin A, a potent PTK blocker. Herbimycin A reduces phosphorylation events induced by the src oncogene in NRK cells. It was also

EGF-dependent

in the presence of this PTK blocker (57). It was suggested that herbimycin A acts on the pp6ov-src protein by irreversibly modifying it through an SH group on the protein (57, 58). This covalent modification of the pp6Osrc protein probably targets the molecule for degradation. This result suggest that irreversible PTK blockers in general may be even more beneficial than reversible blockers if they lead to an irreversible loss of the oncoprotein.

kinase activity

tiproliferative

activity

tiproliferative

activity

in correlation

prompted

us to

in clinically

with their an-

examine

relevant

their

an-

situations.

Tyrphostins inhibit the growth of a human squamous cell carcinoma that overexpresses EGF receptors when implanted into nude mice. Tyrphostins, by intraperitoneal injection, prolonged the survival of the mice and caused shrinking in the size of the tumors. One of the most exciting aspects of this study is the finding that the tyrphostin synergizes with monoclonal antibody 108 against the external

EGF binding

domain

of the EGF receptor

in blocking

tumor

growth (47). This experiment suggests that antibodies against cancer antigens can synergize with PTK blockers to inhibit tumor growth. Antitumor activity of tyrosine kinase inhibitors has also been shown for erbstatin (48, 49). We have also shown that tyrphostins block the proliferation of guinea pig and human keratinocytes grown in culture. The inhibition of cell growth is parallel to the effect of the tyrphostins on DNA synthesis and EGF-induced tyrosine phosphorylation (50). As tyrphostins block EGF-dependent proliferation of normal keratinocytes (50), we examined and obtained evidence that EGFR-directed tyrphostins arrest the growth of psoriatic keratinocytes in culture (A. Levitzki, et al., unpublished results). This observation correlates well with the finding that the psoriatic condition is associated with the overexpression of the TGFa gene (refs 3, 4 and references therein), leading to constitutive secretion of the growth factor and therefore to sustained autocrine stimulation of the psoriatic keratinocytes. The persistent autocrine activation of the EGF receptor by TGFa on the keratinocytes is believed to play a key role in the development and/or maintenance of the proliferative state of the lesion psoriatic condition. Disruption of this autocrine loop by EGFR

kinase-directed

tyrphostins

may

indeed

be of therapeutic

value. We have therefore initiated, in collaboration with Rhone Poulenc Rorer, dinical trials to assess the therapeutic potential of these blockers as inhibitors of psoriasis. The potency of tyrphostins in blocking PDGF-dependent

proliferation of vascular smooth muscle cells grown in culture, which correlates well with its potency to block PDGFdependent phosphorylation of intracellular proteins, points to other

therapeutic

possibilities

(51). PDGF

plays

a key role

in atherosclerotic plaque development (2), pulmonary fibrosis (52), and myelofibrosis (53). We have also identified indole-containing tyrphostins tivity toward PDGF-dependent

that possess significant selecmitogenic effects (IC50 - 20

lead compounds (54, A. Gazit, et al., unpublished results). In human bone marrow fibroblasts grown in culture, the inhibitory dose response for PDGF-dependent phosphorylation correlates with the inhibitory dose response

ILM) as potential

on PDGF-dependent

cell proliferation

(54).

Tyrphostins reverse the transformed phenotype of v-sirtransformed chicken lens cells grown in culture. Increased tyrosine phosphorylation induced by treatment with H2O2/vanadate enhances the transformed phenotype, whereas inhibitors of tyrosine kinase (tyrphostins) reverse the transformed phenotype. Examination of the cytoskeleton of these cells reveals a strong inverse correlation between the

TYRPHOSTINS:

TYROSINE

KINASE BLOCKERS

reported

that the pp60”

PTK BLOCKERS INDUCE ERYTHROID CELLS The

PTK

blocker

ST638,

protein

degrades

at a faster

DIFFERENTIATION

a compound

similar

rate

OF

to tyr-

phostins, in combination with mitomycin (33) and genistein with mitomycin (34), induce differentiation in mouse erythroleukemia (34). Similar results were found with herbimycin A (59). We find that some tyrphostins induce complete differentiation of mouse erythroleukemic cells in the absence of mitomycin (M. Anafi et al., unpublished results). This effect of PTK blockers suggests that in transformed cells, the abnormal enhanced activity of tyrosine kinases interferes with the differentiation program. Similar results have been obtained with the human chronic myleogenous leukemic cell line K562. These cells express an activated form of the c-abl protein kinase: p2lObcrabI, which directly causes the transformed state. PTK inhibitors such as tyrphostins (M. Anafi et al., unpublished results) and herbimycm A (60) induce the erythroid differentiation of these cells. With tyrphostins we can obtain almost complete terminal differentiation, followed by cell death (M. Anafi et al., unpublished results). The potency of tyrphostins and their relative low toxicity suggest that these compounds, as well as other PTK blockers, can be used as antiproliferative agents. Table 1 is a short list of disease states in which PTKs are implicated and therefore may respond to PTK inhibitor therapy. As some tyrphostins were found to be more selective toward the abl kinases than the EGF receptor, efforts are being made to enhance this selectivity. These SAR studies in vitro (27) also revealed a striking selectivity of some tyrphostins that can discriminate by a factor u1p to sevenfold even between the normal abl protein p14O and its oncogenic counterpart p2IobCr.abl. This interesting finding suggests further synthetic opportunities toward even more selective tyrphostins. These results also suggest that the NH2-terminal bcr sequence allosterically influences substrate specificity of the abl kinase domain. This modified specificity may contribute to the oncogenic activity of p2lODCrabI. At the present time, however, we cannot find a straightforward

quantitative correlation between the potency of tyrphostins to block abl kinase activity and their potency to induce differentiation of K562 cells (M. Anafi, Y. Ben Neriah and A. Levitzki, unpublished results). It is plausible that tyrphostins act at downstream targets as well as on the bcr-abl kinase in the p2lO transformed cells. Table 1 lists some disease conditions that can be associated with particular tyrosine kinases. These diseases therefore are candidates for therapy by PTK blockers like tyrphostins. In most proliferative diseases a number of oncoproteins and

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TABLE

1. Diseases associated

with defined PTKs

Disease

PTK

Certain

cancers

Psoriasis

implicated

Mechanism

EGF receptor

(HER 1)

EGF receptor

(HER 1)

of PTK

Amplification of the gene and overexpression of the EGF receptor Overexpression

of the

TGFa

gene leading persistent autocrine

stimulation receptor Mammary

ovary

and carcinomas

neu/c-erb

(certain forms)

Atherosclerosis, pulmonary fibrosis, Chronic

myeloid,

PDGF

be

leukemia

2

(CML)

10b.r-abl

characterized

in terms

of the

oncogenes

and

proto-

oncogenes expressed. This will determine the custom-made cocktail to be designed. This approach can be extended

other

oncoproteins

and

proto-oncoproteins

once

to

suitable

blockers can be synthesized. For example, selective blockers of Ras farnesylation can be added to such custom-made cocktails when activated Ras is identified. Colon carcinoma involves both activated Ras and tyrosine kinases and is therefore a disease that might be treated by such a mixture of signal interceptors.

PTK

INHIBITORS

AS DISSECTORS

OF SIGNALS

Others as well as ourselves have used tyrphostins extensively to examine whether tyrosine phosphorylation is involved in the transmission of certain biochemical signals. For example, we have found that tyrphostins block thrombin-induced platelet aggregation and the release of serotonin in parallel to their inhibitory effect on thrombin-induced pp6O’ tyrosine phosphorylation as well as other intracellular target proteins. The inhibitory effect of tyrphostins on the thrombininduced tyrosine phosphorylation correlates quantitatively with their inhibitory effect on platelet aggregation and

serotonin

release (61, 62). Genistein

was also found to block

platelet

activation by UL46619 (35) and erbstatin was found to block platelet activation factor (PAF)-induced platelet activation in parallel with their PTK blocking activities. Similar results were obtained with erbstatin (63). Similarly, tyrphostins were instrumental in demonstrating the involvement of protein tyrosine phosphorylation in the signaling pathway of B lymphocyte activation (64, 65) by Staphylococcus aureus Cowan I. T cell activation is also blocked by tyrphostins and genistein (32, 46, 66, 67), implicating tyrosine phosphorylation as an essential element in the signaling pathway of T cells. In both the platelet system and the lymphocyte

systems,

affect the signaling of protein

kinase

receptor

it was

pathways C using

shown

elicited

phorbol

that

tyrphostins

do

not

by the direct activation esters.

However,

in other

and

Gene

to

of the EGF

amplification

overexpression HER2 protein

and of the

Stimulation by PDGF after triggering of a pathological process

myelofibrosis

proto-oncoproteins are involved, so it is reasonable to assume that cocktails of tyrphostins will be used for treatment. We would like to suggest that for each situation the disease will

B2(HER2)

malignant

involvement

pl85bCrabI

Chromosome rearrangement that results in fusion between bcr and pl4OCabI leading to enhanced kinase activity

systems phorbol ester can lead to tyrosine kinase phosphorylation. In such a case, tyrphostins can act as blockers of the PKC-PTK pathway. For example, the activation of platelet aggregation by phorbol ester leads to tyrosine phosphorylation. This effect can be blocked by tyrphostins (62). Independently we have shown that cAMP-mediated pathways are not affected by tyrphostins (21) and that protein kinase A is inhibited in vitro by tyrphostin concentrations 300to 10,000-fold higher than those required to block PTK activities of various tyrosine kinases (I. Posner and A. Levitzki, unpublished results). Tyrphostins were instrumental in the elucidation of the signaling pathway leading from EGF receptor activation to the mobilization of intracellular stores of Ca24. It was shown that tyrphostins block EGF receptor-induced phosphoryladon of PLCy (41), and therefore IP3 formation (43) and Ca2 mobilization (41). Tyrphostins have no effect on 1P3 formation or Ca24 mobilization induced by bradykinin and bombesin, which probably activate PLC13 (which is not a substrate for PTK). These results also demonstrate the specificity of tyrphostins and their relative nontoxicity. In B cells it was also shown that the tyrphostins block B cells activation concomitantly with the inhibition of Ca24 mobilization and the formation of 1P3 (64). The involvement of protein tyrosine phosphorylation in the maturation pathway of sea star oocytes has also been clearly demonstrated using tyrphostins and erbstatin (68). In this system, it was shown that tyrosine phosphorylation of 44MuP and the activation of p34cdc2 kinase are inhibited by tyrphostins, thus inhibiting oocyte maturation. Tyrphostins that block insulin receptor kinase (InsR) are also effective in blocking insulin-induced effects such as S6 phosphorylation, glucose uptake, and a-aminoisobutyric acid uptake (Y. Zik et al., unpublished results). Similarly, tyrosine derivatives that block insulin receptor kinase were shown to inhibit glucose uptake by rat fat cells and the insulin-dependent antiipolytic effects (21). In both studies we were able to demonstrate that PTK blockers can selectively block certain insulin actions but not others. The PTK blocker tBoc-tyrosineaminomalonic acid (tBocTyrAm) was found to block both

insulin-induced glucose uptake and the antiipolytic effect of the hormone in rat fat cells. In contrast to tBocTyrAm, other

The FASEB Journal LEVITZKI 3280 Vol. 6 November 1992 ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 17, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe

P1’K

blockers

induced

were

found

to block

only

the

glucose

uptake

by insulin.

A possible clue to understanding this difference is the finding that among the series of Vl’K blockers examined, only tBocTyrAm effectively blocks both InsR autophosphorylation and InsR-catalyzed tyrosine phosphorylation of exogenous substrates. It seems, therefore, that an effective blocker that inhibits InsR autophosphorylation blocks all Insulin-induced actions well; not a surprising result. However, the fact that many blockers that block the kinase activity of the InsR do not block the antiipolytic effect of insulin suggests that antilypolysis and induction of glucose uptake are separate signaling pathways emanating from the phosphorylated InsR. It seems that the pathway leading to antilypolysis is less sensitive to PTK blockers (21). This insensitivity may suggest that the autophosphorylated receptor activates this pathway not by a phosphorylation reaction, but by interacting with another effector system. Another possibility is that the protein whose phosphorylation is essential for the propagation of the antilipolytic signal associates with the InsR with such strong affinity that only the more potent PTK blockers can compete with it effectively. More work is

needed to distinguish between these two possibilities. Another interesting feature of the pleotropic nature of insulin revealed by the use of PTK blockers A few tyrphostins, especially

signaling observation:

The

studies Rorer

Association

described (King

in the article

of Prussia,

Penn.)

were supported by Rhoneand by The Israel Cancer

1. Bishop, J. M. (1987) The molecular genetics of cancer. Science 335, 305-311 2. Ross, R. (1989) Platelet derived growth factor. Lancet 1, 1179-1182 3. Elder, J. T., Fisher, G. J., Lindquist, P. B., Bennet, G. L., Pittelkow, M. R., Coffey, R. J., Ellingsworth, L., Denrynck, R., and Voorhees, J. J. (1989) Overexpression of transforming growth factor a in psoriatic epidermis. Science 243, 811-814 4. Vassar, R., and Fuchs, E. (1991) Transgenic mice provide new insights into the role of TGFa during epidermal development and differentiation. Gen#{128} & Dev. 5, 714-727 5. Slamon, D. J., Godoiphin, W., Jones, L. A., Holt, J. A., Wong, S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J., Ulrich, A., and F. (1989)

Studies

16.

18.

AG18(RG50810),

(Tel Aviv).

M.

15.

17.

REFERENCES

Press,

Naganawa,

is the following

were found to effectively block insulin-induced S6 kinase activation and insulin-dependent glycogen synthesis but had little effect on ppl8O tyrosine phosphorylation. This finding (Y Zick et al., unpublished results) suggests that ppl8O is not part of the signaling pathways leading to activation of S6 kinase and glycogen synthetase. Phosphorylation of ppl8O is one of the hallmarks of insulin responsive cells and probably mediates some actions of insulin but probably not the two discussed above. These and other accumulating examples point to the use of PTKs blockers as useful tools to study signal transduction mechanisms. Poulenc

10. Cushman, M., Nagarthnam, D., Burg, D. L., and Geahlen, R. L. (1991) Synthesis and protein-tyrosine kinase inhibitory activities of flavonoid analogues. j Med. Chem. 34, 798-806 11. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., and Fukami, Y. (1987) Genistein: a specific inhibitor of tyrosine-specific protein kinases. j BioL Chem. 262, 5592-5595 12. Umezawa, H., Imoto, M., Sawar, T, Issihiki, K., Matsuda, N., Uchida, T, Finuma, H., Hamada, M., and Takeuchi, T (1986) Studies on a new epidermal growth factor kinase inhibitor, erbstatin, produced by MH435-hF3. j Antibiot. 39, 170-173 13. Uehara, Y., Murakami, Y., Suzukake-Tsuchiya, K., Moriya, Y., Sano, H., Shibata, K., and Omura, S. (1988) Effects of herbimycin derivatives on src oncogene in relation to antitumor activity. J. Antibiot. (Tokyo) 41, 831-834 14. Onoda, T., Linuma, H., Sasaki, Y., Hamada, M., Isshiki, K.,

of the HER-2/neu

protooncogene

in

human breast and ovarian cancer. Science 244, 707-712 6. Daley, G. Q., and Ben-Neriah, Y. (1991) Implicating the bcr-abl gene in the pathogenesis of Philadelphia chromosome leukemia. Adv. Cancer R#{128}s. 57, 151-184

positive

human

7. Glossmann, H., Presek, P., and Ergenbrodt, E. (1981) Quercetin inhibits tyrosine phosphorylation of the cyclic nucleotide independent transforming protein kinase pp6O. Naunyn &hmie&berg’s Arch. PharmacoL 317, 100-102 8. Graziani, Y., Chayoth, R., Karny, N., Feldman, B., and Levy, J. (1981) Regulation of protein kinases activity by quercetin in ehrlich ascited tumor cells. Biochem. Biophys. Acta 714, 415-421 9. Graziani, Y., Erikson, E., and Erikson, R. L. (1983) The effect of quercetin on the phosphorylation activity of Rous sarcoma virus transforming gene product in vitro and in vivo. Eur. j Biochem. 135, 583-589

19.

H., Takeuchi,

T., Tatsuta,

K., and Umezawa,

K. (1989)

Isolation of a novel tyrosine kinase inhibitor, lavendustin A, from Streptomyces griseolavendus. J. Nat. Prod. 52, 1252-1257 Kreuter, M. H., Leake, R. E., Rinaldi, F., Mueiler-Klieser, W, Maidhof, A., Mueller, E. G., and Schroeder, H. C. (1990) Inhibition of intrinsic protein tyrosine kinase activity of EGF-receptor kinase complex from human breast cancer cells by the marine sponge metabolite (+)-aeroplysinin-1. Comp. Biochem. P/zysiol. 97B, 151-158 Bishop, W. R., Petrin, J., Wang, L., Ramesh, U., and Doll, R. J. (1990) Inhibition of protein kinase C by the tyrosine kinase inhibitor erbastatin. Biochem. Pharmacol. 40, 2129-2135 Knowles, J. R. (1990) Enzyme catalyzed phosphoryl transfer reactors. Annu. Rev. Biochem. 49, 877-919 Posner, I., Engel, M., and Levitzki, A. (1992) Kinetic model of the epidermal growth factor (EGF) receptor tyrosine kinase and a possible mechanism of its activation by EGF. j Biol. Che,n. In press Yaish, P., Gazit, A., Gilon, C., and Levitzki, A. (1988) Blocking of EGF-dependent Science 242,

cell proliferation

by EGF receptor

kinase

inhibitors.

933-935

20. Gazit, A., Yaish, P., Gilon, C., and Levitzki, A. (1989) Tyrphostins I: synthesis and biological activity of protein tyrosine kinase inhibitors. j Med. Chem. 32, 2344-2352 21. Gazit, A., Osherov, N., Posner, I., Yaish, P., Poradosu, E., Gilon, C., and Levitzki, A. (1991) Tyrphostins II: heterocyclic and asubstituted benzylide-nemalononitrile tyrphostins as potent inhibitors of EGF receptor and ErbB2/neu tyrosine kinases. j Med. Chem. 34, 1897-1907 22. Shechter, Y., Yaish, P., Chorev, M., Gilon, C., Braun, S., and Levitzki, A. (1989) Inhibition of insulin dependent lipogenesis and antiipolysis by protein tyrosine kinase inhibitors. EMBOJ 8, 1671-1676 23. Imoto, M., Umezawa, K., Isshiki, K., Kunimoto, S., Sawa, T., Tounio, T, and Umezawa, H. (1987) Kinetic studies of tyrosine kinase inhibition by erbstatin. j Antibiot. (Tokyo) 10, 1471-1473 24. Broom, J. (1989) Rational design of enzyme inhibitors: multisubstrate analogue inhibitors. j Med Che,n. 32, 2-7 25. Ricouart, A., Gesquiere,J. C., Tartar, A., and Segehaert, C. (1991) Design of potent kinase inhibitors using the bisubstrate approach. j Med. Guam. 34, 73-78 26. O’Dell, T J., Kandell, E. R., and Grant, G. V. (1991) Long term potentiation in the hipocampus is blocked by tyrosine kinase inhibitors. Nature (London) 353, 558-560 27. Anafi, M., Gazit, A., Gion, C., Ben-Neriah, Y., and Levitzki, A. (1992) Selective interactions of transforming and normal abl protein with ATP, tyrosine-copolymer substrates and tyrphostins. j BioL Chaos. 267, 4518-4523 28. Markovits, J., Linassier, C., Fosse, P., Couprie, J., Pierre, J., Jacquemin-Sabion, A., Saucier, J. M., Le-Pecq, J. B., and Larsen, A. K. (1989) Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res. 49, 5111-5117 29. Clegg, K. B., and Saxnbhi, M. P. (1989) Inhibition of epidermal growth factor-mediated DNA synthesis by a specific tyrosine kinase inhibitor in vascular smooth muscle cells of the spontaneously hypertensive rat. j Hypertens. 6, s144-s145 30. Linassier, C., Pierre, M., Le Pecq, J. B., and Pierre, J. (1990) Mechanisms of action in NIH-3T3 cells of genistein, an inhibitor of EGF receptor tyrosine kinase activity. Biochem. PharmacoL 39, 187-193 31. Constantinou, A., Kiguchi, K., and Hubernam, E. (1990) Induction of differentiation and DNA strand breakage in human HL-60 and K-562 leukemia cells by genistein. Cancer Rev. 50, 2618-2624 32. Stanley, J. B., Gorczynski, R., Hung, C. K., Love, J., and Mills, G. B. (1990) Tyrosine phosphorylation is an obligatory event in IL-2 secretion. j Immunol. 145, 2189-2198

3281 TYRPHOSTINS: TYROSINE KINASE BLOCKERS ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 17, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe

33. Watanabe, T, Shiraishi, T, Sasaki, H., and Oishi, M. (1989) Inhibitors for protein-tyrosine kinases, ST638 and genistein, induce differentiations of mouse erythroleukemia cells in a synergistic manner. Ezp. Cell. Res. 183, 335-342 34. Watanabe, T, Kondo, K., and Oishi, M. (1991) Induction of in vitro differentiation of mouse erythroleukemia cells by genistein, an inhibitor of tyrosine protein kinases. Cancer Res. 51, 764-768 35. Gaudette, D. C., and Holub, B. J. (1990) Effect of genistein, a tyrosine kinase inhibitor, on U46619-induced phosphinositide phosphorylation in human platelets. Biochem. Biophys. Ret. Commun. 170,

238-242 36. Faaland, C. A., Mermelstein, F. H., Hagashi, J., and Laski, J. D. (1991) Rapid uptake of tyrphostin into A431 human epidermoid cells is followed by delayed inhibition of epidermal growth factor (EGF)stimulated EGF receptor tyrosine kinase activity. Mol. Cell. Biol. 11,

2697-2703

37.

F., Traxler, P., Regenass, U., Murray, B.J., Roesel,J. L., Meyer, T., McGlynn, E., Storni, A., and Lydon, B. (1990) Thiazolidine-Diones biochemical and biological activity of a novel class of tyrosine protein kinase inhibitors. j Biol. Chaos. 265, 22255-22261 38. Hsu, C. Y. J., Persons, R E., Spaola, A. P., Bendar, R. A., Levitzki, Geissler,J.

A., and Zilberstein, Z. (1991) Kinetic analysis of the inhibition of the EGF receptor tyrosine by lavenohistin-A and its analogue. j BioL Chem. 266, 21105-21112

39. Shiraishi, T., Owada, M. K., Tatsuka, M., Fuse, Y., Kiyoshi, W., and Kakunaga, T (1990) A tyrosine-specific protein kinase inhibitor, alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethyl-cinnamamide, blocks the phosphorylation of tyrosine kinase substrate

Jpn. j

in intact

R. M., Zilberstein, A., Gazit, A., Gilon, C., Levitzki, A., and Schlessinger, J. (1989) Tyrphostins inhibit epidermal growth factor (EGF)-receptor tyrosine kinase activity in living cells and EGFstimulated cell proliferation. j Biol. Chaos. 264, 14503-14509 41. Margolis, B., Rhee, S. G., Felder, S., Lyall, R., Levitzki, A., Ullrich, A., Zilberstein, A., and Schlessinger, J. (1989) EGF induces tyrosine 40. Lyall,

of phospholipase C II: a potential signaling. Cell 57, 1101-1107

mechanism

for

42. Schwartz, B., Cagnano, E., Braun, S., and Lamprecht, S. A. (1990) Characterization of tyrosine kinase associated with subcellular components of human colonic ephthelium. Anticancer Rev. 10, 1747-1754 43. Posner, I., Gazit, A., Gion, C., and Levitzki, A. (1989) Tyrphostins inhibit the epidermal growth factor receptor-mediated breakdown of phosphoinositides. FEBS LetI. 257, 287-291 44. Imoto,

M., Shimura,

N., Ui, H., and Umezawa,

K. (1990) Inhibition

208-211 45. Umezawa,

human T cell proliferation. Transplantation 51, 448-450 47. Yoneda, T, Lyall, R., Alsine, M. M., Persons, P. E., Spada, A. P., Levitzki, A., Zilberstein, A., and Mundy, G. R. The antiproliferative effect of tymsine kinase inhibitor of tyrphostin on a human squamous cell carcinoma in vitro and in nude mice. Cancer Res. 51, 4430-4435 48. Imoto, M., Umezawa, K., Kamuro, D., Sawa, T, Takeuchi, T., and Umezawa, H. (1987) Antitumor activity of erbstatin, a tyrosine tein kinase inhibitor. f/in. j Cancer Ret. 78, 329-332

49. Toi, M., Mukaida,

pro-

H., Wada, T, Hirabyashi, N., Toge, T, Hori, effect of erbstatin on hu-

T, and Umezawa, K. (1990) Antineoplastic

man mammary and esophageal tumors in athymic nude mice. Eur. J. Cancer 26, 722-724 50. Dvir, A., Milner, Y., Chomsky, 0., Gion, C., Gazit, A., and Levitzki, A. (1991) The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.j Cell BioL 113, 857-865 51. Rider, G. E., Krawiec, J. A., McVety, K., Gazit, A., Gion, C., Lyall, R., Zilberstein, A., Levitzki, A., Perrone, M. H., and Schreiber, A. B.

DNA

and associPhysiol. 260, C721-

synthesis

C730

H. N., Bravo, M. A., Avila, R. E., Galanopulos, T., J., Maxwell, M., and Selman, M. (1991) Platelet derived growth factor in idiotypic pulmonary fibrosis. j Clin. Invest. Neville-Godden,

86,

1055-106

53. Katoh, 0., Kirmura,

A., Itoh, T, and Kuramoto, A. (1990) PDGF is increased in bone marrow megacaryocytes with myelo proliferative disorders. Am. j Hematol. 35, 145-150 54. Bryckaert, M. C., Eldor, A., Gazit, A., Osherov N., Gilon, C., Fontenay, M., Levitzki, A., and Tobelem, G. (1992) Inhibition of platelet derived growth factor (PDGF) induced mitogenic activity by PDGF receptor tyrosine kinase tyrphostin inhibitors. Exp. CelL Ret. 199, messenger

RNA

255- 261 55. Volberg, T, Zik, Y., Dror, R., Sabanay, I., Gilon, C., Levitzki, A., and Geiger, G. (1992) The effect of tyrosine specific protein phosphorylation on the assembly of adheins type junctions. EMBOJ 11, 1733-1742 56. Umezawa, K., Tanaka, K., Hor, T, Abe, S., Sekizawa, K., and Imoto, M. (1991) Induction of morphological changes by tyrosine kinase inhibitors

in Rous

sarcoma

virus

transformed

rat kidney

cells. FEBS

Lett. 279, 132-136 57. Uehara, Y., Murakami, Y., Sugimoto, Y., and Mizuno, S. (1989) Mechanism of reversion of Rous sarcoma virus transformation by herbimycin A: reduction of total phosphotyrosine levels due to reduced kinase activity and increased turnover of p6O”. Cancer Ret. 49, 780-785

58. Uehara, Y., Fukazawa, H., Murakami, Y., and Mizuno, 5. (1989) Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation

59.

60.

61.

62. 63.

K., Hori, T., Tajima, H., Imoto, M., Issihiki, K., and

Takeuchi, T. (1990) Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors. FEBS LeU. 260, 198-200 46. Atluru, S., and Atluru, D. (1991) Evidence that genistein, a protein tyrosine inhibitor, inhibits CD28 monoclonal antibody stimulated

PDGF-iiiduced

52. Antoniades,

of EGF-induced a tyrosine

phospholipase C activation in A431 cells by erbstatin, kinase inhibitor. Biocham. Biophys. Ret. Commun. 173,

inhibit

ated early events in smooth muscle cells. Am. j

cells.

Cancer Res. 81, 645-652

phosphorylation EGF-receptor

(1991) Tyrphostins

64. 65.

by sulfhydryl

compounds.

Biochem. Biophys. Res.

Commun. 163, 803-809 Kondo, K., Watanabe, T., Sasaki, H., Uehara, Y., and Oishi, M. (1989) Induction of in vitro differentiation of mouse embryonal carcinoma (F9) and erythroleukemia (MEL) cells by herbimycin A, an inhibitor of protein phosphorylation. j Cell Biol. 109, 285-293 Honma, Y., Okabe-Kado, J., Hozumi, M., Uehara, Y., and Mizuno, S. (1989) Induction of erythroid differentiation of K562 human leukemic cells by herbimycin A, an inhibitor of tyrosine kinase activity. Cancer Ret. 49, 331-334 Rendu, F., Eldor, A., Grelac, F., Levy-Toledano, S., and Levitzki, A. (1990) Tyrosine kinase blockers: new platelet activation inhibitors. Blood CoaguL Filimolysis. 1, 713-716 Rendu, F., Eldor, A., Gerlac, F., Gazit, A., Gion, C., Levy-Toledano, S., and Levitzki, A. (1992) Inhibition of platelet activation by tyrosine kinase inhibitors. Biochem. PharmacoL 44, 881-888 Salari, H., Duronio, V., Howard, S. L., Demos, M.,Jones, K., Reany, A., Hudson, A. T., and Pelech, S. L. (1990) Erbstatin blocks PAF induced protein tyrosine kinase phosphox-ylation, polyphosphorisitide hydrolysis, protein kinase activation, serotonin secretion and aggregation of rabbit platelets. FEBS Leti. 263, 104-108 Padeh, S., Levitzki, A., Gazit, A., Mills, G. B., and Roifman, C. M. (1991) Activation of phospholypase C in human B cells is dependent of tyrosine phosphorylation. j Gun. Invest. 87, 1114-1118 Roifman, C., Mills, G. B., Chin, K., Gazit, A., Gilon, C., and

Levitzki, A. (1991) Tyrosine phosphorylation is an essential event in the stimulation of B lymphocytes by Staphylococcus aureus Cowen I. f ImmunoL 146, 2965-2971 66. June, C. H., Fletcher, M. C., Ledbetter,J. A., Schieven, G. L., Siegel, J. N., Phillips, A. F., and Samelson, L. E. (1990) Inhibition of tyrosine phosphorylation prevents T-cell receptor-mediated signal transduction. Proc. Nat!. Acad. &i. USA 87, 7722-7726

67. Travillyan,J.

M., Lu, Y., Durgaprasadarao,

A., Phillips, C. A., and

Bjorndahl,J. M. (1990) Differential inhibition of T cell receptor signal transduction and early activation events by a selective inhibitor of protein-tyrosine kinase. f ImmunoL 145, 3223-3230

68. Daya-Makin,

M., Pelech, S., Levitzki, A., and Hudson,

Erbstatin and tyrphostins block protein meiotic maturation of sea star oocytes.

P. T (1991) and Biochem. Biophys. Acta 1093,

serine

kinase

activation

87-94

3282 Vol. 6 November 1992 The FASEBJournal LEVITZKI ww.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on August 17, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNumbe